TECHNICAL FIELD

[0001] The present invention relates to creating 3 dimensional cell culture scaffolds using a wet-laid manufacturing process and containing a blend of short; staple nanoflbers and microflbers. More specifically, the invention relates to the use of a nonwoven process containing discrete-length polymeric nanoflbers for regulating or promoting tissue modeling, cell growth, function or gene expression as defined herein.

BACKGROUND

[0002] Fibers form, in part or in whole, a large variety of both consumer and industrial materials such as, for example, clothing and other textile materials, medical prostheses, construction materials and reinforcement materials, and barrier, filtration and absorbent materials. There are two main structural classes of fiber materials: woven and non-woven. An advantage of non-woven fiber materials is their lower production cost

[0003] Polymeric nanoflbers are increasingly being investigated for use in various applications. Nanoflbers may attain a high surface area comparable with the finest nanoparticle powders, yet are fairly flexible, and retain one macroscopic dimension which makes them easy to handle, orient and organize. These materials have been constructed to closely represent the size and dimensions of fibers found in the native tissue extracellular matrix (ECM).

[0004] Cells cultured on flat two-dimensional (2D) cell culture surfaces do not necessarily represent an in vivo like atmosphere and often result in artificial 2D sheets of cells. Three-dimensional cell culture is a growing field. There are numerous 3D cell culture products being developed as these can provide in vivo like organization and scale. Approximately 30% of cell culture scientists are predicted to switch from two-dimensional (2D) to 3D cell culture by 2015. Many products have been developed to address the needs of this growing market, including hydrogels, sol gels, ceramic scaffolds, expanded polystyrene supports, permeable membranes, and electrospun nanofiber layers to name a few.

[0005] These current products, although providing either a multilayer or etched surface for cell growth, fail to truly mimic the chaotic, three dimensional fibrous structure of the extracellular matrix deposited by cells growing in living tissue Because of this fact, data gathered of cells growing on material that does not represent the geometry found in vivo may give results different from those found In living tissue.

[0006] Accordingly, an ongoing need remains for developing novel substrates containing polymeric nanofibers designed to emulate the structure found in vivo for 3D cell culture applications.

SUMMARY

[0007] In one aspect; die present invention relates to a cell culture scaffold which comprises a fabric substrate of cotton, synthetic or blend fibers containing wet laid polymeric, staple nanofibers of short cut lengths. The staple polymeric nanofibers can be wet laid onto a fabric substrate of cotton, synthetic or blend fibers, or the nanofibers can be wet laid with other fibers to form a nonwoven substrate, or the nanofibers can be wet laid onto themselves to form a nonwoven containing only nanofibers. The dried substrate can then be cut into discs and placed into individual culture wells or as a flat sheet cut to any size. Culture media and living cells can then be added onto the discs to give the cells a rigid substrate that contains randomly oriented nanofibers throughout the entire thickness of the substrate. Also, the nanofibers mixed with microfibers can be put into slurry form in cell culture medium and provide a structure that can be invaded and remodeled as part of the cell's normal tissue growth.

[0008] A wide variety of polymers may be utilized as starting materials, examples of which are given below.

[0009] A wide variety of fabric substrates of cotton, synthetic or blend fibers may be utilized as starting materials, examples of which are given below.

[0010] Other devices, apparatus, systems, methods, features and advantages of the invention can be understood by one skilled in the art in view of the following figures and detailed description. All such additional systems, methods, features and advantages are within the scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The invention can be better understood by referring to the following figures. The components in the figures are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. In the figures, like reference numerals designate corresponding parts throughout the different views.

[0012] Figure 1 is a schematic of nanofibers and microfibers wet-laid into a composite substrate.

[0013] Figure 2 is a SEM image showing a cross-sectional edge view of a wet laid substrate comprised of 50% CA nanofibers (avg. diameter ~400 nm) and 50% Rayon microfibers (avg. diameter 9 urn).

[0014] Figure 3 Is a SEM image showing a top view of a wet laid substrate comprised of 50% PLA nanofibers (avg. diameter—400 nm) and 50% PET microfibers (avg. diameter 9 um).

[0015] Figure 4 is a SEM image showing a top view of wet laid substrates consisting of 10% cellulose acetate nanofiber/90% PET 1.5 denier microflber (A), 20% cellulose acetate nanofiber/80% PET 1.5 denier microflber (B), 30% cellulose acetate nanofiber/70% PET 1.5 denier microflber (C), 40% cellulose acetate nanofiber/60% PET 1.5 denier microflber (D).

[0016] Figure 5 is a SEM image showing a top view of a colony of MDA-MB-231 breast cancer cells attached to a microflber of a wet laid substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg. diameter ~400 nm).

[0017] Figure 6 is a SEM image showing a top view of a group of MDA-MB-231 breast cancer cells attached to the nanofibers of a wet laid substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg. diameter ~400 nm).

[0018] Figure 7 is an optical inverted fluorescent microscope image showing a top view of a group of green fluorescent protein (GFP)-expressIng MDA-MB-231 breast cancer cells attached to a wet laid substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg, diameter ~400 nm).

[0019] Figure 8 Is a spinning disc confocal fluorescent microscope image of HepG2 cells grown for 5 days and stained with TRITC-phalloidin (red) showing actin filaments and Hoescht 33342 nuclear stain (blue) showing cell growth on die top (main image) and throughout the z-axls (top and right slices) of a substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg. diameter -400 nm).

[0020] Figure 9 is a spinning disc confocal fluorescent microscope image of NIH/3T3 cells grown for 5 days and stained with TRITC-phalloidin (red) showing actin filaments and Hoescht 33342 nuclear stain (blue) showing cell growth on the top (main image) and throughout the z-axis (top and right slices) of a substrate comprised of 80% PET 1.5 denier microfibers (avg. diameter 13 um) and 20% cellulose acetate nanofibers (avg. diameter ~400 nm).

[0021] Figure 10 Is a graph of cell proliferation data of MDA-MB-231 breast cancer cells grown over time on a substrate comprised of 70% PET microfibers (avg. diameter 13 um) and 30% PLA nanofibers (avg. diameter ~400 nm) (Cell Culture Scaffold 1) and 70% Rayon microfibers (avg diameter 11 um) and 30% CA nanofibers (avg. diameter ~400 nm) (Cell Culture Scaffold 2) In addition to 2D cell culture treated plastic

[0022] Figure 11 is a graph of cell proliferation data of NIH 3T3 mouse embroyonic fibroblast cells grown over time on a substrate comprised of 70% PET microfibers (avg. diameter 13 um) and 30% PLA nanofibers (avg diameter -400 nm) (Cell Culture Scaffold 1) and 70% Rayon microfibers (avg. diameter 11 um) and 30% CA nanofibers (avg. diameter ~400 nm) (Cell Culture Scaffold 2) in addition to 2D cell culture treated plastic

[0023] Figure 12 shows the relative expression of matrix metalloproteInase-2 (MMP-2) mRNA in MDA-MB-231 breast cancer cells grown for 3 days. A significant increase (*p = 0.04, Student's T-test) is seen In the cells grown on a substrate comprised of 70% PET microfibers (avg. diameter 13 um) and 30% PLA nanofibers (avg. diameter—400 nm), suggesting an invasive phenotype compared to cells grown on tissue culture treated plastic

[0024] Figure 13 shows mRNA expression of primary human umbilical mesenchymal stem cells (MSCs) grown in a 0.5%wt slurry of PLA nanofibers in growth media and induced to differentiate into chondrocytes to create elastic cartilage. In addition to high elastin expression, the ratio of Collagen II to Collagen I expression (DI) by day 10 (D10) was favorably high (at least greater than 1). Collagen X expression was fairly constant, demonstrating that the chondrocytes are not becoming hypertrophic.

DETAILED DESCRIPTION

[0025] As used herein, the term nanofiber refers generally to an elongated fiber structure having an average diameter ranging from less than 50 nm -5 urn in some examples, in other examples ranging from less than 100 nm - 5 urn, and in other examples ranging from 200 nm -5 um. In further examples, the average diameter ranges from 40 nm -5 um, 40 nm - 2 um, 50 nm ·5 um, 50 nm -2 um, 100 nm - 5 um, 100 nm - 2 um, 200 nm -5 um, or 200 nm -2 um. The "average" diameter may take into account not only that the diameters of individual nanofibers making up a plurality of nanofibers formed by implementing the presently disclosed method may vary somewhat, but also that the diameter of an individual nanofiber may not be uniform over its length in some implementations of the method. In some examples, the average length of the nanofibers may range from 100 nm or greater. In other examples, the average length may range from 100 nm to millions of nm. In some examples, the aspect ratio (length/diameter) of the nanofibers may range from 100 or greater. In other examples, the aspect ratio may range from 20 to millions. In some specific examples, we have demonstrated nanofibers with aspect ratios of at least 10,000. Insofar as the diameter of the nanofiber may be on the order of a few microns or less, for convenience the term "nanofiber" as used herein encompasses both nano-scale fibers and micro-scale fibers (mlcrofibers).

[0026] Polymers encompassed by the present disclosure generally may be any naturally- occurring or synthetic polymers capable of being fabricated into nanofibers. Examples of polymers include many high molecular weight (MW) solution-processable polymers such as polyethylene (more generally, various polyolefins), polystyrene, cellulose, cellulose acetate, poly(L-lactic add) (PLA), poly (lactic-co-gly colic acid) (PLGA), polyacrylonitrile (PAN), polyvinylidene difluoride (PVDF), conjugated organic semiconducting and conducting polymers, biopolymers such as polynucleotides (DNA) and polypeptides, etc

[0027] Other examples of suitable polymers to form nanoflbers include vinyl polymers such as, but not limited to, cellulose acetate propionate, cellulose acetate butyrate, polyethylene, polypropylene, poh/(vlnyl chloride), polystyrene, polytetrafluoroethylene, poly(amethylstyrene), poly(acryllc add), poly(isobutylene), poly(acrylonitrile), poly(methacrylic acid), poly(methyl methacrylate), poly(l- pentene), poly(l,3-butadiene), poly(vinyl acetate), poly(2vinyl pyridine), 1,4-polyisoprene, and 3,4- polychloroprene. Additional examples include nonvinyl polymers such as, but not limited to, poly(ethylene oxide), polyformaldehyde, polyacetaldehyde, poly(3-propionate), poly(10-decanoate), poly(ethylene terephthalate), polycaprolactam, poly(ll-undecanoamide), polyChexamethylene sebacamide), poly(m-phenylene terephthalate), poly(tetramethylene-m-benzenesulfonamide).

Additional polymers include those falling within one of the following polymer classes: polyolefin, polyether (including all epoxy resins, polyacetal, polyetheretherketone, polyetherimide, and

poly(phenylene oxide)), polyamide (including polyureas), polyamideimide, polyaiylate,

polybenzimidazole, polyester (Including polycarbonates), poh/urethane, polyimide, polyhydrazide, phenolic resins, polysilane, polyslloxane, polycarbodiimide, polylmlne, azo polymers, polysulflde, and polysulfone.

[0028] As noted above, the polymer used to form nanoflbers can be synthetic or naturally- occurring. Examples of natural polymers include, but are not limited to, polysaccharides and

derivatives thereof such as cellulosic polymers (e.g., cellulose and derivatives thereof as well as cellulose production byproducts such as lignin) and starch polymers (as well as other branched or nonlinear polymers, either naturally occurring or synthetic). Exemplary derivatives of starch and cellulose include various esters, ethers, and graft copolymers. The polymer may be crossUnkable In the presence of a multifunctional crosslinldng agent or crossUnkable upon exposure to actinic radiation or other type of radiation. The polymer may be homopolymers of any of the foregoing polymers, random copolymers, block copolymers, alternating copolymers, random tripolymers, block tripolymers, alternating tripolymers, derivatives thereof (e.g., graft copolymers, esters, or ethers thereof), and the like.

[0029] Microflber material type in the substrate may include but is not limited to PET, acrylic, aramids, Basofil, PVDF, nylon, PLA, polyethylene, PVA, Rayon, cellulose and cellulose derivatives. The average diameter of the microfibers can be, for example, 2-500 micron, or 3-100 microns, or 5-50 microns, or 8-20 microns. The average length of the microflbers can be, for example, 2-50 mm, or 3-30 mm, or 5-20 mm.

[0030] By "web" is meant a fibrous material capable of being wound into a roll.

[0031] By "nonwoven web" is meant a web of individual fibers or filaments which are Interlaid and positioned in a random manner to form a planar material without identifiable pattern, as opposed to a knitted or woven fabric Nonwoven webs have been in the past formed by a variety of processes known to those skilled in the art such as, for example, meltblown, spunbound, wet-laid, dry-laid, and bonded carded web processes.

[0032] By "in vitro tissue culture" is meant the ability to grow and multiply living cells out of the body in a controlled environment using culture media and synthetic materials.

[0033] A nonwoven or woven fabric substrate or web can be made from natural or synthetic fabrics and may be composed of fibers of cotton, cellulose, Lyocell, acetate, cellulose acetate, rayon, silk, wool, hemp, spandex (including LYCRA), polyolefins (polypropylene, polyethylene, etc), polyamide (nylon 6, nylon 6-6, etc), aramids (eg Kevlar®, TwaronQ, Nomex, etc), acrylic, or polyester

(polyethylene teraphthalate, trlmethylene terephthalate), polyurethane, glass microflbers, fiberglass, etc By "fabric blends" is meant fabrics of two or more types of fibers. Typically these blends are a combination of a natural fiber and a synthetic fiber, but can also include a blend of two natural fibers or two synthetic fibers.

[0034] Nanoflbers can be wet laid deposited onto a non-woven or woven substrate, which is placed on a filter mesh of 27-200 microns pore size.

[0035] Nanoflbers can also be deposited onto themselves without a substrate with basis weights ranging from 4 to 800 GSM or higher. In this case, longer length fibers provide substrate with enhanced Integrity and strength.

[0036] Polymeric nanoflbers can also be wet laid together with other nano-or microflbers to form a nonwoven substrate containing many types of fibers, including borosilicate glass fibers.

[0037] The XanoShear method of producing staple length polymer nanoflbers in a liquid shear process and adding them to a substrate by wet laying techniques is novel and has not been achieved in the prior art as nanofibers are typically produced as long f> 20 cm) dry fibers by electrospinning and meltblowing technologies. The method of creating these discrete fibers is also different from the methods used to manufacture "fibrils" which are formed by phase separation or shear ripping from nanofibers or larger microfibers. In these methods, the diameter of a fibril is generally smaller than the diameter of the nanofiber with which it was associated, and typically smaller by an order of magnitude. Though fibrils may also be characterized as nanofibers, in the present disclosure the term "fibrils" distinguishes these structures from the polymer nanofibers created using the XanoShear method.

[0038] The use of these discrete length fibers in a nano-structured substrate to create a three dimensional scaffold to support in vitro cell growth is also a novel concept Cells growing in the body naturally excrete extracellular matrix made up of a multitude of fibrillar proteins to support the tissue or organ. The diameter of these fibers span a range from lOnm to several micrometers. The

combination of polymeric short-length nanofibers and supporting microfibers presents a material that mimics mat deposited by the cells in vivo. By giving the cells grown in vitro a fibrous support the architecture of which the cell Is familiar with in vivo, true cellular growth and behavior can be studied more realistically than those cells growing on flat plastic, the current industry standard.

[0039] In some embodiment, the 3D cell culture scaffold described herein comprises a nanofiber-microfiber composite. The nanofiber-micro fiber composite can be, for example, a wet-laid composite. The nanofiber-microfiber composite can be, for example, a nonwoven composite. In some embodiment the nanofiber-microfiber composite is not an electrospun or melt-blown composite.

[0040] The nanofiber-microfiber composite can comprise, for example, 5-95 wt% of microfibers and 5-95 wt% nanofibers, or 50-90 wt% of microfibers and 10-50 wt% nanofibers, or about 90 wt% of microfibers and about 10 wL% nanofibers, or about 80 wt.% of microfibers and about 20 wt% nanofibers, or about 70 wt% of microfibers and about 30 wt% nanofibers, or about 60 wt% of microfibers and about 40 wt% nanofibers, or about 50 wt% of microfibers and about 50 wt% nanofibers.

[0041] In some embodiment the nanofibers have an average length of 10-5000 microns, or 50- 2000 microns, or 100-1000 microns, or 200-800 microns. In some embodiment the nanofibers have an average fiber diameter of SO nm to 5 microns, or 100 nm to 1 micron, or 200-800 nm. In some embodiment; the nanofibers have an length to diameter (L:D) ratio of 20-20000, or 50-10000, or 200- 5000, or 500-2000.

[0042] In some embodiment, the 3D cell culture scaffold described herein comprises PET microflbers and cellulose acetate nanofibers. In some embodiment; the 3D cell culture scaffold described herein comprises a nonwoven nanoflber-microfiber composite that comprises, consists essentially of, or consists of PET microflbers and cellulose acetate nanofibers.

[0043] In some embodiment; the 3D cell culture scaffold described herein comprises PET microflbers and PLA nanofibers. In some embodiment; the 3D cell culture scaffold described herein comprises a nonwoven nanofi ber-m icroflber composite that comprises, consists essentially of, or consists of PET microflbers and PLA nanofibers.

[0044] In some embodiment, the 3D cell culture scaffold described herein comprises Rayon microflbers and cellulose acetate nanofibers. In some embodiment; the 3D cell culture scaffold described herein comprises a nonwoven nanoflber-microfiber composite that comprises, consists essentially of, or consists of Rayon microflbers and cellulose acetate nanofibers.

[0045] In some embodiment; the 3D cell culture scaffold described herein comprises Rayon microflbers and PLA nanofibers. In some embodiment; the 3D cell culture scaffold described herein comprises a nonwoven nanoflber-microfiber composite that comprises, consists essentially of, or consists of Rayon microflbers and PLA nanofibers.

[0046] In some embodiment, the 3D cell culture scaffold described herein comprises a nonwoven nanofiber composite that comprises, consists essentially of, or consists of nanofibers wet- laid onto themselves. The nonwoven nanofiber composite can consist essentially of or consist of wet- laid PLA and/or cellulose acetate.

[0047] Working Examples

[0048] Example 1

[0049] Wet laying process (Figure 1): Cellulose acetate (Eastman CA-398-10] nanofibers (average diameter of 400 nm and lengths of ~200-700 μιτι or 2-10 mm) were mixed with 1.5 denier, 6mm length PET microfibers (average diameter 9 um) and added to water in a 10 inch x 10 inch handsheet former. Water is removed by gravity through a 100 mesh stainless steel woven screen at a final basis weight of 50 grams per square meter. The sample is dried on an Emerson Apparatus Speed Dryer at 200 degrees Fahrenheit (Figure 4).

[0050] Example 2

[0051] A wet laid substrate was made consisting of 30% PLA nanofibers and 70% PET 1.5 denier microfibers (13 micron in diameter) or 30% cellulose acetate nanofibers and 70% Rayon 0.8 denier microfibers (11 micron in diameter) by weight The substrate was dried and punched with a die into discs having a diameter of 29/64 inch and one disc was placed into each well of a 48-well plate. The substrate was treated with 5x concentration of penicillln/streptomycin/amphotericin to remove any bacteria or fungus, and washed twice with buffered saline solution. NIH/3T3 or MDA-MB-231 cells were seeded into the wells at a density of 5000/cm ² (4500 cells per well) in DMEM-high glucose containing 10% FBS and lx penicillin/streptomycin. The growth rate of cells was measured over a week using AlamarBhie assay (Life Technologies). The NIH/3T3 (Figure 11) and MDA-MB-231 (Figure 10) cells grew exponentially, doubling in population every 40 hours.

[0052] Example 3

[0053] A wet laid substrate was made consisting of 50% polylactic acid nanofibers and 50% PET 1.5 denier microfibers (13 micron in diameter) by weight (Figure 3). The substrate was dried and punched with a die into discs having a diameter of 29/64 inch and one disc was placed into each well of a 48-well plate. The substrate was treated with 5x concentration of

penicillin/streptomycin/amphoteridn to remove any bacteria or fungus, and washed twice with buffered saline solution. NIH/3T3 or MDA-MB-231 cells were seeded into the wells at a density of 5000/cm ² (4500 cells per well) in DMEM-high glucose containing 10% FBS and lx

penicillin/ streptomycin. The growth rate of cells was measured over a week using AlamarBlue assay (Life Technologies). The NIH/3T3 and MDA-MB-231 cells grew exponentially, doubling in population every 24 hours.

[0054] Example 4 [0055] A wet laid substrate was made consisting of 30% polylactic acid nanoflbers and 70% PET 1.5 denier microfibers (13 micron in diameter) by weight The substrate was punched with a die into discs having a diameter of 13/8 inch and one disc was placed into each well of a 6-well plate. The substrate was treated with 5x concentration of penicillin/streptomycin/amphotericin to remove any bacteria or fungus, and washed twice with buffered saline solution. MDA-MB-231 cells were seeded into either the scaffold wells or standard tissue culture treated plastic at a density of 20000/cm ² (180000 cells per well) in DMEM-high glucose containing 10% FBS and lx penicillin/streptomycin. At days 2, 3, 4 and 7, RNA from separate wells in triplicate was isolated using TRIzol solution (Life Technologies). Gene expression of matrix metalloproteinase-2 (MMP-2) was analyzed by semiquantitative gel electrophoresis in multiplex using ribosomal 18s as a loading standard. MMP-2 is an indicator of extracellular matrix remodeling or migration by the cancer cell It was shown that initially the expression of MMP-2 in cells grown on the scaffold was increased 8 fold over traditional plastic and slowly decreased to a stable 2 fold expression by day 7 (Figure 12).

[0056] Example 5

[0057] A wet laid substrate was made consisting of 20% cellulose acetate nanoflbers and 80% PET 1.5 denier microfibers (13 micron in diameter) by weight The substrate was punched with a die into discs having a diameter of 29/64 inch and one disc was placed into each well of a 48-well plate. The plate was then treated with 19 kGy of gamma radiation to sterilize. HepG2 cells were seeded Into the wells at a density of 15000/cm ² (13500 per well). Cells were grown for 5 days in EMEM containing 10% FBS, Lrglutamine and lx penicillin/streptomycin. The scaffold was then removed and stained with TRITC-conjugated phalloidin to view extracellular actln fibers and Hoescht 33342 (blue nuclear stain). The scaffold was mounted on a slide in Permount (Sigma Aldrlch) and viewed under a fluorescent spinning disc confocal microscope (Figure 9).