METHODS OF PRODUCING THE SAME

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of US Provisional Application No. 62/220,227, filed September 17, 2015, and US Provisional Application No.

62/334,437, filed May 10, 2016, which are incorporated by reference in their entirety for any purpose.

FIELD

[001] This application relates to compositions derived from placental tissue and methods of preparing and using those compositions.

INTRODUCTION AND SUMMARY

[002] This application provides for compositions comprising perforated, unseparated amnion/ chorion; perforated, separated amnion; or perforated, separated chorion and methods of preparing and using those compositions.

[003] Since the early 1900s, grafts derived from the placental membranes have been used in skin transplantation as wound dressings, patch grafts, and in the management of burns and ulcers. Placental grafts have also been used in a variety of other procedures, including tympanoplasty, arthroplasty, in the reconstruction of the oral cavity, bladder, vagina, and in the regeneration of the ocular surface and peripheral nerves.

[004] The placenta includes the amniotic sac, which develops from extraembryonic tissue and separates the developing fetus from the maternal endometrium. The amniotic sac includes a thin but resilient pair of membranes— the amnion and the chorion— separated by an intermediate (or spongy) layer. The amnion is the innermost membrane of the amniotic sac and is in contact with the amniotic fluid, the fetus, and the umbilical cord. The amnion is composed of four layers: an epithelial monolayer, a basement membrane, a compact layer, and a fibroblast layer. The chorion, which is loosely connected to the intermediate layer, forms the outermost layer of the amniotic sac separating the amnion from the maternal endometrium. The chorion is composed of three layers: a reticular layer, a basement membrane, and a trophoblast layer. The chorion is generally several times thicker than the amnion, but the amnion is understood to be stronger and stiffer than the chorion.

[005] It is understood that during the second trimester of pregnancy as the placenta grows, the trophoblast layer of the chorion comes into contact with the capsular decidua layer (the portion of the maternal endometrium facing the uterine cavity). It is also understood that as the capsular decidua degenerates, the cells of the trophoblast layer of the chorion become intermingled with and morphologically indistinguishable from the remaining cells of the capsular decidua. In addition, maternal blood vessels are understood to often be present in the capsular decidua layer. K. Benirschke et aL, Pathology of the Human Placenta, DOI 10.10071978-3-642-23941 - 0_1 1 , © Springer-Verlag Berlin Heidelberg 2012; R.N. Baergen, Manual of Pathology of the Human Placenta, DO1 10.1007 / 978-1 -4419-7494-5 _7, © Springer Science+Business Media, LLC 201 1.

[006] Placental membranes are complicated tissues composed of numerous components, such as growth factors, cells, and extracellular matrix molecules.

Placental membranes are understood to possess many biological properties important for tissue regeneration and wound healing, such as promotion of cell migration and growth; low immunogenicity; and anti-fibrosis, anti- scarring, anti-microbial, antiinflammatory, and anti-pain activities. Placental membranes may also serve as a scaffold for cell proliferation and differentiation.

[007] Current methods of preparing placental membrane grafts involve manually separating the amnion from the chorion to allow access to and removal of blood remnants. For example, after the placenta is recovered, blood clots can form or accumulate inside and on the surfaces of the amnion, intermediate layer, and chorion. Because transmission of blood-borne infectious diseases is a risk associated with tissue transplantation, removal of blood remnants is an important safety

consideration when processing placental membrane grafts.

[008] When the amnion and chorion are separated, the intermediate layer or the reticular layer of the chorion are exposed, risking loss of important membrane components during subsequent processing steps. Even gentle rinsing or cleansing of separated amnion or separated chorion may result in removal of important tissue regeneration and wound healing components, including growth factors, cytokines, other regulatory factors, and extracellular matrix molecules.

[009] A separated amnion graft is thin and can result in difficulties when positioning the graft during transplantation. Such a separated amnion graft may have a tendency to roll up on itself during transplantation or may be difficult to secure in place with sutures. It is understood that one or more additional separated amnion or separated chorion sheets may be layered on top of a separated amnion graft during processing to reinforce the graft, to improve its handling during transplantation, and to increase longevity of the graft post-transplantation.

[0010] A method of preparing a graft such that the amnion and chorion are not separated may be advantageous. Accordingly, a method of preparing a composition comprising at least one perforated, unseparated amnion/ chorion sheet according to the present invention may be advantageous. One potential benefit of perforating unseparated amnion/ chorion during processing may be more efficient removal of blood remnants. For example, a perforated, unseparated amnion/ chorion may allow fluids to pass through the basement membrane of the amnion or the basement membrane of the chorion, and flush out blood remnants from the inner layers of the sheet. In addition, perforating an unseparated amnion/ chorion may allow chemicals to more readily reach the intermediate layer and the inner layers of the amnion and chorion. Other potential benefits of perforating unseparated amnion/ chorion according to the present invention may be increased retention of tissue regeneration and wound healing components or increased preservation of the structure and integrity of the layers compared to processing placental tissue after separating the amnion and chorion. In addition, perforated, unseparated

amnion/ chorion may have increased thickness and improved handling characteristics compared to separated amnion and chorion.

[0011] Moreover, perforating unseparated amnion/ chorion, separated amnion, or separated chorion at a time prior to preservation may result in a product with unique properties compared to grafts that were not perforated during processing. For example, perforating prior to preservation may allow the tissue or certain layers of the tissue (e.g., the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, or the reticular layer of the chorion) to absorb fluids more readily and swell. In addition, unseparated amnion/ chorion, separated amnion, or separated chorion that has been perforated prior to preservation may appear sponge-like or have increased thickness compared to grafts that were not perforated during processing. Those unique properties may result in better handling of the graft during transplant or improved healing capacity.

[0012] In some embodiments, compositions comprising at least one perforated, unseparated amnion/ chorion sheet are provided. In some embodiments, a composition comprises at least one unseparated amnion/ chorion sheet, wherein at least one unseparated amnion/ chorion sheet comprises a perforation.

[0013] In some embodiments, the disclosure provides an unseparated amnion/ chorion sheet comprising a perforation. In some embodiments, the disclosure provides a separated amnion sheet comprising a perforation, wherein the separated sheet comprises an intermediate layer. In some embodiments, the disclosure provides a composition comprising at least one separated amnion sheet, wherein the at least one separated amnion sheet comprises a perforation, and wherein the at least one separated amnion sheet comprises an intermediate layer. In some embodiments, the disclosure provides a separated chorion sheet comprising a perforation, wherein the separated chorion sheet comprises an intermediate layer. In some embodiments, the disclosure provides a composition comprising at least one separated chorion sheet, wherein the at least one separated chorion sheet comprises a perforation, and wherein the at least one separated chorion sheet comprises an intermediate layer.

[0014] In some embodiments, the epithelial layer of the amnion is partially removed or substantially removed from the unseparated amnion/ chorion sheet or from the separated amnion sheet. In some embodiments, the unseparated

amnion/ chorion sheet comprises an intermediate layer.

[0015] In some embodiments, the perforation partially traverses the thickness of the sheet. In some embodiments, the perforation traverses at least one layer of the unseparated amnion/ chorion sheet selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[0016] In some embodiments, the perforation traverses or partially traverses a combination of layers of the unseparated amnion/ chorion sheet, wherein the combination of layers is selected from: the epithelial layer of the amnion and the basement membrane of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, and the compact layer of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, and the reticular layer of the chorion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, and the basement membrane of the chorion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion; the basement membrane of the amnion and the compact layer of the amnion; the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, and the reticular layer of the chorion; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, and the basement membrane of the chorion; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion; the trophoblast layer of the chorion and the basement membrane of the chorion; the trophoblast layer of the chorion, the basement membrane of the chorion, and the reticular layer of the chorion; the trophoblast layer of the chorion, the basement membrane of the chorion, the reticular layer of the chorion, and the intermediate layer; the trophoblast layer of the chorion, the basement membrane of the chorion, the reticular layer of the chorion, the intermediate layer, and the fibroblast layer of the amnion; and the trophoblast layer of the chorion, the basement membrane of the chorion, the reticular layer of the chorion, the intermediate layer, the fibroblast layer of the amnion, and the compact layer of the amnion.

[0017] In some embodiments, the perforation traverses at least one layer of the separated amnion sheet selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer.

[0018] In some embodiments, the perforation traverses or partially traverses a combination of layers of the separated amnion sheet, wherein the combination of layers is selected from: the epithelial layer of the amnion and the basement membrane of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, and the compact layer of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer; the basement membrane of the amnion and the compact layer of the amnion; the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer; the intermediate layer and the fibroblast layer of the amnion; the intermediate layer, the fibroblast layer of the amnion, and the compact layer of the amnion; and the fibroblast layer of the amnion and the compact layer of the amnion.

[0019] In some embodiments, the perforation traverses at least one layer of the separated chorion sheet selected from the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[0020] In some embodiments, the perforation traverses or partially traverses a combination of layers of the separated chorion sheet, wherein the combination of layers is selected from: the trophoblast layer of the chorion and the basement membrane of the chorion; the trophoblast layer of the chorion, the basement membrane of the chorion, and the reticular layer of the chorion; the trophoblast layer of the chorion, the basement membrane of the chorion, the reticular layer of the chorion, and the intermediate layer; the intermediate layer and the reticular layer of the chorion; the intermediate layer, the reticular layer of the chorion, and the basement membrane of the chorion; and the reticular layer and the basement membrane of the chorion.

[0021] In some embodiments, the perforation traverses the basement membrane of the amnion or traverses the basement membrane of the chorion. In some embodiments, the perforation does not traverse the basement membrane of the amnion or does not traverse the basement membrane of the chorion. In some embodiments, the perforation traverses the thickness of the unseparated

amnion/ chorion sheet, the thickness of the separated amnion sheet, or the thickness of the separated chorion sheet.

[0022] In some embodiments, the composition comprises one or more additional sheets, wherein the one or more additional sheets is selected from a perforated, unseparated amnion/ chorion sheet; an imperforated, unseparated amnion/ chorion sheet; a perforated, separated amnion sheet; an unperforated, separated amnion sheet; a perforated, separated chorion sheet; and an unperforated, separated chorion sheet.

[0023] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition is derived from a human placenta.

[0024] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition is dehydrated, cry opre served, or fro2en.

[0025] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition is dehydrated by lyophili2ation, heat-drying, oven-drying, air-drying, or chemical dehydration.

[0026] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition is sterile.

[0027] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition comprises a marking. In some embodiments, the marking is selected from an embossment, a stain, a tint, a stamp, and a cut. In some embodiments, the unseparated

amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition comprises a dye. In some embodiments, the dye is a selected from a natural dye, a vegetable dye, a hypoallergenic dye, and beet juice.

[0028] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition is substantially free of blood remnants.

[0029] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition has a hemoglobin content of less than a value, of greater than a value of, of at least a value, of a value, or ranging from any two values, wherein the value is selected from about 13 μg/ mg, about 12 μg/ mg, about 11 μg/ mg, about 10 μg/ mg, about 9 μg/ mg, about 8 μg/ mg, about 7 μg/ mg, about 6 μg/ mg, about 5 μg/ mg, about 4 μg/ mg, about 3 μg/ mg, about 2 μg/ mg, about 1 μg/ mg, about 0.9 μg/ mg, about 0.8 μg/ mg, about 0.7 μg/ mg, about 0.6 μg/ mg, about 0.5 μg/ mg, about 0.4 μg/ mg, about 0.3 μg/ mg, about 0.2 μg/ mg, about 0.1 μg/ mg, about 0.09 μg/ mg, about 0.08 μg/ mg, about 0.07 μg/ mg, about 0.06 μg/ mg, about 0.05 μg/ mg, about 0.04 μg/ mg, about

0.03 μg/ mg, about 0.02 μg/ mg, and about 0.01 μg/ mg.

[0030] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition has a dry mass of less than a value, of greater than a value, of at least a value, of a value, or ranging from any two values, wherein the value is selected from about 1 mg/ cm ² , about 2 mg/ cm ² , about 3 mg/ cm ² , about 4 mg/ cm ² , about 5 mg/ cm ² , about

6 mg/ cm ² , about 7 mg/ cm ² , about 8 mg/ cm ² , about 9 mg/ cm ² , about 10 mg/ cm ² , about 11 mg/ cm ² , about 12 mg/ cm ² , about 13 mg/ cm ² , about 14 mg/ cm ² , about 15 mg/ cm ² , about 16 mg/ cm ² , about 17 mg/ cm ² , about 18 mg/ cm ² , about

19 mg/ cm ² , about 20 mg/ cm ² , about 21 mg/ cm ² , about 22 mg/ cm ² , about

23 mg/ cm ² , about 24 mg/ cm ² , and about 25 mg/ cm ² .

[0031] In some embodiments, the unseparated amnion/chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition has a water absorption potential of less than a value, of greater than a value, of at least a value, of a value, or ranging from any two values, wherein the value is selected from about 1 mg/ cm ² , about 2 mg/ cm ² , about 3 mg/ cm ² , about 4 mg/ cm ² , about 5 mg/ cm ² , about 6 mg/ cm ² , about 7 mg/ cm ² , about 8 mg/ cm ² , about 9 mg/ cm ² , about 10 mg/ cm ² , about 11 mg/ cm ² , about 12 mg/ cm ² , about 13 mg/ cm ² , about 14 mg/ cm ² , about 15 mg/ cm ² , about 16 mg/ cm ² , about 17 mg/ cm ² , about 18 mg/ cm ² , about 19 mg/ cm ² , about 20 mg/ cm ² , about 21 mg/ cm ² , about 22 mg/ cm ² , about 23 mg/ cm ² , about 24 mg/ cm ² , about 25 mg/ cm ² , about 26 mg/ cm ² , about 27 mg/ cm ² , about 28 mg/ cm ² , about 29 mg/ cm ² , about 30 mg/ cm ² , about 31 mg/ cm ² , about 32 mg/ cm ² , about 33 mg/ cm ² , about 34 mg/ cm ² , about 35 mg/ cm ² , about 36 mg/ cm ² , about 37 mg/ cm ² , about 38 mg/ cm ² , about 39 mg/ cm ² , and about 40 mg/ cm ² .

[0032] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition has a total protein content of less than a value, of greater than a value, of at least a value, of a value, or ranging from any two values, wherein the value is selected from about 25 μg/ cm ² , about 50 μg/ cm ² , about 75 μg/ cm ² , about 100 μg/ cm ² , about

125 μg/ cm ² , about 150 μg/ cm ² , about 175 μg/ cm ² , about 200 μg/ cm ² , about 225 μg/ cm ² , about 250 μg/ cm ² , about 275 μg/ cm ² , about 300 μg/ cm ² , about 325 μg/ cm ² , about 350 μg/ cm ² , about 375 μg/ cm ² , about 400 μg/ cm ² , about 425 μg/ cm ² , about 450 μg/ cm ² , about 475 μg/ cm ² , about 500 μg/ cm ² , about 525 μg/ cm ² , about 550 μg/ cm ² , about 575 μg/ cm ² , about 600 μg/ cm ² , about 625 μg/ cm ² , about 650 μg/ cm ² , about 675 μg/ cm ² , about 700 μg/ cm ² , about 725 μg/ cm ² , about 750 μg/ cm ² , about 775 μg/ cm ² , about 800 μg/ cm ² , about 825 μg/ cm ² , about 850 μg/ cm ² , about 875 μg/ cm ² , about 900 μg/ cm ² , about 925 μg/ cm ² , about 950 μg/ cm ² , about 975 μg/ cm ² , about 1000 μg/ cm ² , about 1025 μg/ cm ² , about 1050 μg/ cm ² , about 1075 μg/ cm ² , about 1100 μg/ cm ² , about 1125 μg/cm ² , about 1150 μg/cm ² , about 1175 μg/cm ² , about 1200 μg/cm ² , about 1225 μg/ cm ² , about 1250 μg/ cm ² , about 1275 μg/ cm ² , about 1300 μg/ cm ² , about 1325 μg/ cm ² , about 1350 μg/ cm ² , about 1375 μg/ cm ² , about 1400 μg/ cm ² , about 1425 μg/ cm ² , about 1450 μg/ cm ² , about 1475 μg/ cm ² , about 1500 μg/ cm ² , about 1525 μg/ cm ² , about 1550 μg/ cm ² , about 1575 μg/ cm ² , about 1600 μg/ cm ² , about 1625 μg/ cm ² , about 1650 μg/ cm ² , about 1675 μg/ cm ² , about 1700 μg/ cm ² , about 1725 μg/ cm ² , about 1750 μg/ cm ² , about 1775 μg/ cm ² , about 1800 μg/ cm ² , about 1825 μg/ cm ² , about 1850 μg/ cm ² , about 1875 μg/ cm ² , about 1900 μg/ cm ² , about 1925 μg/ cm ² , about 1950 μg/ cm ² , about 1975 μg/ cm ² , and about 2000 μg/ cm ² .

[0033] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition has a total protein content of less than a value, of greater than a value, of at least a value, of a value, or ranging from any two values, wherein the value is selected from about 0.1 mg/ cm ² , about 0.2 mg/ cm ² , about 0.3 mg/ cm ² , about 0.4 mg/ cm ² , about 0.5 mg/ cm ² , about 0.6 mg/ cm ² , about 0.7 mg/ cm ² , about 0.8 mg/ cm ² , about 0.9 mg/ cm ² , about 1.0 mg/ cm ² , about 1.1 mg/ cm ² , about 1.2 mg/ cm ² , about 1.3 mg/ cm ² , about 1.4 mg/ cm ² , about 1.5 mg/ cm ² , about 1.6 mg/ cm ² , about 1.7 mg/ cm ² , about 1.8 mg/ cm ² , about 1.9 mg/ cm ² , and about 2.0 mg/ cm ² .

[0034] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition has a mean thickness of less than a value, of greater than a value, of at least a value, of a value, or ranging from any two values, wherein the value is selected from about 10 μίη, about 20 μίη, about 30 μίη, about 40 μίη, about 50 μίη, about 60 μίη, about 70 μίη, about 80 μίη, about 90 μίη, about 100 μίη, about 110 μίη, about 120 μίη, about 130 μίη, about 140 μίη, about 150 μίη, about 160 μίη, about 170 μίη, about 180 μίη, about 190 μίη, about 200 μίη, about 210 μίη, about 220 μίη, about 230 μίη, about 240 μίη, about 250 μίη, about 260 μίη, about 270 μίη, about 280 μίη, about 290 μίη, about 300 μίη, about 310 μίη, about 320 μίη, about 330 μίη, about 340 μίη, about 350 μίη, about 360 μίη, about 370 μίη, about 380 μίη, about 390 μίη, about 400 μίη, about 410 μίη, about 420 μίη, about 430 μίη, about 440 μίη, about 450 μίη, about 460 μίη, about 470 μίη, about 480 μίη, about 490 μίη, about 500 μίη, about 510 μίη, about 520 μίη, about 530 μίη, about 540 μίη, about 550 μίη, about 560 μίη, about 570 μίη, about 580 μηι, about 590 μηι, about 600 μηι, about 700 μηι, about 710 μηι, about 720 μηι, about 730 μηι, about 740 μηι, about 750 μηι, about 760 μηι, about 770 μηι, about 780 μηι, about 790 μηι, about 800 μηι, about 810 μηι, about 820 μηι, about 830 μηι, about 840 μηι, about 850 μηι, about 860 μηι, about 870 μηι, about 880 μηι, about 890 μηι, about 900 μηι, about 910 μηι, about 920 μηι, about 930 μηι, about 940 μηι, about 950 μηι, about 960 μηι, about 970 μηι, about 980 μηι, about 990 μηι, about 1000 μηι (about 1 mm), about 1100 μηι, about 1200 μηι, about 1300 μηι, about 1400 μΐιι, about 1500 μηι, about 1600 μηι, about 1700 μΐιι, about 1800 μηι, about 1900 μΐιι, and about 2000 μηι (about 2 mm).

[0035] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition comprises an intermediate layer having a mean thickness of less than a value, of at least a value, of greater than a value, of a value, or ranging from any two values, wherein the value is selected from about 1 μηι, about 2 μηι, about 3 μηι, about 4 μηι, about 5 μηι, about 10 μΐιι, about 15 μηι, about 20 μηι, about 25 μηι, about 30 μηι, about 35 μηι, about 40 μΐιι, about 45 μηι, about 50 μηι, about 55 μηι, about 60 μηι, about 65 μηι, about 70 μΐιι, about 80 μηι, about 90 μηι, about 100 μηι, about 110 μηι, about 120 μηι, about, about 130 μηι, about 140 μηι, about 150 μηι, about 160 μηι, about 170 μηι, about 180 μΐιι, about 190 μηι, about 200 μηι, about 210 μηι, about 220 μΐιι, about 230 μΐη, about 240 μΐιι, about 250 μηι, about 260 μηι, about 270 μΐη, about 280 μηι, about 290 μΐιι, about 300 μηι, about 310 μηι, about 320 μΐη, about 330 μηι, about 340 μΐη, about 350 μηι, about 360 μηι, about 370 μΐη, about 380 μηι, about 390 μΐη, about 400 μΐιι, about 410 μηι, about 420 μΐη, about 430 μηι, about 440 μΐιι, about 450 μΐη, about 460 μηι, about 470 μΐιι, about 480 μηι, about 490 μΐη, about 500 μηι, about 510 μΐιι, about 520 μΐη, about 530 μηι, about 540 μΐη, about 550 μηι, about 560 μΐη, about 570 μΐιι, about 580 μηι, about 590 μΐη, and about 600 μΐη.

[0036] In some embodiments, the unseparated amnion/ chorion sheet, the separated amnion sheet, the separated chorion sheet, or the composition has a surface area of less than a value, of greater than a value, of at least a value, of a value, or ranging from any two values, wherein the value is selected from about 1 mm ² , about 2 mm ² , about 3 mm ² , about 4 mm ² , about 5 mm ² , about 6 mm ² , about 7 mm ² , about 8 mm ² , about 9 mm ² , about 10 mm ² (or about 1 cm ² ), about 2 cm ² , about 3 cm ² , about 4 cm ² , about 5 cm ² , about 6 cm ² , about 7 cm ² , about 8 cm ² , about 9 cm ² , about 10 cm ² , about 11 cm ² , about 12 cm ² , about 13 cm ² , about 14 cm ² , about 15 cm ² , about 16 cm ² , about 17 cm ² , about 18 cm ² , about 19 cm ² , about 20 cm ² , about 30 cm ² , about 40 cm ² , about 50 cm ² , about 60 cm ² , about 70 cm ² , about 80 cm ² , about 90 cm ² , about 100 cm ² , about 10 dm ² , about 20 dm ² , about 30 dm ² , about 40 dm ² , and about 40 dm ² .

[0037] In some embodiments, the perforation comprises one or more holes having a depth of less than a value, of greater than a value, of at least a value, of a value, or ranging from any two values, wherein the value is selected from about 1 μηι, about 2 μΐη, about 3 μηι, about 4 μηι, about 5 μηι, about 6 μηι, about 7 μηι, about 8 μΐιι, about 9 μηι, about 10 μηι, about 11 μηι, about 12 μηι, about 13 μηι, about 14 μΐη, about 15 μηι, about 16 μηι, about 17 μηι, about 18 μηι, about 19 μηι, about 20 μΐη, about 25 μηι, about 30 μηι, about 40 μηι, about 50 μηι, about 60 μηι, about 70 μΐη, about 80 μηι, about 90 μηι, about 100 μηι, about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about

5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, and about 10 mm.

[0038] In some embodiments, the perforation comprises one or more holes having an opening with an area of less than a value, of greater than a value, of at least a value, of an value, or ranging from any two values, wherein the value is selected from about 1 μηι ² , about 2 μηι ² , about 3 μηι ² , about 4 μηι ² , about 5 μηι ² , about

6 μηι ² , about 7 μηι ² , about 8 μηι ² , about 9 μηι ² , about 10 μηι ² , about 11 μηι ² , about 12 μηι ² , about 13 μηι ² , about 14 μηι ² , about 15 μηι ² , about 16 μηι ² , about 17 μηι ² , about 18 μηι ² , about 19 μηι ² , about 20 μηι ² , about 25 μηι ² , about 30 μηι ² , about

40 μηι ² , about 50 μηι ² , about 60 μηι ² , about 70 μηι ² , about 80 μηι ² , about 90 μηι ² , about 100 μηι ² , about 0.1 mm ² , about 0.2 mm ² , about 0.3 mm ² , about 0.4 mm ² , about 0.5 mm ² , about 0.6 mm ² , about 0.7 mm ² , about 0.8 mm ² , about 0.9 mm ² , about 1 mm ² , about 2 mm ² , about 3 mm ² , about 4 mm ² , about 5 mm ² , about 6 mm ² , about 7 mm ² , about 8 mm ² , about 9 mm ² , about 10 mm ² , about 1 cm ² , about 2 cm ² , about 3 cm ² , about 4 cm ² , about 5 cm ² , about 6 cm ² , about 7 cm ² , about 8 cm ² , about 9 cm ² , and about 10 cm ² .

[0039] In some embodiments, the perforation comprises a plurality of holes, wherein the combined area of the openings of the holes is less than a percentage of the surface area of the sheet or the composition, greater than a percentage of the surface area of the sheet or the composition, at least a percentage of the surface area of the sheet or the composition, a percentage of the surface area of the sheet or the composition, or a range of any two percentages of the sheet or the composition, wherein the percentage is selected from about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, and about 50%.

[0040] In some embodiments, the disclosure provides a product prepared by powderizing a sheet or composition disclosed herein.

[0041] In some embodiments, the disclosure provides a method of preparing a composition comprising perforating a tissue selected from an unseparated

amnion/ chorion tissue, a separated amnion tissue, and a separated chorion tissue. In some embodiments, the disclosure provides a method of preparing a composition comprising perforating a sheet selected from an unseparated amnion/ chorion sheet, a separated amnion sheet, and a separated chorion sheet.

[0042] In some embodiments, the tissue or the sheet comprises an

intermediate layer. [0043] In some embodiments, the perforating comprises making one or more holes that partially traverse the thickness of the tissue or the sheet.

[0044] In some embodiments, the perforating comprises making one or more holes that traverse at least one layer of the unseparated amnion/ chorion tissue or at least one layer of the unseparated amnion/ chorion sheet selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[0045] In some embodiments, the perforating comprises making one or more holes that traverse or partially traverse a combination of layers of the unseparated amnion/ chorion tissue or of the unseparated amnion/ chorion sheet, wherein the combination of layers is selected from: the epithelial layer of the amnion and the basement membrane of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, and the compact layer of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, and the reticular layer of the chorion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, and the basement membrane of the chorion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion; the basement membrane of the amnion and the compact layer of the amnion; the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, and the reticular layer of the chorion; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, and the basement membrane of the chorion; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion; the trophoblast layer of the chorion and the basement membrane of the chorion; the trophoblast layer of the chorion, the basement membrane of the chorion, and the reticular layer of the chorion; the trophoblast layer of the chorion, the basement membrane of the chorion, the reticular layer of the chorion, and the intermediate layer; the trophoblast layer of the chorion, the basement membrane of the chorion, the reticular layer of the chorion, the intermediate layer, and the fibroblast layer of the amnion; and the trophoblast layer of the chorion, the basement membrane of the chorion, the reticular layer of the chorion, the intermediate layer, the fibroblast layer of the amnion, and the compact layer of the amnion.

[0046] In some embodiments, the perforating comprises making one or more holes that traverse at least one layer of the separated amnion tissue or the separated amnion sheet selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer.

[0047] In some embodiments, the perforating comprises making one or more holes that traverse or partially traverse a combination of layers of the separated amnion tissue or the separated amnion sheet, wherein the combination of layers is selected from: the epithelial layer of the amnion and the basement membrane of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, and the compact layer of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion; the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer; the basement membrane of the amnion and the compact layer of the amnion; the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion; the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, and the intermediate layer; the intermediate layer and the fibroblast layer of the amnion; the intermediate layer, the fibroblast layer of the amnion, and the compact layer of the amnion; and the fibroblast layer of the amnion and the compact layer of the amnion.

[0048] In some embodiments, the perforating comprises making one or more holes that traverse at least one layer of the separated chorion tissue or the separated chorion sheet selected from the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[0049] In some embodiments, the perforating comprises making one or more holes that traverse or partially traverse a combination of layers of the separated chorion tissue or the separated chorion sheet, wherein the combination of layers is selected from: the trophoblast layer of the chorion and the basement membrane of the chorion; the trophoblast layer of the chorion, the basement membrane of the chorion, and the reticular layer of the chorion; the trophoblast layer of the chorion, the basement membrane of the chorion, the reticular layer of the chorion, and the intermediate layer; the intermediate layer and the reticular layer of the chorion; the intermediate layer, the reticular layer of the chorion, and the basement membrane of the chorion; and the reticular layer and the basement membrane of the chorion.

[0050] In some embodiments, the perforating comprises making one or more holes that traverse the basement membrane of the amnion or traverse the basement membrane of the chorion. In some embodiments, the perforating comprises making one or more holes that do not traverse the basement membrane of the amnion or do not traverse the basement membrane of the chorion. In some embodiments, the perforating comprises making one or more holes that traverse the thickness of the tissue or the sheet.

[0051] In some embodiments, the tissue, sheet, or composition is derived from a human placenta.

[0052] In some embodiments, the perforating comprises using a perforator to make the one or more holes that traverse or partially traverse a tissue, a sheet, a layer, or a combination of layers, wherein the perforator is selected from a needle, a roller with one or more pins, a stamper with one or more pins, a hole punch, a punch and a die, a die cutter, a drill and a bit, a blade, scalpel, a sharp object, a skin graft mesher with one or more blades, a high-pressure water gun, a high-pressure air gun, and a laser.

[0053] In some embodiments, the method comprises washing the tissue or the sheet with at least one suitable washing medium. In some embodiments, the washing is under vacuum, under pressure, by agitating, or by soaking. In some embodiments, the agitating is performed using a rocker, shaker, stir plate, or rotating mixer.

[0054] In some embodiments, the method comprises at least one washing step, at least two washing steps, at least three washing steps, at least four washing steps, at least five washing steps, at least six washing steps, at least seven washing steps, at least eight washing steps, at least nine washing steps, at least ten washing steps, or ten or more washing steps, and wherein at least one washing step comprises washing the tissue or the sheet with at least one suitable washing medium. In some embodiments, at least one washing step comprises exchanging the at least one washing medium at least once, once, at least twice, twice, at least thrice, thrice, at least four times, four times, at least five times, five times, at least six times, six times, at least seven times, seven times, at least eight times, eight times, at least nine times, nine times, at least ten times, ten times, or ten or more times. [0055] In some embodiments, the exchanging comprises transferring the tissue or the sheet to the at least one washing medium or transferring the at least one washing medium to a container comprising the tissue or the sheet.

[0056] In some embodiments, the at least one washing step comprises heating or cooling the tissue, the sheet, or the at least one suitable washing medium. In some embodiments, the heating or cooling comprises placing the tissue, the sheet, or the at least one suitable washing medium in an environment having a temperature above, at, or below a refrigeration temperature, a room temperature, or a heated temperature.

[0057] In some embodiments, the heating or cooling comprises placing the tissue, the sheet, or the at least one suitable washing medium in an environment, wherein the environment has less than a temperature of, greater than a temperature of, at least a temperature of, a temperature of, or a range of any two temperatures, wherein the temperature is selected from about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, and about 50°C.

[0058] In some embodiments, the tissue, the sheet, or the at least one suitable washing medium is placed in an environment for less than a period of time, greater than a period of time, at least a period of time, a period of time, or a range of any two periods of time, wherein the period of time is selected from about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about

5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 64 hours, and about 72 hours.

[0059] In some embodiments, the temperature of the environment changes over time. In some embodiments, the temperature of environment does not change over time.

[0060] In some embodiments, the at least one suitable washing medium is selected from water, a sodium chloride solution, a sodium hypochlorite solution, a hydrogen peroxide solution, Lactated Ringer's solution, Ringer's solution, phosphate- buffered saline (PBS), tris-buffered saline (TBS), Hank's balanced salt solution (HBSS), Dulbecco's phosphate-buffered saline (DPBS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced salt solution (GBSS), and a cell culture medium. In some embodiments, the sodium chloride solution is a solution containing a w/v concentration of NaCl of less than a value, of greater than a value, of at least a value, a value, or a range of any two values, wherein the value is selected from about 0.45%, about 0.9%, about 1.8%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 34%, about 35%, and about 36%. In some embodiments, the sodium chloride solution comprises one or more ingredients selected from phosphate, potassium, magnesium, calcium, dextrose, glucose, citrate, lactate, tris, and HEPES. In some embodiments, the cell culture medium is Delbecco's Modified Eagle Medium (DMEM) or Minimum Essential Media (MEM).

[0061] In some embodiments, the method comprises pretreating the tissue or the sheet before perforating, before washing, or between any two washing steps. In some embodiments, the pretreating comprises picking material away from the tissue or sheet, or dabbing, rubbing, or massaging the tissue or sheet. [0062] In some embodiments, the method comprises separating the chorion from the amnion and intermediate layer before perforating, after perforating, before washing, after washing, before at least one wash step, or after at least one wash step. In some embodiments, the method comprises separating the amnion from the chorion and intermediate layer before perforating, after perforating, before washing, after washing, before at least one wash step, or after at least one wash step.

[0063] In some embodiments, the method comprises layering on a surface of the tissue or the sheet at least one separated amnion sheet, at least one perforated, separated amnion sheet, at least one unperforated, separated amnion sheet, at least one separated chorion sheet, at least one perforated, separated chorion sheet, at least one unperforated, separated chorion sheet, or at least one perforated, unseparated amnion/ chorion sheet.

[0064] In some embodiments, the method comprises marking the tissue, the sheet, or the composition. In some embodiments, the marking is embossing, staining, tinting, stamping, or cutting the tissue, the sheet, or the composition. In some embodiments, the method comprises marking the tissue, the sheet, or the

composition with a dye. In some embodiments, the dye is a selected from a natural dye, a vegetable dye, a hypoallergenic dye, and beet juice.

[0065] In some embodiments, the method comprises preserving the composition, the tissue, or the sheet. In some embodiments, the preserving comprises dehydrating, cryopre serving, or free2ing the composition, the tissue, or the sheet. In some embodiments, the dehydrating comprises lyophili2ing, heat-drying, oven-drying, air-drying, and or chemically dehydrating the composition, the tissue, or the sheet.

[0066] In some embodiments, the method comprises cutting the tissue into one or more sheets or cutting the sheet into a plurality of sheets.

[0067] In some embodiments, the method comprises sterili2ing the

composition, the tissue, or the sheet(s). In some embodiments, the method comprises sterili2ing the composition, the tissue, or the sheet(s) by exposure to gamma radiation, E-beam radiation, ethylene oxide, ethylene oxide with a stabilizing gas (such as carbon dioxide or hydrochlorofluorocarbons (HCFC)), peracetic acid, hydrogen peroxide gas plasma, or ozone.

[0068] In some embodiments, the disclosure provides a composition produced according to a method disclosed herein.

[0069] In some embodiments, the disclosure provides a method of healing a wound of a subject comprising applying a sheet, composition, or product disclosed herein to the wound of the subject. In some embodiments, the disclosure provides a use of a sheet, composition, or product disclosed herein for healing a wound of a subject.

[0070] In some embodiments, the wound is a wound of the eye, a wound of the skin, a wound of a nerve, a wound of a tendon, a wound of the dura. In some embodiments, the eye wound is a burn, a laceration, a corneal ulceration, a conjunctival lesion, or a surgical wound. In some embodiments, the skin wound is a burn, a laceration, a diabetic ulcer, a venous ulcer, an arterial ulcer, a decubitus ulcer, or a surgical wound.

[0071] In some embodiments, the composition is applied so that the amnion side of the unseparated amnion/ chorion sheet, the chorion side of the unseparated amnion/ chorion sheet, the epithelial layer of the amnion, the basement membrane of the amnion, the intermediate layer, the reticular layer of the chorion, or the trophoblast layer of the chorion faces the wound.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0072] As used herein, the term "about" refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values (e.g., +/ - 5-10% of the recited range) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). When terms such as at least and about precede a list of numerical values or ranges, the terms modify all of the values or ranges provided in the list. In some instances, the term about may include numerical values that are rounded to the nearest significant figure.

[0073] As used herein, "a" or "an" means "at least one" or "one or more" unless specified otherwise. As used herein, the term "or" means "and/ or" unless specified otherwise. In the context of a multiple dependent claim, the use of "or" when referring back to other claims refers to those claims in the alternative only.

A. Exemplary Embodiments of Products Made from the Amnion,

Intermediate Layer, and Chorion

[0074] The amnion, intermediate layer, and chorion are composed of numerous components, including cells, growth factors, extracellular matrix molecules, and other biomolecules important for tissue regeneration and wound healing. For example, the amnion, intermediate layer, or chorion may contain cells, such as epithelial cells, fibroblasts, and trophoblasts; growth factors, such as fibroblast growth factors, epidermal growth factor, transforming growth factor (TGF) beta, platelet-derived growth factors, etc.; extracellular matrix molecules such as collagens, elastins, proteoglycans, non-proteoglycan polysaccharides, fibronectins, laminins, nidogens, etc.; and other biomolecules, such as cytokines (e.g., interleukins, TGF- beta, etc.), metalloproteinases, tissue inhibitors of metalloproteinases (TIMPs), etc.

[0075] The amnion consists of four layers: an epithelial monolayer, a basement membrane, a compact layer, and a fibroblast layer. The epithelial layer of the amnion is composed of a single layer of epithelial cells arranged on the basement membrane. Amniotic epithelial cells may include amniotic stem cells or have stem cell-like characteristics. For example, the cells of the epithelial layer of the amnion may be capable of differentiating into or producing, in the presence of a

differentiation-inducing factor, at least one different cell lineage, such as osteogenic, adipogenic, chondrogenic, myogenic, neurogenic, epithelial, or other cell lineage. Amniotic stem cells or cells with stem cell-like characteristics may also be found in other layers of the amnion or chorion. [0076] Amniotic epithelial cells may lack HLA-A, HLA-B (Class IA) and HA-DR (Class II) on their surfaces suggesting that these cells may be

immunologically inert and have reduced risk of rejection or immune reaction upon transplantation.

[0077] The amnion/ chorion may be processed such that the epithelial layer of the amnion is maintained (intact), partially removed, or substantially removed. The epithelial layer may be removed, for example, by using a mechanical procedure or detergent-based procedure. In one embodiment, the epithelial cells are gently scraped away using a cell scraper with the aid of a microscope. In other embodiments, the epithelial layer is removed using an ionic detergent, a nonionic detergent, or a 2witterionic detergent. In yet a further embodiment, after removal of the epithelial layer, the other layers of the amnion/ chorion are retained. As used herein, "partially removed" refers to a percentage of the epithelial layer having been removed in the range of about 1% to about 89% and "substantially removed" refers to a percentage of the epithelial layer having been removed in the range of about 90% to about 99%. In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the epithelial layer is removed.

[0078] The base of the epithelial cell layer is in contact with the basement membrane of the amnion. The basement membrane of the amnion is a thin layer comprising extracellular matrix components, including collagen types III, IV, and V, noncollagenous glycoproteins (e.g., laminins, fibronectins, and nidogens), and proteoglycans (e.g., perlecans). The compact layer of the amnion is a dense, fibrous network comprising extracellular matrix components, including collagens (e.g., collagen types I, III, V, and VI) and fibronectins, and is almost devoid of cells. The fibroblast layer is the thickest layer of the amnion and comprises fibroblasts and extracellular matrix components, such as collagens (e.g., collagen types I, III, and VI) and noncollagenous glycoproteins (e.g., laminins, fibronectins, and nidogens). [0079] The intermediate layer, or spongy layer, is the interface between the amnion and the chorion. The intermediate layer comprises extracellular matrix components, such as collagens (e.g., collagen types I, III, and IV), proteoglycans, and glycoproteins. In some embodiments, "intermediate layer" includes cells or extracellular matrix derived from an intermediate layer. For example, in some embodiments, when an amnion and a chorion are separated along an intermediate layer, the cells or extracellular matrix derived from the intermediate layer may remain associated with the fibroblast layer of the amnion or may remain associated with the reticular layer of the chorion.

[0080] The chorion is several times thicker than the amnion and is composed of three layers: a reticular layer, a basement membrane, and a trophoblast layer. The reticular layer is in contact with the intermediate layer and comprises extracellular components, such as collagens (e.g., collagen types I, III, IV, V, and VI) and proteoglycans. The basement membrane is between the reticular layer and

trophoblast layer of the chorion. Components of the basement membrane of the chorion comprise collagens (e.g., collagen type IV), laminins, and fibronectins. The trophoblast layer comprises several layers of trophoblasts and is in contact with the maternal endometrium. As used herein, the term "trophoblast layer" includes cells, extracellular matrix, or blood vessels that may be present and that are derived from the capsular decidua, the portion of the maternal endometrium facing the uterine cavity.

B. Exemplary Embodiments for Collecting and Evaluating Donor

Placenta

[0081] Placenta may be collected from a human or other mammal, including but not limited to a primate, artiodactyl, peris sodactyl, cow, bison, horse, pig, goat, or the like. Placenta may be recovered from a human during a full-term or near full-term Cesarean (C-section) birth. Potential donor mothers may be screened for risk factors to determine whether the placenta is safe and suitable for donation or processing. In one embodiment, a donor mother is tested for one or more viruses or bacteria using serological tests, which can include without limitation antibody, nucleic acid, or culture testing. The viral or bacterial screen may include screening for the human immunodeficiency virus type 1 or type 2 (HIV-l and HIV-2), the hepatitis B virus (HBV), the hepatitis C virus (HCV), human T-lymphotropic virus type I or type II (HTLV-I and HTLV-II), CMV, or Treponema pallidum (a bacterium that causes syphilis). The placenta of a donor mother may be considered acceptable based on review of her health information or any screening test results.

[0082] A donor placenta may be recovered during an elective C-section procedure performed in a sterile operating room environment. A recovered placenta may be placed in a labeled, sterile container or bag and submerged in a suitable storage medium. For example, a suitable storage medium includes a sodium chloride solution, such as a solution containing a w/v concentration of NaCl of about 0.45%, about 0.9%, about 1.8%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, or at a w/ v concentration of NaCl less than any of the

concentrations listed above, greater than any of the concentrations listed above, of at least any of the concentrations listed above, or a range bounded by any two of the concentrations listed above, such as about 0.45% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 20% to about 25%, about 25% to about 30%, about 30% to about 36%, about 0.45% to about 15%, about 15% to about 33%, or about 15% to about 36%.

[0083] In addition, a suitable storage medium includes a sodium chloride solution as described above that also contains one or more other ingredients, such as phosphate, potassium, magnesium, calcium, dextrose, glucose, citrate, lactate, tris, HEPES, etc. In some embodiments, the suitable storage medium comprises a buffer. Other suitable storage mediums include water (e.g., purified water, sterile water, or water for injection), Lactated Ringer's solution, Ringer's solution, phosphate -buffered saline (PBS), tris-buffered saline (TBS), Hank's balanced salt solution (HBSS), Dulbecco's phosphate -buffered saline (DPBS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced salt solution (GBSS), cell culture mediums (e.g., Delbecco's Modified Eagle Medium (DMEM), Minimum Essential Media (MEM), etc.), or other medium suitable for storing tissue.

[0084] The container or bag may be shipped on wet ice to a processing laboratory for processing and evaluation. If the donor mother's health information and screening tests are satisfactory (i.e., indicate no risk or acceptable level of risk to human handling or use), the donor placenta may be processed for human medical applications.

[0085] After opening the shipment at the processing laboratory, personnel may verify that the sterile container or bag is still sealed and in coolant, and that the donor number on the accompanying paperwork matches the number on the sterile container. Processing of donor placenta for human medical applications may be conducted in a controlled, aseptic environment, such as in a hood or clean room.

C. Exemplary Embodiments for Dissecting or Cutting

[0086] A placenta may be processed by dissecting the unseparated

amnion/ chorion from the placental disc and umbilical cord. As used herein,

"unseparated amnion/ chorion" and "unseparated amnion/ chorion tissue" are used interchangeably throughout to refer to the membrane portion of a placenta, irrespective of whether the membrane portion is attached to or detached from the placental disc and umbilical cord. An unseparated amnion/ chorion may include an intermediate layer or may not include an intermediate layer, the inclusion or exclusion of which may depend on the characteristics of the donor placenta. In addition, unseparated amnion/ chorion, as used herein, includes unseparated amnion/ chorion processed by any step or any combination of steps selected from dissecting, cutting, perforating, washing, sterilizing, and preserving. An unseparated amnion/ chorion, as used herein, includes unseparated amnion/ chorion having an epithelial layer of amnion (also referred to as intact) and unseparated amnion/ chorion in which the epithelial layer of the amnion has been partially removed or substantially removed.

[0087] Amnion and chorion may be separated before, during, or after any step or combination of steps selected from dissecting, cutting, perforating, and washing. Amnion and chorion may be separated by any method understood by a person of ordinary skill in the art, including, but not limited to, gently separating the amnion and chorion by hand. As used herein, "separated amnion" and "separated amnion tissue" interchangeably refer to amnion that has been separated from chorion. A separated amnion may include an intermediate layer or may not include an intermediate layer. For example, in some embodiments, the intermediate layer is substantially intact on the separated amnion. As used herein, "separated chorion" and "separated chorion tissue," interchangeably refer to chorion that has been separated from amnion. A separated chorion may include an intermediate layer or may not include an intermediate layer. For example, in some embodiments, the intermediate layer is substantially intact on the separated chorion. As used herein, an intermediate layer that is substantially intact may include an intermediate layer that appears to the naked eye to cover the separated amnion or the separated chorion without visible holes. For example, the separated amnion or the separated chorion may lack intermediate layer at the periphery of the tissue. An intermediate layer that is substantially intact may also include an intermediate layer that appears to the naked eye to cover at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the separated amnion or the separated chorion.

[0088] An unseparated amnion/ chorion may be dissected away from the placental disc and umbilical cord or cut using any number of methods known to those of ordinary skill in the art, for example by using a scalpel, a pair of surgical scissors, a rotary blade, etc. In one embodiment, a placenta may be transferred to a surface suitable for dissection, such as a soft, nonporous mat, and the unseparated amnion/ chorion dissected away from the rest of the placenta, e.g., using surgical scissors or a scalpel.

[0089] An unseparated amnion/ chorion, a separated amnion, or a separated chorion may be cut into one or more sheets before, during, or after any step or any combination of steps selected from dissecting, perforating, washing, sterling, and preserving. For example, an unseparated amnion/ chorion, a separated amnion, or a separated chorion may be cut into 1 sheet, 2 sheets, 3 sheets, 4 sheets, 5 sheets, 6 sheets, 7 sheets, 8 sheets, 9 sheets, 10 sheets, 11 sheets, 12 sheets, 13 sheets, 14 sheets, 15 sheets, 16 sheets, 17 sheets, 18 sheets, 19 sheets, 20 sheets, or less than any number of sheets listed above, greater than any number of sheets listed above, at least any number of sheets listed above, or a range of sheets bounded by any two numbers listed above. In addition, in other embodiments, an unseparated

amnion/ chorion sheet, a separated amnion sheet, or a separated chorion sheet may be cut into one or more additional sheets before, during, or after any step or any combination of steps selected from dissecting, perforating, washing, sterilizing, and preserving. As used herein, "sheet" includes any three-dimensional conformation that may be formed from the sheet, including but not limited to, a cylindrical shape (e.g., sleeve), a cone shape, etc.

[0090] An unseparated amnion/ chorion, a separated amnion, or a separated chorion may be cut to any shape or size sheet that the tissue may accommodate. For example, the tissue may be cut to or may have a regular or irregular shape. In some embodiments, the tissue may be cut to or may have the shape of a square, rectangle, oval, or circle, etc. In other embodiments, the tissue may be cut to have or may have one or more straight or curved edges.

[0091] An unseparated amnion/ chorion, a separated amnion, or a separated chorion may be cut to or may have any diameter that the tissue may accommodate, such as a diameter of about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm (or about 1 cm), about 2 cm, about 3 cm, about 4 cm, about 5 cm, about 6 cm, about 7 cm, about 8 cm, about 9 cm, about 10 cm, about 11 cm, about 12 cm, about 13 cm, about 14 cm, about 15 cm, about 16 cm, about 17 cm, about 18 cm, about 19 cm, about 20 cm, or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 mm to about 5 mm, about 1 cm to about 5 cm, about 10 cm to about 15 cm, about 1 mm to about 20 cm, about 1 mm to about 10 mm, about 10 mm to about 10 cm, about 10 cm to about 20 cm.

[0092] In addition, unseparated amnion/ chorion, a separated amnion, or a separated chorion may be cut to or may have any length or width that the tissue may accommodate, such as a length or width of about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm (or about 1 cm), about 2 cm, about 3 cm, about 4 cm, about 5 cm, about 6 cm, about 7 cm, about 8 cm, about 9 cm, about 10 cm, about 11 cm, about 12 cm, about 13 cm, about 14 cm, about 15 cm, about 16 cm, about 17 cm, about 18 cm, about 19 cm, about 20 cm, or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 mm to about 5 mm, about 1 cm to about 5 cm, about 10 cm to about 15 cm, about 1 mm to about 20 cm, about 1 mm to about 10 mm, about 10 mm to about 10 cm, about 10 cm to about 20 cm.

[0093] Further, unseparated amnion/ chorion, a separated amnion, or a separated chorion may be cut to or may have any surface area that the tissue may accommodate, including a surface area of about 1 mm ² , about 2 mm ² , about 3 mm ² , about 4 mm ² , about 5 mm ² , about 6 mm ² , about 7 mm ² , about 8 mm ² , about 9 mm ² , about 10 mm ² (or about 1 cm ² ), about 2 cm ² , about 3 cm ² , about 4 cm ² , about 5 cm ² , about 6 cm ² , about 7 cm ² , about 8 cm ² , about 9 cm ² , about 10 cm ² , about 11 cm ² , about 12 cm ² , about 13 cm ² , about 14 cm ² , about 15 cm ² , about 16 cm ² , about 17 cm ² , about 18 cm ² , about 19 cm ² , about 20 cm ² , about 30 cm ² , about 40 cm ² , about 50 cm ² , about 60 cm ² , about 70 cm ² , about 80 cm ² , about 90 cm ² , about 100 cm ² (about 10 dm ² ), about 20 dm ² , about 30 dm ² , about 40 dm ² , or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 mm ² to about 5 mm ² , about 1 cm ² to about 5 cm ² , about 10 cm ² to about 20 cm ² , about 50 cm ² to about 20 dm ² , about 1 mm ² to about 10 mm ² , about 1 mm ² to about 10 cm ² , about 10 cm ² to about 100 cm ² , about 40 cm ² to about 40 dm ² , or about 1 mm ² to about 40 dm ² .

D. Exemplary Embodiments for Perforating

[0094] An unseparated amnion/ chorion, a separated amnion, or a separated chorion may be perforated before, during, or after any step or any combination of steps selected from dissecting, cutting, washing, sterling, and preserving. When used as a verb herein, "perforate," and other forms (e.g., perforated, perforating, etc.) refer to making one or more holes having any shape or pattern. When used as an adjective herein, "perforated" refers to having one or more holes having any shape or pattern. When used as a noun herein, "perforation" refers to one or more holes having any shape or pattern. "Perforate" and any other form of the word as used herein does not refer to a tear or break.

[0095] As used herein, "perforate," and other forms (e.g., perforated, perforating, etc.) includes, but is not limited to, perforating by making one or more holes that traverse the entire thickness of one or more membrane(s) or layer(s). In some embodiments, methods of perforating include making one or more holes that traverse the entire thickness of an unseparated amnion/ chorion, a separated amnion, or a separated chorion. In other embodiments, methods of perforating an

unseparated amnion/ chorion, a separated amnion, or a separated chorion include making one or more holes traversing any one layer or combination of layers selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion. In a further embodiment, a method of perforating an unseparated amnion/ chorion or separated amnion includes making one or more holes traversing the epithelial layer of the amnion and the basement membrane of the amnion. In another embodiment, a method of perforating an unseparated amnion/ chorion or separated chorion includes making one or more holes traversing the trophoblast layer of the chorion and the basement membrane of the chorion. In further embodiments, methods of perforating an unseparated amnion/ chorion, a separated amnion, or a separated chorion includes making one or more holes that do not traverse any one layer or any combination of layers selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[0096] "Perforate," and other forms (e.g., perforated, perforating, etc.) as used herein also includes making one or more holes that partially traverse one or more membrane(s) or layer(s). For example, in some embodiments, methods of perforating an unseparated amnion/ chorion or separated amnion include making one or more holes that partially traverse the amnion. In other embodiments, methods of perforating an unseparated amnion/ chorion or separated chorion include making one or more holes that partially traverse the chorion. In further embodiments, methods of perforating an unseparated amnion/ chorion, a separated amnion, or a separated chorion include making one or more holes that partially traverse any one or any combination of layers selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[0097] "Perforate," and other forms (e.g., perforated, perforating, etc.) as used herein also includes making one or more holes that traverse or partially traverse the one or more membrane(s) or layer(s) across one or more perforations. For example, in some embodiments, methods of perforating an unseparated amnion/ chorion, a separated amnion, or a separated chorion include making one or more holes that traverse the entire thickness of the tissue in one portion and one or more holes that partially traverse the tissue in another portion. In other

embodiments, methods of perforating an unseparated amnion/ chorion include making one or more holes that traverse the amnion and chorion in one portion, and making one or more holes that traverse the amnion and partially traverses the chorion in another portion. In further embodiments, methods of perforating an unseparated amnion/ chorion include making one or more holes that traverse the chorion in one portion, and making one or more holes that traverse the chorion and partially traverses the amnion in another portion. In additional embodiments, methods of perforating unseparated amnion/ chorion, separated amnion, or separated chorion include making one or more holes that traverse or partially traverse any one or any combination of layers in one portion, and making one or more holes that traverse or partially traverse any one or any combination of layers to a different extent in another portion. The any one or any combination of layers referred to may be selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[0098] As used herein, "perforated" includes, but is not limited to, having one or more holes traversing the entire thickness of one or more membrane(s) or layer(s). For example, in some embodiments, a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion has one or more holes traversing one or more layers of the tissue. In other embodiments, a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion has one or more holes traversing any one layer or combination of layers selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion. In a further embodiment, a perforated, unseparated amnion/ chorion or a perforated, separated amnion has one or more holes traversing the epithelial layer of the amnion and the basement membrane of the amnion. In another embodiment, a perforated, unseparated amnion/ chorion or a perforated, separated chorion has one or more holes traversing the trophoblast layer of the chorion and the basement membrane of the chorion. In a further embodiment, a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion has one or more holes that do not traverse any one layer or any combination of layers selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[0099] Perforated, as used herein, may also include having one or more holes that partially traverse one or more membrane(s) or layer(s). In some embodiments, a perforated, unseparated amnion/ chorion or a perforated, separated amnion has one or more holes that partially traverse the amnion. In other embodiments, a perforated, unseparated amnion/ chorion or a perforated separated chorion has one or more holes that partially traverse the chorion. In other embodiments, a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion has one or more holes that partially traverse any one or any combination of layers selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[00100] Perforated, as used herein, may also include having one or more holes that traverse or partially traverse one or more membrane(s) or layer(s) differently across one or more perforations. For example, a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion may have one or more holes that traverse the entire thickness of the tissue in one portion and one or more holes that partially traverse the tissue in another portion. In one embodiment, a perforated, unseparated amnion/ chorion has one or more holes that traverse the amnion and chorion in one portion, and has one or more holes that traverse the amnion and partially traverses the chorion in another portion. In another embodiment, a perforated, unseparated amnion/ chorion has one or more holes that traverse the chorion in one portion and one or more holes that traverse the chorion and partially traverses the amnion in another portion. In some embodiments, a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion has one or more holes that traverse or partially traverse any one or any combination of layers in one portion and one or more holes that traverse or partially traverse any one or any combination of layers to a different extent in another portion. The any one or any combination of layers referred to herein may be selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[00101] As used herein, "perforation" includes, but is not limited to, one or more holes traversing the entire thickness of one or more membrane(s) or layer(s). For example, a perforation may traverse the entire thickness of an unseparated amnion/ chorion, a separated amnion, or a separated chorion. In other embodiments, a perforation may traverse one or more layers of an unseparated amnion/ chorion, a separated amnion, or a separated chorion. In one embodiment, a perforation traverses the entire thickness of an unseparated amnion/ chorion, a separated amnion, or a separated chorion. In other embodiments, a perforation traverses any one layer or any combination of layers selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion. In a further embodiment, a perforation traverses the epithelial layer and basement membrane of the amnion. In another embodiment, a perforation traverses the trophoblast layer and basement membrane of the chorion. In another embodiment, a perforation does not traverse any one layer or any combination of layers selected from epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[00102] A perforation, as used herein, may also include one or more holes partially traversing one or more membrane(s) or layer(s). In one embodiment, a perforation is one or more holes that partially traverses the amnion. In another embodiment, a perforation is one or more holes that partially traverses the chorion. In some embodiments, a perforation is one or more holes that partially traverses any one or any combination of layers selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[00103] A perforation, as used herein, may also include one or more holes traversing or partially traversing one or more membrane(s) or layer(s) differently across the perforation. For example, a perforation may have one or more holes that traverse the entire thickness of an unseparated amnion/ chorion, a separated amnion, or a separated chorion in one portion and have one or more holes that partially traverse the tissue in another portion. In one embodiment, a perforation traverses the amnion and chorion in one portion, and traverses the amnion and partially traverses the chorion in another portion. In another embodiment, a perforation traverses the chorion in one portion and traverses the chorion and partially traverses the amnion in another portion. In some embodiments, a perforation traverses or partially traverses any one or any combination of layers in one portion and traverses or partially traverses any one or any combination of layers to a different extent in another portion. The any one or any combination of layers referred to herein may be selected from the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion.

[00104] As previously stated, "perforation," as used herein, refers to one or more holes having any shape or pattern. As used herein, a "hole" may have any depth ranging from less than a micron to greater than several millimeters. The depth of a hole may be determined by measuring the distance between one opening of the hole to the other opening of the hole. In some embodiments, the depth of a hole may be about 1 μηι, about 2 μηι, about 3 μηι, about 4 μηι, about 5 μηι, about 6 μηι, about 7 μΐιι, about 8 μηι, about 9 μηι, about 10 μηι, about 11 μηι, about 12 μηι, about 13 μΐη, about 14 μηι, about 15 μηι, about 16 μηι, about 17 μηι, about 18 μηι, about 19 μΐη, about 20 μηι, about 25 μηι, about 30 μηι, about 40 μηι, about 50 μηι, about 60 μΐη, about 70 μηι, about 80 μηι, about 90 μηι, about 100 μηι (about 0.1 mm), about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 μηι to about 5 μηι, about 5 μηι to about 10 μηι, about 15 μηι to about 25 μηι, about 50 μηι to about 0.1 mm, about 0.1 mm to about 0.5 mm, about 1 mm to about 10 mm, about 1 μηι to about 100 μηι, about 0.1 mm to about 10 mm, or about 1 μηι to about 10 mm.

[00105] A hole may have an area ranging from less than a square micron to greater than several square centimeters. The area of a hole may be determined by measuring the area spanning an opening of the hole. In some embodiments, a hole may have an area of about 1 μηι ² , about 2 μηι ² , about 3 μηι ² , about 4 μηι ² , about 5 μηι ² , about 6 μηι ² , about 7 μηι ² , about 8 μηι ² , about 9 μηι ² , about 10 μηι ² , about 11 μηι ² , about 12 μηι ² , about 13 μηι ² , about 14 μηι ² , about 15 μηι ² , about 16 μηι ² , about 17 μηι ² , about 18 μηι ² , about 19 μηι ² , about 20 μηι ² , about 25 μηι ² , about 30 μηι ² , about 40 μηι ² , about 50 μηι ² , about 60 μηι ² , about 70 μηι ² , about 80 μηι ² , about 90 μηι ² , about 100 μηι ² (about 0.1 mm ² ), about 0.2 mm ² , about 0.3 mm ² , about 0.4 mm ² , about 0.5 mm ² , about 0.6 mm ² , about 0.7 mm ² , about 0.8 mm ² , about 0.9 mm ² , about 1 mm ² , about 2 mm ² , about 3 mm ² , about 4 mm ² , about 5 mm ² , about 6 mm ² , about 7 mm ² , about 8 mm ² , about 9 mm ² , about 10 mm ² (about 1 cm ² ), about 2 cm ² , about 3 cm ² , about 4 cm ² , about 5 cm ² , about 6 cm ² , about 7 cm ² , about 8 cm ² , about 9 cm ² , about 10 cm ² , or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 μηι ² to about 10 μηι ² , about 10 μηι ² to about 100 μηι ² , about 100 μηι ² to about 1 mm ² , about 1 mm ² to about 10 mm ² , or about 1 cm ² to about 10 cm ² , about 1 μηι ² to about 100 μηι ² , about 0.1 mm ² to about

10 mm ² , or about 1 μηι ² to about 10 cm ² .

[00106] The distance between any two adjacent holes may range from less than a micron to greater than several millimeters. The distance between any two adjacent holes may be determined by measuring the distance between the two closest points on the boundaries of the respective holes. In some embodiments, two holes may be separated by a distance of about 1 μηι, about 2 μηι, about 3 μηι, about 4 μηι, about 5 μΐη, about 6 μηι, about 7 μηι, about 8 μηι, about 9 μηι, about 10 μηι, about

11 μΐη, about 12 μηι, about 13 μηι, about 14 μηι, about 15 μηι, about 16 μηι, about 17 μΐη, about 18 μηι, about 19 μηι, about 20 μηι, about 25 μηι, about 30 μηι, about 40 μΐη, about 50 μηι, about 60 μηι, about 70 μηι, about 80 μηι, about 90 μηι, about 100 μηι (about 0.1 mm), about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about

2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 μηι to about 10 μηι, about 10 μηι to about 50 μηι, about 50 μηι to about 100 μηι, about 0.1 mm to about 1 mm, about 1 mm to about 10 mm, about 1 μηι to about 10 mm, about 1 μηι to about 100 μηι, or about 0.1 mm to about 10 mm.

[00107] A perforation may comprise a plurality of holes having a total area less than the surface area of the sheet. In some embodiments, the total area of a plurality of holes may be less than the surface area of the sheet by about 1%, by about 2%, by about 3%, by about 4%, by about 5%, by about 6%, by about 7%, by about 8%, by about 9%, by about 10%, by about 11%, by about 12%, by about 13%, by about 14%, by about 15%, by about 16%, by about 17%, by about 18%, by about 19%, by about 20%, by about 21%, by about 22%, by about 23%, by about 24%, by about 25%, by about 26%, by about 27%, by about 28%, by about 29%, by about 30%, by about 31%, by about 32%, by about 33%, by about 34%, by about 35%, by about 36%, by about 37%, by about 38%, by about 39%, by about 40%, by about 41%, by about 42%, by about 43%, by about 44%, by about 45%, by about 46%, by about 47%, by about 48%, by about 49%, by about 50%, or less than any percentage listed above, greater than any percentage listed above, at least any percentage listed above, or a range of percentages bounded by any two of the percentages listed above, such as by about 1% to by about 5%, by about 5% to by about 10%, by about 10% to by about 15%, by about 15% to by about 20%, by about 25% to by about 30%, by about 30% to by about 40%, by about 40% to by about 50%, by about 1% to by about 50%, by about 1% to by about 25%, or by about 25% to by about 50%.

[00108] An unseparated amnion/ chorion, a separated amnion, or a separated chorion may be perforated using any number of perforators. As used herein, a "perforator" refers to a device that is capable of making a perforation including, but not limited to, a needle, a roller with one or more pins, a stamper with one or more pins, a hole punch, a punch and die, a die cutter (e.g., a rotary die cutter), a drill and bit, a blade, scalpel or other sharp object, a skin graft mesher with blades, high-pressure water or air gun, a laser, etc. A perforating device may have one or multiple penetrators of the same or different type.

[00109] A perforator may be used to make perforations that traverse or partially traverse one or more layers or membranes. For example, in some

embodiments, a perforator may be applied to the surface of an unseparated amnion/ chorion, a separated amnion, or a separated chorion, and portion of the perforator penetrating the surface (also referred to herein as a "penetrator") permitted to penetrate the entire thickness of the tissue or sheet or to only penetrate to a particular thickness or a particular layer. As used herein, a "penetrator" includes, but is not limited to, a needle, punch, pin, die, bit, blade, laser beam, water stream, or air stream. A penetrator or penetrators may be adapted to penetrate a tissue or sheet to a particular thickness or a particular layer, for example, by adjusting the extent of penetration by controlling the pressure applied to the penetrator(s) or by controlling the length of the penetrator(s). "Air" includes any suitable gas, such as atmospheric air, oxygen, or an inert gas such as nitrogen or argon.

[00110] A penetrator or penetrators may be of any length, dimension, or pattern that is capable of making a perforation having any one of the depths or areas, or any one of the ranges of depths or areas, recited above. In some embodiments, the distance between any two adjacent penetrators may be of any distance that is capable of making a perforation having a distance between any two adjacent holes as recited above.

[00111] A perforating device may have a penetrator or penetrators capable of making one or more holes having an opening with an area ranging from less than a square micron to greater than several square centimeters. In some embodiments, the penetrator or penetrator(s) are capable of making one or more holes having an area of about 1 μηι ² , about 2 μηι ² , about 3 μηι ² , about 4 μηι ² , about 5 μηι ² , about 6 μηι ² , about 7 μηι ² , about 8 μηι ² , about 9 μηι ² , about 10 μηι ² , about 11 μηι ² , about 12 μηι ² , about 13 μηι ² , about 14 μηι ² , about 15 μηι ² , about 16 μηι ² , about 17 μηι ² , about 18 μηι ² , about 19 μηι ² , about 20 μηι ² , about 25 μηι ² , about 30 μηι ² , about 40 μηι ² , about 50 μηι ² , about 60 μηι ² , about 70 μηι ² , about 80 μηι ² , about 90 μηι ² , about 100 μηι ² (about 0.1 mm ² ), about 0.2 mm ² , about 0.3 mm ² , about 0.4 mm ² , about 0.5 mm ² , about 0.6 mm ² , about 0.7 mm ² , about 0.8 mm ² , about 0.9 mm ² , about 1 mm ² , about 2 mm ² , about 3 mm ² , about 4 mm ² , about 5 mm ² , about 6 mm ² , about 7 mm ² , about 8 mm ² , about 9 mm ² , about 10 mm ² (about 1 cm ² ), about 2 cm ² , about 3 cm ² , about 4 cm ² , about 5 cm ² , about 6 cm ² , about 7 cm ² , about 8 cm ² , about 9 cm ² , about 10 cm ² , or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 μηι ² to about 10 μηι ² , about 10 μηι ² to about 100 μηι ² , about 100 μηι ² to about 1 mm ² , about 1 mm ² to about 10 mm ² , or about 1 cm ² to about 10 cm ² , about 1 μηι ² to about 100 μηι ² , about 0.1 mm ² to about 10 mm ² , or about 1 μηι ² to about 10 cmm ² . A perforating device may have more than one penetrator capable of making holes of the same or different sizes. For example, half of the penetrators of a perforator may be capable of making holes ranging from about 20 μηι ² to 1 mm ² and the other half of the penetrators may be capable of making holes ranging from about 0.4 mm ² to about 10 mm ² .

[00112] A perforating device may have a penetrator or penetrators having a length corresponding to the thickness of at least one layer or membrane, or ranging from less than a micron to greater than several millimeters. In some embodiments, the length of a penetrator or penetrators may be about 1 μηι, about 2 μΐιι, about 3 μηι, about 4 μηι, about 5 μηι, about 6 μηι, about 7 μηι, about 8 μηι, about 9 μΐη, about 10 μηι, about 11 μηι, about 12 μηι, about 13 μηι, about 14 μηι, about 15 μΐη, about 16 μηι, about 17 μηι, about 18 μηι, about 19 μηι, about 20 μηι, about 25 μΐη, about 30 μηι, about 40 μηι, about 50 μηι, about 60 μηι, about 70 μηι, about 80 μΐη, about 90 μηι, about 100 μηι (about 0.1 mm), about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 μηι to about 10 μηι, about 10 μηι to about 50 μηι, about 50 μηι to about 100 μηι, about 0.1 mm to about 1 mm, about 1 mm to about 10 mm, about 1 μηι to about 10 mm, about 1 μηι to about 100 μηι, or about 0.1 mm to about 10 mm. A

perforating device may have more than one penetrator of the same or different lengths. For example, half of the penetrators of a perforator may have a length of about 10 μηι and the other half of the penetrators may have a length of about 1 mm.

[00113] Examples of perforating devices that may be used to make perforations that traverse or partially traverse one or more layers or membranes include a rolling device having a cylindrical core and multiple penetrators extending from the core or a stamping device having a platform and multiple penetrators extending from the platform. A rolling device or a stamping device may have a handle. The penetrators of these exemplary perforating devices may be of the same length or of varying lengths. The length of the penetrators may correspond to the desired hole depth, correspond to the thickness of the layer(s) sought to be perforated, or range from less than a micron to greater than several millimeters.

[00114] Other examples of perforating devices that may be used to make perforations that traverse or partially traverse one or more layers or membranes include a perforating bed of penetrators. The perforating bed may be made of a rigid or flexible material. The penetrators may have the same length or different lengths or be capable of making holes of the same or different si2es. In one embodiment, a tissue or sheet is placed on a perforating bed and pressure is applied from above. For example, a bladder (e.g., balloon, etc.) may be used to apply pressure to the surface of the tissue or sheet. The pressure applied may be consistent across the perforating bed or may vary from one area of the bed to another. Multiple perforating beds could be used to make perforations originating from both sides of a tissue or sheet. E. Exemplary Embodiments for Washing or Pretreating

[00115] As used herein, "washing" refers to trying to remove material

(e.g., blood remnants or other debris) from the surface or interior of an unseparated amnion/ chorion, a separated amnion, or a separated chorion in the presence of a suitable washing medium by any number of methods, including but not limited to flushing, immersing, perfusing, soaking, or agitating in the presence or absence of pressure or vacuum. In some embodiments, the agitating is performed using a rocker, shaker, stir plate, rotating mixer, or other equipment capable of agitating. In some embodiments, an unseparated amnion/ chorion, a separated amnion, or a separated chorion may be washed before, during, or after any step or any combination of steps selected from dissecting, separating, pretreating, cutting, and perforating. For example, in some embodiments, an unseparated amnion/ chorion, a separated amnion, or a separated chorion may be washed before or after being perforated.

[00116] As used herein, "pretreating" is one or more steps intended to remove material (e.g., blood remnants or other debris) from the surface or loosen material from the interior of an unseparated amnion/ chorion, a separated amnion, or a separated chorion without disrupting the tissue by any number of methods, including but not limited to picking, dabbing, rubbing, or massaging with a fingertip, swab, or gau2e. Pretreating may be performed manually. In some embodiments, the pretreating is performed in the presence of a suitable washing medium. In some embodiments, an unseparated amnion/ chorion, a separated amnion, or a separated chorion may be pretreated before, during, or after any step or any combination of steps selected from dissecting, separating, cutting, perforating, and washing. For example, in some embodiments, an unseparated amnion/ chorion, a separated amnion, or a separated chorion may be pretreated before or after being perforated.

[00117] In some embodiments, the material sought to be removed from the surface or interior of an unseparated amnion/ chorion, a separated amnion, or a separated chorion is blood remnants (e.g., blood clots, whole blood, intact blood cells, blood cell remnants, etc.) or other debris. In some embodiments, e.g., following pretreatment or washing, an unseparated amnion/ chorion, a separated amnion, or a separated chorion is substantially free of blood remnants. In other embodiments, e.g., following pretreatment or washing, a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion is substantially free of blood remnants. As used herein, "substantially free" refers to a significantly reduced amount of an agent referenced. In some embodiments, the reduction in amount is greater than about 50%. In some embodiments, the reduction in amount is greater than about 60%, 70%, 80%, 90%, or 95%. In some embodiments,

substantially free of blood remnants means that the hemoglobin content is less than about 13 μg hemoglobin per mg of the tissue or sheet ('^g/mg"). In some

embodiments, substantially free of blood remnants means that the hemoglobin content is less than about 12 μg/ mg, about 11 μg/ mg, about 10 μg/ mg, about 9 μg/ mg, about 8 μg/ mg, about 7 μg/ mg, about 6 μg/ mg, about 5 μg/ mg, about 4 μg/ mg, about 3 μg/ mg, about 2 μg/ mg, about 1 μg/ mg, about 0.9 μg/ mg, about 0.8 μg/ mg, about 0.7 μg/ mg, about 0.6 μg/ mg, about 0.5 μg/ mg, about 0.4 μg/ mg, about 0.3 μg/ mg, about 0.2 μg/ mg, about 0.1 μg/ mg, about 0.09 μg/ mg, about 0.08 μg/ mg, about 0.07 μg/ mg, about 0.06 μg/ mg, or about 0.05 μg/ mg.

[00118] In one embodiment, a suitable washing medium includes a sodium chloride solution, such as a solution containing a w/ v concentration of NaCl of about 0.45%, about 0.9%, about 1.8%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 34%, about 35%, about 36%, or less than any percentage listed above, greater than any percentage listed above, at least any percentage listed above, or a range of percentages bounded by any two of the percentages listed above, such as about 0.45% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 20% to about 25%, about 25% to about 30%, about 30% to about 36%, about 0.45% to about 15%, about 15% to about 33%, or about 15% to about 36%.

[00119] In addition, a suitable washing medium includes a sodium chloride solution as described above that also contains one or more other ingredients, such as phosphate, potassium, magnesium, calcium, dextrose, glucose, citrate, lactate, tris, HEPES, etc. Other suitable washing mediums include water (e.g., purified or sterile), Lactated Ringer's solution, Ringer's solution, phosphate -buffered saline (PBS), tris-buffered saline (TBS), Hank's balanced salt solution (HBSS), Dulbecco's phosphate -buffered saline (DPBS), Earle's balanced salt solution (EBSS), standard saline citrate (SSC), HEPES-buffered saline (HBS), Gey's balanced salt solution (GBSS), cell culture mediums (e.g., Delbecco's Modified Eagle Medium (DMEM), Minimum Essential Media (MEM), etc.), hydrogen peroxide solutions, sodium hypochloride solutions, or other medium suitable for washing tissue.

[00120] A tissue or a sheet may be washed in at least one suitable washing medium over one or more washing steps. For example, a tissue or sheet may be washed in at least one washing step, at least two washing steps, at least three washing steps, at least four washing steps, at least five washing steps, at least six washing steps, at least seven washing steps, at least eight washing steps, at least nine washing steps, at least ten washing steps, or ten or more washing steps.

[00121] In some embodiments, at least one washing step includes exchanging the at least one suitable washing medium at least once, once, at least twice, twice, at least thrice, thrice, at least four times, four times, at least five times, five times, at least six times, six times, at least seven times, seven times, at least eight times, eight times, at least nine times, nine times, at least ten times, ten times, or ten or more times. A suitable washing medium may be exchanged by transferring a tissue or a sheet from old medium to fresh medium of the same or different type, or by removing some or all of the old medium and transferring fresh medium of the same or different type. [00122] In other embodiments, at least one washing step includes comprises heating or cooling a tissue, sheet, or suitable washing medium. For example, a tissue, sheet, or suitable washing medium may be placed in an

environment having a temperature above, at, or below a refrigeration temperature, a room temperature, or a heated temperature. As used herein, "refrigeration

temperature" refers to a temperature of from about 1°C to about 12°C, "room temperature" refers to a temperature of from about 13°C to about 25°C, and "heated temperature" refers to a temperature of from about 26°C to about 50°C.

[00123] In some embodiments, a tissue, sheet, or suitable washing medium is placed in an environment having a temperature of about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21 °C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31°C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as refrigeration temperature, room temperature, or heated temperature.

[00124] A tissue, sheet, or suitable washing medium may be placed in any of the environments listed above for any period of time ranging from minutes to hours. For example, the period of time may be about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 48 hours, or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 18 hours, about 18 hours to about 36 hours, about 36 hours to about 60 hours, about 60 hours to about 90 hours, about 5 minutes to about 24 hours, about 24 hours to about 48 hours, or about 5 minutes to about 48 hours.

[00125] The temperature of an environment in which a tissue, sheet, or suitable washing medium is placed may change or may not change over time. As used herein, a temperature of an environment "changes over time" when the environment (e.g., the interior of a container such as a bottle or bag) is placed in surroundings (e.g., a room, oven, refrigerator, free2er, lyophili2er, or water bath) such that the

environment and its surroundings are not initially at thermal equilibrium. As used herein, a temperature of an environment "does not change over time" when the environment is at thermal equilibrium with its surroundings. Thermal equilibrium includes a temperature differential of less than about 1°C. In some embodiments, thermal equilibrium includes a temperature differential of less than about 2°C or 3 °C.

F. Exemplary Embodiments for Sterilizing or Preserving

[00126] Any tissue, sheet, or composition described herein may be sterili2ed before, during, or after processing, including after final packaging.

Sterilizing may be performed using one or more of any number of techniques, including but not limited to exposure to gamma radiation, E-beam radiation, ethylene oxide with a stabilizing gas (such as carbon dioxide or hydrochlorofluorocarbons (HCFC)), peracetic acid, hydrogen peroxide gas plasma, or ozone.

[00127] Any tissue, sheet, or composition as described herein may be stored in a suitable preservation medium at a suitable temperature for a suitable amount of time. In one embodiment, a tissue, sheet, or composition is placed in a container containing a suitable preservation medium, such as water, a saline solution, petrolatum, petroleum jelly, Vaseline, soft paraffin, glycerol, or Ringer's solution. In another embodiment, a tissue, sheet, or composition is stored at refrigeration temperature for a limited time. In a further embodiment, a tissue, sheet, or composition is stored at refrigeration temperature in a container containing a suitable preservation medium, such as those listed above. For example, a tissue, sheet, or composition produced according to the methods described herein may be stored at a temperature of about 1°C to about 12°C for about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, or about 90 hours, less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 6 hours to about 36 hours, about 36 hours to about 60 hours, about 60 hours to about 90 hours, or about 6 hours to about 90 hours.

[00128] A tissue, sheet, or composition as described herein may also be preserved by cryopreservation, refrigeration, free2ing, or dehydration. A tissue, sheet, or composition may be preserved at any processing step.

[00129] A tissue, sheet, or composition may be cryopreserved by free2ing at e.g., liquid nitrogen or dry ice temperature, or a temperature of

about -200°C to about -40°C, -200°C to about -70°C, about -200°C to about -190°C, or -86°C to about -78°C and storing at liquid nitrogen temperatures for up to about 5 years. For example, a tissue, sheet, or composition may be exposed to 5% dimethylsulf oxide (DMSO) and 5% serum in DMEM and fro2en slowly or rapidly.

[00130] A tissue, sheet, or composition may be stored at refrigeration temperature for a limited time. For example, a tissue, sheet, or composition may be stored at a temperature of about 1°C to about 12°C or about 1°C to about 5°C for a time of about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, about 90 hours, or less than any time listed above, longer than any time listed above, at least any time listed above, or a range of times bounded by any two of the times listed above, such as about 6 hours to about 36 hours, about 36 hours to about 60 hours, about 60 hours to about 90 hours, or about 6 hours to about 90 hours.

[00131] A tissue, sheet, or composition produced according to the methods described herein may be dehydrated, for example by lyophilization, by dehydration in an oven, using chemicals, or by any number of methods known in the art.

[00132] In some embodiments, a tissue, sheet, or composition may be dehydrated by lyophilization. See, e.g., US 4,001,994 for a discussion of freeze-drying techniques. In some embodiments, a product may be frozen then lyophilized. For example, a tissue, sheet, or composition may be quickly frozen by 100% ethanol and dry ice before lyophilizing or frozen less rapidly before lyophilizing. In some embodiments, a tissue, sheet, or composition may be stored for a period of time at a freezing temperature before lyophilization, such as for about 5 minutes, about 15 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 54 hours, about 60 hours, about 66 hours, about 72 hours, about 78 hours, about 84 hours, or about 90 hours, less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 5 minutes to about 30 minutes, about 30 minutes to about 1 hour, about 1 hour to about 18 hours, about 18 hours to about 36 hours, about 36 hours to about 60 hours, about 60 hours to about 90 hours, about 5 minutes to about 24 hours, about 24 hours to about 90 hours, or about 5 minutes to about 90 hours.

[00133] A tissue, sheet, or composition may also be dehydrated by chemical dehydration, for example by using a dehydration fluid that decreases the water content of the product. A dehydration fluid may be a fluid comprising an alcohol, an organic solvent, a hydrophilic polymer (e.g., polyoxyethylene oxide), a polysaccharide (such as a cellulose derivative or dextrose, etc.), or a salt. [00134] In some embodiments, a tissue, sheet, or composition may be stored for an extended period at a free2ing, refrigeration, or room temperature. As used herein, a "free2ing temperature" refers to a temperature of about 0°C or lower (e.g., a free2ing temperature may be a range of from about 0°C to about -25°C, of from about 0°C to about -45°C, of from about 0°C to about -86°C, etc.). As used herein, a "refrigeration temperature" refers to a temperature of from about 1°C to about 12°C, and a "room temperature" refers to a temperature of from about 13°C to about 25°C. For example, a tissue, sheet or composition may be stored at a temperature of about -85°C to about 25°C for weeks to years depending on the storage temperature. In a further embodiment, a tissue, sheet, or composition may be stored for an extended period at liquid nitrogen temperatures, for example for a period of up to about 5 years.

G. Exemplary Embodiments for Characterizing Tissue or Sheets

[00135] The dry mass of a tissue or a sheet prepared according to the methods described herein may range from less than about one milligram per square centimeter to greater than about 25 milligrams per square centimeter. The dry mass per surface area (mg/cm ² ) of a tissue or sheet may be calculated by dividing the mass of the tissue or sheet by its surface area. For example, a tissue or a sheet may have a dry mass of about 1 mg/ cm ² , about 2 mg/ cm ² , about 3 mg/ cm ² , about 4 mg/ cm ² , about 5 mg/ cm ² , about 6 mg/ cm ² , about 7 mg/ cm ² , about 8 mg/ cm ² , about 9 mg/ cm ² , about 10 mg/ cm ² , about 11 mg/ cm ² , about 12 mg/ cm ² , about 13 mg/ cm ² , about 14 mg/ cm ² , about 15 mg/ cm ² , about 16 mg/ cm ² , about 17 mg/ cm ² , about 18 mg/ cm ² , about 19 mg/ cm ² , about 20 mg/ cm ² , about 21 mg/ cm ² , about

22 mg/ cm ² , about 23 mg/ cm ² , about 24 mg/ cm ² , or about 25 mg/ cm ² , or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 mg/ cm ² to about 25 mg/ cm ² , about 1 mg/ cm ² to about 12 mg/ cm ² , about 12 mg/ cm ² to about 25 mg/ cm ² , about 1 mg/ cm ² to about 5 mg/ cm ² , about 5 mg/ cm ² to about 10 mg/ cm ² , about 10 mg/ cm ² to about 15 mg/ cm ² , about 15 mg/ cm ² to about 20 mg/ cm ² , about 20 mg/ cm ² to about 25 mg/ cm ² .

[00136] The water absorption potential of a tissue or a sheet prepared according to the methods described herein may range from less than about one milligram per square centimeter to greater than about 40 milligrams per square centimeter. The water absorption potential of a tissue or a sheet (e.g., a dehydrated tissue or a dehydrated sheet) may be determined by subtracting the mass of the tissue or sheet before hydrating from its mass after hydrating and dividing by the surface area of the tissue or sheet. For example, a tissue or a sheet may have a water absorption potential of about 1 mg/ cm ² , about 2 mg/ cm ² , about 3 mg/ cm ² , about 4 mg/ cm ² , about 5 mg/ cm ² , about 6 mg/ cm ² , about 7 mg/ cm ² , about 8 mg/ cm ² , about 9 mg/ cm ² , about 10 mg/ cm ² , about 11 mg/ cm ² , about 12 mg/ cm ² , about 13 mg/ cm ² , about 14 mg/ cm ² , about 15 mg/ cm ² , about 16 mg/ cm ² , about

17 mg/ cm ² , about 18 mg/ cm ² , about 19 mg/ cm ² , about 20 mg/ cm ² , about

21 mg/ cm ² , about 22 mg/ cm ² , about 23 mg/ cm ² , about 24 mg/ cm ² , about

25 mg/ cm ² , about 26 mg/ cm ² , about 27 mg/ cm ² , about 28 mg/ cm ² , about

29 mg/ cm ² , about 30 mg/ cm ² , about 31 mg/ cm ² , about 32 mg/ cm ² , about

33 mg/ cm ² , about 34 mg/ cm ² , about 35 mg/ cm ² , about 36 mg/ cm ² , about

37 mg/ cm ² , about 38 mg/ cm ² , about 39 mg/ cm ² , or about 40 mg/ cm ² , or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 mg/ cm ² to about 40 mg/ cm ² , about 1 to about 20 mg/ cm ² , about 20 to about 40 mg/ cm ² , about 1 mg/ cm ² to about 5 mg/ cm ² , about 5 mg/ cm ² , to about 10 mg/ cm ² , about 10 mg/ cm ² to about 15 mg/ cm ² , about 15 mg/ cm ² to about 20 mg/ cm ² , about 20 mg/ cm ² to about 30 mg/ cm ² , about 30 mg/ cm ² to about 40 mg/ cm ² .

[00137] Total protein content of a tissue or a sheet prepared according to the methods described herein may range from less than about 25 microgram per square centimeter to greater than about 2 milligrams per square centimeter. For example, a tissue or a sheet may have a total protein content of about 25 μg/ cm ² , about 50 μg/ cm ² , about 75 μg/ cm ² , about 100 μg/ cm ² , about 125 μg/ cm ² , about 150 μg/ cm ² , about 175 μg/ cm ² , about 200 μg/ cm ² , about 225 μg/ cm ² , about 250 μg/ cm ² , about 275 μg/ cm ² , about 300 μg/ cm ² , about 325 μg/ cm ² , about 350 μg/ cm ² , about 375 μg/ cm ² , about 400 μg/ cm ² , about 425 μg/ cm ² , about 450 μg/ cm ² , about 475 μg/ cm ² , about 500 μg/ cm ² , about 525 μg/ cm ² , about 550 μg/ cm ² , about 575 μg/ cm ² , about 600 μg/ cm ² , about 625 μg/ cm ² , about 650 μg/ cm ² , about 675 μg/ cm ² , about 700 μg/ cm ² , about 725 μg/ cm ² , about 750 μg/ cm ² , about 775 μg/ cm ² , about 800 μg/ cm ² , about 825 μg/ cm ² , about 850 μg/ cm ² , about 875 μg/ cm ² , about 900 μg/ cm ² , about 925 μg/ cm ² , about 950 μg/ cm ² , about 975 μg/ cm ² , about 1000 μg/ cm ² , about 1025 μg/ cm ² , about 1050 μg/ cm ² , about 1075 μg/ cm ² , about 1100 μg/ cm ² , about 1125 μg/ cm ² , about 1150 μg/ cm ² , about 1175 μg/ cm ² , about 1200 μg/ cm ² , about 1225 μg/ cm ² , about 1250 μg/ cm ² , about 1275 μg/ cm ² , about 1300 μg/ cm ² , about 1325 μg/ cm ² , about 1350 μg/ cm ² , about 1375 μg/ cm ² , about 1400 μg/ cm ² , about 1425 μg/ cm ² , about 1450 μg/ cm ² , about 1475 μg/ cm ² , about 1500 μg/ cm ² , about 1525 μg/ cm ² , about 1550 μg/ cm ² , about 1575 μg/ cm ² , about 1600 μg/ cm ² , about 1625 μg/ cm ² , about 1650 μg/ cm ² , about 1675 μg/ cm ² , about 1700 μg/ cm ² , about 1725 μg/ cm ² , about 1750 μg/ cm ² , about 1775 μg/ cm ² , about 1800 μg/ cm ² , about 1825 μg/ cm ² , about 1850 μg/ cm ² , about 1875 μg/ cm ² , about 1900 μg/ cm ² , about 1925 μg/ cm ² , about 1950 μg/ cm ² , about 1975 μg/ cm ² , or about 2000 μg/ cm ² , or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 25 μg/ cm ² to about 100 μg/ cm ² , about 100 μg/ cm ² to about 500 μg/ cm ² , about 500 μg/ cm ² to about 1000 μg/ cm ² , about 1000 μg/ cm ² to about 1500 μg/ cm ² , about 1500 μg/ cm ² to about 2000 μg/ cm ² , about 25 μg/ cm ² to about 500 μg/ cm ² , about 500 μg/ cm ² to about 1500 μg/ cm ² , about 1000 μg/ cm ² to about 2000 μg/ cm ² , or about 25 μg/ cm ² to about 2000 μg/ cm ² . [00138] A tissue or a sheet may have a total protein content of about

0.1 mg/ cm ² , about 0.2 mg/ cm ² , about 0.3 mg/ cm ² , about 0.4 mg/ cm ² , about 0.5 mg/ cm ² , about 0.6 mg/ cm ² , about 0.7 mg/ cm ² , about 0.8 mg/ cm ² , about 0.9 mg/ cm ² , about 1 mg/ cm ² , about 1.1 mg/ cm ² , about 1.2 mg/ cm ² , about

1.3 mg/ cm ² , about 1.4 mg/ cm ² , about 1.5 mg/ cm ² , about 1.6 mg/ cm ² , about 1.7 mg/ cm ² , about 1.8 mg/ cm ² , about 1.9 mg/ cm ² , or about 2 mg/ cm ² , or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 0.1 mg/ cm ² to about 1 mg/ cm ² , about 1 mg/ cm ² to about 1.5 mg/ cm ² , about 1.5 mg/ cm ² to about 2 mg/ cm ² , or about 0.1 mg/ cm ² to about 2 mg/ cm ² . A procedure for determining total protein content is provided in Example 17 below.

[00139] Hemoglobin is a major component of erythrocytes (or red blood cells) and is responsible for the cells' characteristic red color. The hemoglobin content of a tissue or a sheet prepared according to the methods described herein may range from less than about 13 micrograms per milligram of dry mass of the tissue or sheet to about 0.01 microgram per milligram of dry mass of the tissue or sheet. The hemoglobin content of a tissue or a sheet is the ratio of hemoglobin mass to mass of the tissue or sheet. For example, a tissue or a sheet may have a

hemoglobin content of about 13 μg/ mg, about 12 μg/ mg, about 11 μg/ mg, about 10 μg/ mg, about 9 μg/ mg, about 8 μg/ mg, about 7 μg/ mg, about 6 μg/ mg, about 5 μg/ mg, about 4 μg/ mg, about 3 μg/ mg, about 2 μg/ mg, about 1 μg/ mg, about 0.9 μg/mg, about 0.8 μg/mg, about 0.7 μg/mg, about 0.6 μg/mg, about 0.5 μg/mg, about 0.4 μg/ mg, about 0.3 μg/ mg, about 0.2 μg/ mg, about 0.1 μg/ mg, about 0.09 μg/ mg, about 0.08 μg/ mg, about 0.07 μg/ mg, about 0.06 μg/ mg, about

0.05 μg/ mg, about 0.04 μg/ mg, about 0.03 μg/ mg, about 0.02 μg/ mg, or about 0.01 μg/ mg, or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 0.01 μg/ mg to about 0.05 μg/ mg, about 0.05 μg/ mg to about 0.1 μg/ mg, about 0.1 μg/ mg to about 0.5 μg/ mg, about 0.5 μg/ mg to about 1 μg/ mg, about 1 μg/ mg to about 5 μg/ mg, about 5 μg/ mg to about 10 μg/ mg, about 10 μg/ mg to about 13 μg/ mg, about 0.01 μg/ mg to about 13 μg/ mg, about 0.01 μg/ mg to about 0.1 μg/ mg, about 0.1 μg/ mg to about 1 μg/ mg, about 1 μg/ mg to about 10 μg/ mg, or about 1 μg/ mg to about 13 μg/ mg. A procedure for determining hemoglobin content is provided in Example 18 below.

[00140] Thickness of placental tissue and the thickness of layers within the tissue can vary from donor to donor and can vary across the same placental tissue obtained from the same donor. In addition, thickness of placental tissue and its layers may be greater in some species than others. Mean thickness of a tissue or a sheet prepared according to the methods described herein may range from less than about 10 micrometers to greater than about 2 millimeters. Mean thickness of the intermediate layer of a tissue or sheet prepared according to the methods described herein may range from less than about one micrometer to greater than about

600 micrometers. Mean thickness may be determined by measuring the approximate thickness of the tissue or sheet or the approximate thickness of the intermediate layer at two or more representative areas. A procedure for determining mean thickness is provided in Example 19 below.

[00141] For example, a tissue or sheet may have a mean thickness of about 10 μίη, about 20 μίη, about 30 μίη, about 40 μίη, about 50 μίη, about 60 μίη, about 70 μίη, about 80 μίη, about 90 μίη, about 100 μίη, about 110 μίη, about 120 μίη, about 130 μίη, about 140 μίη, about 150 μίη, about 160 μίη, about 170 μίη, about 180 μίη, about 190 μίη, about 200 μίη, about 210 μίη, about 220 μίη, about 230 μίη, about 240 μίη, about 250 μίη, about 260 μίη, about 270 μίη, about 280 μίη, about 290 μίη, about 300 μίη, about 310 μίη, about 320 μίη, about 330 μίη, about 340 μίη, about 350 μίη, about 360 μίη, about 370 μίη, about 380 μίη, about 390 μίη, about 400 μίη, about 410 μίη, about 420 μίη, about 430 μίη, about 440 μίη, about 450 μίη, about 460 μίη, about 470 μίη, about 480 μίη, about 490 μίη, about 500 μίη, about 510 μίη, about 520 μίη, about 530 μίη, about 540 μίη, about 550 μίη, about 560 μίη, about 570 μηι, about 580 μίη, about 590 μίη, about 600 μίη, about 700 μίη, about 710 μίη, about 720 μίη, about 730 μίη, about 740 μηι, about 750 μίη, about 760 μίη, about 770 μηι, about 780 μίη, about 790 μηι, about 800 μίη, about 810 μίη, about 820 μηι, about 830 μίη, about 840 μηι, about 850 μίη, about 860 μίη, about 870 μίη, about 880 μίη, about 890 μηι, about 900 μίη, about 910 μίη, about 920 μηι, about 930 μίη, about 940 μηι, about 950 μίη, about 960 μίη, about 970 μηι, about 980 μίη, about 990 μηι, about 1000 μίη (about 1 mm), about 1100 μίη, about 1200 μίτι, about 1300 μΐιι, about 1400 μίη, about 1500 μίη, about 1600 μΐη, about 1700 μίτι, about 1800 μΐιι, about 1900 μίη, about 2000 μίη (about 2 mm), or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 10 μίη to about 50 μίη, about 50 μίη to about 100 μίη, about 10 μίη to about 100 μίη, about 100 μίη to about 250 μίη, about 10 μίη to about 250 μίη, about 10 μίη to about 500 μίη, about 250 μίη to about 500 μίη, about 500 μίη to about 750 μίη, about 750 μίη to about 1 mm, about 1 mm to about 2 mm, about 1000 μίη to about 1500 μίη, about 10 μίη to about 2 mm, about 10 μίη to about 1 mm, or about 10 μίη to about 2 mm. In addition, a composition may comprise a tissue or a sheet having a mean thickness of any value or range of values listed above.

[00142] In addition, the intermediate layer of a tissue or sheet may have a mean thickness of about 1 μίη, about 2 μίη, about 3 μίη, about 4 μίη, about 5 μίη, about 10 μίη, about 15 μίη, about 20 μίη, about 25 μίη, about 30 μίη, about 35 μίη, about 40 μίη, about 45 μίη, about 50 μίη, about 55 μίη, about 60 μίη, about 65 μίη, about 70 μίη, about 80 μίη, about 90 μίη, about 100 μίη, about 110 μίη, about 120 μίη, about, about 130 μίη, about 140 μίη, about 150 μίη, about 160 μίη, about 170 μίη, about 180 μίη, about 190 μίη, about 200 μίη, about 210 μίη, about 220 μίη, about 230 μίη, about 240 μΐιι, about 250 μίη, about 260 μίη, about 270 μm, about 280 μίη, about 290 μΐιι, about 300 μίη, about 310 μίη, about 320 μΐη, about 330 μίη, about 340 μΐη, about 350 μίη, about 360 μίη, about 370 μΐη, about 380 μίη, about 390 μΐιι, about 400 μίη, about 410 μίη, about 420 μΐη, about 430 μίη, about 440 μΐιι, about 450 μίη, about 460 μίη, about 470 μΐιι, about 480 μίη, about 490 μΐιι, about 500 μΓη, about 510 μηι, about 520 μηι, about 530 μηι, about 540 μηι, about 550 μηι, about 560 μηι, about 570 μηι, about 580 μηι, about 590 μηι, or about 600 or less than any value listed above, greater than any value listed above, at least any value listed above, or a range of values bounded by any two of the values listed above, such as about 1 μηι to about 5 μηι, about 5 μηι to about 25 μηι, about 25 μηι to about 50 μηι, about 50 μηι to about 100 μηι, about 100 μηι to about 250 μηι, about 250 μηι to about 500 μηι, about 100 μηι to about 600 μηι, about 1 μηι to about 600 μηι, about 1 μηι to about 100 μηι, about 10 μηι to about 100 μηι, or about 50 μηι to about 500 μηι. Furthermore, a composition may comprise a tissue or a sheet with an intermediate layer having a mean thickness of any value or range of values listed above.

[00143] The presence of nucleated cells, extracellular matrix molecules, growth factors, and other biomolecules in a tissue or sheet prepared according to the methods described herein may be determined. ELISA kits and staining procedures for identifying some components are provided in Example 20. In some

embodiments, a tissue or sheet comprises bFGF, EGF, TGF beta 1, PDGF-AA, PDGF-BB, TIMP-1, TIMP-2, TIMP-4, or hyaluronic acid. In some embodiments, a tissue or sheet comprises nucleated cells and extracellular matrix molecules such as collagens, glycosaminoglycan-containing molecules (e.g., proteoglycans, etc.), or elastins.

H. Exemplary Embodiments for Layering Sheets

[00144] In some embodiments, another sheet may be layered on a surface of a perforated, unseparated amnion/ chorion; a perforated, separated amnion, or a perforated, separated chorion before or after preservation using methods understood by those of ordinary skill in the art. For example, one or more perforated or unperforated, unseparated amnion/ chorion may be layered on a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion. In other embodiments, one or more perforated or unperforated separated amnion may be layered on a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion. In further embodiments, one or more perforated or unperforated separated chorion may be layered on a perforated, unseparated amnion/ chorion; a perforated, separated amnion; or a perforated, separated chorion. Moreover, perforated and unperforated separated amnion and chorion sheets can be layered on a perforated, unseparated amnion/ chorion sheet in any number, combination, order, direction, or orientation.

I. Exemplary Embodiments for Powderizing

[00145] A tissue, sheet, or composition prepared according to the methods described herein may also be powderi2ed. As used herein, "powderi2ing" refers to milling, mincing, grinding, pulveri2ing, or any combination thereof to produce a powder.

[00146] In some embodiments, a tissue, sheet, or composition is milled, minced, ground, pulveri2ed, or any combination thereof into a powder product by any method known in the art. For example, a cryopreserved tissue, sheet, or composition may be powderi2ed using a cryogenic impact grinder (e.g., the 6770, 6870, 6970D, or 6970EFM Spex Free2er/Mill ^® (Metuchen, NJ)).

J. Exemplary Embodiments for Marking

[00147] A tissue, sheet, or composition prepared according to the methods described herein may be transparent or whitish in color, and it may be difficult to discern orientation of the tissue, sheet, or composition. In some embodiments, the tissue, sheet, or composition may have a marking, such as an embossment, a stain, a tint, a stamp, or a cut. The stain, tint, or stamp may be of any hue, including any shade of pink, red, blue, violet, orange, yellow, or green. Such a marking may be used to orient the user to as to the direction or orientation of the tissue, sheet, or composition. In some embodiments, the marking is made using one or more dyes (e.g., a natural dye, a vegetable dye, a hypoallergenic dye, beet juice, etc.). The dye may be of any hue, including any shade of pink, red, blue, violet, orange, yellow, or green. Any one or more layers of the tissue, sheet, or composition may be dyed, including, but not limited to, the epithelial layer of the amnion, the basement membrane of the amnion, the compact layer of the amnion, and the fibroblast layer of the amnion, the intermediate layer, the reticular layer of the chorion, the basement membrane of the chorion, and the trophoblast layer of the chorion. The dye may be applied to the tissue, sheet, or composition using any number of methods, including stamped on, painted on, or diffused into the tissue, sheet, or composition.

K. Exemplary Embodiments for Wound Healing

[00148] The compositions and products described herein may be used in wound healing and applied to the eye, the skin, a nerve, a tendon, or the dura of a subject in need of treatment. For example, the compositions and products may be applied to a wound of the eye, such as a burn, a laceration, a corneal ulceration, a conjunctival lesion, or a surgical wound. The compositions and products may be applied to a wound of the skin, for example, a burn, a laceration, a diabetic ulcer, a venous ulcer, an arterial ulcer, a decubitus ulcer, or a surgical wound. Techniques for applying the compositions and products to a wound of the eye, skin, nerve, tendon, or dura are understood by those of ordinary skill in the art.

EXAMPLES

Example 1. Preparing Perforated, Unseparated Amnion/ Chorion Sheets

[00149] A placenta collected from a donor mother is placed in sealed bag containing a suitable storage medium and is shipped on wet ice to the laboratory. After receipt, the placenta is stored at refrigeration temperature. The placenta is removed from its bag for processing at room temperature. The placenta is transferred to a metal tray. Using a pair of surgical scissors, the unseparated amnion/ chorion is dissected from the placental disc and umbilical cord. The unseparated

amnion/ chorion sheet is transferred to a 1 L Nalgene® plastic screw-top bottle containing about 750 mL of a sodium chloride solution and placed on a rocker platform and gently agitated for approximately 1 hour at room temperature.

[00150] The unseparated amnion/ chorion is transferred to a cutting mat and cut into multiple sheets using a rotary blade. An unseparated amnion/ chorion sheet is placed on a rubber or silicon mat (or other soft, nonporous surface that will allow a perforator to penetrate the tissue) with the epithelial layer of the amnion facing up. The unseparated amnion/ chorion sheet is perforated, for example, by rolling a perforator device (e.g., a device having a wooden handle and steel roller with a series pins arranged in multiple rows) across the top surface of the sheet such that multiple series of holes are made that traverse the thickness of the amnion/ chorion sheet.

[00151] The sheet is transferred to another 1 L bottle containing about

750 mL of a sodium chloride solution and placed on the rocker and gently agitated for about 1 hour at room temperature. The sheet is then transferred to a 1 L bottle containing about 750 mL of sterile water or a sodium chloride solution and placed on the rocker at room temperature with gentle agitation for about 1.5 hours followed by no agitation for another about 1.5 hours without changing the solution.

[00152] The sheet is then transferred to a 1L bottle containing about

750 mL of sterile water or a sodium chloride solution and stored at refrigeration temperature for a period of up to several days before lyophili2ation. After storage and before lyophilization, the sheet is transferred to a 1 L bottle containing about 750 mL of sterile water or a sodium chloride solution and placed on the rocker at room temperature with gentle agitation for about 1.5 hours followed by no agitation for an additional about 1.5 hours without changing the solution. The sheet is then stored in sterile water at a freezing temperature overnight and then lyophilized until dry.

Example 2. Preparing Perforated, Unseparated Amnion/ Chorion Layered with a Separated Sheet

[00153] Following collection of a placenta, dissection of the unseparated amnion/ chorion away from the placental disc, and washing in a sodium chloride solution, the unseparated amnion/ chorion is cut into multiple sheets using a rotary blade as described in Example 1. Two sheets are placed on a mat with the epithelial layer of the amnion facing down. The chorion is gently separated from the amnion of each sheet by hand and placed on the mat. One of the separated amnion sheets and one of the separated chorion sheets is perforated, for example, by rolling a perforator device across the sheets such that several holes are made that traverse the thickness of the sheets. The other separated amnion sheet and the other separated chorion sheet are not perforated.

[00154] The perforated and unperforated separated amnion sheets and chorion sheets are transferred to a 1 L bottle containing about 750 mL of a suitable washing medium (e.g., sterile water or a sodium chloride solution), placed on a rocker, and gently agitated for about 1 hour at room temperature. The suitable washing medium is replaced with about 750 mL of the same or a different suitable washing medium and gently agitated at room temperature for about 1.5 hours followed by no agitation for another about 1.5 hours without changing the solution. The sheets are then transferred to a 1L bottle containing about 750 mL of a suitable washing medium and stored at refrigeration temperature for a period of up to several days.

[00155] Four perforated, unseparated amnion/ chorion sheets are prepared according to the method of Example 1 through the step of storing at refrigeration temperature and before lyophilization. After transferring the perforated, unseparated amnion/ chorion sheets; the perforated, separated amnion sheet; the perforated, separated chorion sheet; the unperforated, separated amnion sheet; and the unperforated, separated chorion sheet to about 750 mL of a suitable washing medium, the sheets are gently agitated at room temperature for about 1.5 hours followed by no agitation for an additional about 1.5 hours without changing the solution.

[00156] The four perforated, unseparated amnion/ chorion sheets are each placed on a solid surface with either the epithelial layer of the amnion facing upward or downward. A separated sheet is layered on top of one of the perforated, unseparated amnion/ chorion sheets such that either side of the separated sheet is facing up. After storing the layered sheets at a free2ing temperature overnight, they are lyophili2ed until dry.

Example 3. Preparing Partial Perforations of Unseparated Amnion/ Chorion

[00157] Following collection of a placenta, dissection of the unseparated amnion/ chorion away from the placental disc, and washing in a suitable washing medium, the unseparated amnion/ chorion is cut into two sheets using a rotary blade as described in Example 1. The sheets are then placed on a mat with the epithelial layer of one sheet facing up and the epithelial layer of the other sheet facing down.

[00158] A perforating roller device having pins of varying length is rolled across the surface of the sheets to create perforations of varied depth. For example, a roller having pins with a length ranging from 0.1 μηι or less to 1 mm or greater may be used to create partial perforations.

Example 4. Process A

[00159] Donor placentas were logged and quarantined in the free2er until results from serological testing were received indicating that the placentas were nonreactive. Each placenta was processed separately according to the following procedure. The bag containing the donor placenta was placed on a tray. The placenta was transferred from the bag to another tray by grasping the umbilical cord. The unseparated amnion/ chorion was then carefully dissected away from the placental disc. The umbilical cord and placental disc were then discarded. Using fingertips, some visible, removable blood clots were gently lifted away from the outer surfaces of the unseparated amnion/ chorion to reduce the amount of blood entering the first washing medium.

[00160] A portion of the unseparated amnion/ chorion tissue was placed in a 1 L Nalgene ^® screw-top bottle to which about 750 mL of a first suitable washing medium was added. For Process A, the first washing medium used was 26% sodium chloride maintained at room temperature. The bottle was closed and gently agitated for 1 hour at room temperature. In this example and the following examples, gentle agitation was achieved by using a rocker at about 30 RPM.

[00161] The unseparated amnion/ chorion was then removed and laid flat on a soft, nonporous mat with the epithelial layer of the amnion facing up. The first washing medium was discarded. Using fingertips, additional visible, removable blood clots were gently lifted away from the tissue's outer surfaces. Blood-tinted areas of the unseparated amnion/ chorion tissue were gently massaged using fingertips with care so as not to separate the tissue.

[00162] To perforate the unseparated amnion/ chorion, a perforating roller was placed on top of the center of the tissue and rolled outwards over the surface of the tissue. The perforating roller used had a metal roller head with 24 x 7 rows of metal pins spaced about 6 mm apart.

[00163] Next, the perforated, unseparated amnion/ chorion was placed back into the empty bottle to which about 750 mL of a second suitable washing medium was added. For Process A, the second washing medium used was purified water maintained at room temperature. The bottle was closed and gently agitated for 1.5 hours at room temperature.

[00164] The second washing medium was decanted and replaced with about 750 mL of a third suitable washing medium. For Process A, the third washing medium used was 26% sodium chloride maintained at room temperature. After closing the bottle, it was gently agitated for 1 hour at room temperature.

[00165] After removing the third washing medium, about 750 mL of a fourth suitable washing medium was added. For Process A, the fourth washing medium used was 26% sodium chloride maintained at room temperature. The sample was then refrigerated (at about 4°C) overnight.

[00166] The next morning, the fourth washing medium was decanted and replaced with about 750 mL of a fifth suitable washing medium. For Process A, the fifth washing medium used was purified water maintained at room temperature. After closing the bottle, it was gently agitated for 1 hour at room temperature. [00167] The tissue was removed and placed in a closed container and stored overnight at a free2ing temperature (about -20°C). The fro2en tissue was then lyophili2ed overnight at about -46°C. The lyophili2ed tissue was placed into a container, labeled, and sealed.

Example 5. Process B

[00168] The steps of Process A were performed with the exception that

(1) the perforating step was performed after the placental disc and umbilical cord were removed and prior to the step involving the first washing medium; (2) the first washing medium used was purified water instead of 26% sodium chloride; (3) the second washing medium used was 26% sodium chloride instead of purified water; and (4) the overnight refrigeration step involving the third washing medium and the gentle agitation step involving the fourth washing medium were performed in reverse order.

Example 6. Process C

[00169] The steps of Process A were performed with the exception that prior to the addition of each of the first, second, third, fourth, and fifth washing medium, the mediums were preheated in a water bath maintained at 37°C.

Example 7. Process D

[00170] The steps of Process A were performed with the exception that the overnight refrigeration step involving the third washing medium and the gentle agitation step involving the fourth washing medium were performed in reverse order. Example 8. Process E

[00171] The steps of Process A were performed with the exception that

(1) the overnight refrigeration step involving the third washing medium and the gentle agitation step involving the fourth washing medium were performed in reverse order, and (2) prior to the addition of each of the first, second, and fourth washing mediums (the washing steps performed during the first day for Process E), the mediums were preheated in a waterbath maintained at 37°C. Example 9. Process F

[00172] The steps of Process A were performed with the exception that

(1) the overnight refrigeration step involving the third washing medium and the gentle agitation step involving the fourth washing medium were performed in reverse order, and (2) prior to the addition of each of the first, second, third, fourth, and fifth washing mediums, the mediums were preheated in a waterbath maintained at 37°C. Example 10. Process G

[00173] The steps of Process A were performed with the exception that the step involving a third washing medium was not performed.

Example 11. Process H

[00174] The steps of Process A were performed with the exception that

(1) the step involving a third washing medium was not performed, and (2) prior to the addition of each of the first, second, fourth, and fifth washing mediums, the mediums were preheated in a waterbath maintained at 37°C.

Example 12. Process I

[00175] The steps of Process A were performed with the exception that

(1) the step involving a third washing medium was not performed, and (2) for the step involving the first washing medium, the bottle containing the tissue was gently agitated for 2 hours at room temperature.

Example 13. Processes J _a -Ji

[00176] The steps of Processes A-I were performed with the exception that the unseparated amnion/ chorion tissue was not perforated. "Process J _a " refers to the process in which the steps were as in Process A except that the unseparated amnion/ chorion tissue was not perforated; "Process Jb" refers to the process in which the steps were as in Process B except that the unseparated amnion/ chorion tissue was not perforated; and so on.

Example 14. Processes Ka-Ki and Kj _a -Kji

[00177] The steps of Processes A-I and J _a -Ji were performed with the exception that instead of free2ing the tissue overnight followed by lyophilizing the tissue overnight, the sample was oven dried overnight at about 35°C. "Process K _a " refers to the process in which the steps were as in Process A except that instead of free2ing the tissue overnight followed by lyophilizing the tissue overnight, the sample was oven dried overnight at about 35°C; "Process Kb" refers to the process in which the steps were as in Process B except that instead of free2ing the tissue overnight followed by lyophili2ing the tissue overnight, the sample was oven dried overnight at about 35°C; and so on. "Process Kj _a " refers to the process in which the steps were as in process J _a except that instead of free2ing the tissue overnight followed by lyophili2ing the tissue overnight, the sample was oven-dried overnight at about 35°C; "Process Kjb" refers to the process in which the steps were as in Process Jb except that instead of free2ing the tissue overnight followed by lyophili2ing the tissue overnight, the sample was oven dried overnight at about 35°C; and so on.

Example 15. Dry Mass

[00178] The dry mass per surface area (mg/ cm ² ) of processed, unseparated amnion/ chorion tissue was measured and compared to that of unprocessed, unseparated amnion/ chorion tissue. The results are summari2ed in

Table 1.

[00179] Table 1

Example 16. Water Absorption

[001 80] The water absorption potential (mg of water absorbed per cm ² of tissue) of processed, unseparated amnion/ chorion tissue was compared to fresh, unprocessed, unseparated amnion/ chorion tissue. A 3 cm x 3 cm sheet of each sample was weighed before and after rehydration. Rehydration was achieved by placing the sample in a dish and adding 100 mL distilled water over the tissue. After 80 seconds of remaining in the distilled water, the tissue was removed, laid out on a mat, and the excess surface water wicked away using a wipe. The water absorption potential was calculated by subtracting the mass of the dried sample from the mass of the rehydrated sample and dividing by the surface area of the tissue. The water absorption potential of 3 samples of fresh, unprocessed, unseparated

amnion/ chorion tissue was undetectable (LOD= 0. 1 mg), presumably because the tissue was already fully hydrated. The results of the processed samples are

summari2ed in Table 2.

[001 81] Table 2

Example 17. Protein content

[001 82] Total protein content was determined for samples of processed, unseparated amnion/ chorion tissue and for fresh, unprocessed, unseparated amnion/ chorion tissue. Total protein was extracted from the tissue samples, quantified using a Pierce™ BCA Protein Assay Kit (Thermo Scientific™, Product No. 23227) , and normali2ed to the surface area of the sample. The results are summari2ed in Table 3. [00183] Table 3

Example 18. Hemoglobin

[00184] The hemoglobin content of unprocessed, unseparated amnion/ chorion tissue was compared to that of processed, unseparated

amnion/ chorion tissue that were either perforated or unperforated. Hemoglobin content was determined using a RayBio® Human Hemoglobin ELISA kit

(RayBiotech, Product No. ELH-Hgbl) and normalized to the dry mass of the sample. The mean and ranges of hemoglobin content (based on the ratio of hemoglobin mass to tissue mass) are summarized in Table 4.

[00185] The limit of detection (LOD) for the hemoglobin assay as performed was considered to be 0.043 ug/mg, based on lysis of the sample in 1 mL lysis buffer per 35 mg sample and the lowest detectable hemoglobin concentration for the RayBio® Human Hemoglobin ELISA kit being 1.5ug/ mL.

[00186] Table 4

(LOD: limit of detection.) [00187] Both (i) the difference in hemoglobin content between unprocessed and processed, perforated, unseparated amnion/ chorion tissue and (ii) the difference in hemoglobin content between unprocessed and processed, unperforated, unseparated amnion/ chorion tissue were statistically significant (p-values < 0.01). The significant reduction in hemoglobin content observed in processed, unseparated amnion/ chorion tissue compared to unprocessed tissue indicates a significant reduction in red blood cell remnants.

Example 19. Thickness

[00188] The thickness of processed, unseparated amnion/ chorion tissue was compared to that of fresh, unprocessed, unseparated amnion/ chorion tissue. The thickness of the intermediate layer of the tissues were also compared. A portion of each tissue was embedded, sectioned, mounted, and stained with hematoxylin and eosin (H & E). A digital image of each slide was obtained. Approximate thickness of the tissues and approximate thickness of the intermediate layer of the tissues were measured at five or six representative areas under x20 magnification. Mean thicknesses of each tissue were determined by calculating the average of the measured values. The mean and range of mean thicknesses were determined for each group of samples and are summari2ed in Table 5.

[00189] Table 5

Example 20. Growth factors, extracellular matrix molecules, and other biomolecules

[00190] The presence of various growth factors and other biomolecules in processed, unseparated amnion/ chorion tissues was determined using ELISA kits that assay for bFGF, EGF, TGF beta 1, PDGF-AA, PDGF-BB, TIMP-1, TIMP-2, and TIMP-4 (RayBiotech, RayBio® ELISA kits Product Nos. ELH-bFGH, ELH- EGF, ELH-TGFbl, ELH-PDGFAA, ELH-PDGFBB, ELH-TIMPl, ELH-TIMP2, and ELH-TIMP4), and for hyaluronic acid (R&D Systems, Hyaluronan Quantikine ELISA Product No. DHYAL0). Samples of processed, unseparated amnion/ chorion tissue were found to contain bFGF, EGF, TGF beta 1, PDGF-AA, PDGF-BB, TIMP-1, TIMP-2, TIMP-4, and hyaluronic acid regardless of whether they were perforated or unperforated, and of whether they were lyophilized or oven-dried.

[00191] The presence of nucleated cells and extracellular matrix molecules in processed, unseparated amnion/ chorion tissue was determined by standard histological staining. Unseparated amnion/ chorion tissue was prepared according to Process IQ and Kji. A portion of each tissue was embedded, sectioned, mounted, and stained. The presence of nucleated cells (H & E), collagens (Masson's Trichrome), glycosaminoglycan-containing molecules (e.g., proteoglycans, etc.) (Alcan Blue), and elastins (VanGieson) was observed and the intensity of staining for each layer of the tissues is summari2ed in Table 7.

[00192] Table 7

Scoring: +, ++, + + + indicates slight, moderate, and heavy staining, respectively. * indicates not quantified.

Example 21. Chorion Separation from Amnion and Intermediate Layer

[00193] Unseparated amnion/ chorion having an intermediate layer is dissected away from the placental disc and placed in a tray with the chorion facing up. The edge of the chorion is gently pulled up using one hand while the other hand secures the amnion with the intermediate layer on the tray. With flattened fingers of one hand on the amnion/ intermediate layer securing it to the base of the tray, the other hand holds the chorion close to the line of separation and pulls upwards slowly such that the chorion pulls away from the amnion/ intermediate layer over a short distance. This process of peeling away of the chorion is slowly continued by repeating the same securing and pulling actions along the line of separation between the intermediate layer and the chorion until the chorion is separated away leaving the intermediate layer substantially intact on the separated amnion.

[00194] The separated amnion with intermediate layer or the separated chorion may be processed using any one of Processes A-K. In addition, the chorion may be separated from the amnion and intermediate layer before, during, or after dissecting, cutting, perforating, or washing.

Example 22. Amnion Separation from Chorion and Intermediate Layer

[00195] Unseparated amnion/ chorion having an intermediate layer is dissected away from the placental disc and placed in a tray with water to swell the intermediate layer. The excess water is drained away and the unseparated

amnion/ chorion is oriented with the amnion facing up on the tray. Using forceps, the edge of the amnion is pulled up slightly to visualize the line of separation between the intermediate layer and the amnion. The flat end of a scalpel is then used to gently scrape from the forceps to the line of separation while gently and slowly pulling up with the forceps. This process of scraping and pulling is slowly repeated along the line of separation between the intermediate layer and the amnion until the amnion is separated away leaving the intermediate layer substantially intact on the separated chorion. [00196] The separated chorion with intermediate layer or the separated amnion may be processed using any one of Processes A-K. In addition, the amnion may be separated from the chorion and intermediate layer before, during, or after dissecting, cutting, perforating, or washing.