Methods of producing constructs for tissue reconstruction

Abstract

Provided herein according to some aspects is a method of culture for expanding smooth muscle cells, said method comprising: growing said smooth muscle cells in a medium, said medium comprising: amino acids, vitamins, and inorganic salts; wherein said amino acids comprise two or more selected from the group consisting of: L-Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic acid, and L-Proline. The expanded smooth muscle cells may be used in forming a seeded scaffold for tissue reconstruction.

Description

[0001] METHODS OF PRODUCING CONSTRUCTS FOR TISSUE RECONSTRUCTION

[0002] Related Applications

[0003] This application claims the benefit of United States Provisional Patent Application Serial No. 63 / 583,935, filed September 20, 2023, the disclosure of which is incorporated by reference herein in its entirety.

[0004] Field

[0005] The present invention is generally related to methods of producing constructs for tissue reconstruction and cell culture methods useful for the same.

[0006] Background

[0007] Vaginal reconstruction improves the quality of life in women with primary congenital vaginal abnormalities or women who experience secondary conditions. Such abnormalities and conditions include, e.g., cloacal exstrophy, congenital mullerian duct anomaly, intersex disorders, cancer, trauma, infection, inflammation, and iatrogenic injuries that lead to vaginal tissue damage or loss. Current methodologies in vaginal reconstruction often use non-vaginal tissue substitutes. These tissue substitutes, including segments of the large intestine or skin, are not ideal because they do not adequately replicate vaginal histology.

[0008] Atlantida M Raya-Rivera et al., Tissue-engineered autologous vaginal organs in patients: a pilot cohort study, Lancet 384:329-36 (2014), reports on a pilot study in which vaginal constructs were created from autologous epithelial cells and smooth muscle cells.

[0009] While the autologous vaginal constructs reduce the potential for immunological rejection and more accurately replicate the vaginal histology without severe complications, the methods thus far are limited in reproducibility and robustness. Accordingly, improved methods of isolating, culturing, and seeding autologous cells are needed, without sacrificing the benefits of the resulting constructs. Summary

[0010] Provided herein according to some aspects is a method of culture for expanding smooth muscle cells (SMC), said method comprising: growing said smooth muscle cells in a medium, said medium comprising: a) amino acids, b) vitamins, and c) inorganic salts; wherein said amino acids comprise two or more selected from the group consisting of: L- Alanine, L- Asparagine, L-Aspartic acid, L-Glutamic acid, and L-Proline.

[0011] In some aspects, the vitamins comprise Vitamin B12 and / or d-Biotin.

[0012] In some aspects, the medium further comprises a nucleobase or a nucleoside (e.g., Adenine, Thymidine).

[0013] In some aspects, the medium further comprises an antioxidant (e.g., DL-alpha Lipoic Acid).

[0014] In some aspects, the medium further comprises a buffer (e.g., HEPES).

[0015] In some aspects, the medium further comprises a polyamine (e.g., Putrescine).

[0016] In some aspects, the medium further comprises one or more selected from the group consisting of: fetal bovine serum, a growth factor, an anticoagulant, an albumin, and an antibiotic. In some aspects, the medium comprises human albumin.

[0017] In some aspects, the medium comprises one or more growth factors selected from the group consisting of: basic fibroblast growth factor, epidermal growth factor, and insulin-like growth factor 1.

[0018] In some aspects, the smooth muscle cells are provided by enzymatic digestion of a biopsy of a subject (e.g., a human subject).

[0019] In some aspects, the enzymatic digestion comprises a protease (e.g., BP Protease, Collagenase HA, trypLE, or a combination thereof).

[0020] In some aspects, the growing comprises passaging said smooth muscle cells in vitro (e.g., from 2 or 3 to 5 or 6 passages).

[0021] In some aspects, the method further comprises seeding the smooth muscle cells onto a scaffold material, to form a seeded scaffold. In some aspects, the scaffold material comprises a collagen -based extracellular matrix (e.g., small intestinal submucosa). In some aspects, the extracellular matrix comprises a decellularized tissue.

[0022] In some aspects, the method further comprises seeding epithelial cells onto an opposite side of said scaffold material.

[0023] In some aspects, the epithelial cells are seeded onto the scaffold material prior to the seeding of the smooth muscle cells (e.g., from 1 or 2 to 3, 4 or 5 days prior to the SMC seeding).

[0024] In some aspects, the epithelial cells are provided by enzymatic digestion of a biopsy of a subject (e.g., a human subject, optionally the same human subject of the smooth muscle cells).

[0025] In some aspects, the seeded scaffold is implantable in vivo. Also provided in the use of a seeded scaffold as taught herein for tissue reconstruction in a subject in need thereof.

[0026] In some aspects, the seeded scaffold comprises a vaginal construct, and said method further comprises forming the seeded scaffold over a vaginal stent. Also provided is the use of a vaginal construct as described herein for vaginal reconstruction. In some aspects, the vaginal construct has Mucinl (MUC1) secretion.

[0027] Brief Description of the Drawings

[0028] FIG. 1A and FIG. IB show representative images of SMC cultures. FIG. 1 A is the culture of Sample ID TEV-004, SMC PO, 10 days after isolation cultured in DMEM (left panel) or M200 media (right panel). FIG. IB is the culture of Sample ID TEV-005, SMC PO, 10 days after isolation cultured in DMEM (left panel) or M200 media (right panel).

[0029] FIG. 2 presents a graph of doubling times of SMC in the DMEM versus M200 culture media. P-value = 0.005, which is considered significant.

[0030] Detailed Description of Embodiments

[0031] Embodiments of the present invention are now described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0032] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0033] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0034] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0035] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a construct comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

[0036] As used herein, the transitional phrase "consisting essentially of' (and grammatical variants) is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0037] The term "about," as used herein when referring to a measurable value such as an amount or concentration and the like, is meant to encompass variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified value as well as the specified value. For example, "about X" where X is the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value includes any range and / or individual value therein, which variations are encompassed by the instant disclosure. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0038] A "medium" or "media," sometimes referred to as a "culture medium" or "culture media," as used herein, refers to an aqueous based solution that is provided for the growth, viability, and / or maintenance of cells used in carrying out the present invention. A media or culture media may be natural or artificial. A media or culture media may include a base media and may be supplemented with nutrients or other components (e.g., one or more salts, amino acids, vitamins, trace elements, antioxidants, nucleobases, nucleosides, polyamines) to promote the desired cellular activity, such as cell viability, growth, proliferation, and / or differentiation of the cells cultured in the media.

[0039] A "base media," or "basal media" as used herein, refers to a basal salt nutrient or an aqueous solution of salts and other elements that provides cells with water and inorganic ions that are needed for normal cell metabolism and maintaining intracellular and / or extracellular osmotic balance. In some embodiments, a base media may include a buffering system to maintain the medium within a physiological pH range of about 7 to about 8, or a pH range of about 7.2 to about 7.8, or a pH of about 7.3 to about 7.5. Known base media include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), and Medium 200 (also known as Human Large Vessel Endothelial Cell Basal Medium).

[0040] A "complete media" as used herein, refers to a base media supplemented with nutrients and / or other components such as antimicrobial agents.

[0041] As used herein, "growth factors" include molecules that promote the regeneration, growth and survival of cells or tissue. Examples of growth factors include, but are not limited to, basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and insulin-like growth factor 1 (IGF-1).

[0042] In some embodiments, the complete media includes serum. Serum may support the growth of cells by providing nutrition, hormones, growth factors and / or attachment factors. Examples of serum include, but are not limited to, fetal bovine serum, bovine serum, chicken serum, goat serum, horse serum, sheep serum, newborn calf serum, porcine serum, and rabbit serum. In other embodiments, the complete media is serum free. Examples of components that may be included in a complete media include, but are not limited to, amino acids, vitamins, inorganic salts, buffers, antioxidants, antibiotics, and other additives.

[0043] "Amino acids" include, for example, Glycine, L-Alanine, L-Arginine, L-Asparagine, L- Aspartic acid, L-Cysteine, L-Glutamic acid, L-Glutamine, L-Histidine, L-Isoleucine, L-Leucine, L-Lysine, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L- Tyrosine, and L-Valine.

[0044] "Vitamins" include, for example, vitamin B12, d-biotin, choline chloride, D-pantothenic acid, folinic acid, myo-inositol, niacinamide, pyridoxal hydrochloride, and thiamine hydrochloride.

[0045] "Inorganic salts" include, but are not limited to, ammonium metavanadate, ammonium molybdate, calcium chloride, cupric sulfate, ferric sulfate, magnesium sulfate, manganese sulfate, nickelous chloride, potassium phosphate, sodium bicarbonate, sodium chloride, sodium meta silicate, sodium selenite, tin chloride, and zinc sulfate.

[0046] "Buffers" may be used in cell culture to maintain the pH of the media in the presence of about 4-10% carbon dioxide. Buffers include, for example, HEPES, sodium bicarbonate, MOPS, MES, tricine, etc.

[0047] As used herein, the term "antioxidant" refers to those compounds capable of scavenging free radicals (e.g., reactive oxygen species (ROS) or free oxygen radicals such as superoxide and hydroxyl radicals), and those compounds that inactivate antioxidant enzymes (e.g., SOD, catalase) under physiological conditions. Antioxidants include naturally-occurring as well as synthetic antioxidants, and further encompass both water-soluble and lipid-soluble antioxidants. Antioxidants include, but are not limited to, the tocopherols (especially a -tocopherol, or vitamin E), ascorbic acid (vitamin C), ascorbyl palmitate, vitamin A and the carotenes (including betacarotene), glutathione and its precursors, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), isopropyl citrate, riboflavin, manganese, selenium, zinc, uric acid, bilirubin, the isoflavones, flavonoids, lycopene, and ubiquinol; and the chemical derivatives, analogs, and precursors thereof.

[0048] "Antibiotics" may be used in cell culture media for preventing contamination, maintaining aseptic conditions, and / or selecting for cells containing genetic modifications. Antibiotics include, for example, penicillin, streptomycin, Amphotericin B, gentamicin, puromycin, etc. Other media additives may include, but are not limited to, a nucleobase or a nucleoside (e.g., Adenine, Thymidine), a polyamine (e.g., Putrescine), albumin (e.g., human albumin), an anticoagulant (e.g., heparin), etc.

[0049] "Cell" or "cells" as used herein are, in general, mammalian cells, such as dog, cat, cow, goat, horse, sheep, mouse, rabbit, rat, etc. cells. In some preferred embodiments the cells are human cells. "Isolated" as used herein signifies that the cells are placed into conditions other than their natural environment. Tissue or cells are "harvested" when initially isolated from a subject, e.g., a biopsy. Cells are "autologous" when initially isolated from a genetically identical donor. For example, autologous cells may be extracted from a patient and returned back to the same, genetically identical patient.

[0050] In some embodiments, harvested cells are disaggregated by enzymatic digestion before the primary culture, which may include incubation with a protease such as trypsin or trypLE; dispase, Dispase II, or BP Protease; a collagenase such as Collagenase HA, or a combination thereof.

[0051] "Cell culture" or "culture" is the growth or proliferation of cells in vitro. The "primary culture" is the first culture to become established after seeding disaggregated cells or primary explants into a culture vessel. "Expanding" or "expansion" as used herein refers to an increase in number of viable cells. Expanding may be accomplished by, e.g., "growing" the cells through one or more cell cycles, wherein at least a portion of the cells divide to produce additional cells. "Growing" as used herein includes the culture of cells such that the cells remain viable, and may or may not include expansion and / or differentiation of the cells.

[0052] "Passaged in vitro" or "passaged" refers to the transfer or subculture of a cell culture to a second culture vessel, usually implying mechanical or enzymatic disaggregation, reseeding, and often division into two or more daughter cultures, depending upon the rate of proliferation. A cell culture may be referred to reflecting the number of times it has been passaged (e.g., Pl for one passage). In some embodiments, the cells are passaged from 1 to 9 times, such as from 2 or 3 to 4, 5, 6 or 7 passages. If a cell population is selected for a particular genotype or phenotype, then the culture becomes a "cell strain" upon subculture, i.e., the culture is homogeneous and possesses desirable characteristics (e.g., the ability to express a certain protein or marker).

[0053] "Smooth muscle cells" or "SMC" are differentiated cells normally found in the walls of hollow organs, such as the intestines, bladder, uterus and stomach, in the walls of passageways, such as blood and lymph vessels, and in the tracts of the respiratory, urinary, and reproductive systems. SMC are typically spindle-shaped and have a single nucleus. The major function of SMC is contraction, which occurs due to being largely composed of filaments, such as actin, tropomyosin, and myosin-containing filaments. SMC are also capable of synthesizing extracellular matrix.

[0054] "Epithelial cells" or "EPC" are differentiated cells found on the surfaces of the body (e.g., skin), organs, and blood vessels, as well as on the lining of cavities of major organs, such as the lungs, gastrointestinal tract, and genitourinary system. EPC are tightly bound together via various cellular arrangements, e.g., squamous, cuboidal, and columnar. Their cellular function changes based on their location within the body and the type of cellular arrangement of the epithelial tissue. For example, EPC on the skin surface provide protection, whereas EPC lining the gut have secretory and absorptive properties.

[0055] "Scaffolds" on which cells may be seeded and grown to produce cultured tissue include any suitable support. See, e.g., U.S. Patent Nos. 6,998,418; 6,485,723; 6,206,931; 6,051,750; and 5,573,784. Preferably, the scaffold is configured to support the attachment, proliferation and / or differentiation of cells thereon.

[0056] The scaffold may be formed from any suitable material, including, but not limited to, synthetic or natural polymers, other biopolymers, and combinations thereof. In some embodiments, scaffolds include collagen supports or decellularized tissue supports. In some embodiments, scaffolds include a polymeric matrix (e.g., collagen, a hydrogel, etc.). In some embodiments, scaffolds include fibrin or fibrinogen.

[0057] Biodegradable scaffolds that may be used include, e.g., natural or synthetic polymers, such as collagen (e.g., acellular extracellular matrix from human or porcine dermis or from porcine intestine or bladder submucosa), poly(alpha esters) such as poly(lactate acid), poly(glycolic acid), polyorthoesters and polyanhydrides and their copolymers, which can be degraded by hydrolysis at a controlled rate and are reabsorbed. Examples of other suitable materials are provided in U.S. Patent No. 7,186,554.

[0058] In some embodiments, the scaffold has epithelial and / or smooth muscle cells seeded thereon. "Seeding" is the placement of cells to be seeded onto or into a scaffold (e.g., the matrix of a scaffold) intended for the cells to attach and / or infiltrate. In some embodiments, the cells are mixed in media and then applied directly onto the scaffold. A "seeded scaffold" comprises a scaffold and cells that are attached and / or infiltrated onto and / or into the scaffold. In some embodiments, a seeded scaffold is implanted in the body of the recipient subject in need thereof, whereby the seeded cell populations on the scaffold facilitate the formation of functional tissue structures. In some embodiments, the seeded scaffold is formed over a stent (e.g., a vaginal stent) and / or implanted along with a stent. In some embodiments, the functional tissue structure has MUC1 secretion.

[0059] "Implant" refers to a product configured to repair, augment or replace (at least a portion of) a natural tissue of a subject (e.g., for veterinary or medical (human) applications). The term "implantable" means the device can be inserted, embedded, grafted or otherwise chronically attached or placed on or in a subject or patient.

[0060] "Subjects" are generally human subjects and include, but are not limited to, "patients." Subjects also include animal subjects, particularly mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, non-human primates, etc., for, e.g., veterinary medicine, laboratory research and / or pharmaceutical drug development purposes.

[0061] EXAMPLES

[0062] Example 1: Isolation of Smooth Muscle Cells

[0063] Tissue from a vaginal biopsy was disinfected by incubating in a series of antibiotic washes and gentamicin washes. The washed biopsy tissue was then cut into four pieces to test four methods for isolating vaginal smooth muscle cells: 1) Explant method; 2) Collagenase II, 30 minutes; 3) Collagenase HA + Protease, 30 minutes; and 4) Collagenase II, 24 hours. For methods 3 and 4, biopsy pieces were incubated in Dispase solution at 4°C for 24 hours before proceeding with the enzymatic digestion. Next, the muscular layer and the epithelial layer of each biopsy piece were separated using scissors and forceps. Each tissue portion was then subjected to the assigned isolation method.

[0064] For method 1, the muscular layer was cut into small pieces (Ixlxl mm3), and the small pieces were placed, spread out, in a petri dish. The pieces were placed in an incubator to dry, and once dry, DMEM complete media (DMEM-Low Glucose, 10% FBS, 2 pM L-Glutamate, 5 pg / mL Gentamicin) was added to the petri dish. The dish was incubated as explant culture for 72 hours at 37°C. The cells were then lifted by enzymatic disassociation. For methods 2-4, the tissue portion was incubated in the appropriate solution for the given amount of time at 37°C (method 2: Collagenase II for 30 minutes, method 3: Collagenase HA + Protease for 30 minutes, method 4: Collagenase II for 24 hours). The solutions were then neutralized and centrifuged. The resulting cell pellets were resuspended in DMEM complete media.

[0065] The isolated cells from each method were then seeded into culture vessels, cultured in DMEM complete media, and evaluated for viability and cell morphology. The results are shown in Table 1, below. Table 1. Summary of SMC isolation and culture

[0066] Enzymatic digestion with Collagenase HA and BP protease was determined to be the most effective method for isolating smooth muscle cells from vaginal biopsy tissue. The cells isolated with this method retained the morphology of smooth muscle cells (long, spindle shaped with one nucleus). In addition, cells isolated with Collagenase HA and BP protease had significantly higher viability and survived more passages than the cells isolated with Collagenase II. Further testing determined that there was no difference between pre-treatment of the tissue with Dispase, as required for epithelial cell isolation, and no pre-treatment.

[0067] Example 2: Media for Smooth Muscle Cell Culture

[0068] Two media were tested for efficacy in expanding smooth muscle cells: DMEM complete media (DMEM-Low Glucose, 10% FBS, 2 pM L-Glutamate, 5 pg / mL Gentamicin) and SMC complete media (Medium 200, lx Gibco Smooth Muscle Growth Supplement, 5 pg / mL Gentamicin).

[0069] Isolated smooth muscle cells from vaginal biopsy tissue were split into two groups: one was cultured with DMEM complete media, and the other group was cultured with SMC complete media. Each group was cultured at standard culture conditions (37°C, 5% CO2). Cells were observed at 72 hours after seeding and were passaged each time they reached 80% confluence. At each passage, the number of cells, cell viability, and cell size were recorded. Results are shown in Table 2. Doubling time calculations, performed using Formula 1, are shown in Table 3.

[0070] Formula 1: Doubling time = duration (hours) x log 2 log (Final Concentration) - log (Initial Concentration)

[0071] Table 2. Cell Culture Summary

[0072] Table 2- summary of culture information in the two SMC growth media

[0073] Table 3. Doubling Time As reflected in the data above, and also shown in FIG. 1A,B and FIG. 2, using SMC complete media significantly decreased the doubling time for the smooth muscle cells (about 1 day vs. about 1 week with DMEM complete media). In addition, it supported greater survival and improved cell morphology. Cells grown in SMC complete media maintained the SMC phenotype

[0074] (long, thin cells), whereas cells grown in DMEM complete media failed to proliferate and showed signs of senescence. Example 3: Albumin

[0075] Supplements were investigated for their ability to support cell growth and proliferation of vaginal derived SMC for use in tissue engineered vagina construct seeding. Medium 200 was supplemented to compare a) Control Media: Gibco™ Smooth Muscle Growth Supplement (SMGS), b) Media 1: FBS, bFGF, EGF, IGF-1, heparin, and pharmaceutical grade Human Albumin (HA), and c) Media 2: FBS, bFGF, EGF, IGF-1, heparin, and Bovine Serum Albumin (BSA). All three media had 5 pg / mL gentamicin. For b) and c), FBS was added to a final concentration of 4.9%; heparin to 5 ng / mL, hbFGF to 2 ng / mL, hEGF to 0.5 ng / mL, IGF-1 to 0.01 pg / mL, and albumin to 0.2 pg / mL. Equal numbers of cells were cultured in the different growth media for three passages. The number of viable cells, cell viability, and cell size were recorded. Results are shown in Table 4. Doubling time calculations, performed using Formula 2, are shown in Table 5.

[0076] Formula 2: Doubling time = _ culture duration (hours) x log 2 _ log (Final Cell count) - log (Initial Cell Count)

[0077] Table 4. Cell Culture Summary Table 5. Average Doubling Time of Cultured SMC

[0078] Once the culture had expanded to 4.6-5 x 107cells, the cells were seeded on a small intestine submucosa (SIS) scaffold. The SIS was incubated for one hour in the corresponding media prior to being seeded with cells at a density of IxlO6cells / cm2. Each seeded SIS construct was cultured in the corresponding SMC media group for three days. Then, epithelial cells were seeded on the other side of each SIS. The constructs were cultured for four more days to mature using 1 :1 EPC media: SMC media. The viability of the SMC on matured constructs was evaluated using a Live / Dead assay. Metabolic activity of the SMC was measured using a PrestoBlue assay.

[0079] The viability and size of the SMC cultured in all three SMC growth media were not significantly different. In addition, the immunophenotype was also comparable between SMC grown in the different media as shown with flow cytometry. The SMC on the matured constructs also did not have a significant difference in cell viability or metabolic activity. Thus, it was found that using human albumin or bovine serum albumin in Media 1 or Media 2 does not have a negative effect on SMC growth and construct maturation compared to SMGS.

[0080] Example 4: Seeding the scaffold

[0081] The order of cell seeding was investigated. Cultures of vaginal epithelial cells (EPC) and smooth muscle cells (SMC) were expanded to 7.0 x 107cells. Cells were seeded by enzymatically disassociating the cells from the culture flasks, counting the cells, then seeding them onto one side of an SIS scaffold with a seeding density of 0.25-0.5 x 106 / cm2. The seeded constructs were incubated under standard conditions (37°C, 5% CO2) without culture media for two hours to allow for cell attachment to the scaffolds. At the end of the cell attachment period, complete media for the respective cell type was added to each dish, and the culture was placed back into the incubator. The media was changed after 24 hours. Following the seeding of the second cell type, complete mix media was added. Complete Mix media was prepared by combining the complete media for each cell type with a 1 : 1 ratio. For method 1 , SMC was seeded first on the non-luminal side of a SIS scaffold and cultured for three days. The, EPC were seeded on the other, luminal side of the same scaffold with the same cell seeding density. For method 2, EPC were seeded first on the non-luminal side of the SIS scaffold and cultured for three days, then the SMC were seeded on the other, luminal side of the same scaffold with the same cell seeding density. For both methods, the entire constructs were matured in culture for a total of 7 days. On day 7, the constructs’ viability was evaluated by Live / Dead assay. In addition, epithelial secretory protein mucin 1 (MUC1) levels were measured by ELISA. MUC l is a glycoprotein secreted by epithelial cells that can be correlated to the number of functional EPC.

[0082] Results of the Live / Dead assay showed the presence of live EPC and SMC on both sides of the constructs matured following both methods. While the number of SMC on the mature constructs from both methods look similar, the number of EPC on mature constructs from method 2 seemed to be much higher than from method 1.

[0083] The ELISA results showed that the level of secretory MUC1 is significantly higher when the mature construct from method 2 (P-value = 0.04).

[0084] The results show that seeding EPC first, followed by seeding SMC three days later, provides improved EPC attachment and growth on the final construct, while not adversely affecting SMC attachment and growth.

[0085] Example 5: Forming the construct

[0086] Mature seeded SIS is be washed with Hank’s Balanced Salt Solution (HBSS) and transported under sterile conditions to the clinical site. The construct is formed according to the Mclndoe procedure as described in Urogynecology and Reconstructive Pelvic Surgery, Walters and Karram (2021). The seeded SIS are formed into the correct shape using a cylindrical vaginal stent (Allura) as the mold. The standard Mclndoe procedure is modified by using two pieces (about 10 cm x 7 cm each) of the seeded SIS to form the sides of the construct, and a smaller piece of seeded SIS (about 7 cm x 7 cm) is used to form the top of the construct (instead of a typical skin graft).

[0087] The level of MUC 1 secretion on the maturing construct may be measured to show the expansion and functionality of epithelial cells on the construct. The measuring may be performed, for example, with a clinical MUC1 ELIZA kit (Catalog # EHMUC1, Thermofisher Scientific). The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

That Which is Claimed Is:

1. A method of culture for expanding smooth muscle cells, said method comprising: growing said smooth muscle cells in a medium, said medium comprising: a) amino acids, b) vitamins, and c) inorganic salts; wherein said amino acids comprise two or more selected from the group consisting of: L- Alanine, L-Asparagine, L-Aspartic acid, L-Glutamic acid, and L-Proline.

2. The method of claim 1, wherein said vitamins comprise Vitamin B12 and / or d- Biotin.

3. The method of claim 1 or 2, wherein said medium further comprises a nucleobase or a nucleoside (e.g., Adenine, Thymidine).

4. The method of any one of claims 1-3, wherein said medium further comprises an antioxidant (e.g., DL-alpha Lipoic Acid).

5. The method of any one of claims 1-4, wherein said medium further comprises a buffer (e.g., HEPES).

6. The method of any one of claims 1-5, wherein said medium further comprises a polyamine (e.g., Putrescine).

7. The method of any one of the preceding claims, wherein said medium further comprises one or more selected from the group consisting of: fetal bovine serum, a growth factor, an anticoagulant, an albumin, and an antibiotic.

8. The method of claim 7, wherein said albumin comprises human albumin.

9. The method of claim 7 or 8, wherein said growth factors comprise one or more selected from the group consisting of: basic fibroblast growth factor, epidermal growth factor, and insulin-like growth factor 1.

10. The method of any one of the preceding claims, wherein said smooth muscle cells are provided by enzymatic digestion of a biopsy from a subject (e.g., a human subject).

11. The method of claim 10, wherein the enzymatic digestion comprises a protease (e.g., BP Protease, Collagenase HA, trypLE, or a combination thereof).

12. The method of any one of the preceding claims, wherein said growing comprises passaging said smooth muscle cells in vitro (e.g., from 2 or 3 to 5 or 6 passages).

13. The method of any one of the preceding claims, further comprising seeding the smooth muscle cells onto a scaffold material to form a seeded scaffold.

14. The method of claim 13, wherein said scaffold material comprises a collagen- based extracellular matrix (e.g., small intestinal submucosa).

15. The method of claim 14, wherein said extracellular matrix comprises a decellularized tissue.

16. The method of any one of claims 13-15, further comprising seeding epithelial cells onto an opposite side of said scaffold material.

17. The method of claim 16, wherein said epithelial cells are seeded onto the scaffold material prior to the seeding of the smooth muscle cells (e.g., from 1 or 2 to 3, 4 or 5 days prior to the smooth muscle cell seeding).

18. The method of claim 16 or claim 17, wherein said epithelial cells are provided by enzymatic digestion of a biopsy of a subject (e.g., a human subject, optionally the same human subject of the smooth muscle cells).

19. The method of any one of claims 13-18, wherein the seeded scaffold is implantable in vivo.

20. The method of claim 19, wherein said seeded scaffold comprises a vaginal construct, and said method further comprises forming the seeded scaffold over a vaginal stent.

21. The method of claim 20, wherein said vaginal construct has MUC1 expression.

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