WO2022098861A1 - Compositions and methods for preparing reconstructed multilayered epithelial cell sheets - Google Patents

Abstract

The present disclosure demonstrates the feasibility and successful production of rabbit cGMP-certified cultured oral mucosal epithelial cell sheet (cG-CAOMECS), designed to reconstruct corneal surface of patients with bilateral limbal stem cell deficiency. The production makes use of epithelial progenitor cells differentiated in a feeder cell-free, serum-free, medium. In addition to reconstruct corneal epithelium, the present technology can also be used to construct skin tissues and esophageal epithelia, without limitation.

Description

COMPOSITIONS AND METHODS FOR PREPARING RECONSTRUCTED MULTILAYERED EPITHELIAL CELL SHEETS CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit under 35 U.S.C. § 119(e) of United States Provisional Application Serial Number 63/110,140, filed November 5, 2020, the content of which is incorporated by reference in its entirety into the present disclosure. BACKGROUND [0002] Treatment of corneal surface with oral mucosal epithelial stem cells was initially reported in the twentieth century. These studies documented the possibility of reconstructing corneal surface with epithelial cells from a non-ocular surface. In 2003, the expansion and engineering of oral mucosal epithelial cell sheet using cell culture was reported. Several other groups reported the use of oral mucosal epithelial cell sheet to reconstruct corneal surface and treat corneal diseases. [0003] Oral mucosal epithelial stem cells are somatic stem cells distributed all over the buccal tissue. Upon cultivation and grafting, the cells mimic the corneal epithelium as they maintain their stemness at the ectopic site and can replace the lost limbal stem cells of the host with limbal stem cell deficiency (LSCD). The cells in the resulting differentiated cell sheet should express corneal specific markers such as Pax6 and deltaNp63, as well as others corneal epithelium markers as cytokeratins CK3 and K12. The regenerative spectrum of cultured oral mucosal epithelial cell sheet (CAOMECS) is not limited to reconstruct corneal surface. CAOMECS can be used in various epithelial disorders, such as severe burns, giant congenital nevi, and esophagus regeneration. [0004] However, with all the above listed successful literature reporting the therapeutic role that oral mucosal epithelial cell sheets offer, cell culture methods used to produce these cell sheets are to date utilizing animal origin products. Xenogeneic products present risks of immunologic rejection and potential introduction of infections across species barriers. The inherent possibility of infection or unknown pathogen transmission from animal-derived materials and reagents need to be addressed. [0005] The biggest challenge that cell sheet technology is facing is to produce a multilayered epithelium-like cell sheet without the use of fetal bovine serum and murine 3T3 fibroblasts feeder cells. The use of murine feeder cells and animal derived products is a major concern in regenerative medicine. These xenogeneic products can cause pathogen transmissions and immune reactions in humans. Adhering to FDA regulations of xeno-free cell culture conditions for the isolation and expansion of OMECS without any feeder cells, will make the clinical application of oral mucosal epithelial cell sheet safe and transplantable for human use.

[0006] Existing cell culture media contain some form or derivative of xenogeneic products that are unapproved by U.S regulatory agencies for human clinical applications. Cholera toxin, animal derived cytokines, and digestive enzymes are examples of such reagents that need to be phased out and replaced with human derived equivalents. Furthermore, recombinant proteins and growth factors used to produce multilayered oral mucosal epithelium cell sheets need to be manufactured in a cGMP facility and certified for clinical use. The entire process including reagents, consumables and cultureware needs to be cGMP certified for clinical application with regard to enhancing the efficacy of the graft pertaining to the patient’s safety.

SUMMARY

[0007] The present disclosure demonstrates the feasibility and successful production of rabbit cGMP-certified cultured oral mucosal epithelial cell sheet (cG-CAOMECS), designed to reconstruct corneal surface of patients with bilateral limbal stem cell deficiency. The production makes use of epithelial progenitor cells differentiated in a feeder cell-free, serum- free, medium. In addition to reconstruct corneal epithelium, the present technology can also be used to construct skin tissues and esophageal epithelia, without limitation.

[0008] One embodiment of the present disclosure provides a method for preparing a multilayered epithelial cell sheet, comprising culturing a plurality of epithelial progenitor cells on a surface coated with an extracellular matrix substrate, and in a medium not containing animal feeder cells, wherein the medium is a basal medium supplemented with non-animal serum replacement, albumin, and isoproterenol.

[0009] In some embodiments, the epithelial stem cells comprise oral mucosal epithelial cells. In some embodiments, the oral mucosal epithelial cells have been isolated with trypsin. In some embodiments, the multilayered epithelial cell sheet comprises corneal epithelial cells. In some embodiments, the multilayered epithelial cell sheet comprises skin cells. In some embodiments, the multilayered epithelial cell sheet comprises esophageal epithelial cells. [0010] In some embodiments, the basal medium is Dulbecco’s Modified Eagle Medium (DMEM). In some embodiments, the medium comprises DMEM supplemented with 4-15% (v/v) non-animal serum replacement, 0.1-1% (w/v) albumin, and 0.1-2 pg/mL isoproterenol.

[0011] In some embodiments, the medium further comprises a Rho-associated protein kinase (ROCK) inhibitor, epithelial growth factor (EGF) and keratinocyte growth factor (KGF). In some embodiments, the medium comprises 2-50 pM of the ROCK inhibitor, 1-100 ng/mL EGF and 2-50 ng/mL KGF.

[0012] In some embodiments, the medium is changed every 1, 2 or 3 days. In some embodiments, the culturing is carried out for 5-30 days.

[0013] In some embodiments, the method further comprises detaching the multilayered epithelial cell sheet from the coated surface with collagenase.

[0014] Also provided, in one embodiment, is a multilayered epithelial cell sheet prepared by the method of the present disclosure. In some embodiments, the multilayered epithelial cell sheet is a corneal epithelium. In some embodiments, the multilayered epithelial cell sheet is a skin. In some embodiments, the multilayered epithelial cell sheet is an esophageal epithelium.

[0015] Also provided are methods for using the reconstructed epithelial cell sheets. In some embodiments, a method for reconstructing a corneal in a patient in need thereof, comprising implanting the reconstructed corneal epithelium. In some embodiments, the patient suffers limbal stem cell deficiency (LSCD).

[0016] In another embodiment, provided is a culture medium, comprising a basal culture medium, non-animal serum replacement, albumin, and isoproterenol. In some embodiments, the basal culture medium is Dulbecco’s Modified Eagle Medium (DMEM).

[0017] In some embodiments, the medium comprises DMEM supplemented with 4-15% (v/v) non-animal serum replacement, 0.1-1% (w/v) albumin, and 0.1-2 pg/mL isoproterenol. In some embodiments, the medium further comprises a Rho-associated protein kinase (ROCK) inhibitor, epithelial growth factor (EGF) and keratinocyte growth factor (KGF). In some embodiments, the medium comprises 2-50 pM of the ROCK inhibitor, 1-100 ng/mL EGF and 2-50 ng/mL KGF. BRIEF DESCRIPTION OF THE FIGURES

[0018] FIG. 1 shows the preparation of rabbit oral mucosal epithelial cell sheets cG- CAOMECS. A, D and G are the same cell sheet pictured at D5 and D17 of culture. B, E and H are the same cell sheet pictured at D9 and D18 of culture. C, F and I are the same cell sheet examined at DIO and D17 of culture. The three cell sheets were produced with KaFa medium on 6MWP coated with CELLstart® and respectively harvested on day 17, 18 and 17. Detachment of cell sheet was accomplished using collagenase treatment.

[0019] FIG. 2 shows immunofluorescent staining of cross section of cG-CAOMECS. Green indicates staining of: A is DeltaNp63, B is Pax6 staining C is PCNA, D is K3 and E is K4 staining. Red indicates nuclear staining. Mag x40.

[0020] FIG. 3 shows the effects of KaFa® medium on the expression of cG-CAOMECS progenitor stem cell DeltaNp63 and Pax-6 (A and B), corneal keratins K3/K12 (C and D), adherens molecules, E-cadherin and beta catenin (E and F), gap junction protein Cnx43 (G) and beta-actin levels measurement for gel loading control (D). Beta-actin was used to control the amount of protein loaded on the gels (H).

[0021] FIG. 4. shows detachment of the cell sheet. A, B and C showed the PVDF membrane used to lift the cell sheet after incubation with collagenase. Cross sections of harvested cell sheet stained positive for phosphorylated FAK (D), integrin beta 1 and 4 (E and F). The levels of expression of intergrins is shown in G and F. I is the loading control for the semi quantitative measurements.

DETAILED DESCRIPTION

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As used herein the following terms have the following meanings.

[0023] As used in the specification and claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of cells, including mixtures thereof.

[0024] As used herein, the term “comprising” or “comprises” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of’ when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) claimed. “Consisting of’ shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0025] As used herein, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated polynucleotide is separated from the 3’ and 5’ contiguous nucleotides with which it is normally associated in its native or natural environment, e.g., on the chromosome. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart. An isolated cell is a cell that is separated from tissue or cells of dissimilar phenotype or genotype.

[0026] As used herein, “stem cell” defines a cell with the ability to divide for indefinite periods in culture and give rise to specialized cells. Non-limiting examples of types of stem cells include somatic (adult) stem cells, embryonic stem cells, parthenogenetic stem cells, and/or induced pluripotent stem cells (iPS cells or iPSCs).

[0027] As used herein, the term “culturing” refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation or division of cells.

[0028] As used herein, the term “propagate” means to grow or alter the phenotype of a cell or population of cells. The term “grow” or “expand” refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell type. In one embodiment, the growing/expansion of cells results in the regeneration of tissue. [0029] As used herein and as set forth in more detail below, “conditioned medium” is medium which was cultured with a mature cell that provides cellular factors to the medium such as cytokines, growth factors, hormones, extracellular matrix, and some materials that would facilitate cell growth, development, and differentiation.

[0030] As used herein, the term “differentiation” describes the process whereby an unspecialized cell acquires the features of a specialized cell such as a skin, heart, liver, or muscle cell. “Directed differentiation” refers to the manipulation of stem cell culture conditions to induce differentiation into a particular cell type. “Dedifferentiated” defines a cell that reverts to a less committed position within the lineage of a cell. As used herein, the term “differentiates or differentiated” defines a cell that takes on a more committed (“differentiated”) position within the lineage of a cell.

[0031] As used herein, the “lineage” of a cell defines the heredity of the cell, i.e. its predecessors and progeny. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. As used herein, “a cell that differentiates into a mesodermal (or ectodermal or endodermal) lineage” defines a cell that becomes committed to a specific mesodermal (or ectodermal or endodermal) lineage, respectively. Examples of cells that differentiate into a mesodermal lineage or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic, leiomyogenic, chondrogenic, cardiogenic, dermatogenic, hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic, or stromal. Examples of cells that differentiate into ectodermal lineage include, but are not limited to epidermal cells, neurogenic cells, and neurogliagenic cells. Examples of cells that differentiate into endodermal lineage include, but are not limited to cells that give rise to the pancreas, liver, lung, stomach, intestine, and thyroid.

[0032] As used herein, the term “pluripotent stem cells” refers to cells that are: (i) capable of indefinite proliferation in vitro in an undifferentiated state; (ii) maintain a normal karyotype through prolonged culture; and (iii) maintain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture. Non- limiting examples of currently available pluripotent stem cells include embryonic stem cells and iPSCs. As used herein, the term “embryonic-like stem cells” refers to cells derived from tissues, organs, or blood, possessing pluripotent characteristics of embryonic stem cells. [0033] As used herein, the term “pharmaceutically acceptable” indicates that the indicated material does not have properties that would cause a reasonably prudent medical practitioner to avoid administration of the material to a patient, taking into consideration the disease or conditions to be treated and the respective route of administration. For example, it is commonly required that such a material be essentially sterile.

[0034] As used herein, the term “pharmaceutically acceptable carrier (or medium)”, which may be used interchangeably with the term “biologically compatible carrier (or medium)”, refers to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers suitable for use in the present invention include liquids, semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds and matrices, tubes sheets and other such materials as known in the art and described in greater detail herein). These semi-solid and solid materials may be designed to resist degradation within the body (non-biodegradable) or they may be designed to degrade within the body (biodegradable, bioerodable). A biodegradable material may further be bioresorbable or bioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids (water-soluble implants are one example), or degraded and ultimately eliminated from the body, either by conversion into other materials or breakdown and elimination through natural pathways. For topical use, the pharmaceutically acceptable carrier is suitable for manufacture of creams, ointments, jellies, gels, solutions, suspensions, etc. Such carriers are conventional in the art, e.g., for topical administration with polyethylene glycol (PEG). These formulations may optionally comprise additional pharmaceutically acceptable ingredients such as diluents, stabilizers, and/or adjuvants.

[0035] As used herein, the term “solution” refers to solutions, suspensions, emulsions, drops, ointments, liquid wash, sprays, and liposomes, which are well known in the art. In some embodiments, the liquid solution contains an aqueous pH buffering agent which resists changes in pH when small quantities of acid or base are added.

[0036] As used herein, the term “pH buffering agent” refers to an aqueous buffer solution which resists changes in pH when small quantities of acid or base are added to it. pH buffering solutions typically comprise a mixture of weak acid and its conjugate base, or vice versa. For example, pH buffering solutions may comprise phosphates such as sodium phosphate, sodium dihydrogen phosphate, sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate, disodium hydrogen phosphate dodecahydrate, potassium phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate; boric acid and borates such as, sodium borate and potassium borate; citric acid and citrates such as sodium citrate and disodium citrate; acetates such as sodium acetate and potassium acetate; carbonates such as sodium carbonate and sodium hydrogen carbonate, etc. pH adjusting agents can include, for example, acids such as hydrochloric acid, lactic acid, citric acid, phosphoric acid and acetic acid, and alkaline bases such as sodium hydroxide, potassium hydroxide, sodium carbonate and sodium hydrogen carbonate, etc. In some embodiments, the pH buffering agent is a phosphate buffered saline (PBS) solution (i.e., containing sodium phosphate, sodium chloride and in some formulations, potassium chloride and potassium phosphate).

[0037] As used herein, the term “formulated” or “formulation” refers to the process in which different substances, including one or more pharmaceutically active ingredients, are combined to produce a dosage form. In certain embodiments, two or more pharmaceutically active ingredients can be co-formulated into a single dosage form or combined dosage unit, or formulated separately and subsequently combined into a combined dosage unit. A sustained release formulation is a formulation which is designed to slowly release a therapeutic agent in the body over an extended period of time, whereas an immediate release formulation is a formulation which is designed to quickly release a therapeutic agent in the body over a shortened period of time.

[0038] As used herein, the term “treating” refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and/or halting one or more clinical symptoms of a disease or disorder prior to, during, and/or after an injury or intervention.

[0039] As used herein, the term “patient” or “subject” refers to animals, including mammals, such as murine, canine, equine, bovine, porcine, simian, or humans, who are treated with the pharmaceutical compositions or in accordance with the methods described herein.

[0040] As used herein, the term “delivery” refers to routes, approaches, formulations, technologies, and systems for transporting a pharmaceutical composition in the body as needed to safely achieve its desired therapeutic effect. The route of delivery can be any suitable route, including but not limited to, intravascular, intravenous, intraarterial, intramuscular, cutaneous, subcutaneous, percutaneous, intradermal, and intraepidermal routes. In some embodiments, an effective amount of the composition is formulated for applying on the skin or delivery into the skin of a patient. In some embodiments, an effective amount of the composition is formulated for delivery into the blood stream of a patient.

[0041] As used herein, the term “effective amount” refers to a concentration or amount of composition or a reagent, such as a composition as described herein, cell population or other agent, that is effective for producing an intended result, including cell growth, cell division, and/or differentiation in vitro or in vivo, or for the treatment of a disease, disorder or condition in a patient in need thereof. It will be appreciated that the number of cells to be administered will vary depending on the specifics of the disorder to be treated, including but not limited to size or total volume/surface area to be treated, as well as proximity of the site of administration to the location of the region to be treated, among other factors familiar to the medicinal biologist and/or treating physician.

[0042] As used herein, the terms “effective period (or time)” and “effective conditions” refer to a period of time or other controllable conditions (e.g., temperature, humidity for in vitro methods), necessary or preferred for an agent or composition to achieve its intended result, e.g., the differentiation of cells to a pre-determined cell type.

[0043] As used herein, the term “control” or “control group” refers to an alternative subject or sample used in an experiment for comparison purpose. A control can be “positive” or “negative”.

[0044] As used herein, the term “target tissue” or “target organ” refers to an intended site for accumulation of the stem cells as disclosed herein and/or the differentiated cells derived from the stem cells as disclosed herein, following administration to a subject. For example, the methods as disclosed herein involve a target tissue or a target organ that has been damaged (e.g., by ischemia or other injury) in some embodiments.

[0045] As used herein, the terms “autologous transfer”, “autologous transplantation”, “autograft” and the like refer to treatments wherein the cell donor is also the recipient of the cell replacement therapy. The terms “allogeneic transfer”, “allogeneic transplantation”, “allograft” and the like refer to treatments wherein the cell donor is of the same species as the recipient of the cell replacement therapy, but is not the same individual. A cell transfer in which the donor’s cells have been histocompatibly matched with a recipient is sometimes referred to as a syngeneic transfer. The terms xenogeneic transfer, xenogeneic transplantation, xenograft and the like refer to treatments wherein the cell donor is of a different species than the recipient of the cell replacement therapy.

[0046] As used herein, the term “lineage markers” or “Lin” refers to characteristic molecules for cell lineages, e.g. cell surface markers, mRNAs, or internal proteins. Lineage-positive (Lin⁺) cells refer to a mix of cells expressing mature cell lineage markers. Lineage- negative (Lin-) cells include stem and progenitor cells, which are not differentiated mature cells. In one aspect, “Lin” refers to a panel of markers. As used herein, the FITC anti-human lineage antibody cocktail is optimized for the detection of human peripheral blood T cells, B cells, NK cells, monocytes, and neutrophils. This cocktail is composed of CD3, CD14, CD16, CD19, CD20, and CD56.

Media for Preparing Multilayered Epithelial Cell Sheets

[0047] The accompanying experimental examples have demonstrated the feasibility and successful production of rabbit cG-CAOMECS (cGMP-certified cultured oral mucosal epithelial cell sheet), useful for reconstructing corneal surface of patients with bilateral limbal stem cell deficiency.

[0048] To produce a safe, xeno-free and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP- certified cell culture surface coated with GMP grade Extracellular Matrix. A newly designed clinical-grade xeno-free medium (KaFa® medium, Table 1) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment.

[0049] Cells attached onto the surface and self-assembled into colony forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells differentiated into a multilayered epithelial cell sheet that was harvested after 17-19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 and Pax-6 were expressed in the basal cells and K3/K12 were expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, such as E-cadherin, beta-catenin and Cnx43, were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of Integrin - betal and beta4 confirmed that the Collagenase treatment used for detaching and harvesting the cell sheet did not have any adverse effects. Thus, these results show that cG-CAOMECS is a promising translational technology for human application.

[0050] It is worth noting that the instant cell sheet reconstruction technology was implemented without the use of animal feeder cells and without fetal bovine serum. Only FDA-approved GMP grade cell culture reagents were utilized. This technology, therefore, can be the solution for bilateral total loss of limbal stem cells as it poses no problem of immunosuppression and graft failure. Oral mucosal epithelium can be easily harvested with a small biopsy. Moreover, the regenerative spectrum of cultured oral mucosal epithelial cell sheet is not limited to reconstruct corneal surface, but can be used in various epithelial disorders, such as severe burns, giant congenital nevi, and esophagus regeneration.

[0051] In accordance with one embodiment of the present disclosure, provided is a medium useful for expanding and/or differentiating progenitor cells. The medium is exemplified by the KaFa® medium (Table 1). In one embodiment, the medium of the present disclosure includes a basal culture medium supplemented with a non-animal serum replacement.

[0052] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle’s Minimum Essential Medium (EMEM) medium, aMEM medium, Dulbecco’s modified Eagle’s Medium (DMEM) medium, Ham’s F12 medium, RPMI 1640 medium, Fischer’s medium, StemPro34, RPMI-base medium, mTeSRl, or a mixed medium thereof. In a preferred embodiment, the basal medium is DMEM.

[0053] A non-animal serum replacement, or simply a serum replacement, is combination of chemically defined ingredients that provides a serum- free and xeno-free approach for cell culture. Commercial products are available, such as the Knockout™ Serum Replacement (KSR) from Thermo Fisher.

[0054] In some embodiments, the serum replacement is present in the medium in a concentration of about 1% to 20% (v/v, volume of commercial stock solution (e.g., Thermo Fisher Scientific Catalog number: 10828010) over total volume of the medium). In some embodiments, the concentration is about 2%-15% (v/v), 3%-12% (v/v), 4%- 11 % (v/v), 5%- 10% (v/v), 6%-9% (v/v), 7%-8% (v/v) or about 7.5% (v/v). [0055] In some embodiments, the medium further includes albumin. In some embodiments, the albumin is human albumin. Human albumin products are available commercially. For instance, Albutein (25% albumin) is a sterile, aqueous solution for single dose intravenous administration containing 25% human albumin (weight/ volume). Albutein is prepared by a cold alcohol fractionation method from pooled human plasma obtained from venous blood. The product is stabilized with 0.08 millimole sodium caprylate and 0.08 millimole sodium acetyltryptophanate per gram of protein. Albutein is manufactured from Source Plasma collected from FDA approved plasmapheresis centers in the United States.

[0056] In some embodiments, the concentration of albumin in the medium is about 0.05%- 5% (w/v). In some embodiments, the concentration of albumin in the medium is about 0.05%-4%, 0.1%-3%, 0.1%-2%, 0.1%-l%, 0.1%-0.8%, 0.15%-0.6%, 0.15%-0.5%, 0.15%- 0.4%, 0.2%-0.3%, or about 0.25% (w/v).

[0057] In some embodiments, the medium further includes isoproterenol. Isoproterenol, also known as isoprenaline, has a chemical name of (RS)-4-[ 1 -hydroxy-2-(isopropylamino)ethyl ] benzene- 1,2-diol. It is a non-selective P adrenoceptor agonist that is the isopropylamine analog of epinephrine (adrenaline). Isoproterenol as a medication has been marketed under many different brands. An example is Isuprel which includes 0.2 mg isoproterenol hydrochloride in each mL solution.

[0058] In some embodiments, the medium includes about 0.1 pg/mL to 2 pg/mL isoproterenol or a salt thereof. In some embodiments, the medium includes about 0.1 -1.5 pg/mL, 0.15-1.2 pg/mL, 0.15-1 pg/mL, 0.15-0.8 pg/mL, 0.15-0.7 pg/mL, 0.2-0.7 pg/mL, 0.3-0.6 pg/mL, 0.3-0.5 pg/mL, 0.35-0.45 pg/mL, or 0.4 pg/mL isoproterenol or a salt thereof.

[0059] In some embodiments, the medium includes basal medium DMEM supplemented with 4-15% (v/v) non-animal serum replacement, 0.1-1% (w/v) albumin, and 0.1-2 pg/mL isoproterenol. In some embodiments, the medium includes basal medium DMEM supplemented with 5-10% (v/v) non-animal serum replacement, 0.1 -0.4% (w/v) albumin, and 0.2-0.6 pg/mL isoproterenol.

[0060] In some embodiments, the medium further includes a Rho-associated protein kinase (ROCK) inhibitor, epithelial growth factor (EGF) and/or keratinocyte growth factor (KGF). [0061] ROCK inhibitors (rho-associated protein kinase inhibitor or ROCK inhibitor) are a series of compounds that target rho kinase (ROCK) and inhibit the ROCK pathway. ROCK has multiple functions, including regulation of smooth muscle cell contraction, cell migration, and maintenance of cell viability and morphology, in part by regulating stress fibers and focal adhesions. ROCK inhibitors can limit cellular death and dedifferentiation. Example ROCK inhibitors include AT-13148, BA-210, [LElemene, Chroman 1, DJ4, Fasudil, GSK-576371, GSK429286A, H-1152, Hydroxyfasudil, Ibuprofen, LX-7101, Netarsudil, RKI-1447, Ripasudil, TCS-7001, Thiazovivin, Verosudil, Y-27632, Y-30141, Y-33075, and Y-39983.

[0062] In some embodiments, the medium includes 1-50 pM of the ROCK inhibitor. In some embodiments, the medium includes 1-50 pM, 2-45 pM, 3-40 pM, 4-30 pM, 5-20 pM, 6-20 pM, 7-15 pM, 8-12 pM, or about 10 pM of the ROCK inhibitor.

[0063] In some embodiments, the medium includes about 1-100 ng/mL EGF, such as human EGF. In some embodiments, the medium includes about 2-90 ng/mL, 3-80 ng/mL, 5-70 ng/mL, 5-60 ng/mL, 5-50 ng/mL, 10-90 ng/mL, 10-80 ng/mL, 10-50 ng/mL, 10-40 ng/mL, 10-30 ng/mL, 5-20 ng/mL, 5-15 ng/mL, 20-80 ng/mL, or 40-60 ng/mL EGF.

[0064] In some embodiments, the medium includes about 1-100 ng/mL KGF, such as human KGF. In some embodiments, the medium includes about 2-90 ng/mL, 3-80 ng/mL, 5-70 ng/mL, 5-60 ng/mL, 5-50 ng/mL, 10-90 ng/mL, 10-80 ng/mL, 10-50 ng/mL, 10-40 ng/mL, 10-30 ng/mL, 5-20 ng/mL, or 5-15 ng/mL KGF.

[0065] In some embodiments, the medium further includes D-Glucose, at a concentration of about 1-20 g/L, or 1-15 g/L, 1-12 g/L, 1-10 g/L, 1-8 g/L, 2-7 g/L, 3-6 g/L, or 4-5 g/L, or about 4.5 g/L.

[0066] In some embodiments, the medium further includes a serum-free B-27™ supplement (e.g., Thermo Fisher Scientific catalog number: 17504044). In some embodiments, the medium further includes L-glutamine or a GlutaMAX™ supplement. In some embodiments, the concentration of GlutaMAX is about 0.1%-10% (w/v), or about 0.1%-10%, 0.2%-9%, 0.3%-7%, 0.4%-6%, 0.5%-5%, 0.6%-4%, 0.7%-3%, 0.8%-2%, 0.9%-1.5%, or at about 1% (w/v).

[0067] In some embodiments, the medium further includes sodium pyruvate. In some embodiments, the concentration of sodium pyruvate is about 0.05 mM to 5 mM, or about 0.1 mM to 4 mM, 0.1 mM to 3 mM, 0.1 mM to 2 mM, 0.2 mM to 2 mM, 0.2 mM to 1 mM, 0.2 mM to 0.8 mM, 0.3 mM to 0.7 mM, 0.4 mM to 0.6 mM, 0.45 mM to 0.55 mM, or about 0.5 mM.

[0068] In some embodiments, the medium further includes hydrocortisone. In some embodiments, the concentration of hydrocortisone is about 0.05 pg/mL to 5 |ig/mL, 0.05 |ig/mL to 4 |ig/mL, 0.05 pg/mL to 2 |ig/mL, 0.1 pg/mL to 2 |ig/mL, 0.1 pg/mL to 1 |ig/mL, 0.1 pg/mL to 0.9 |ig/mL, 0.2 |ig/mL to 0.8 |ig/mL, 0.2 |ig/mL to 0.7 |ig/mL, 0.3 pg/mL to 0.6 |ig/mL, 0.3 pg/mL to 0.5 |ig/mL, or about 0.4 |ig/mL.

[0069] In some embodiments, the medium further includes T3 (Triostat, 3,3’,5-Triiodo-L- thyronine sodium salt). In some embodiments, the concentration is about O.lxlO^-9 to lOxlO^-9 M, or 0.2xl0’⁹ to 8xl0’⁹ M, 0.3xl0’⁹ to 8xl0’⁹ M, 0.5xl0’⁹ to 8xl0’⁹ M, 0.5xl0’⁹ to 5xl0’⁹ M, 0.5xl0’⁹ to 4xl0’⁹ M, IxlO’⁹ to 4xl0’⁹ M, IxlO’⁹ to 3xl0’⁹ M, 1.5xl0’⁹ to 2.5xl0’⁹ M, or about 2xl0^-9 M.

[0070] In some embodiments, the medium further includes insulin, transferrin, and/or selenium. In some embodiments, the medium includes calcium chloride. In some embodiments, the medium includes an antibiotic and/or antimycotic.

Preparation of Multilayered Epithelial Cell Sheets

[0071] Methods for preparing multilayered epithelial cell sheets are also provided, in some embodiments. In one embodiment, the method entails culturing a plurality of epithelial progenitor cells in a medium as disclosed herein. In some embodiments, the culturing is on a surface coated with an extracellular matrix substrate.

[0072] Extracellular matrix substrates are typically made of extracellular matrix (ECM) proteins. They can be coated to a surface, such as a glass or polymer surface, by an agent such as (3-aminopropyl) triethoxysilane. Commercial products are available for ECM coating, such as CELLstart® substrate (Thermo Fisher Scientific Catalog number: A1014201).

[0073] Epithelial progenitor cells are progenitor cells of the epithelial lineage. Epithelial progenitor cells can be prepared from stem cells such as induced pluripotent stem cells (iPSC), or isolated from sources such as oral mucosal epithelial cells from a biopsy of a buccal tissue. It is known that epithelial progenitor cells are concentrated in the occlusal plane in the back of the cheek. In some embodiments, the the oral mucosal epithelial cells have been isolated with trypsin.

[0074] In some embodiments, the culturing is carried out without the use of feeder cells. Feeder cells are commonly used in cell culture, as a layer of non-dividing cells to help another cell to proliferate. Feeder cells are typically animal cells that provide direct cell-cell interactions, produce a bioactive matrix for cell-ECM interactions, and release nutrients/growth factors into the culture medium to maintain the pluripotency of progenitor cells during cultivation and prevent spontaneous differentiation.

[0075] Feeder layers are viewed as an integral part of deriving and culturing human pluripotent stem cells (Leonardo et al., “Chapter 2 - Preparation of Mouse Embryonic Fibroblast Feeder Cells” in Human Stem Cell Manual (Second Edition) 2012, Academic Press, Pages 15-27). The present technology, however, does not rely on the use of feeder cells. In some embodiments, the epithelial progenitor cells in the culture is not in contact, directly or indirectly though a medium, to an animal feeder cell.

[0076] In some embodiments, the medium is exemplified by the KaFa® medium (Table 1). In one embodiment, the medium used in the method includes a basal culture medium supplemented with a non-animal serum replacement.

[0077] Examples of the basal medium include IMDM medium, Medium 199 medium, Eagle’s Minimum Essential Medium (EMEM) medium, aMEM medium, Dulbecco’s modified Eagle’s Medium (DMEM) medium, Ham’s F12 medium, RPMI 1640 medium, Fischer’s medium, StemPro34, RPMI-base medium, mTeSRl, or a mixed medium thereof. In a preferred embodiment, the basal medium is DMEM.

[0078] A non-animal serum replacement, or simply a serum replacement, is combination of chemically defined ingredients that provides a serum- free and xeno-free approach for cell culture. Commercial products are available, such as the Knockout™ Serum Replacement (KSR) from Thermo Fisher.

[0079] In some embodiments, the serum replacement is present in the medium in a concentration of about 1% to 20% (v/v, volume of commercial stock solution (e.g., Thermo Fisher Scientific Catalog number: 10828010) over total volume of the medium). In some embodiments, the concentration is about 2%-15% (v/v), 3%-12% (v/v), 4%- 11 % (v/v), 5%- 10% (v/v), 6%-9% (v/v), 7%-8% (v/v) or about 7.5% (v/v).

[0080] In some embodiments, the medium further includes albumin. In some embodiments, the albumin is human albumin. Human albumin products are available commercially. For instance, Albutein (25% albumin) is a sterile, aqueous solution for single dose intravenous administration containing 25% human albumin (weight/ volume). Albutein is prepared by a cold alcohol fractionation method from pooled human plasma obtained from venous blood. The product is stabilized with 0.08 millimole sodium caprylate and 0.08 millimole sodium acetyltryptophanate per gram of protein. Albutein is manufactured from Source Plasma collected from FDA approved plasmapheresis centers in the United States.

[0081] In some embodiments, the concentration of albumin in the medium is about 0.05%- 5% (w/v). In some embodiments, the concentration of albumin in the medium is about 0.05%-4%, 0.1%-3%, 0.1%-2%, 0.1%-l%, 0.1%-0.8%, 0.15%-0.6%, 0.15%-0.5%, 0.15%- 0.4%, 0.2%-0.3%, or about 0.25% (w/v).

[0082] In some embodiments, the medium further includes isoproterenol. Isoproterenol, also known as isoprenaline, has a chemical name of (7?S)-4-[ 1 -hydroxy-2-(isopropylamino)ethyl ] benzene- 1,2-diol. It is a non-selective P adrenoceptor agonist that is the isopropylamine analog of epinephrine (adrenaline). Isoproterenol as a medication has been marketed under many different brands. An example is Isuprel which includes 0.2 mg isoproterenol hydrochloride in each mL solution.

[0083] In some embodiments, the medium includes about 0.1 pg/mL to 2 pg/mL isoproterenol or a salt thereof. In some embodiments, the medium includes about 0.1 -1.5 pg/mL, 0.15-1.2 pg/mL, 0.15-1 pg/mL, 0.15-0.8 pg/mL, 0.15-0.7 pg/mL, 0.2-0.7 pg/mL, 0.3-0.6 pg/mL, 0.3-0.5 pg/mL, 0.35-0.45 pg/mL, or 0.4 pg/mL isoproterenol or a salt thereof.

[0084] In some embodiments, the medium includes basal medium DMEM supplemented with 4-15% (v/v) non-animal serum replacement, 0.1-1% (w/v) albumin, and 0.1-2 pg/mL isoproterenol. In some embodiments, the medium includes basal medium DMEM supplemented with 5-10% (v/v) non-animal serum replacement, 0.1 -0.4% (w/v) albumin, and 0.2-0.6 pg/mL isoproterenol. [0085] In some embodiments, the medium further includes a Rho-associated protein kinase (ROCK) inhibitor, epithelial growth factor (EGF) and/or keratinocyte growth factor (KGF).

[0086] ROCK inhibitors (rho-associated protein kinase inhibitor or ROCK inhibitor) are a series of compounds that target rho kinase (ROCK) and inhibit the ROCK pathway. ROCK has multiple functions, including regulation of smooth muscle cell contraction, cell migration, and maintenance of cell viability and morphology, in part by regulating stress fibers and focal adhesions. ROCK inhibitors can limit cellular death and dedifferentiation. Example ROCK inhibitors include AT-13148, BA-210, P-Elemene, Chroman 1, DJ4, Fasudil, GSK-576371, GSK429286A, H-1152, Hydroxyfasudil, Ibuprofen, LX-7101, Netarsudil, RKI-1447, Ripasudil, TCS-7001, Thiazovivin, Verosudil, Y-27632, Y-30141, Y-33075, and Y-39983.

[0087] In some embodiments, the medium includes 1-50 pM of the ROCK inhibitor. In some embodiments, the medium includes 1-50 pM, 2-45 pM, 3-40 pM, 4-30 pM, 5-20 pM, 6-20 pM, 7-15 pM, 8-12 pM, or about 10 pM of the ROCK inhibitor.

[0088] In some embodiments, the medium includes about 1-100 ng/mL EGF, such as human EGF. In some embodiments, the medium includes about 2-90 ng/mL, 3-80 ng/mL, 5-70 ng/mL, 5-60 ng/mL, 5-50 ng/mL, 10-90 ng/mL, 10-80 ng/mL, 10-50 ng/mL, 10-40 ng/mL, 10-30 ng/mL, 5-20 ng/mL, 5-15 ng/mL, 20-80 ng/mL, or 40-60 ng/mL EGF.

[0089] In some embodiments, the medium includes about 1-100 ng/mL KGF, such as human KGF. In some embodiments, the medium includes about 2-90 ng/mL, 3-80 ng/mL, 5-70 ng/mL, 5-60 ng/mL, 5-50 ng/mL, 10-90 ng/mL, 10-80 ng/mL, 10-50 ng/mL, 10-40 ng/mL, 10-30 ng/mL, 5-20 ng/mL, or 5-15 ng/mL KGF.

[0090] In some embodiments, the medium further includes D-Glucose, at a concentration of about 1-20 g/L, or 1-15 g/L, 1-12 g/L, 1-10 g/L, 1-8 g/L, 2-7 g/L, 3-6 g/L, or 4-5 g/L, or about 4.5 g/L.

[0091] In some embodiments, the medium further includes a serum-free B-27™ supplement (e.g., Thermo Fisher Scientific catalog number: 17504044). In some embodiments, the medium further includes L-glutamine or a GlutaMAX™ supplement. In some embodiments, the concentration of GlutaMAX is about 0.1%-10% (w/v), or about 0.1%-10%, 0.2%-9%, 0.3%-7%, 0.4%-6%, 0.5%-5%, 0.6%-4%, 0.7%-3%, 0.8%-2%, 0.9%-1.5%, or at about 1% (w/v). [0092] In some embodiments, the medium further includes sodium pyruvate. In some embodiments, the concentration of sodium pyruvate is about 0.05 mM to 5 mM, or about 0.1 mM to 4 mM, 0.1 mM to 3 mM, 0.1 mM to 2 mM, 0.2 mM to 2 mM, 0.2 mM to 1 mM, 0.2 mM to 0.8 mM, 0.3 mM to 0.7 mM, 0.4 mM to 0.6 mM, 0.45 mM to 0.55 mM, or about 0.5 mM.

[0093] In some embodiments, the medium further includes hydrocortisone. In some embodiments, the concentration of hydrocortisone is about 0.05 pg/mL to 5 pg/mL, 0.05 pg/mL to 4 pg/mL, 0.05 pg/mL to 2 pg/mL, 0.1 pg/mL to 2 pg/mL, 0.1 pg/mL to 1 pg/mL, 0.1 pg/mL to 0.9 pg/mL, 0.2 pg/mL to 0.8 pg/mL, 0.2 pg/mL to 0.7 pg/mL, 0.3 pg/mL to 0.6 pg/mL, 0.3 pg/mL to 0.5 pg/mL, or about 0.4 pg/mL.

[0094] In some embodiments, the medium further includes T3 (Triostat, 3,3’,5-Triiodo-L- thyronine sodium salt). In some embodiments, the concentration is about O.lxlO^-9 to lOxlO^-9 M, or 0.2xl0’⁹ to 8xl0’⁹ M, 0.3xl0’⁹ to 8xl0’⁹ M, 0.5xl0’⁹ to 8xl0’⁹ M, 0.5xl0’⁹ to 5xl0’⁹ M, 0.5xl0’⁹ to 4xl0’⁹ M, IxlO’⁹ to 4xl0’⁹ M, IxlO’⁹ to 3xl0’⁹ M, 1.5xl0’⁹ to 2.5xl0’⁹ M, or about 2xl0^-9 M.

[0095] In some embodiments, the medium further includes insulin, transferrin, and/or selenium. In some embodiments, the medium includes calcium chloride. In some embodiments, the medium includes an antibiotic and/or antimycotic.

[0096] The duration of the culture is generally from a few days to 4-6 weeks. It is observed herein that the cultured cells attached onto the surface and self-assembled into colony forming units (CFUs) during the first 5 days, and a monolayer cell sheet formed in less than 10 days. Cells differentiated and formed a multilayered epithelial cell sheet after 17-19 days.

[0097] Differentiation of the cells can also be monitored with optical imaging or immunostaining. For instance, deltaNp63 and Pax-6 expression in the basal cells and K3/K12 expression in the apical cells can be used to indicate the presence of corneal epithelial-like cells in the produced cell sheet.

[0098] Upon formation of the multilayered epithelial cell sheet, the cell sheet can be detached from the surface, e.g., with collagenase (e.g., at 0.5 mg/mL final concentration). Collagenase treatment, it is noted, is not required when the conventional feeder cell technology is used. [0099] As noted, the epithelial progenitor cells can be differentiated into multilayered cell sheets of different cell types. In one embodiment, the cells form a corneal epithelium. In one embodiment, the cells form a skin tissue. In one embodiment, the cells form an esophageal epithelium. Differentiation into the different epitheliums can be directed with different growth factors, as generally known in the art. Also provided, in some embodiments, are the reconstructed epitheliums.

Therapeutic Use of Multilayered Epithelial Cell Sheets

[0100] The present technology can be used to reconstruct epithelial tissues of different types, such as a corneal epithelium, an esophageal epithelium, or a skin. The reconstructed epithelium can be transplanted to a patient in need of, such as those that have limbal stem cell deficiency (LSCD), severe burns, or giant congenital nevi, or in need of esophagus regeneration. In some embodiments, the cell sheet is reconstructed from epithelial progenitor cells isolated from the patient which thus constitutes autologous cell transplant.

[0101] In one embodiment, the cell sheet is a reconstructed corneal epithelium. A method is provided for using such a reconstructed corneal epithelium in a patient in need thereof. In some embodiments, the patient is in need of replacement of the corneal epithelium. In some embodiments, the patient suffers limbal stem cell deficiency (LSCD) or is at risk of developing LSCD. In some embodiments, the cell sheet is transplanted to the eye of the patient.

[0102] In one embodiment, the cell sheet is a reconstructed skin. A method is provided for using such a reconstructed skin in a patient in need thereof. In some embodiments, the patient is in need of replacement of the skin. In some embodiments, the patient suffers severe bums. In some embodiments, the patient has a congenital nevi or tattoo that desires removal. In some embodiments, the cell sheet is transplanted to the skin of the patient.

[0103] In one embodiment, the cell sheet is a reconstructed esophageal epithelium. A method is provided for using such a reconstructed esophageal epithelium in a patient in need thereof. In some embodiments, the patient is in need of replacement of a portion of the esophageal epithelium. In some embodiments, the patient suffers severe bums to the esophagus or esophageal degeneration. In some embodiments, the cell sheet is transplanted to the esophagus of the patient. EXAMPLES

Example 1. cG-CAOMECS - Clinical Grade Cultured Autologous Oral Mucosal Epithelial Cell Sheet, for Corneal Surface Reconstruction

[0104] This example shows the feasibility and successful production of rabbit cG- CAOMECS, designed to reconstruct corneal surface of patients with bilateral limbal stem cell deficiency.

[0105] To produce a safe, xeno-free and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMPcertified cell culture surface coated with GMP grade Extracellular Matrix. A newly designed clinical-grade xeno-free medium (KaFa® medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using Collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells differentiated into a multilayered epithelial cell sheet that was harvested after 17-19 days in culture.

Immunostaining and Western blot analyses showed that deltaNp63 and Pax-6 were expressed in the basal cells and K3/K12 were expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, such as E- cadherin, beta-catenin and Cnx43, were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of Integrin - betal and beta4 confirmed that the Collagenase treatment used for detaching and harvesting the cell sheet did not have any adverse effects. Thus, this example shows that cG-CAOMECS is a promising translational technology for human application.

Material and Methods

[0106] Animals. New Zealand white rabbits weighing between 2.5 - 3 kg were used. The animals were maintained according to the Guidelines of Animal Care, as described by the National Academy of Sciences and published by the Institute of Laboratory Animal Resources Commission on Life Sciences National Research Council. Buccal tissue biopsy was performed on healthy control rabbits from an existing protocol, approved by the IACUC. The rabbits used for the oral biopsy were deceased from surgical complications (sacrificed for a different experiment). This adopted approach prevents animal purchase and sacrifice, just for securing a small buccal biopsy.

[0107] Oral mucosa buccal biopsy. A buccal biopsy was performed on the oral cavity of the rabbits after disinfection by povidone-iodine. After sanitization of the oral cavity, buccal mucosa biopsy was performed below the occlusal plane in the back of the cheek. The progenitor stem cells are more concentrated in this area. The mucosa was grasped with tissue forceps and tissue piece was removed using dissecting scissors. The biopsied tissue was transported in cell culture basal medium at 4°C from the vivarium to cell culture room for processing.

[0108] Feeder cells preparation. The effect of co-culture with feeder cells was investigated on the generation of CAOMECS by using two type of feeder cells. The two types of feeder cells used for comparison were the Cell line NIH3T3 mouse embryonic fibroblasts (ATCC) and the FibroGRO human fibroblasts (Millipore, St. Louis, MO). Both the type of feeder cells were mitotically inactivated with Mitomycin C. Both the types of feeder cells were plated 24 hours before OMECS seeding.

[0109] Oral mucosal epithelial cells isolation and cell culture. Tissue biopsy was washed in sterile saline, placed in 5% povidone iodine solution for 1 min, washed again in sterile saline, and then in cell culture medium K0DMEMF12. The tissue biopsy was cut into smaller pieces after being cleaned from any excess of fat and muscle tissue. Tissue pieces were incubated with Dispase® I (neutral protease, grade I. Roche Diagnostics GmbH, Mannheim, Germany) for one hour at 37 °C, which allowed the epithelium to be separated from the lamina propria.

[0110] Next, these epithelium pieces were subjected to trypsin digestion to extract and isolate the oral mucosal epithelial cells. Trypsin digestion was inactivated using our newly designed clinical grade KaFa® medium (Table 1). Isolated cells were re-suspended in a small volume of KaFa® medium and were seeded at a density of 0.3-05 10⁵ cells/cm² on GMP certified 6- multiwell plate (6MWP) previously coated with GMP grade extra cellular matrix substrate CELLStart®. The cells were cultured for about 3 weeks at 37°C in a humidified atmosphere containing 5% CO2. Cell Culture medium change was scheduled for every other day in the first week, and every day in the last 2 weeks. After a multilayered cell sheet was observed under the microscope, harvesting was performed using collagenase treatment (0.5 mg/mL final concentration - Collagenase NB-6; Amsbio, Cambridge, MA). Table 1. Composition of the KaFa® medium

[0111] Immunohistochemistry. Harvested cell sheets were fixed using 10% neutral buffered formalin and histologically processed via dehydration to be finally paraffin embedded. Cross sections of cell sheet were cut and tissue sections were stained for morphological analysis. Tissue sections were immunofluorescent stained using DeltaNp63 (Biocare Medical, Concord, CA), Pax-6 (Millipore, St. Louis, MO), PCNA (DAKO, Carpinteria, CA), K3 (ImmuQuest, Cleveland, UK) , K4 and K12 (Santa Cruz Inc., Santa Cruz CA). E-cadherin and beta catenin (BD bioscience, San Jose, CA) to examine the expression levels of junctional complexes.

[0112] Alexa Fluor® 488 and Alexa Fluo® 568 donkey anti-rabbi t/mouse/goat fluorophore conjugated secondary antibodies (Santa Cruz Biotechnology) were used. DAPI or propidium iodide (Invitrogen, Eugene, OR) was used for nuclear staining of the cell nuclei. A Nikon 400 fluorescent microscope was used to analyze the slides. The images were analyzed and processed using Adobe Photoshop CS5.

[0113] Western blot analysis. The harvested cell sheet was homogenized in PBS and the protein concentration measured. Two pg of total protein from sample homogenates were separated by SDS-PAGE gels and transferred to a PVDF membrane (Bio-Rad, Hercules, CA) for one hour in 25mM Tris-HCl (pH=8.3), 192mM Glycine and 20% methanol. Membranes were probed with primary antibodies against deltaNp63, Pax-6, K3, K12, E-cadherin, Beta catenin, Cnx43 (Abeam, Cambridge, MA), and Beta actin (Millipore, St. Louis, MO). The levels of expression of phosphorylated focal adhesion kinase (FAK from Millipore, St. Louis, MO) as well as integrins betal and beta4 (R&D system Minneapolis, MN) were also measured.

[0114] Statistics. Statistics data were calculated using at least three separate set of experiments. Bars represent mean values ± SEM. P values are determined by one-way ANOVA and Student-Newman Keuls for multiple group comparisons (Sigma-Stat softdish, San Francisco, CA). Statistical significance is set at p= or < to 0.05. Bar graphs were shown as Mean+/- SEM, n=12-15.

RESULTS

[0115] Safety of cell culture products remains the most important criterion for translational applications. For human translational studies, the FDA regulations encourage the use of xeno- free cell culture conditions to minimize the risk of transmitting pathogens or causing human immune reactions. A goal of this study was to isolate oral mucosal epithelial cells (OMECS) from a buccal biopsy and expand them under clinical grade condition for use in future translational applications.

[0116] Rabbit oral mucosal epithelial cells were isolated from a biopsy of buccal tissue. The harvested buccal tissue biopsies were transported to a cell culture hood in a tube containing cold sterile basal medium. The tissue was enzymatically dissociated and OMECS were isolated and cultured at 37 °C in 5% CO2 in a high humidity environment to grow a multilayered cell sheet.

[0117] Experiments were conducted to develop the cell culture medium that will fully support the production of cell sheet without feeder cells and animal originated reagents. The reagents of the basal medium were carefully chosen to ensure that each component was certified for clinical application. KaFa® medium composition was formulated and proved efficient in maintaining rabbit OMECS cell growth when used with CELLstart® as the matrix. Cell culture surfaces were also investigated by testing several clinical grade certified cell culture surfaces. Certified GMP grade 6-multiwell plate (6MWP) were pre-coated with extra cellular matrix substrate CELLStart® before OMECS seeding. After OMECS seeding at a density of 4 to 5xl0⁵, cell culture medium was changed every two days during the initial week, and every day henceforth until the cell sheet was completely formed. Live cell imaging and visual morphological analysis were used to examine cell growth, cell morphology and cell sheet formation. It was observed that the cells attached onto the surface and selfassembled into colony forming units (CFUs). These CFUs formed during the first 5 days (FIG. 1A), and a monolayer cell sheet formed in less than 10 days (FIG. IB and C). Cells differentiated and formed a multilayered epithelial cell sheet that was harvested after 17 - 19 days (FOG. 1D-I).

[0118] The harvested cell sheets were processed and paraffin embedded for immunofluorescent staining and analysis. FIG. 2 shows that DeltaNp63 was expressed in the basal cells of cell sheet, indicating the positive expression of progenitor stem cells (FIG. 2A). Pax6, an important transcription factor for regulating the growth and differentiation of limbal and corneal epithelial stem cells was positively expressed. Expression of Pax6 is associated with undifferentiated pluripotent stem cells.

[0119] The cultured cell sheet showed a positive expression of Pax-6, indicating that the cell sheet had most of the self-renewing progenitor stem cells observed in normal corneal epithelium (FIG. 2B). PCNA staining performed also indicated the level of proliferative capacity of the cells and showed that basal and supra-basal cells were positive for PCNA, indicating that cells were entering S phase of cell cycle (FIG. 2C).

[0120] FIG. 2D shows that cell sheet stained positive for K3, a corneal epithelial cellspecific keratin that pairs with K12 to form keratin filaments specific to corneal epithelial cells. These results indicated that KaFa® medium and our cell culture conditions successfully differentiated oral mucosal epithelial cells into corneal epithelial cells. FIG. 2E shows that few apical cells were still positive for K4, an oral mucosal epithelial cell-specific keratin that pairs with KI 3.

[0121] In order to examine the role of cell culture media, this example compared cell sheets grown with KaFa® medium to those grown with AOM (Animal Origin Media), with and without inactivated feeder cells (NIH3T3 and/or HuFFs - Human Foreskin Fibroblasts). Quantitative experiments were also conducted to measure the levels of DeltaNp63, Pax-6 and K3 expression, comparing cell sheet grown with KaFa® medium to those grown with AOM containing FBS (Fetal Bovine Serum) and animal origin reagents. Results showed a similar positive detection of DeltaNp63, Pax-6 in all conditions, indicating the expression of progenitor stem cell markers in the cell sheets. Cell sheets grown without feeder and with xeno-free medium stained positive for DeltaNp63, as well as Pax-6, similarly to cell sheets grown with AOM and feeder, indicating that KaFa® medium perfectly supported the production of the cell sheet. Cell sheets grown on a matrix of CellStart using KaFa® media showed similar levels of expression of progenitor stem cells when compared to cell sheets grown with AOM and feeder cells (FIG. 3A and B). These results confirmed that a comprehensively designed compositionally complete chemically defined cell culture medium can support tissue-like production without the use of feeder cells or fetal bovine serum or any other animal origin supplements.

[0122] The results also showed that cell sheet grown with KaFa® medium similarly expressed K3 as compared to cell sheet grown with AOM and feeder cells. K3 expression was significantly higher in KaFa® cell sheets compared to those in AOM cell sheets (FIG. 3C). K3 positive expression indicated that the produced cell sheet with KaFa® had differentiated into corneal epithelial-like cells. The expression of K12 was higher in KaFa® than in the other conditions but the difference was insignificant.

[0123] Junctional complexes were also investigated for documentation of the epithelial integrity of the produced cell sheets. The levels of E-cadherin, beta catenin, and Cnx43 expression were measured and results showed that E-cadherin was significantly high in cell sheets produced with KaFa® medium compared to those grown on feeder cells or with AOM alone (FIG. 3E). E-cadherin recruits beta-catenin in downstream signaling to promote cell adhesion and epithelial integrity. FIG. 3F shows that total beta-catenin is low in cell sheets grown with KaFa® medium in comparison to cells grown with AOM and feeder cells with no significant difference. However, similar to E-cadherin, Cnx-43 - a gap junction protein - was significantly high in cell sheets grown with KaFa® medium (FIG. 3G) compared to all other conditions, indicating a strong adhesion and integrity of the epithelium-like tissue of the cell sheet.

[0124] Every time AOM was used, the need to use collagenase to detach the cell sheet did not arise. A pre-cut PVDF ring membrane and a plastic spatula to gently manipulate and mechanically detach and lift the cell sheet was utilized. When KaFa® medium was used; it was not possible to manually detach an intact cell sheet from any surface of cell culture. Collagenase treatment had to be used to release the cell sheet from the surface of cell culture dish. The expression levels of extracellular matrix components in cell sheet produced with KaFa® medium were then compared to those in cell sheets produced with AOM.

[0125] Specifically, the effects of collagenase on the extracellular matrix of cell sheets grown with KaFa® medium was investigated. FIG. 4A shows a cell sheet produced with KaFa® medium and treated with collagenase for one hour at 37°C. The edges of the cell sheet were free and detached. Pre-cut PVDF membrane was used to fold over the free edges and gently lift the cell sheet (FIG. 4B). In the final step, the cell sheet was released from the PVDF membrane (FIG. 4C) to be processed for morphology and proteomic analysis.

[0126] The effect of collagenase was investigated by measuring the levels of expression of extra cellular matrix proteins such as integrins. FIG. 4D shows that cell sheet produced with KaFa® medium similarly expressed integrin beta 1 as compared to cell sheet produced without collagenase treatment.

[0127] Harvested cell sheets were stained for phosphorylate focal adhesion molecule (FIG. 4D) and for integrin betal and beta 4 (FIG. 4E and F). Results show that in the cell sheets harvested with collagenase, phosphorylated FAK was detected in the basal side of the cell sheet (FIG. 4D, arrow). FAK phosphorylation indicates that focal adhesion was functional and that integrin beta 1 was recruited/activated via the cytoplasmic membrane for better adhesion with extra cellular matrix molecules such as collagen, fibronectin and laminin. FIG. 4E and FIG. 4F show that both integrin beta 1 and integrin beta 4 were positively expressed on the cell membrane. The semi quantitative measurements showed that both integrins were highly expressed in the cell sheets produced with KaFa® medium and harvested with collagenase, as compared to cell sheets harvested without collagenase (FIG. 4G and H).

[0128] Currently, Limbal stem cell deficiency (LSCD) is treated with donor corneal graft tissue. Oral mucosal epithelial cells (OMEC) have successfully been used as a non-limbal cell source to treat unilateral and bilateral LSCD. However, the animal-derived growth supplements utilized for CAOMECS (Cultured Autologous Oral Mucosal Epithelial Cell Sheet) manufacturing may lead to clinical complications. A goal of this example was to design a clinical grade cell culture medium that completely supports the growth of the cell sheet without any type of feeder cells and without any animal-derived growth supplements. Development of a chemically defined xeno-free cell culture medium reduces the risk of transmitting inherent organism specific carrier diseases and improves the consistency of the results. KaFa® medium was formulated and developed, solely to produce clinical grade corneal epithelial cell sheets, consequently, eliminating inherent biological variability due to the use of feeder cells and animal origin reagents.

[0129] Experiments designed, and tested the effects of clinical grade reagents. Every reagent added in the preparation of KaFa® cell culture medium had to be animal-free and produced under GMP guidelines. Each supplement used in the developed medium had to be certified for clinical application and included the MDF number listed by the FDA to ensure the clinical quality of the produced cell sheet.

[0130] To eliminate the mouse NIH3T3 feeder cells, we tested our newly designed clinical grade KaFa® medium with human foreskin fibroblast (HuFFs) as a feeder cell (data not shown ) for comparison with the previously produced cell sheet with mouse NIH3T3 feeder and animal-origin medium (AOM). The results showed that both feeder cells supported cell sheet growth in a similar way. However, the effect of the type of media used made a major impact on cell sheet growth. Regardless of feeder cell type, cell sheets grown with AOM quickly formed a multilayered graft and were sturdy for easy manipulation.

[0131] Cell sheets produced with KaFa® medium needed more time (24 - 72 hours more) to grow and to form a multilayered cell sheet. This discovery geared our efforts towards further focusing on designing the cell culture medium. Specifically, we needed to supplement the medium with chemically defined FDA/GMP grade substitutes to fetal bovine serum (FBS) that could sustain cell growth, and for CAOMECS to qualify as an autologous graft compliant with FDA regulations for human clinical application. We also investigated each reagents’ suitability with respect to oral mucosal epithelial stem cells to direct them to differentiate towards corneal epithelial cells. Epithelial stem cells intrinsically require a matrix to attach in the absence of feeder cells. We chose CELLstart® matrix for cell attachment as it is GMP certified. The surface of the 6MWP was treated for an hour with CELLstart® coating prior to OMECS seeding. Cells were seeded using KaFa® medium, and the medium was changed every two days during the first week and every day in the following weeks.

[0132] Colony forming units (CFU) formed in the first 3 - 6 days of culture, reflecting the existence of epithelial stem cells and their expansion. Towards the end of week two of cell culture, the CFUs disappeared and integrated in the differentiated multilayered epithelial cell sheet. This result was important as it suggested that the newly designed KaFa® medium was sufficient to support the growth of the cell sheet - cG-CAOMECS. This experiment was successfully reproduced, and the cultured cell sheet was harvested intact. Collagenase was used for cell sheet harvesting. A completely grown cell sheet was harvested and processed for morphology and proteomic analysis. Microscopic imaging analysis showed that the harvested cell sheet grown with KaFa® medium was a multilayered cell sheet similar to cell sheets grown with feeder cells and AOM medium.

[0133] Tissue processing and immunofluorescent staining of cell sheet cross sections confirmed that the cell sheets produced with KaFa® medium were multilayered with at least three layers of epithelial cells. The presence of deltaNp63 positive cells in a corneal graft is essential for a successful clinical outcome for the treatment of ESCD. Similar to limbal stem cells, our cG-CAOMECS showed a great number of nuclei positive for delataNp63 in the basal layer of the cell sheet, reflecting the existence of progenitor stem cells and the proliferative capacity of these cells for future grafting onto the Bowman’ s membrane of denuded corneas. Pax-6 and PCNA staining were also used to examine the proliferative capacity of cG-CAOMECS. Pax-6 is a well-known transcription factor that plays a central role in eye development; its expression in proliferated cells re-epithelizes and restores corneal epithelium. Morphological analysis also showed a positive expression of corneal epithelial biomarkers keratin 3 (K3) reflecting that the cultured OMECS differentiated into corneal epithelial cells. The designed cell culture conditions proved to be adequate for differentiation of OMECS into corneal epithelial progenitor cells.

[0134] The persistency of oral mucosal epithelial keratin pattern was documented using K4 staining. Results show a positive staining in the apical squamous mature cells. It is possible that after grafting back onto cornea and after a period of time; the K4 positive cells will shed off the surface. Similar to conjunctival epithelial cells, oral mucosal epithelia cells are characterized by the expression of keratins K4 and KI 3. The effects of corneal injuries on the expression of these keratins as well as the effects of clinical grade cell culture conditions are yet to be determined in the preclinical model of LSCD.

[0135] Proteomic semi-quantitative analysis was conducted to compare cG-CAOMECS to CAOMECS produced with AOM and feeder cells. Results showed that cG-CAOMECS expressed similar levels with no significant differences of epithelial progenitor stem cells (deltaNp63 and Pax-6) and corneal marker K3/K12 when compared to CAOMECS grown with feeder cells. Furthermore, cG-CAOMECS expressed significantly high levels of K3 when compared to CAOMECS grown with only AOM and no feeder cells, validating the efficacy of our newly designed cell culture conditions.

[0136] In addition, the expression of E-cadherin was significantly decreased in cell sheet produced with AOM and no feeder, while E-cadherin expression in cG-CAOMECS was similar to cell sheets grown with feeder cells and AOM. Total beta-catenin expression decreased in cG-CAOMECS, which may reflect a low level of beta-catenin translocation to the nucleus for canonical WNT/TCF activation and low levels of pro-survival gene expression. The expression levels of Cnx43, a gap junction protein was significantly greater in cG-CAOMECS, which corroborated the results obtained from E-cadherin and beta-catenin analysis.

[0137] cG-CAOMECS produced with KaFa® medium is a multilayered epithelial cell sheet that expressed epithelial markers required for normal corneal epithelial barrier function. Results indicate that KaFa® medium with GMP grade extracellular matrix replaces and exceeds the efficiency of feeder cells and AOM supplements in producing cG-CAOMECS.

[0138] This example shows that the combination of the newly designed clinical grade cell culture conditions and KaFa® medium resulted in the successful production of cG- CAOMECS, which in the clinical setting, using autologous human cells shall qualify as an efficient and safe clinically feasible tissue suitable for grafting back onto an LSCD patient’s corneal surface.

* * *

[0139] The present disclosure is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the disclosure, and any compositions or methods which are functionally equivalent are within the scope of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made in the methods and compositions of the present disclosure without departing from the spirit or scope of the disclosure. Thus, it is intended that the present disclosure cover the modifications and variations of this disclosure provided they come within the scope of the appended claims and their equivalents. [0140] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

CLAIMS What is Claimed is:

1. A method for preparing a multilayered epithelial cell sheet, comprising culturing a plurality of epithelial progenitor cells on a surface coated with an extracellular matrix substrate, and in a medium not containing animal feeder cells, wherein the medium is a basal medium supplemented with non-animal serum replacement, albumin, and isoproterenol.

2. The method of claim 1, wherein the epithelial stem cells comprise oral mucosal epithelial cells.

3. The method of claim 2, wherein the oral mucosal epithelial cells have been isolated with trypsin.

4. The method of any one of claims 1-3, wherein the multilayered epithelial cell sheet comprises corneal epithelial cells.

5. The method of any one of claims 1-4, wherein the basal medium is Dulbecco’s Modified Eagle Medium (DMEM).

6. The method of any one of claims 1-5, wherein the medium comprises DMEM supplemented with 4-15% (v/v) non-animal serum replacement, 0.1-1% (w/v) albumin, and 0.1-2 pg/mL isoproterenol.

7. The method of any one of claims 1-6, wherein the medium further comprises a Rho- associated protein kinase (ROCK) inhibitor, epithelial growth factor (EGF) and keratinocyte growth factor (KGF).

8. The method of claim 7, wherein the medium comprises 2-50 pM of the ROCK inhibitor, 1-100 ng/mL EGF and 2-50 ng/mL KGF.

9. The method of any one of claims 1-7, wherein the medium is changed every 1, 2 or 3 days.

10. The method of any one of claims 1-9, wherein the culturing is carried out for 5-30 days.

11. The method of any one of claims 1-10, further comprising detaching the multilayered epithelial cell sheet from the coated surface with collagenase.

12. A multilayered epithelial cell sheet prepared by the method of any one of claims 1-11.

13. The multilayered epithelial cell sheet of claim 12, which is a corneal epithelium.

14. A method for reconstructing a corneal in a patient in need thereof, comprising implanting the corneal epithelium of claim 13 to the patient.

15. The method of claim 14, wherein the patient suffers limbal stem cell deficiency (LSCD).

16. A culture medium, comprising a basal culture medium, non-animal serum replacement, albumin, and isoproterenol.

17. The culture medium of claim 16, wherein the basal culture medium is Dulbecco’s Modified Eagle Medium (DMEM).

18. The culture medium of claim 17, wherein the medium comprises DMEM supplemented with 4-15% (v/v) non-animal serum replacement, 0.1-1% (w/v) albumin, and 0.1-2 pg/mL isoproterenol.

19. The culture medium of any one of claims 16-18, further comprising a Rho-associated protein kinase (ROCK) inhibitor, epithelial growth factor (EGF) and keratinocyte growth factor (KGF).

20. The culture medium of claim 19, wherein the medium comprises 2-50 pM of the ROCK inhibitor, 1-100 ng/mL EGF and 2-50 ng/mL KGF.