Cross-References

This application claims the benefit of provisional U.S. Patent Application No. 62/200,087 (filed 2 August 2015), which is incorporated by reference herein in its entirety.

Technical Field

This invention relates to bioengineered corneal tissue.

Background

This invention relates to the engineering of corneal tissue. FIG. 1 shows a cross-section of the cornea of the human eye. The corneal epithelium is a thin layer of non-keratinized stratified squamous epithelium. This thin layer is composed of several layers of cells which are shed constantly on the exposed layer and are regenerated by multiplication in the basal layer. Bowman's layer (also known as the anterior limiting membrane) is the subepithelial basement membrane. The corneal stroma is a thick, transparent, middle layer of the cornea. The stroma constitutes up to 90% of the corneal thickness and is constructed of highly regular collagenous lamellae and extracellular matrix components along with sparsely distributed, interconnected keratocytes. The stroma has about 300 to 500 such collagenous lamellae, which are generally parallel to the corneal surface. Corneal keratocytes are specialized fibroblasts residing in the stroma. They are the principal cell component of corneal stroma and are responsible for the stromal matrix. Through cell-cell dendritic connections, the keratocytes have an extensive 3D network. The cornea stroma is relatively avascular and receives its essential nutrients primarily by diffusion. The corneal keratocytes produce the extracellular matrix containing collagen, proteoglycans (such as keratocan), and glycosaminoglycans (such as keratan sulfate or dermatan sulfate). The keratocytes constitute approximately 5 - 10% of the stromal volume and are distributed in a helical network from the anterior to the posterior surface. Descemet's membrane (also posterior limiting membrane) is a thin acellular layer that serves as the modified basement membrane of the corneal endothelium, from which the cells are derived. The corneal endothelium is a monolayer of simple squamous or low cuboidal cells. These cells are responsible for regulating fluid and solute transport between the aqueous and corneal stromal compartments.

Summary

In one aspect, the invention is method of constructing cornea tissue. The method comprises: (a) using a cell culture substrate having a surface, wherein the surface has a grooved pattern and comprises a temperature-responsive polymer in which its hydrophilicity or hydrophobicity can be controlled by a change in temperature; (b) culturing functional keratocytes on the substrate at a first temperature; (c) lowering the temperature to a second temperature to make the temperature-responsive polymer more hydrophilic; (d) detaching the functional keratocytes and extracellular matrix from the substrate surface as a sheet; (e) harvesting the sheet of functional keratocytes and extracellular matrix. In some embodiments, the harvested sheet is transparent. In some embodiments, the functional keratocytes are non-randomly oriented. In some embodiments, the method comprises culturing the functional keratocytes until they have a non-random orientation. In some embodiments, the mean orientation angle of the functional keratocytes on the sheet is 30° or less. In some embodiments, at least 75% of the functional keratocytes on the sheet have an orientation angle of 30° or less.

In some embodiments, the collagen fibrils in the extracellular matrix are non-randomly oriented. In some embodiments, the alignment of the collagen fibrils in the extracellular matrix are influenced by the direction of the grooves. In some embodiments, the method comprises culturing the functional keratocytes until they produce extracellular matrix comprising collagen fibrils having a non-random orientation. In some embodiments, at least 50% of the collagen fibrils in the extracellular matrix have an orientation angle of 30° or less. In some embodiments, at least 75% of the collagen fibrils have a diameter in the range of 15 - 90 nm. In some embodiments, the fibril-to-fibril spacing of the collagen fibrils are spaced with an average spacing of less than 400 nm.

In some embodiments, the functional keratocytes are cultured until they stratify to form multiple layers of functional keratocytes. In some embodiments, the functional keratocytes are cultured for at least two weeks. In some embodiments, the sheet is detached and harvested prior to the functional keratocytes reaching full confluency.

In some embodiments, the functional keratocytes are corneal keratocytes or corneal stromal cells that are from an autologous or donor source. In some embodiments, the method further comprises: seeding the substrate with progenitor cells or stem cells; and inducing the cells to differentiate into functional keratocytes.

In some embodiments, the substrate surface is a hydrogel comprising the temperature- responsive polymer. In some cases, the hydrogel is non-soluble in an aqueous solution. In some cases, the temperature-responsive polymer is covalently cross-linked. In some cases, the temperature-responsive polymer is covalently grafted onto the substrate. In some embodiments, between steps (b) and (c), the method further comprises: (i) lowering the temperature to make the temperature-responsive polymer more hydrophilic and cause it to swell; and (ii) before the sheets completely detach, raising the temperature to make the temperature-responsive polymer more hydrophobic and cause it to shrink. In some cases, the method further comprises repeating steps (i) and (ii) one or more times. In some embodiments, the method further comprises repeating steps (a) - (e) to create multiple sheets and stacking the sheets on top of one another to form a multi-layer tissue construct. This repeating of steps (a) - (e) could be performed according to any suitable time schedule (e.g. in parallel, series, staggered timing, etc.) to produce the multiple cell sheets.

In some embodiments, the method further comprises culturing the multi-layer tissue construct until at least some of the functional keratocytes migrate from one layer of the multilayer tissue construct to another layer. In some embodiments, the method further comprises culturing the multi-layer tissue construct until at least some of the functional keratocytes interconnect with a functional keratocyte on a different layer of the multi-layer tissue construct. In some embodiments, the multi-layer tissue construct is cultured for a further duration of at least five days.

In some embodiments, the method further comprises inducing the functional keratocytes to undergo apoptosis. In some cases, the functional keratocytes are exposed to an apoptosis-inducing agent that induces apoptosis of the functional keratocytes. In some cases, the functional keratocytes in a cultured sheet are induced to undergo apoptosis prior to detaching and harvesting the cell sheet. In some cases, the functional keratocytes in a multilayer tissue construct are induced to undergo apoptosis. In some embodiments, compression is applied onto the cultured sheet or the multi-layer tissue construct in order to induce keratocyte apoptosis.

In another aspect, the invention is a cell sheet comprising functional keratocytes and extracellular matrix. In some embodiments, the cell sheet has a round, oval, or elliptical shape. In some embodiments, the cell sheet is scaffold-free. In some embodiments, the cell sheet is sufficiently sturdy to hold a suture. In some embodiments, the functional keratocytes occupy an area of less than 50% of the area of the sheet.

In some embodiments, the functional keratocytes on the cell sheet are aligned in a non- random orientation. In some embodiments, the collagen fibrils in the extracellular matrix are aligned in a non-random orientation.

In another aspect, the invention is a tissue construct comprising two or more stacked sheets of functional keratocytes and extracellular matrix. In some embodiments, the tissue construct has a round, oval, or elliptical shape. In some embodiments, the tissue construct is scaffold-free. In some embodiments, the tissue construct is sufficiently sturdy to hold a suture. In some embodiments, at least some of the functional keratocytes are in physical contact with a functional keratocyte located in a different sheet. In some embodiments, each sheet has functional keratocytes that are aligned in a non- random orientation. In some embodiments, each sheet has an extracellular matrix comprising collagen fibrils that are aligned in a non-random orientation In some embodiments, for at least one of the sheets, the alignment orientation of the functional keratocytes are at an angle with respect to the alignment orientation of the functional keratocytes in an adjacent sheet. In some embodiments, for at least one of the sheets, the alignment orientation of the collagen fibrils in the extracellular matrix are at an angle with respect to the alignment orientation of the collagen fibrils in the extracellular matrix in an adjacent sheet.

In some embodiments, the functional keratocytes occupy a volume of less than 50% of the volume of the multi-layer tissue construct. In some embodiments, the average density of the functional keratocytes in the multi-layer tissue construct is less than 75,000 cells/mm ³ . In some embodiments, the average density of the functional keratocytes in the multi-layer tissue construct is less than 38,000 cells in a 1 mm ² column.

In another aspect, the invention is a method of performing a corneal replacement surgery in a patient. The method comprises: removing cornea tissue from the patient's eye, and implanting a bioengineered cornea tissue construct described herein into the patient's eye. In some embodiments, the bioengineered cornea tissue construct is optically transparent. In some embodiments, the step of implanting comprises suturing the bioengineered cornea tissue construct into the patient's eye. In some embodiments, the method comprises, after implanting the bioengineered cornea tissue construct, applying a contact lens on the eye over the implanted tissue construct to result in keratocyte apoptosis in the tissue construct. Brief Description of the Drawings

FIG. 1 shows a cross-section of the cornea of the human eye.

FIG. 2 shows an enlarged view of a portion of an example substrate surface having a parallel groove/ridge topography.

FIG. 3 shows how the pitch, depth, width of grooves, and width of ridges are measured. FIGs. 4A and 4B show an example of how cell attachment and detachment on a temperature-responsive surface can be controlled by changes in temperature. FIGs. 5A and 5B show an example of how a cell sheet can be cultured and harvested from a temperature-responsive surface.

FIG. 6 shows an example of how a thermoresponsive polymer film can be applied onto a patterned substrate.

FIG. 7 shows an example of forming a pattern directly onto a layer of thermoresponsive polymer.

FIG. 8 shows an example of how alignment of the cells can be measured.

FIG. 9 shows keratocytes being cultured on a grooved substrate.

FIG. 10 shows how stromal sheets can be stacked from multiple separately cultured sheets.

FIG. 11 shows an example of a multi-layer corneal tissue construct.

FIGs. 12A - 12C show an example of a corneal replacement surgery.

Detailed Description

A sheet of cornea stromal tissue can be made by in vitro culturing of functional keratocytes on a substrate such that they deposit extracellular matrix components including collagen fibrils. "Functional keratocytes" are cells that have the ability to deposit an organized transparent extracellular matrix similar to that of the native corneal stroma. As such, they would produce corneal stromal extracellular matrix components, such as collagen fibrils (type I, type V, or type l/V hybrid), proteoglycans (e.g. keratocan), or glycosaminoglycans (e.g. keratan sulfate and dermatan sulfate).

In some embodiments, the functional keratocytes are corneal keratocytes as they natively exist in the human cornea. However, "functional keratocytes" may or may not be corneal keratocytes per se as they exist in the human body with precisely the same phenotypic markers as natively found in the human body. They may be corneal stromal cells or

differentiated cells that are not literally keratocytes, but exhibiting the ability to function as keratocytes in their ability to deposit organized extracellular matrix components, such as collagen, keratan sulfate, or keratocan. I n addition to their ability to produce extracellular matrix in an organized manner, there are other markers that can be used to identify cells as being functiona l keratocytes. For example, functional keratocytes may also be characterized by their cytoplasmic content of crystallins or aldehyde dehydrogenase that contribute to the transparency of the cornea. Other characteristic markers that can be used to identify functional keratocytes are described in

[Ruberti et a I, "The keratocyte: Corneal stromal cell with variable repair phenotypes" (2006) Int J Biochem Cell Biol., 38(10):1625-1631].

I n this invention, the identification of the cells as "functional keratocytes" may occur at any point during the culturing the process. I n some embodiments, the functional keratocytes are seeded onto the substrate at the beginning of culturing. I n some embodiments, corneal stromal cells (such as quiescent keratocytes, activated keratocytes, fibroblasts, repair fibroblasts, or myofibroblasts) are seeded onto the substrate at the beginning of culturing. Under appropriate culture conditions, fibroblasts, repair fibroblasts, or myofibroblasts can be changed into keratocytes. See [Wu et a I, "Corneal Stromal Stem Cells versus Corneal Fibroblasts in Generating Structurally Appropriate Corneal Stromal Tissue" (2014 Mar) Exp Eye Res., 120:71-81], which is incorporated by reference herein.

I n some embodiments, more primitive cells (i.e. at an earlier step along a developmental progression to functional keratocytes) such as progenitor cells or stem cells (including induced stem cells) are seeded onto the substrate, which are then subsequently made to differentiate into functional keratocytes during culturing (e.g. by culturing in a keratocyte differentiation medium). That is, the functional keratocytes may result from the differentiation of progenitor cells or stem cells that are seeded onto the substrate.

Progenitor cells are early descendants of stem cells that can differentiate to form one or more kinds of cells, but cannot divide and reproduce indefinitely. A progenitor cell, being more specific than a stem cell, is often more limited than a stem cell in the kinds of cells it can become. I n some embodiments of the invention, the progenitor cell is a mesenchymal fibroblast cell or a primary corneal fibroblast (usually a de-differentiated corneal keratocyte extracted from donor tissue). Some keratocyte progenitor cells are known to express the ocular development gene Pox6, which is not expressed by the resident stromal keratocytes. This may be one possible way to identify a progenitor cell for a functional keratocyte.

Examples of stem cells that can be used are human corneal stromal stem cells, human adipose-derived stem cells, and human dental pulp stem cells. See [Syed-Picard et al, "Dental pulp stem cells: a new cellular resource for corneal stromal regeneration" (2015 Mar) Stem Cells Transl Med., 4(3):276-85]. In some embodiments, the stem cells are stem cells that originate from the cranial neural crest (i.e. of cranial neural crest lineage). The stem cells of neural crest lineage can be co-cultured with mouse PA6 fibroblasts to induce differentiation into adult stromal stem cells, which can then be induced to differentiate into corneal keratocytes by culturing in serum-free medium containing ascorbate. See [Syed-Picard et al, supra]. In some embodiments, the stem cells are mesenchymal stem cells (e.g. neural-crest derived mesenchymal stem cells).

The cells seeded onto the substrate may be functional keratocytes (including native keratocytes, quiescent keratocytes, and activated keratocytes), progenitor cells, stem cells, corneal stromal cells (including fibroblasts, repair fibroblasts, and myofibroblasts that can be induced to differentiate into keratocytes), or corneal mesenchymal cells. The source of the cells may be from any suitable source, including autologous source, allogeneic source (e.g. from a donor), or xenogeneic. Because cornea stroma is relatively immunologically privileged, there is some flexibility in the selection of the source. The cells may be cultured in a culture medium that maintains the viability of the cells or supports their growth. In some embodiments, the culture medium contains an agent that enhances production of extracellular matrix. In some embodiments, the culture medium contains an agent that induces differentiation of the cell into a functional keratocyte (i.e.

differentiation medium). Examples of agents that can do one or both include ascorbic acid or one of the more stable derivatives or analogs of ascorbic acid having the same functional activity as a co-factor in collagen synthesis, such as ascorbyl 2-phosphate, ascorbyl 2-sulfate, ascorbyl 2-glucoside, 2-O-a-D-glucopyranosyl-L-ascorbic acid, ethyl ascorbic acid, and 6-0- alkanoyl ascorbic acids such as ascorbyl palmitate, ascorbyl stearate, ascorbyl octanoate, and ascorbyl dipalmitate. Additional ascorbic acid derivatives that may be used are described in patent publications US 2003/0045572 (Niyiro et al), US 2006/0100178 (Masatsuji et al), and US 2006/0030621 (Inaoka et al), which are incorporated by reference herein. Additional ascorbic acid derivatives that may be used are described in [Moribe et al, "Drug Nanoparticle

Formulation Using Ascorbic Acid Derivatives" (2011) Journal of Drug Delivery, Article ID

138929].

The composition of the culture medium may remain the same or change throughout the culturing of the cells. The culture medium may comprise growth factors such as fibroblast growth factor-2 (FGF-2 or basic fibroblast growth factor), transforming growth factor such as TGF- 2 and TGF- 3, platelet-derived growth factor (such as PDGF BB), insulin, or insulin-like growth factor 2 (IGF-2). Culture medium containing insulin or IGF-2 (particularly in combination with ascorbate) has been shown to promote cell proliferation while maintaining keratocyte cell morphology. See [Musselmann, "Developing culture conditions to study keratocyte phenotypes in vitro" (2006) Graduate Thesis at University of South Florida; see http://scholarcommons. usf.edu/etd/641].

In some embodiments, the culture medium contains heparin (optionally, along with FGF-2). In some embodiments, the culture medium contains bovine serum albumin. In some embodiments, the culture medium contains corneal extract or corneal stromal extract. Any suitable technique for making corneal extract or corneal stromal extract may be used, including the technique described in [Musselmann, supra].

The culture medium may contain serum (e.g. fetal bovine serum or platelet poor horse serum). However, fetal bovine serum is known to induce the conversion of keratocytes from their quiescent phenotype to a fibroblastic phenotype and fibroblasts may be reversed to a keratocyte-like morphology by removal of the serum in the medium. In some embodiments, the culture medium is serum-free.

To enhance corneal transparency, the harvested sheets may have relatively lower cellular content. In some embodiments, the functional keratocytes occupy an area of less than 50% of the area of the sheet. Relatively lower cellular content can be achieved by culturing the cells sufficiently long for them to produce extracellular matrix, but before the cells become fully confluent. In some cases, the sheet of functional keratocytes are detached and harvested before the functional keratocytes reach confluency of 85% (e.g. upon reaching confluence of 68%); in some cases, before reaching confluency of 70%.

In some embodiments, the functional keratocytes are cultured until they stratify to form multiple (two or more) layers of the cells in the sheet. In some embodiments, the functional keratocytes are cultured for at least two weeks; in some cases, at least three weeks; and in some cases, at least four weeks. In some embodiments, the harvested sheet is scaffold-free (at the time when the sheet is harvested, the sheet contains only cultured cells and the material produced by the cells).

The substrate on which the cells are grown has a pattern of grooves and ridges. Surface patterns are known to play a role in cell alignment. On grooved substrates, cells generally align in the direction of the grooves on the substrate, known as "contact guidance." See [Gil et al, "Response of Human Corneal Fibroblasts on Silk Film Surface Patterns" (2010 June) Macromol Biosci., 10(6):664-673]. The substrate may be part of articles such as microplates, culture dishes, microscope slides, chips, etc. The substrate may be constructed from any suitable material, including plastic, metal, glass, or ceramic. Examples of materials for constructing the substrate include poly(dimethylsiloxan), polystyrene, poly(lactic acid), poly(glycolic acid), poly(hydroxybutyrate), polycarbonate, polycaprolactone, polymethylmethacrylate, or other thermoplastic polymers.

The substrate topography can be fabricated by any suitable micro- or nano-patterning technique, such as photolithography, electron beam lithography, soft lithography (including capillary force lithography), etching (e.g. laser etching, chemical etching, dry etching, etc.), stamping, imprinting, embossing, vapor deposition, inkjet printing, etc. Examples of such patterning techniques are described in WO 2013/151755 (Kim et al, Univ. of Washington), [Kim et al, "Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs" (2010 Jan) Proc Natl Acad Sci., 107(2):565-570], and [Fuj ^' ita et al, "Time-lapse observation of cell alignment on nanogrooved patterns" (2009) 7. R. Soc. Interface, 6:S269- S277]. FIG. 2 shows an enlarged view of a portion of an example substrate surface having a parallel groove/ridge topography. FIG. 3 shows how the pitch, depth, width of grooves, and width of ridges are measured. In some embodiments, the pitch of the grooves is between 400 - 4,000 nm. In some embodiments, the depth of the groove is between 100 - 1,000 nm. In some embodiments, the width of the groove is between 70 - 2,000 nm. In some embodiments, the width of the ridge is between 70 - 2,000 nm. The dimensions of the grooves and ridges (e.g. pitch, width, or depth) may or may not be uniform across the entirety of the substrate. In some embodiments, the grooves are substantially parallel. In some embodiments, the grooves are in a uniformly repeating pattern. As an example, one way to create the surface topography is to make a master containing the patterned relief structure prepared via traditional photolithography techniques. Cast an elastomer, such as polydimethylsiloxane (PDMS), against the master to make a mold or stamp. Use the elastomer stamp for hot embossing against a thermoplastic polymer (e.g.

polystyrene). Press the patterned mold against a thin sheet of polystyrene and heat it above the glass transition temperature of polystyrene. This will transfer the pattern on the mold to the polystyrene substrate.

The substrate on which the cells are grown has a surface that comprises a temperature- responsive polymer (also commonly known as thermoresponsive polymers), which undergoes a drastic and discontinuous change in hydrophilicity/hydrophobicity with a change in

temperature (in contrast to the more gradual change characteristic of temperature-sensitive polymers). Temperature-responsive polymers undergo this reversible change below and above a lower critical solution temperature (LCST). The temperature-responsive polymer changes to become more hydrophilic as temperature falls below its LCST and becomes more hydrophobic as temperature rises above the LCST. This allows the hydrophilicity/hydrophobicity of the polymer to be controlled by a change in temperature. Because cells tend to attach more readily to hydrophobic surfaces, this allows for the control of cell attachment and detachment to produce intact cell sheets.

Conventional detaching of cells using digestive enzymes, such as trypsin, or physical scraping can cause damage to the extracellular matrix (ECM), cell-ECM connections, or cell-cell connections. By growing the cells on a temperature-responsive surface, intact sheets of cells can be harvested with reduced damage.

FIGs. 4A and 4B show an example of how cell attachment and detachment on a temperature-responsive surface can be controlled by changes in temperature. The substrate 12 is covered with a film of poly(N-isopropylacrylamide) ('poly-NIPAAm'), which is covalently grafted onto the substrate. Poly-NIPAAm has an LCST of 32° C and is relatively hydrophobic at temperatures above its LCST. FIG. 4A shows the culture-substrate interaction at the 37° C temperature under which the cells are cultured. At this temperature, poly-NIPAAm is in a relatively hydrophobic state and the polymer chains 14 are collapsed on the substrate surface. This allows the surface to support cell attachment and growth, similar to conventional culture dishes.

In FIG. 4B, the temperature is reduced to room temperature below poly-NIPAAm's 32° C LCST such that the polymer 14 becomes more hydrophilic. Because the poly-NIPAAm is grafted onto the substrate surface, this polymer layer also acts like a gel and becomes swollen as it becomes hydrated. With this transition, the polymer chains 14 expand and swell to create a hydration layer between the cultured cells 10 and the substrate surface, allowing for cell detachment.

The cells are grown on the substrate at a temperature in which the temperature- responsive polymer is relatively hydrophobic such that the cells attach to the surface. When the cell sheet is ready to be harvested, the temperature is reduced to cause the temperature- responsive polymer to become relatively more hydrophilic. This change in the temperature- responsive polymer causes the cells to detach. Cells are harvested from the substrate as an intact sheet comprising the cells and their associated extracellular matrix.

FIGs. 5A and 5B show an example of how a cell sheet can be cultured and harvested from a temperature-responsive surface. Shown here is a cross-section side view of a sheet of cells being cultured on a temperature-responsive surface of poly-NIPAAm. FIG. 5A shows a monolayer of cells 20 grown on the substrate 24 along with its extracellular matrix 22. At 37° C, the cells attach to the temperature-responsive substrate. As shown in FIG. 5B, when the temperature is reduced to 25° C, the temperature-responsive substrate 24 becomes hydrophilic, causing the cells to detach. The monolayer of cells 20 along with the extracellular matrix 22 can be detached and harvested as an intact sheet without disturbing cell-cell and cell- ECM connections. As seen here, the temperature-responsive substrate 24 is left intact.

Moreover, when harvested, the monolayer of cells 20 is free of any scaffolding.

Examples of thermoresponsive polymers that can be used are described in [Ward et al, "Thermoresponsive Polymers for Biomedical Applications" (2011) Polymers, 3:1215-1242]. Particularly useful thermoresponsive polymers are those that show thermoresponsivity in aqueous solution. Particularly useful thermoresponsive polymers are those that have its LCST in the range of 0 to 100° C, and more particularly, at a relatively low temperature, i.e. in the range of 15 - 35° C. Thermoresponsive polymers that could be used include poly(/V,/V- diethylacrylamide) with an LCST over the range of 25 - 32° C, poly(/V-vinlycaprolactam) with an LCST over the range 25 - 35° C, poly[2-(dimethylamino)ethyl methacrylate)] with a LCST of around 50° C, and poly(ethylene glycol) with a LCST of around 85° C, poly(vinylmethyl ether), poly(hydroxyethyl methacrylate), and hydroxypropylcellulose.

The properties of the thermoresponsive polymer can be tuned by selecting its backbone com position (e.g. incorporating different hydrophilic or hydrophobic co-monomers or copolymers), its molecular weight, side groups or grafted groups, or the architecture of the polymer network (e.g. cross-linking, etc.). Properties that can be altered include its phase temperature (such as LCST), pH responsiveness, swelling/deswelling kinetics, speed of thermoresponsiveness, and degradability. In particular, it may be useful to tune the phase temperature of the polymer to around physiological temperature (37° C) to tailor its use for biological applications. In the case of poly-NI PAAm, its LCST can be tailored by incorporating hydrophilic or hydrophobic co-monomers into the polymer structure. See [Tekin et a I,

"Thermoresponsive Platforms for Tissue Engineering and Regenerative Medicine" (2011 Dec) AIChE Journal, 57(12):3249-3258]. Other examples of poly-N IPAAm that can be used are described in [Ta ng et al, "Recent development of temperature-responsive surfaces and their application for cell sheet engineering" (2014 Nov) Regenerative Biomaterials, 1(1):91-102]. For more examples, the LCST of poly-N IPAAm can be modified by incorporating hydrophilic co- monomers such as acrylamide, /V-methyl-/V-vinylacetamide, /V-vinylacetamide, and /V-vinyl-2- pyrrolidinone via free-radical polymerization. Incorporation of ionic co-monomers into poly- NIPAAm can raise the LCST. Copolymers of poly-NIPAAm with two distinct LCSTs can be fabricated by using oligomers, such as carboxy-terminated oligo-NIPAAm, oligo(/V- vinylcaprolactam), and a random co-oligomer of NIPAAm and acrylamide.

In some embodiments, the thermoresponsive polymer is formed into a hydrogel layer. Hydrogels are polymer networks dispersed in an aqueous solution such that they take on semisolid states containing a high water-to-polymer ratio. Hydrogels can be either a physical gel or a covalently cross-linked gel. See [Ward et al, supra]. In cross-linked gels, the polymer chains are covalently cross-linked together. Physical gels, on the other hand, are formed by the physical entanglement of polymer chains in solution and not from covalently linked polymer chains. Both of these gels, crosslinked and physical, have the ability to swell in a solvent depending on their compatibility with the solvent. However, whereas a physical gel will eventually dissolve in the appropriate solvent if given sufficient time and space, a cross-linked gel will not. Cross- linked networks of thermoresponsive polymers swell/shrink with hydration in response to temperature, whereas physical gels show a sol-gel transition with hydration. A cross-linked hydrogel swells in an aqueous solution but does not dissolve therein. Another way to make the thermoresponsive polymers insoluble in an aqueous solution and give them gel-like properties is to covalently graft them onto the substrate. The substrate surface can be made with a temperature-responsive polymer in any suitable way. Examples of techniques for making the temperature-responsive polymer layer are described in WO 2013/151755 (Kim et al, Univ. of Washington), [Jiao et al, "Thermoresponsive Nanofabricated Substratum for the Engineering of Three-Dimensional Tissues with Layer-by- Layer Architectural Control" (2014 May) ACS Nano, 8(5):4430-9], [Elloumi-Hannachi et al, "Cell sheet engineering: a unique nanotechnology for scaffold-free tissue reconstruction with clinical applications in regenerative medicine" (2009) Journal of Internal Medicine, 267:54-70],

[Isenberg et al, "A thermoresponsive, microtextured substrate for cell sheet engineering with defined structural organization" (2008 Jun) Biomaterials, 29(17):2565-2572], [Tekin et al, "Responsive Microgrooves for Formation of Harvestable Tissue Constructs" (2011 May) Langmuir, 27(9):5671-5679], and [Tang et al, supra].

One possible way to make a patterned thermoresponsive surface is to apply the thermoresponsive polymer onto a patterned substrate surface. For example, FIG. 6 shows a thermoresponsive polymer film 30 deposited onto a grooved foundation 32. This

thermoresponsive polymer film 30 constitutes the surface of the substrate. This can be performed by various techniques including in-situ polymerization of precursor monomers, depositing a film of the thermoresponsive polymer onto the substrate surface, or grafting the thermoresponsive polymer onto the substrate surface. Coating methods that can be used include electrostatic coating and physical coating. One possible technique for in-situ

polymerization and grafting is UV irradiation. For example, this can be performed by applying a thin layer of NIPAAm monomer and benzophenone (a photoinitiator) dissolved in a solvent (e.g. 2-propanol) onto a textured polystyrene surface. UV irradiation causes polymerization of the NIPAAm monomers and grafting onto the polystyrene surface. [Tang et a I, supra]. The grafted polymer thickness can be controlled by monomer concentrations and radiation energy.

Another possible technique for in-situ polymerization and grafting is electron beam (EB) irradiation. This can be performed by applying a thin layer of NIPAAm monomer solution spread on a textured polystyrene surface. Exposing the monomer solution to electron beam irradiation causes polymerization of the NIPAAm monomers and grafting onto the polystyrene surface. The grafted polymer thickness can be controlled by monomer concentrations and radiation energy.

Other possible techniques are free-radical initiated polymerization, chemical vapor deposition, reversible addition-fragmentation chain transfer radical polymerization (RAFT), and plasma treatment. Other possible techniques are the "grafting to" and "grafting from" and surface-initiated living radical polymerization techniques described in [Nagase et al,

"Temperature-responsive intelligent interfaces for biomolecular separation and cell sheet engineering" (2009 Jun) J R Soc Interface, 6(Suppl 3):S293-S309].

Another possible way to make the layer of thermoresponsive polymer is to directly form the desired pattern on the layer of thermoresponsive polymer. For example, FIG. 7 shows a thermoresponsive polymer bed 40 on a glass dish base 42. The grooves are imprinted directly into the thermoresponsive polymer bed 40. This direct patterning of the thermoresponsive polymer bed 40 can be performed by any of a variety of possible techniques including soft lithography, photolithography, photopolymerization, micromoulding, embossing, or any of the patterning techniques described above.

The temperature-responsive polymer layer may have any suitable thickness (e.g. in the range of 5 - 200 nm). Among the factors that may affect the cell adhesion and detachment are film thickness, surface wettability, polymer chain mobility, grafting density, and swelling ratio. These parameters can be optimized for the best effect. In addition to the temperature- responsive polymer, the substrate surface may comprise other materials to tune the properties of the surface, including biodegradable materials such as peptides, proteins (e.g. zein protein), alginate, and gelatin. Such additional materials may be incorporated into the network of temperature-responsive polymers without chemical cross-linking.

The functional keratocytes are cultured on the grooved substrate to influence the alignment of the functional keratocytes. The alignment of the cells can be measured by their orientation angle, which is the angle between the longest axis 48 of the cell boundary and the groove direction, ranging from 0 to 90°. For example, as shown in FIG. 8, an ellipse 36 can be drawn around the cell 38 to approximate the shape of the cell 38. The long axis 48 of the approximated ellipse can be considered the long axis of the cell 38. The orientation angle could also be measured between the long axis of the cell nucleus 46 and the direction of the grooves. The orientation angle of the cells can be determined by any other suitable cell alignment analysis, such as those described in [Koo et al, "Human Corneal Keratocyte Response to Micro- and Nano-Gratings on Chitosan and PDMS" (2011 Sept) Cellular and Molecular Bioengineering, 4(3):399-410], [Teixeira et a I, "Responses of human keratocytes to micro- and nanostructured substrates" (2004 Dec) J Biomed Mater Res A, 71(3):369-376], [Pot et al, "Nanoscale

Topography-Induced Modulation of Fundamental Cell Behaviors of Rabbit Corneal Keratocytes, Fibroblasts, and Myofibroblasts" (2010 Mar) Invest Ophthalmol Vis Sci, 51(3):1373-1381], and [Gil et al, supra]. In some embodiments, the functional keratocytes are non-randomly oriented (i.e.

generally tending to be oriented in a particular axial direction). In some embodiments, the method comprises culturing the functional keratocytes until they have a non-random orientation. In some embodiments, the mean orientation angle of the functional keratocytes on the sheet is 30° or less. The orientation angle of the cells can also be characterized by the distribution of the cell orientation angles. In some embodiments, at least 75% of the functional keratocytes on the sheet have an orientation angle of 30° or less.

In addition to influencing the alignment of the functional keratocytes, the grooved substrate may also promote the alignment of extracellular matrix components produced by the functional keratocytes. This may help imitate the structure of the stromal lamellae in which the collagen fibrils are tightly packed, aligned, and parallel. Proper organization of the extracellular matrix components is thought to contribute to optical transparency. The collagen fibrils in the extracellular matrix produced by the cells may imitate the lattice arrangement in the native stroma in one or more ways, such as fibril diameter size, fibril diameter uniformity, fibril-to- fibril spacing, etc.

In some embodiments, the collagen fibrils (type I, type V, or type l/V hybrid) in the extracellular matrix are non-randomly oriented (i.e. generally tending to be oriented in a particular axial direction). In some embodiments, the method comprises culturing the functional keratocytes until they produce extracellular matrix comprising collagen fibrils (type I, type V, or type l/V hybrid) having a non-random orientation. In some embodiments, at least

50% of the collagen fibrils (type I, type V, or type l/V hybrid) in the extracellular matrix have an orientation angle in the range of 0 - 30°. For example, FIG. 9 shows keratocytes 50 being cultured on a grooved substrate. The keratocytes 50 have produced extracellular matrix 52 that contains collagen fibrils 54, among other components. The keratocytes 50 are generally aligned in the direction of the parallel grooves 56. Likewise, the collagen fibrils 54 are generally aligned in the direction of the parallel grooves 56.

Collagen fibril measurements can be performed by any suitable technique, such as those described in [Karamichos et al, "TGF- 3 Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells" (2013 Oct) Investigative Ophthalmology & Visual Science, 54:6612-6619], [Ka ramichos et al., "A Role for Topographic Cues in the Organization of Collagenous Matrix by Corneal Fibroblasts and Stem Cells" (2014 Jan) PLoS ONE, 9(l):e86260], [Wu et al, "The engineering of organized human corneal tissue through the spatial guidance of corneal stromal stem cells" (2012 Feb) Biomoteriols, 33(5):1343-1352], and [Gil et al, supra] .

The collagen fibrils (type I, type V, or type l/V hybrid) may have a relatively small diameter (less than 250 nm) and be monodisperse in diameter size distribution. I n some embodiments, at least 75% of the collagen fibrils have a diameter in the range of 15 - 90 nm. The fibril-to-fibril spacing of the collagen fibrils may be spaced with an average spacing of less than 400 nm; but other spacing distances are possible as well.

One or more features described above may contribute to the sheet being transparent. I n some embodiments, the sheet is transparent. In some cases, the transparent sheet transmits at least 80% of incident white light.

The harvested cell sheets can be used individually, or may be stacked on top of one another to form a multi-layer (two or more) tissue construct. Each sheet may be 1 - 15 μιη in thickness, but other thicknesses are possible as well. Many of such sheets (e.g. up to 400) may be stacked to form the multi-layer tissue construct. In some embodiments, the multi-layer tissue construct is transparent. In some cases, the multi-layer tissue construct transmits at least 60% of incident white light.

Adjacent sheets in the stack may be oriented at an angle (non-parallel) relative to each other (e.g. orthogonally). The direction of orientation for a sheet may be the mean axis of orientation for the functional keratocytes or the collagen fibers, or it may be the orientation of the grooves on the substrate on which the cell sheets are cultu red.

The functional keratocytes in different layers may form interconnections with each other (e.g. dendrites of neighboring cells contacting each other). The functional keratocytes may also migrate to different layers (i.e. vertical migration). The multi-layer tissue construct may be maintained in culture for a sufficient duration for such interconnections or cell migrations to occur. In some embodiments, the multi-layer tissue construct is further cultured for at least five days to allow for such interconnections or cell migrations. In some embodiments, compression is applied to the multi-layer tissue construct in order to promote adhesion between the layers. This can be performed in any suitable way. For example, the below-described cell sheet manipulator can be used to apply gentle compression on the multi-layer tissue construct.

The cell sheets may be harvested or stacked using any suitable technique or apparatus. The cells sheets may be harvested and stacked using a sheet manipulator device, such as those described in [Tang et al, supra] and the automated robotic apparatus described in [Kikuchi et al, "Automatic fabrication of 3-dimensional tissues using cell sheet manipulator technique" (2014) Biomoteriols, 35:2428-35].

As an example, FIG. 10 shows how stromal sheets can be stacked from multiple separately cultured sheets. For recovering a cell sheet from the temperature-responsive cell culture dish, construct a cell sheet manipulation device using a plunger 60 and a gelatin hydrogel matrix 64 coated on the bottom of the plunger 60. Prepare the gelatin gel with gelatin powder from porcine skin dissolved in solution. Neutralize with NaOH and sterilize the gelatin solution by filtration and pour into a silicone rubber mold. Put the plunger 60 in the mold. Cool the plunger 60 on ice to complete gelation. This results in a gelatin gel coating 64 on the bottom surface of the plunger 60.

Cell sheets #1, #2, and #3 are simultaneously and independently cultured at 37° C on cell culture dishes 61, 62, and 63. When the cell sheets are ready to be harvested, place the hydrogel-coated plunger 60 on the surface of cell sheet #1 and reduce the temperature to 23° C for harvesting. Harvest the cell sheet by lifting the plunger 60. This will recover the cell sheet while avoiding shrinking or folding of the sheet. Moving to culture dish 62 for cell sheet #2, place the plunger 60 on cell sheet #2 so that cell sheet #1 is transferred on top of cell sheet #2. Sheet orientation can be controlled by rotating the plunger 60 as desired (e.g. so that the two sheets are orthogonal to each other). Continue incubating at 37° C to promote adhesion between the two cell sheets. Then reduce the temperature to 23° C for harvesting and lift the plunger 60 to harvest a double-layer sheet 66 of cell sheets #1 and #2. Repeat this cycle another time for cell sheet #3. After harvesting a triple-layer sheet 68, place the plunger 60 on a receiving dish 65. Incubate at 37° C to melt the gelatin gel. With the gelatin coating 64 melted, a triple-layer construct 68 remains. As seen here, the triple-layer construct 68 is free of any scaffolding.

An epithelium or endothelium can also be added to the multi-layer tissue construct, which could be incorporated into the multi-layer tissue construct in any suitable way. One possible way is to separately grow the epithelium cell layer and stack the epithelium cell layer on the harvested stromal sheets, or the reverse; similarly for the endothelium layer. Another possible way is to seed epithelium or endothelial cells on top of the stack of stromal sheets. For example, corneal endothelial cells could be seeded on top of the stromal stack and cultured in endothelial growth medium. Then limbal epithelial cells could be seeded on the other side of the stromal stack and cultured in epithelial cell medium.

FIG. 11 shows an example of a multi-layer corneal tissue construct made according to the invention. Three stromal sheets of functional keratocytes are stacked on top of one another such that they are oriented at an angle relative to each other. This stack of stromal sheets is sandwiched between a sheet of epithelial cells and a sheet of endothelial cells. To enhance corneal transparency, the multi-layer tissue construct may have relatively lower cellular content. I n some embodiments, the functional keratocytes occupy a volume of less than 50% of the volume of the multi-layer tissue construct. I n some embodiments, the average density of the functional keratocytes in the multi-layer tissue construct is less than 75,000 cells/mm ³ . In some embodiments, the average density of the functional keratocytes in the multi-layer tissue construct is less than 38,000 cells in a 1 mm ² column.

Mechanical stimuli may enhance the organization, strength, or optical properties of corneal tissue. See [Ruberti et a I, "Prelude to corneal tissue engineering - Gaining control of collagen organization" (2008 Sept) Prog Retin Eye Res., 27(5):549-577] . In some embodiments, the cell sheet or the multi-layer tissue construct is subject to mechanical stimuli.

Swelling/shrinking properties of the temperature-responsive polymer could be exploited to apply the mechanical stimuli. For example, the temperature could be reduced to cause the substrate's temperature-responsive surface to swell, applying mechanical stress on the sheet. Before the cell sheet completely detaches, the temperature could then be raised back to physiologic temperature to cause the temperature-responsive polymer to shrink and continue culturing the cell sheet. This cycle could be repeated multiple times while culturing the cell sheet to repeatedly apply mechanical stress to the sheet.

After production of the extracellular matrix, the functional keratocytes may be induced to undergo apoptosis. This may reduce the number or density of cells in the cell sheet or the multi-layer tissue construct, which may enhance optical transparency. Any suitable way of inducing apoptosis can be used, including exposing the functional keratocytes to an apoptosis- inducing agent. One example of an apoptosis-inducing agent is a proinflammatory cytokine, such as interleukin-1 (e.g. IL-la), platelet-activating factor (PAF), FAS-ligand (also known as FasL or CD95L), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-a), which have been shown to cause apoptosis in keratocytes. See [Musselmann, supra].

Another example of an apoptosis-inducing agent is a growth factor. Certain growth factors such as FGF-2 (basic fibroblast growth factor) and PDGF-BB (platelet-derived growth factor) have been shown to induce apoptosis. Another potential apoptosis-inducing agent is nitric oxide.

In some embodiments, the functional keratocytes are exposed to an apoptosis-inducing agent that induces apoptosis of the functional keratocytes. In some embodiments, the functional keratocytes in a cell sheet are induced to undergo apoptosis. In some embodiments, the functional keratocytes in a multi-layer tissue construct are induced to undergo apoptosis. This may involve exposing the functional keratocytes to an apoptosis-inducing agent.

Eye rubbing has been shown to cause keratocyte apoptosis. [Kallinikos, "On the Etiology of Keratocyte Loss during Contact Lens Wear" (2004 Sept) Investigative Ophthalmology & Visual Science, 45:3011-3020]. In some embodiments, compression is applied onto the sheet or the multi-layer tissue construct in order to promote keratocyte apoptosis. This can be performed in any suitable way. For example, the above-described cell sheet manipulator can be used to apply gentle compression on the cell sheet or the multi-layer tissue construct.

The harvested sheet or the multi-layer tissue construct could be used for implantation into a patient's eye. The harvested sheet or the multi-layer tissue construct may be processed into a structure that is suitable for implantation. To facilitate implantation into the eye, the harvested sheet or the multi-layer tissue construct could be made or processed to have a round, oval, or elliptical shape (e.g. by cutting or punching).

The harvested sheet or multi-layer tissue construct could be used in corneal

replacement surgery, such as full thickness corneal transplantation (penetrating keratoplasty) or partial thickness corneal transplantation, such as lamellar keratoplasty, replacement of the stroma only, or replacement of the stroma and endothelium. The harvested sheet or the multilayer tissue construct may be sufficiently sturdy to be sutured into place (i.e. sufficiently sturdy to hold a suture). In some embodiments, implanting the harvested sheet or the multi-layer tissue construct comprises suturing it into the patient's eye.

For example, FIGs. 12A - 12C show a corneal replacement surgery using an example engineered corneal tissue of the invention. FIG. 12A shows a cloudy and scratched cornea 70 that needs replacement. As shown in FIG. 12B, the surgeon cuts an incision 74 and removes the damaged cornea 70. As shown in FIG. 12C, the engineered corneal tissue 76 is implanted into the eye 72 and anchored into place with sutures 78.

The functional keratocytes may be induced to undergo apoptosis after implantation. Applying a contact lens onto the eye has been shown to cause keratocyte apoptosis. See

[Kallinikos, supra]. In some embodiments, the method further comprises applying a contact lens on the eye over the implanted corneal tissue.

Experimental

Substrate fabrication. Fabricate the cell culture substrate as described in [Jiao et al, supra] and [Kim et al (2010), supra], which are incorporated by reference herein. In more detail, make the substrate with polyurethane-acrylate:polyglycidyl-methacrylate (pUA-pGMA), imprint the grooved pattern by capillary force lithography, and apply a coating of poly-NIPAAm onto the substrate by grafting.

Tissue collection and cell culture. Isolate human corneal keratocytes and passage them as previously described in [Karamichos et al, "TGF-βΒ Stimulates Stromal Matrix Assembly by Human Corneal Keratocyte-Like Cells" (2013 Oct) Investigative Ophthalmology & Visual Science, 54:6612-6619], which is incorporated by reference herein. Seed the corneal keratocytes on the polyNIPAAm-coated grooved substrates at the appropriate optimized density (e.g. 5,000 - 15,000 cells/cm ² ). Culture the cells in Eagle's Minimum Essential Medium with fetal bovine serum, TGF- 3, and 2-O-a-D-glucopyranosyl-L-ascorbic acid. Continue the culture for a suitable duration (e.g. 4 weeks). Examine the morphology of the cultures under a microscope (e.g. brightfield or phase contrast microscopy).

Cell alignment analysis. At the appropriate time (e.g. on the second or third day of culture), perform phase contrast microscopy and analyze the orientation of the keratocytes with Image-Pro or similar software. Calculate the orientation angle as the angle difference between the longest direction within the cell borders and the direction of the grooves.

Immunofluorescence staining. Fix the samples for staining and imaging following the same procedures as given in [Wu et al (2014), supra]. For staining the extracellular matrix, label collagen type-l with primary antibodies. For staining of cytoskeleton, label F-actin with phalloidin conjugated to Alexa Fluor 488 and label a-smooth muscle actin (SMA) with primary antibodies. Examine under confocal microscope.

Multi-layer construct. On multiple thermoresponsive grooved substrates,

simultaneously culture multiple separate sheets of human corneal keratocytes. Construct a gelatin-coated, plunger tool as described in [Haraguchi et al., "Fabrication of functional three- dimensional tissues by stacking cell sheets in vitro" (2012) Nature Protocols, 7:850-858], which is incorporated by reference herein. After culturing for an appropriate optimized time at 37° C temperature and when ready for transfer, place the plunger tool onto the cell sheet and incubate at 20° C while waiting for detachment of the cell sheet and adhesion onto the gelatin. Gently lift up the plunger tool holding the cell sheet. Gently place the plunger tool holding the first cell sheet onto another one of the cell sheets cultured on the thermoresponsive grooved substrate. Incubate at 20° C while waiting for detachment of the bottom cell sheet and adhesion between the two cell sheets. Gently lift up the plunger tool holding the double- layered cell sheet. Repeat stacking of the sheets on the plunger tool as desired to create a multi-layer construct. Recover the multi-layered cell sheet from the plunger tool by placing the the multi-layer construct on another culture dish. Add prewarmed (37° C) culture medium to the dish and incubate at 37° C. This will melt the gelatin. Wash the dish with warmed Hank's balanced salt solution to remove the melted gelatin solution and add fresh medium for continued cultivation.

Cell sheet adhesion. If improved cell sheet adhesion is needed, this can be achieved by adding an adhesive mixture into the gelatin solution. Add to the gelatin solution, fibrinogen (for example, about 20% wt/vol) and riboflavin-5'-phosphate (for example, about 2.5% wt/vol). After the three sheets are stacked, irradiate with long wavelength UV-A light (365 nm) at 3 mW/cm ² intensity. This treatment protocol has been shown to adhere stromal layers in a corneal flap. See [Littlechild et a I, "Fibrinogen, riboflavin, and UVA to immobilize a corneal flap — conditions for tissue adhesion" (2012 Jun) Invest Ophthalmol Vis Sci., 53(7):4011-20].

Alternate technique for cell sheet harvesting. For an alternate way of harvesting the cell sheets and making a multilayer tissue construct, use the gelatin cast technique described in [Jiao et al, supra], which is incorporated by reference herein. Cell sheet adhesion can be improved by adding a fibrinogen/riboflavin adhesive mixture into the gelatin solution and irradiating with long wavelength UV-A light, as described above.

Alternate cell source using corneal stromal stem cells. Isolate human corneal stromal stem cells as previously described in [Wu et al, "The engineering of organized human corneal tissue through the spatial guidance of corneal stromal stem cells" (2012 Feb) Biomaterials, 33(5):1343-1352], which is incorporated by reference herein. From donor corneas, isolate human corneal stromal stem cells by collagenase-digestion of limbal stromal tissue. Expand the population of corneal stromal stem cells in stem cell growth medium (SCGM) containing DMEM (Dulbecco's Modified Eagle Medium)/MCDB-201 with fetal bovine serum, epidermal growth factor, platelet-derived growth factor, insulin, transferrin, selenous acid, linoleic acid-bovine serum albumin, ascorbic acid-2-phosphate, dexamethasone, penicillin, streptomycin, gentamicin, and cholera toxin. Passage the cells several times to use in the experiments.

Seed the human corneal stromal stem cells onto the thermoresponsive grooved substrates at the appropriate optimized density (e.g. 7.0 ^χ 10 ⁴ cells/cm ² ) and incubate in SCGM. After three days incubation in SCGM, change the culture medium to keratocyte differentiation medium consisting of Advanced DMEM (Life Technologies) supplemented with basic fibroblast growth factor, ascorbic acid-2-phosphate, L-glutamine, gentamicin, and penicillin. Change the media twice weekly and continue culture for a suitable duration (e.g. up to six weeks).

The foregoing description and examples have been set forth merely to illustrate my invention and are not intended to be limiting. Each of the disclosed aspects and embodiments of my invention may be considered individually or in combination with other aspects, embodiments, and variations of my invention. In addition, unless otherwise specified, the steps of the methods of my invention are not confined to any particular order of performance.

Modifications of the disclosed embodiments incorporating the spirit and substance of my invention may occur to persons skilled in the art, and such modifications are within the scope of my invention.

Any use of the word "or" herein is intended to be inclusive and is equivalent to the expression "and/or," unless the context clearly dictates otherwise. As such, for example, the expression "A or B" means A, or B, or both A and B. Similarly, for example, the expression "A, B, or C" means A, or B, or C, or any combination thereof.