Cultured leather scaffold

Abstract

This invention relates to the field of cell culture. In particular, this invention relates to the culture of cells such as fibroblasts for obtaining collagen sheets, and to collagen sheets obtained by such culture. This invention also relates to leather obtained by tanning such collagen sheets.

Description

[Technical Field]

[0001] This invention relates to the field of cell culture. In particular, this invention relates to cells such as fibroblasts for obtaining collagen sheets, and to collagen sheets obtained by such culture. This invention also relates to leather obtained by tanning such collagen sheets. [Background technology]

[0002] Leather is used in many applications, including clothing, shoes, fashion accessories, upholstery, bags, and automotive applications. It is generally made from animal hides. Historically, these hides were taken from animals hunted for sustenance, and leather was an easily accessible source of a highly durable and functional fabric. With the advent of plastic, leather is now seen as a luxury item, primarily used for fashion and a sense of luxury. Current sourcing of leather can be undesirable for both sustainability and ethical reasons. For example, the carbon footprint of cowhide can be considerably greater than that of synthetic fabrics. Furthermore, traditional leather production requires the killing of animals, which can be considered ethically undesirable.

[0003] Cultured leather is known in the art. It relies on cell and tissue engineering techniques to create tissue similar to the subepidermal skin (dermis). International Publication No. 2017193058 describes a method for producing cultured leather, comprising culturing multiple layers, each layer containing cells, and each layer of the multiple layers being bonded to adjacent layers by nucleotide adhesion molecules. This requires special techniques to provide layers of cellular material of sufficient thickness.

[0004] Jakab et al. (Materials Today Sustainability, 5 (2019) 100018, DOI: 10.1016 / j.mtsust.2019.100018) describes the in vitro production of collagen sheets for tanning. For this production, it is necessary to seed the next cell layer on top of the previous layer. This requires repeated seeding of cells in order to obtain a collagen sheet of the desired thickness without controlling the direction or boundary of cell growth. Similarly, WO 2014 / 201406 pamphlet requires stacking multiple layers of cells in successive rounds of culture.

[0005] EP 1589098 describes the use of collagen materials as scaffolds for cell culture. The use of waste as a scaffold having a related risk of contamination is described. Other sources of collagen are animals in nature, which can reduce the cost-effectiveness of the process. SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0006] There is a need for an improved method for providing a collagen sheet, particularly a collagen sheet suitable for tanning. An improved in vitro leather is also needed. There is a need for an improved collagen sheet with controlled properties. There is a need for an improved scaffold used in cell culture, particularly for the production of collagen sheets. MEANS FOR SOLVING THE PROBLEMS

[0007] The present invention provides a method for providing a collagen sheet, comprising: i) providing a glass fiber sheet containing solid fibers; ii) placing the glass fiber sheet in a bioreactor; iii) seeding fibroblasts on the glass fiber sheet in the bioreactor; iv) culturing the fibroblasts under conditions conducive to collagen production to form a collagen sheet; optionally v) isolating the collagen sheet. Step i) preferably includes autoclaving the glass fiber sheet. The fibroblasts are preferably skin fibroblasts, more preferably bovine skin fibroblasts. In a preferred embodiment, in step ii), a plurality of glass fiber sheets are placed in the bioreactor to form a multi-layer scaffold. Preferably, the glass fibers have an average diameter of about 1 to about 25 μm, preferably about 5 to about 15 μm, and / or the fiber density in the glass fiber sheet is about 1 to about 10 mg / cm 2 2, preferably about 1.5 to about 8 mg / cm 2 ; and / or the gap size between the fiber densities in the glass fiber sheet is about 10 to about 140 μm, preferably about 25 to about 125 μm; and / or the glass fiber sheet has a thickness of about 20 to 1000 μm.

[0008] In that method, in step iii), about 1×10 2 to about 1×10 4 fibroblasts per 1 cm 8 of the glass fiber sheet, preferably about 1×10 5 to about 1×10 7 fibroblasts / cm 2 , more preferably about 5×10 5 to about 5×10 6 fibroblasts / cm 2 are preferably seeded. Preferably, the culture in step iv) is carried out in DMEM / F-12 medium optionally containing 2 to 20% serum such as 10% fetal bovine serum and optionally containing 2-phospho-L-ascorbic acid. The culture can be carried out for about 2 to 6 weeks, preferably about 2 to 5 weeks, more preferably about 3 to 5 weeks. Preferably, the culture is carried out under gentle stirring, for example, using an orbital shaker.

[0009] The present invention also provides the use of glass fibers in the production of leather in vitro. Collagen sheets containing glass fibers are also provided. Such collagen sheets are preferably available by the method according to the present invention.

[0010] A method for producing in vitro leather, comprising the step of tanning a collagen sheet according to the present invention, is also provided. In vitro leather obtainable by this method is also provided. Consumer products containing or comprising such in vitro leather are further provided. [Modes for carrying out the invention]

[0011] The inventors have surprisingly discovered that using a glass fiber sheet as a scaffold for cell culture improves cell seeding and collagen production. This makes the production of leather in vitro more efficient, and the resulting leather has attractive properties. Therefore, the present invention provides a method for providing a collagen sheet: i) A process of providing a glass fiber sheet containing solid fibers; ii) The process of placing a fiberglass sheet inside the bioreactor; iii) A step of seeding fibroblasts onto a glass fiber sheet in a bioreactor; iv) A step of culturing fibroblasts under conditions that aid in collagen production to form a collagen sheet; optionally, v) A method is provided which includes the step of isolating a collagen sheet.

[0012] Such a method is referred to herein as the method according to the present invention. The collagen sheet produced by this method has a good collagen content and can be tanned to produce high-quality in vitro leather. In a preferred embodiment, the steps are carried out in numerical order. In a preferred embodiment, step v) is optional. In other embodiments, step v) is not performed.

[0013] Provision of glass fiber sheets In step i), a glass fiber sheet is provided, the sheet containing solid fibers. In a preferred embodiment, substantially all fibers of the glass fiber sheet are solid fibers. The glass fiber sheet preferably does not contain sol-gel fibers or fibers derived from the sol-gel method. The fibers are preferably derived from a molten material, for example, by heating the required elements (e.g., silica, boron, aluminum, calcium, magnesium) until they melt (1100°C < ΔT < 1500°C), followed by extrusion molding, such as pressure extrusion molding of single fibers. A suitable supply form may be 1.5m × 20m (w × l). Preferred solid fibers are quartz glass fibers. Preferably, the glass fibers contain glass containing SiO 5-70%, BO 5-40%, and CaO 1-50% by mass% of oxide conversions as the glass composition. The glass fibers may contain at least Si, B, and Ca, preferably at least Si, B, Ca, and Mg, or Si, B, Ca, and Al, more preferably Si, B, Ca, Al, and Mg. Optionally, P may also be present.

[0014] In some embodiments, silica, boron, and calcium may each be present in the composition in amounts of about 1% to about 99% relative to the weight of the glass. In further embodiments, silica, boron, and calcium may each be present in the composition in amounts of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In certain embodiments, silica, boron, and calcium may be present in the composition at concentrations of about 5–10%, about 10–15%, about 15–20%, about 20–25%, about 25–30%, about 30–35%, about 35–40%, about 40–45%, about 45–50%, about 50–55%, about 55–60%, about 60–65%, about 65–70%, about 70–75%, about 75–80%, about 80–85%, about 85–90%, about 90–95%, or about 95–99%, respectively. Some embodiments may substantially contain each of silica, boron, calcium, aluminum, and magnesium, and, if present, trace amounts of other elements, preferably none other than the necessary elements such as H or O.

[0015] Glass fibers may further contain one or more silicates, borosilicates, borates, or calcium, such as CaO, P2O5, SiO2, and B2O3. Suitable glass types are widely known. Examples include A glass, C glass, D glass, E glass, R glass, and S glass. Most preferably, the glass is E glass, which is an aluminoborsilicate glass containing less than 1% (w / w) of alkali oxides. The glass is preferably alkali-free, which in this context means an alkali oxide content of less than 1% (w / w). The glass is preferably acid / base resistant.

[0016] The form or shape of the sheet is not important. It may be convenient to have fiberglass sheets of approximately the same shape as the internal space available in the intended bioreactor. Most fiberglass sheets can be cut to a specific size using scissors. Those skilled in the art can select an appropriate size. Preferred fiberglass sheets used as scaffolds are rectangular or substantially rectangular, such as squares. Preferably, the sides of the fiberglass sheet are about 0.1 to 50 cm, more preferably about 1 to 40 cm, more preferably about 5 to 30 cm, and even more preferably 10 to 25 cm. For smaller assays, 1 cm 2 The following area may be appropriate: 100 cm² for the final production of in vivo leather. 2 (10 x 10 cm) or 625 cm 2 (25 x 25 cm) or most preferably 900 cm 2 An area of ​​(30 x 30 cm) may be appropriate. Other preferred scaffolding sizes are 4 x 4 cm and 10 x 10 cm.

[0017] The glass fiber sheet can be any suitable type of sheet. Examples of glass fiber sheets include woven fabrics, knitted fabrics, unidirectional fabrics, or chopped strand mats. Chopped strand mats have been found to provide an attractive spatial distribution of fibers for culture, and therefore, the glass fiber sheet is preferably a chopped strand mat. In a preferred embodiment, the glass fiber sheet is not a woven fabric, and preferably not a woven or knitted fabric. In a chopped strand mat, the glass fibers are randomly overlapped with each other. The chopped strand mat further contains a binder, and in a preferred embodiment, the binder is washed off the sheet before cell culture. The chopped strand mat is preferably isotropic. Generally, the fibers in a chopped strand mat are substantially of equal length. Chopped strand mats are widely available from commercial suppliers (see, for example, Examples). A suitable mat from a commercial supplier is a chopped strand mat used in the sea, more preferably containing a GRP resin (a commonly used polyester resin).

[0018] For reference, chopped strand mats are often seen in fiberglass that can be used, for example, in repair work on boat hulls. Such mats generally consist of glass fibers and a binder. The mats are usually processed using a hand lay-up technique in which a sheet of material is placed in a mold and coated with resin. The material conforms to different shapes when wet, and the binder conveniently dissolves in the resin, leaving the glass fibers behind. After the resin hardens, a solid product remains. Thus, commercially available fiberglass chopped strand mats are a convenient raw material for the glass fiber sheets used in the present invention, and the non-glass components can be easily washed away, leaving the glass fiber sheets. In a preferred embodiment, the glass fiber sheet is a washed fiberglass sheet. In a preferred embodiment, the glass fiber sheet is essentially made of glass fibers or consists of glass fibers.

[0019] Cell seeding and proliferation were found to be optimal for glass fibers of a specific diameter, with a specific density and a specific interfiber distance. In a preferred embodiment, - The glass fibers have an average diameter of approximately 1 to 25 μm, preferably approximately 5 to 15 μm; - The fiber density of the glass fiber sheet is approximately 1 to 30 mg / cm³. 2 For example, approximately 10 to 25 mg / cm³ 2 Preferably about 13 to about 22 mg / cm³ 2 For example, approximately 14-16 mg / cm³ 2 , or for example, about 19-21 mg / cm³ 2 It is either approximately 1 to approximately 10 mg / cm³. 2 Preferably about 1.5 to about 8 mg / cm³ 2 and; - The gap size between the fibers of the glass fiber sheet is approximately 10 to approximately 140 μm, preferably approximately 25 to approximately 125 μm; and / or - The present invention provides a method in which the glass fiber sheet has a thickness of approximately 10 to 3000 μm, for example, approximately 1000 to 2000 μm, preferably 1250 to 1750 μm, more preferably approximately 1400 to 1600 μm, or approximately 20 to 1000 μm.

[0020] The dimensions of the glass fibers in the glass fiber sheet are preferably determined using a microscope or calipers.

[0021] More preferably, all four of the above conditions apply. The glass fiber diameter is preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μm. The glass fiber diameter is preferably up to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 μm. Preferably, the diameter is about 3 to about 20 μm, more preferably about 7 to about 18 μm, and more preferably about 10 to about 14 μm. The fiber diameter may be measured using any known technique, a suitable example being measurement by scanning electron microscopy. The fiber diameter is preferably the radius of the cylinder that best represents the fiber, and is generally the minimum diameter of the fiber.

[0022] The fiber density is preferably at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.5, 5.6, 5.7, or 5.8 mg / cm³ 2 The fiber density is preferably up to 10, 9.5, 8, 7.5, 7, 6.5, 6.4, 6.3, 6.2, 6.1, 6, 5.9, or 5.8 mg / cm³. 2 Therefore, thicker sheets generally have a higher density (mg / cm³). 2 ) has a further preferred density of approximately 2 to approximately 7 mg / cm³. 2 , or approximately 3.5 to 6.5 mg / cm³ 2 The fiber density can be determined by conventional weighing of fragments of known dimensions.

[0023] The size of the gaps between fibers can affect the available dimensions for cells, if they are mobile or during proliferation. It is known that the dimensions of the surrounding matrix can affect cell behavior and therefore collagen production. Good results were obtained with glass fiber sheets having gap sizes of approximately 10–140 μm, preferably approximately 15–130 μm, more preferably approximately 20–120 μm, more preferably approximately 30–110 μm, even more preferably approximately 40–100 μm, more preferably approximately 50–90 μm, and more preferably approximately 60–80 μm. The gap size can be determined by scanning electron microscopy.

[0024] The glass fiber sheets preferably have a thickness of 1000 μm or less. If a thicker scaffold is desired, multiple sheets may be placed on top of each other. For mechanical strength, the sheet thickness is preferably not less than 20 μm. The sheets may have a thickness of 50 to 800 μm, preferably 100 to 750 μm, more preferably 150 to 700, 200 to 650, 250 to 600, 300 to 550, 350 to 500 μm, or most preferably about 400 to 450 μm. The thickness may be determined by scanning electron microscopy or using high-precision calipers. The glass fiber scaffold is preferably not crosslinked.

[0025] The fiberglass sheet may be further pre-treated before fibroblasts are seeded onto it. This pre-treatment may be performed before or inside the bioreactor. Since bioreactors are conveniently often filled with or drained of various liquids, the latter is convenient if the pre-treatment includes a washing step. Pre-treatment before placing the sheet in the bioreactor is convenient if it includes steps such as heating or pressurizing the sheet. Preferably, the fiberglass sheet is pre-treated.

[0026] Appropriate pretreatment may include washing, heating, drying, or prewetting steps. Washing may be performed one, two, three, four, five times, or more times. Washing may be carried out for one, five, ten, fifteen, twenty, twenty-five, thirty minutes, or longer. Washing may be carried out in any suitable medium, saline buffer, or organic solvent. Water is preferably ultrapure water. Saline buffer is preferably phosphate-buffered saline. Organic solvents are preferably C1-C4 alcohols, such as methanol, ethanol, and isopropyl alcohol. Organic solvents may be miscible with water. A preferred washing medium is 70% C1-C4 alcohol in water, more preferably ethanol. The washing medium may be decanted, discharged, or aspirated, preferably aspirated.

[0027] Heating may be to temperatures of 37°C, 50 or 55°C, 80°C, 100°C, 120°C, or higher. Drying may be in a vacuum or preferably under an air flow, for example, under a nitrogen flow. Drying may be carried out for 15, 30, 45, 60, 90, 120, 150, 180, 210 minutes, or longer. Drying under an air flow can conveniently be carried out in a flow cabinet. Drying is preferably carried out after all washing steps are completed. Pressurization may be carried out under any suitable conditions, for example, using PBS (preferably pH about 7-8, e.g., about 7.2). Good results are obtained when the fiberglass sheet is pressurized as part of the pretreatment, and therefore in a preferred embodiment, step i) includes pressurizing the fiberglass sheet. Preferably, this is pressurization in PBS, more preferably pH 7-8, even more preferably about 7.2.

[0028] Pre-wetting has been found to improve the performance of glass fiber sheets, particularly when pre-wetting is performed with a protein-containing medium. This makes the cell medium a superior pre-wetted medium. Pre-wetting is preferably performed using a cell medium such as DMEM or SF medium. The medium may be a complete medium (e.g., FBS such as 10% FBS, and optionally DMEM containing an antibiotic such as penicillin-streptomycin-amphotericin B, also known as P / S / A). Pre-wetting may be performed for about 0.5 to 48 hours, preferably about 1 to 36 hours, more preferably about 2 to 24 hours, more preferably about 4 to 20 hours, more preferably about 6 to 18 hours, more preferably about 8 to 16 hours, more preferably about 10 to 14 hours, for example, about 12 hours. Pre-wetting is completed by removing the pre-wetted medium and then optionally performing the washing step described above. Improved cell seeding was observed when pre-wetting was performed using a cell medium, preferably a complete cell medium.

[0029] One example of pretreatment is drying after three washes with 70% C1-C4 alcohol (preferably ethanol) in water. A more preferred example is pressurized sterilization of the fiberglass sheet followed by three washes with 70% C1-C4 alcohol (preferably ethanol) in water, followed by drying. These examples preferably involve pre-wetting in cell culture medium.

[0030] In step ii), the glass fiber sheet is placed in a bioreactor. This can be any bioreactor suitable for culturing fibroblasts, and those skilled in the art can select a suitable bioreactor. Examples include culture flasks such as T-75, Petri dishes, or multi-well plates such as 6-well plates, 24-well plates, or 48-well plates. It is convenient to open the bioreactor to facilitate the final isolation of the collagen sheet. If a disposable bioreactor such as a flask is used, the flask can also be cut open. Generally, this can be an ioreactor cell culture dish, flask, roller, chamber (e.g., a rotating chamber), etc. Dishes, especially glass dishes, are preferred.

[0031] The bioreactor is preferably a low-adhesion bioreactor, such as a silicon-treated bioreactor. The bioreactor is preferably sterilized before the fiberglass sheet is placed. After the sheet is placed, the scaffold in the bioreactor may be sterilized for 10 to 40 minutes, preferably 20 to 30 minutes, by ethanol washing, such as 70% ethanol incubation. Before seeding the cells, the bioreactor and / or fiberglass sheet are preferably washed with saline solution such as PBS.

[0032] In some embodiments, in step ii), a number of glass fiber sheets are placed in the bioreactor to form a multi-layer scaffold. Preferably, when a number of glass fiber sheets are used, the sheets are substantially equal in size or of equal size. In some embodiments, two sheets are used. In some embodiments, three sheets are used. In some embodiments, four, five, six, seven, or eight sheets are used. As described above, the glass fiber sheets may be placed in the bioreactor before any of the pretreatments. Steps i) and ii) may be carried out simultaneously, for example, by providing a bioreactor that already contains glass fiber sheets.

[0033] Cell seeding and culture In steps iii) and iv), cells are cultured on a glass fiber sheet in a bioreactor. The cells secrete collagen, forming a collagen sheet suitable for the production of in vitro leather, for example, by tanning. Conventional animal hides are mainly formed of collagen, a fibrous protein. Collagen is a general term for a family of at least 28 different collagen types; animal skin is usually type 1 collagen, and therefore type 1 collagen is preferred. Other types of collagen exist and can also be used in leather formation. Collagen is composed of amino acids, such that approximately one-third of the amino acid residues in collagen are glycine, -(Gly-XY) nIt is characterized by the repetition of a triplet of -, where X is often proline and Y is often hydroxyproline. Structurally, collagen can consist of three wrapped units of peptide chains of different lengths. Different animals, and therefore different cells, can produce different amino acid compositions of collagen, resulting in different properties. Collagen fiber monomers can be produced from α chains about 1050 amino acids long, so the triple helix takes the form of a rod about 300 nm long and 1.5 nm in diameter. Triple helix monomers can be synthesized in the production of the extracellular matrix by cells such as fibroblasts, and the monomers can self-assemble into fibers. These triple helices can be held together by salt bonds, hydrogen bonds, hydrophobic bonds, and covalent bonds. The triple helices can be joined together in bundles called fibrils, and these fibril bundles come together to form fibers. Fibers usually spread throughout the layers of skin and connect to one another. Strength can be imparted to the material by variations in crosslinking or linking.

[0034] In step iii), cells are seeded onto a fiberglass sheet. The cells are preferably animal cells, more preferably mammalian or reptile cells, most preferably mammalian cells, such as bovine, sheep, goat, horse, bison, pig, and aquatic mammals, such as seals and crocodiles. Good results have been obtained using bovine cells. The cells are preferably collagen-releasing cells, more preferably fibroblasts. Mixtures of different cells may also be used. Fibroblasts are a type of cell that synthesizes extracellular matrix and collagen, producing the structural framework (crust) of animal tissues. Fibroblasts are the most common cells in connective tissue in animals. Preferred fibroblasts are dermal fibroblasts, more preferably bovine dermal fibroblasts.

[0035] In some preferred embodiments, the cells are primary cells, preferably fibroblasts. The cells may originate from tissue extracts / explants, or modified (gene-transformed) cell lines, or modified forms thereof. In some modified forms, the cells may be grown to full confluence (e.g., 100% confluence), at which point further growth may be suppressed, but the cells may continue to produce collagen, or be stimulated to produce and release collagen. In some modified forms, the cells may not be grown to full confluence (e.g., approximately 99% confluence, 95% confluence, 90% confluence, 85% confluence, 80% confluence, etc.). The cells may be cultured to confluence above 80%, above 85%, above 90%, above 90%, above 95%, and / or under full (100%) confluence. Primary fibroblasts can be used after initial elongation, preferably after about 3 to 10 passages, more preferably after about 4 to 9 passages, and even more preferably after about 5 to 8 passages, for example after 6 passages (and therefore using P6 cells).

[0036] Before seeding, cells can be grown in a suitable medium. Preferred media are DMEM or SF medium. The medium may be a complete medium (generally containing 10% serum), a serum-free medium, or a medium containing 5% serum. Preferably, the medium for growth before seeding is a serum-free medium or contains 5% serum. A preferred medium for growth before seeding is combined with 50% serum-free growth medium based on DMEM, such as DMEM-F12, and contains 1-5 mg / mL of protein such as albumin, e.g., about 2.5 mg / mL; 1-10 μg / mL of hormone such as insulin, e.g., 5 μg / mL; 2-10 μg / mL of iron source such as transferrin, e.g., about 5.5 μg / mL; and 2-12 μg / mL of trace elements such as selenium, e.g. A 50% DMEM medium such as DMEM-F12 + 10% serum such as FBS + 1% antibiotic such as P / S / A is supplemented with 6.7 μg / mL of vitamin C, preferably 100-500 mg, e.g., about 250 nM vitamin C and 2-phospho-L-ascorbic acid, and 1-100 ng / mL of growth factors such as 10 ng / mL of basic fibroblast growth factor (bFGF) and 50 ng / mL of platelet-derived growth factor (PDGF), and 0.02-0.5 mM, e.g., 0.1 mM amino acids such as glutamine. This serum-free growth medium can also be used alone. Another preferred medium for growth before seeding is FBS medium. Another preferred medium for growth before seeding is SF medium. In any of the media described herein, gentamicin, e.g., 50 μg / mL of gentamicin, may be used as the antibiotic.

[0037] The cells can be seeded at any appropriate density. 1 × 10 6 cells / cm 2 Good results were achieved. Therefore, the cells are preferably about 1 × 10 4 ~Approx. 1×10 8 cells / cm 2 Glass fiber sheet, preferably about 1 x 10 5 ~Approx. 1×10 7 cells / cm 2 Comfortably approximately 5 x 10 5 ~Approx. 5×10 6 cells / cm2 They are sown at a density of . In a preferred embodiment, in step iii) about 1 × 10 4 ~Approx. 1×10 8 Fibroblasts / cm 2 Glass fiber sheet, preferably about 1 x 10 5 ~Approx. 1×10 7 Fibroblasts / cm 2 Comfortably approximately 5 x 10 5 ~Approx. 5×10 6 Fibroblasts / cm 2 A method is provided for sowing seeds.

[0038] Cells can be seeded from a concentrated suspension. Cells can also be seeded by dropping a certain amount of cell suspension onto the center of the scaffold. Cells can also be seeded by multiple droplets, which can be spatially distributed throughout the scaffold. After seeding, before adding additional culture medium, the cells can be attached to a fiberglass sheet. This can be for 1 to 3 hours, preferably 1.5 to 2 hours.

[0039] Multiple seedings may be performed to improve cell density. In some embodiments, two, three, four, five, or six seedings are performed, preferably two, three, four, or five seedings, more preferably three or four seedings, for example four seedings. Further seedings may be performed in exactly the same manner as the initial seeding. Seedings are preferably separated within about 1 to 4 days of culturing, more preferably within 2 to 3 days of culturing. This allows the original cells to adhere. The ratio of adhered cells to suspended cells may be called seeding efficiency. This is preferably determined 24 hours after seeding. During the seeding process, culturing is preferably carried out under static conditions rather than under gentle agitation.

[0040] In step iv), cells are cultured under conditions that aid in collagen production to form a collagen sheet. Those skilled in the art know how to culture cells such as fibroblasts. Generally, cells are cultured at 35-40°C, preferably about 37°C. Generally, cells are cultured in a controlled CO2 atmosphere, for example, at CO2 3-8%, preferably about 5%. Preferably, cells are cultured in a humidified environment. Conventional cell incubators can be used to provide these conditions. The culture medium is preferably changed every 2-3 days.

[0041] In preferred embodiments, the culture is carried out for about 1 to 6 or 2 to 6 weeks, preferably about 2 to 5 weeks, more preferably about 3 to 5 weeks. After about 35 days, the collagen sheet is found to be suitable for isolation, and therefore the culture can be about 30 to about 40 days. In some embodiments, the culture is carried out for about 2 to 3 weeks and has been found to be suitable for developmental purposes. In some embodiments, the culture is carried out for about 1 to 9 weeks, or preferably 6 to 8 weeks, for example, 7 weeks. This has been found to be beneficial for producing large sheets.

[0042] Cells can be cultured in any suitable medium. If the glass fiber sheet has been pre-moistened, the cells are preferably cultured in the same medium used for pre-moistening. DMEM and SF have been found to be very suitable for collagen production, with DMEM being preferred. Preferably, the culture in step iv) is carried out in DMEM / F-12 medium optionally containing 2-20% serum, such as 10% fetal bovine serum, and optionally containing 2-phospho-L-ascorbic acid. In some embodiments, the culture in step iv) is carried out in a medium optionally containing 2-20% serum, such as 10% fetal bovine serum, and optionally containing 2-phospho-L-ascorbic acid. In some embodiments, the culture in step iv) is carried out in a medium containing 2-phospho-L-ascorbic acid.

[0043] If the culture medium contains 2-20% serum, it preferably contains 4-18%, more preferably 5-16%, more preferably 7-14%, even more preferably 8-12%, and also more preferably 9-11%, and most preferably 10%. Those skilled in the art can select suitable serum, examples of which are platelet lysates, such as human platelet lysates, fetal bovine serum, horse serum, and neonatal calf serum. Fetal bovine serum is most preferred.

[0044] 2-phospho-L-ascorbic acid was found to promote collagen production. When the culture medium contains 2-phospho-L-ascorbic acid, it preferably contains 20-2000 μM, more preferably 50-1500 μM, even more preferably 100-1250 μM, and more preferably 150-1000 μM. Good results were obtained at 200-750 μM, for example, 250-500 μM.

[0045] Culture can be carried out under static conditions without a bioreactor, glass fiber scaffold, or cell medium agitation. Culture can also be carried out with gentle agitation, for example, using an orbital shaker. The orbital shaker may be set to a gentle setting such as 2 to 50 rpm, preferably 5 to 35 rpm, for example 10 rpm. Gentle agitation such as with an orbital shaker at the above settings is generally 0.01 to 5 Pa or 0.1 to 50 dynes / cm². 2 This includes shear stress in the range of 0.1 to 3 Pa or 1 to 40 dynes / cm². In some embodiments, the shear stress is 0.1 to 3 Pa or 1 to 40 dynes / cm². 2 The range is as follows: In some embodiments, the shear stress is 0.5 to 2 Pa or 5 to 30 dynes / cm 2 The range is as follows: Preferably, the shear stress is 1 to 1.5 Pa or 10 to 20 dynes / cm². 2 This is within the range. In some embodiments, cultivation is first carried out under static conditions, followed by cultivation under gentle stirring. Preferably, the first 10-40%, more preferably 15-30%, for example 20% of the total cultivation period is carried out under static conditions, and the remainder is carried out under gentle stirring. Alternately, seeding is carried out under static conditions, followed by cultivation under gentle stirring.

[0046] Collagen sheets and their use In step v), the formed collagen sheet is isolated. The sheet was found to maintain its shape, whereas collagen sheets cultured without the glass fiber sheet were found to shrink when removed from the bioreactor. Therefore, the present invention provides a means for obtaining a form-fast collagen sheet using readily available materials as a scaffold. The collagen sheet can be fixed before or after its isolation. Means for fixing tissue are well known, and those skilled in the art can select an appropriate method. For example, the collagen sheet can be fixed using formaldehyde, for example, 4% paraformaldehyde in water. The collagen sheet can be isolated using any suitable means, for example, by hand, with a spatula, a scraper, or preferably with tweezers.

[0047] The present invention provides a collagen sheet containing glass fibers. This collagen sheet is preferably obtained by a method according to the present invention. In this specification, the collagen sheet may contain up to 50% by weight of glass fibers, preferably 0.5 to 40% by weight, more preferably 1 to 30% by weight, more preferably 2 to 25% by weight, even more preferably 3 to 20% by weight, and more preferably 4 to 15% by weight, and most preferably 5 to 10% by weight. These collagen sheets do not shrink, or preferably maintain their dimensions for at least 1, 2, 3, 4, 5, 6, or 7 days after the sheet is formed. In this context, maintaining dimensions means that the maximum reduction in dimensions is such that the shrinkage is such that the maximum reduction in dimensions is 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the original dimensions. The dimensions of the collagen sheet are preferably defined with respect to the glass fiber sheet.

[0048] Hydroxyproline content is an excellent measure of collagen content. In a preferred embodiment, the collagen sheet contains at least 1 μg / mg of hydroxyproline, more preferably at least 2 μg / mg, more preferably at least 3 μg / mg, and most preferably at least 4 μg / mg. Using known techniques, for example by measuring absorbance in a pigment productivity assay, conveniently using commercially available kits such as those described in the examples, the hydroxyproline content can be assayed. Hydroxylysine content is another measure of collagen content. In a preferred embodiment, the collagen sheet contains at least 0.05 μg / mg of hydroxylysine, more preferably at least 0.1 μg / mg, more preferably at least 0.5 μg / mg, and most preferably at least 1 μg / mg of hydroxylysine. Using known techniques, for example by measuring absorbance in a pigment productivity assay, the hydroxylysine content can be assayed.

[0049] In vitro leather The present invention provides the use of glass fibers in the production of in vitro leather. The use is substantially as described above, and the glass fibers are used as a glass fiber sheet in the method according to the present invention. The resulting collagen sheet is then used to provide in vitro leather. Accordingly, the present invention also provides a method for producing in vitro leather, comprising the step of tanning a collagen sheet, wherein the collagen sheet is as defined above.

[0050] Tanning is an ancient technique well known to those skilled in the art. It is generally understood to be the process of treating animal skins for the production of leather. Tanning can be carried out in many well-known ways, such as vegetable tanning (using tannins, e.g.), chromium tanning (using chromium salts such as chromium sulfate), aldehyde tanning (using glutaraldehyde or oxazolidine compounds), and syntan (using synthetic tannins, aromatic polymers). Tanning can proceed through preparation steps, such as preservation, soaking (rehydration), limeing, splitting, reliquation, demineralization, fermentation (protein decomposition), degreasing, frizzing, bleaching, pickling, and de-picking. Tanning may include pH adjustment to improve the tanning process, preferably lowering the pH, more preferably adjusting it to pH 2.8-3.2. Preferably, tanning is carried out using a chromium-based tanning method.

[0051] Tanning can be followed by post-tanning processes, also known as crusting, which include wetting (rehydration), squeezing (drying), thinning (dividing into thinner layers), shaving, neutralization (adjusting the pH to a more neutral level), retanning, dyeing (coloring), fatliquoring, filling, stuffing, stripping, whitening, fixing of unbound chemicals, stretching, conditioning, softening, and buffing.

[0052] After tanning, in vivo leather is obtained. Thus, the present invention provides in vivo leather that can be obtained by the method according to the present invention. The in vivo leather may have a thickness of 0.01 to 0.5 mm, preferably 0.015 to 0.4 mm, more preferably 0.2 to 0.3 mm, and most preferably 0.24 mm after the tanning and drying process. The in vivo leather may consist of multiple layers, for example, about 2 to about 50 layers, 2 to about 40 layers, 3 to about 30 layers, 4 to about 20 layers, 5 to about 10 layers, or about 7 to 8 layers, in order to achieve the desired thickness. The in vivo leather may be colored or dyed using, for example, one or more colorants or pigments. It may also be patterned, for example, after an animal skin pattern.

[0053] Exoplastic leather is an attractive alternative to leather derived from animal hides. Therefore, it can be used in consumer products. Examples of consumer products include clothing such as jackets, vests, hats, dresses, trousers, and gloves; footwear such as shoes, sandals, flip-flops, and boots; accessories such as belts, suitcases, bracelets, watch straps, handbags, coin purses, keychains, cell phone holders, cell phone cases, laptop cases, eyeglass cases, electronic device cases, wallets, card holders, billfolds, briefcases, and monocular cases. Products include: cases, luggage tags, and portfolio holders; automotive applications such as car seat covers, steering wheel covers, dashboards, armrests, headrests, doors, motorcycle saddles, and motorcycle handlebars; household and office supplies such as sofas, lap cushions, footrests, chair backs, armrests, leather decorative wall hangings, mats, carpets, lampshades, and pillows; sports equipment such as softballs, sports footwear, shin guards, sports gloves, costumes, racing suits, masks, knee and elbow protection, bicycle seats, boxing gloves, helmets, roller skates, and punching bags; protective products such as heat-resistant aprons and gloves, steering wheel covers, holsters, quivers, and knife sheaths; musical instrument products such as percussion instruments, guitar straps, and instrument cases; and pet accessories such as collars, chains, muzzles, harnesses, saddles, bridles, leather straps, and pet furniture.

[0054] general definition In this specification and in the claims, the verb “includes” and its conjugations are used in a non-restrictive sense, meaning that the items following the word are included, but not excluded, items not specifically mentioned. Furthermore, the verb “consist of” may be replaced with “essentially consisting of,” meaning that the products or compositions or nucleic acid molecules, peptides or polypeptides of nucleic acid constructs or vectors or cells as defined herein may contain additional components other than the specifically identified components; such additional components do not alter the inherent characteristics of the invention. Furthermore, references to elements with the indefinite article “a” or “an” do not preclude the possibility of multiple elements unless the context explicitly requires the presence of only one or one of the elements. Thus, the indefinite article “a” or “an” usually means “at least one.” When used with a number (e.g., about 10), the words “about” or “approximately” preferably mean that the value may be more than 10% of a given value (of 10) or less than 10% of that value.

[0055] Whenever a parameter or substance is considered in the context of the present invention, unless otherwise specified, it is assumed that the parameter is determined, measured, or revealed under physiological conditions. Physiological conditions are known to those skilled in the art and include an aqueous solvent system, atmospheric pressure, pH value of 6–8, a temperature range of room temperature to about 37°C (about 20°C to about 40°C), and appropriate concentrations of buffer salts or other components.

[0056] All patents and references described herein are incorporated herein by reference in their entirety. Unless otherwise specified, each embodiment described herein may be combined with other embodiments described herein. The following examples are provided for illustrative purposes only and are not intended in any way to limit the scope of the invention. [Brief explanation of the drawing]

[0057] [Figure 1A]The bright-field microscope observation of the glass fiber sheet from Example 1.1.1 is shown. The magnification is as indicated. [Figure 1B] Scanning electron microscopy images of various glass fiber sheets are shown. The magnifications are as indicated, with the 40x scale bar representing 500 μm and the 250x scale bar representing 100 μm. The top two images show the EsportsMJJ mat, the two middle images show the Utek mat, and the two bottom images show the GuoTai mat. [Figure 2] The results for live / dead cells (Et-H1-ethidium homodimer-1) of cells seeded on a scaffold (from FBS medium) after undergoing preliminary treatment 2, followed optionally by pre-moistening with SF medium (as indicated SF) or FBS medium (as indicated FBS) are shown. [Figure 3] The seeding efficiency determined by the PicoGreen assay is shown. Pre-treatment 1 performed better than all pre-treatment 2 options, demonstrating the beneficial effects of pressure sterilization. [Figure 4] Fluorescence microscopy reveals excellent cell behavior on glass fiber scaffolds. [Figure 5] The image shows a bright-field image (5x magnification) of the instructed cell culture. The filling of the gaps can be observed over time. [Figure 6] Z-stack imaging of cell nuclei (Hoechst stained blue) and collagen (CNA35 stained red) on a glass fiber scaffold, as well as staining of live / dead cells 5 weeks after culture. The small prototype shows staining throughout the sample and high variability in cell distribution throughout the sample. Samples from the center or corners of the scaffold are shown as shown in the figure. Viability is high. Some cell death is observed. Low background staining is observed in the blank control (data not shown). Biopsy punch diameter: 6 mm. [Figure 7] This shows extracorporeal leather produced by tanning collagen sheets cultured on a fiberglass scaffold. [Examples]

[0058] Example 1 - Glass fiber sheet as a scaffold for cell culture 1.1 Materials and Methods 1.1.1 Provision of glass fiber sheets A 1x5 meter roll of 30g chopped strand emulsion mat fiberglass was purchased from the product supplier (FEVERWORK 1x5m 30g CSM Fibreglass Mat Fibreglass Strand Pads Fibreglass Roll for Marine GRP resin; Product name: EsportsMJJ; Ean: 6953105511954; Part number: 511954). The white mat contained 30g of alkali-free fiberglass and was a fiber-reinforced polymer produced by pulping and winding. Fiberglass mat is a sheet-like product manufactured by continuous strands or surfaces bonded non-directionally to each other by chemical or mechanical action. It was characterized by uniform fiber distribution, good tensile strength, and good flexibility. The type of glass was alkali-free, type E, acid / base resistant, melting point: 1100℃ < ΔT < 1500℃. Notably, 20m 2 The cost per unit is less than 20 euros; 0.001 euros / cm 2 It was less than [amount missing].

[0059] Using a ruler and a small knife, cut the mat to 25cm. 2 0.25cm 2 One piece (for 48-well plates) or 1 cm 2 The sample was cut into two pieces (for 24-well plates) and subjected to one of two different pretreatment methods. Pre-treatment 1: For each wash, use 70% ethanol ( aq Wash (x3) for 20 minutes Pre-treatment 2: Pre-treatment 1 follows pressure sterilization.

[0060] Pre-treatment 2 was performed by pressure sterilization in 1 × PBS; pH 7.2. Pressure-sterilized materials can be stored in a desiccator before use. 1 cm 2Place the scaffolding on an ultra-low adhesion (ULA) 24MW plate (or 48MW plate, or other as needed), followed by 70% ethanol ( aq It was washed once, suctioned, and then dried in a BSL2 flow cabinet under a constant flow rate.

[0061] Optionally, pretreatment included pre-wetting, in which case it included washing with ethanol, completely drying, and then incubation overnight in complete medium [DMEM + 10% FBS + 1% P / S / A]. For comparison, pre-wetting was also performed overnight in SF medium or 1×PBS. Before cell seeding, the scaffolds were washed with ethanol (×3), vacuum-dried, and air-dried in a flow cabinet under constant airflow for at least 3 hours.

[0062] Glass fiber sheets were analyzed using bright-field microscopy or scanning electron microscopy. Sheets from additional suppliers (e.g., Utek or GuoTai) were compared. The results are shown in Figure 1. Good results were also obtained with other scaffolding sizes, such as 4×4cm and 10×10cm.

[0063] 1.1.2 Serum-free growth medium Cells (primary bovine fibroblasts) were grown in serum-free medium. The medium was based on DMEM / F12 medium supplemented with albumin 2.5 mg / mL, insulin 5 μg / mL, transferrin 5.5 μg / mL, selenium 6.7 μg / mL, 250 nM vitamin C, basic fibroblast growth factor (bFGF) 10 ng / mL, platelet-derived growth factor (PDGF) 50 ng / mL, and 0.1 mM glutamine.

[0064] 1.1.3 Preparation of Petri dishes and sterilization of scaffolding The glass petri dishes were treated with Sigmacote silicone according to the manufacturer's instructions and then sterilized. The fiberglass scaffolds were then placed in the petri dishes within the flow cabinet. The scaffolds were sterilized by incubation in 70% (v / v) ethanol for 20–30 minutes. Any remaining solvent was washed off with PBS (×1). To dry the scaffolds, as much as possible was removed using a pipette vacuum aspirator. The resulting scaffolds were sterile and ready for use in the cell seeding process.

[0065] 1.1.4 Seed sowing on scaffolding Primary bovine fibroblasts were seeded using the 125 μL droplet seeding method in the center of the scaffold, with a sample size of 1 × 10⁶. 6 cells / cm 2 Cells were seeded onto the scaffold at a density of 250,000 cells per scaffold (cells grown in FBS medium or SF medium). The 24MW plates containing the seeded scaffolds were carefully transferred to a 5% CO2 37°C humidified incubator and incubated for 1.5–2 hours. After the cells had attached to the scaffolds, 500 μL of complete medium was gently added to each well. The medium was changed every 2–3 hours; the total culture period was 7 days. All samples were prepared in triplicates; a representative scaffold without cells was used as a blank.

[0066] Alternately, 1 x 10 6 cells / cm 2 Using the droplet seeding method, PBF cells were seeded onto scaffolds; 30-40 μL of cell-containing medium was immediately seeded onto the scaffolds and placed on 48MW plates. The 48MW plates with seeded scaffolds were carefully transferred to a 5% CO2 37°C humidified incubator for 1.5-2 hours. After the cells had adhered to the scaffolds, 500 μL of medium was gently added to the wells. The medium used was either complete medium (+250 μM ascorbic acid 2-phosphate) or SF, depending on (matching) the pre-wetting of the scaffolds. Cells seeded on PBS-rinsed scaffolds were cultured in SF. The medium was changed every 24 hours; the total culture time was 3 days. All samples were performed in triplicates; a representative scaffold without cells was used as a blank.

[0067] 1.1.5 Analysis of seeding efficiency Seeding efficiency counts, DNA PicoGreen assays, and Live / DEAD Hoechst / ethidium homodimer-1) staining were performed 24 hours after seeding. Seeding efficiency was performed using the PicoGreen assay, and cell content was estimated as a function of total DNA content. Briefly, representative scaffolds (2-3) / conditions were homogenized for 2 minutes in ice-cold Tris buffer (pH 8) according to the manufacturer's protocol. Estimates were based on a DNA content of 6 pg per single cell.

[0068] The doubling of cell numbers over 72 hours was also performed using the DNA Picogreen assay. The doubling was calculated as the relative fluorescence unit (RFU)-induced [DNA] at 24 hours compared to the RFU [DNA] at 72 hours = (72-hour cell number) / (24-hour cell number).

[0069] Representative samples / conditions were seeded and stained with Live / DEAD (Hoechst / ethidium homodimer-1) 24 hours later. On the final day, samples were stained with Hoechst / phalloidin-FITC / CNA35-AlexaFluor(trademark) 568 and analyzed by fluorescence microscopy. The scaffolds were washed with warm culture medium, and the cells were fixed with 4% paraformaldehyde (PFA, pH 7.2) at room temperature for 15 minutes, or overnight in the refrigerator. Following the standard protocol, the scaffolds were first stained with Hoechst / CNA35 at room temperature for 15-30 minutes (for 5 mL of staining solution in medium / PBS: 5 μL of stock Hoechst ([Hoechst] FINAL =1 μg / mL; Stock EthD-1 1.6 μL ([EthD-1] FINAL =650nM; Add enough staining solution to cover the sample and incubate in the dark for 15 minutes; wash once with culture medium, then wash with 1×PBS (pH 7.2) (×3). Next, treat the scaffold with permeabilization buffer (0.1% tergitol in PBS) at room temperature for 15-30 minutes, wash with 1×PBS (pH 7.2) (×3), and add one drop of phalloidin-FITC per well.

[0070] 1.1.6 Hoechst / Phalloidin / CNA35 staining Cells were fixed with PFA to stain the nucleus, actin, and collagen from the cells. The cell walls were then permeabilized with 0.5% Tween20 in PBS and incubated for 1 hour. Staining solution in PBS (in the same tube): • Hoechst (blue): 1 μL / 1000 μL • Phalloidin (green): 1 μL / 1000 μL • CNA35 (red): 1 μL / 100 μL The sample was completely covered with staining solution, approximately 500 μL relative to the aforementioned scaffold. The sample was incubated in the dark for 1 hour and washed twice with PBS.

[0071] 1.2 Results 1.2.1 Characteristics of glass fiber sheets Bright-field microscopy revealed a sheet height of 150–200 μm, determined by a Tanner's gauge. Fiber diameters were 5–10 μm, or 10 μm ± 1.1 μm, as determined by SEM. Sheets with the fiber diameters shown in Table 1 were provided by other manufacturers. Representative microscopic images are shown in Figure 1. Pressure sterilization was found not to affect the mechanical structure of the glass fiber sheets.

[0072] [Table 1]

[0073] No fibers with a diameter of less than 5 μm were identified. Density: 5.8 mg / cm³ 2 Sheets with an average diameter of 14-17 μm and a thickness of 0.5 mm were also used. The glass fiber sheets were not brittle, and no fibers transferred to the hands / tweezers during handling. The sheets were flexible and robust, and the decomposition products were silica, boron, aluminum, calcium, and magnesium.

[0074] 1.2.2 Live / Dead Assay Live / Dead assays for all permutations in Pre-treatment 1 showed no viable cells at 24 hours. Live / Dead assay results for Pre-treatment 2 (pressure-sterilized in 1×PBS) indicated a relatively high level of cell death. Less cell death was observed under "non-wet" conditions compared to scaffolds pre-treated with culture medium. This was confirmed from spinner flasks for cells from FBS medium and SF medium. Figure 2 shows microscopic images.

[0075] 1.2.3 Sowing Efficiency The seeding efficiency of Pretreatment 2 on the glass fiber scaffold was satisfactory. The best seeding conditions for all cell origins (FBS or SF medium) were when the scaffold was pre-moistened in complete serum medium or SF medium. A decrease in seeding efficiency was observed for pre-moistened scaffolds and for all conditions of Pretreatment 1. Seeding efficiency was determined using the PicoGreen assay. Pretreatment 2 (pre-moistened) and Pretreatment 1 (without pre-moistening) had low seeding efficiency for FBS-grown cells, and Live / DEAD staining indicated the absence of live cells on the Pretreatment 1 scaffold. Fluorescent labeling was performed on day 7, and cell distribution, cytoskeleton, and collagen production were evaluated (Figure 4). Good results were observed for all Pretreatment 2 scaffolds. Cells adhered, proliferated, and produced collagen for at least 7 days.

[0076] 1 x 10 6 cells / cm 2 The seeding efficiency 24 hours after seeding was 118% for pre-wetting and culture in complete medium, 79% for pre-wetting and culture in SF medium, and 64% for pre-wetting in PBS and culture in SF medium. Therefore, pre-wetting in the cell medium improves cell seeding efficiency.

[0077] When evaluated with n=6, it was evident that cell density doubled over 72 hours, while culturing in complete medium resulted in a slight decrease in cells (73%). Culturing in SF pre-moistened scaffolds and in SF increased cell density (111% when pre-moistened in SF or 144% when pre-moistened in PBS). Exposure of cells to complete medium may result in a short burst of growth due to exposure to a richer medium, which could lead to overestimation at t=24 hours. The low seeding efficiency of PBS-moistened scaffolds may be related to the lack of surface inactivation due to the absence of relevant biomolecules in PBS. In any case, serum-free cells from subcultures (spinner flasks) readily attached to and grew on fiberglass scaffolds.

[0078] For comparative experiments, different glass fiber materials were used (sol-gel fibers with a fiber diameter of 30-70 μm; gap size of 150-250 μm; fiber density of approximately 12 mg / cm³). 2 The seeding efficiency was also determined at a thickness of approximately 450-700 μm. Seeding was approximately 80% of that achieved on the scaffolding according to the present invention.

[0079] Example 2 - Production of leather in vitro 2.1 - Scaffolds, cell culture, and seeding The fiberglass scaffolding was prepared as described above. EsportsMJJ alkali-free, E-type, acid / base resistant fiberglass with a melting point of 1100°C < ΔT < 1500°C was cut into 4cm x 4cm pieces to create the scaffolding. Two layers of scaffolding were used (dimensions: 4cm x 4cm, thickness approximately 0.2μm, V=320mm). 3 The scaffolds were pressure-sterilized in PBS. The scaffolds were sterilized with 70% (v / v) ethanol and dried overnight on silicone-coated glass petri dishes.

[0080] Primary bovine fibroblasts (passage P6) were used. During elongation (P5), cells were cultured in "50 / 50" medium (50% DMEM-F12 + 10% FBS + 1% P / S / A (see Section 1.1.2)) combined with 50% serum-free growth medium. P6 cells were seeded four times on a scaffold at intervals of 2-3 days. Each seeding yielded 32 × 10⁶ cells. 6Cells were concentrated in 1.6 mL, the cell suspension was spread over the scaffold, and incubated for 1 hour before adding 25 mL of medium (DMEM-F12 + 10% (v / v) FBS + gentamicin 50 μg / mL + 500 μM L-ascorbic acid diphosphate sesquimagnesium salt aqueous compound (vitamin C)). During the seeding process, the scaffold was cultured under static conditions for 1 week. After this process, the petri dishes were then placed in dynamic culture using an orbital shaker at 35 RPM for the remaining 4 weeks of culture (shear stress 0.01-5 Pa or 0.1-50 dynes / cm²). 2 (Induces collagen formation). Orbital shakers were found to promote collagen formation and improve overall collagen deposition; in experiments where a fiberglass scaffold was used under static conditions or on an orbital shaker (30 rpm), static conditions produced only about 50% more collagen compared to culture on an orbital shaker. Seeding efficiency was measured as the number of cells in suspension 24 hours after each seeding step. After the final seeding step was completed, 50% of the medium was replaced weekly.

[0081] Cell proliferation was primarily accessed by bright-field imaging (5x magnification). On the final day of culture, a 6 mm biopsy punch was collected and stained LIVE / DEAD (see Example 1.1.5). The remaining scaffold was fixed with 4% (v / v) PFA, cut in half (2 cm × 4 cm) for analysis, and the other half was tanned. Samples were collected and stained with Hoechst and CNA35 fluorescent dyes to evaluate the cell and collagen distribution on the scaffold, respectively (see Example 1.1.6). Z-stack imaging (16 slices, 10 μm steps) was performed using ImageExpress Pico (molecular devices).

[0082] 2.2-Hydroxyproline content Collagen content can be assessed based on the hydroxyproline content of the scaffold. For quantitative analysis, a hydroxyproline assay was performed at the end of culture with approximately 10 mg of immobilized sample for each blank scaffold. The protocol for the commercially available kit followed the OH-proline assay (Sigma-Aldrich kit #MAK357). Briefly, before starting, the kit components were warmed to room temperature and mixed by vortex before use. Dried sample tissue was weighed. Homogenized the sample by adding 100 μL of dH2O per 10 mg of tissue (using 100 μL increments, not using volumes smaller than 100 μL, and rounding to the nearest multiple of 100). The homogenate was transferred to a sample container with a heat-resistant and PTFE screw cap. 100 μL of NaOH (10M) was added to the homogenate and thoroughly mixed. The sample was pressure-sterilized at 121°C for 1 hour, then cooled to room temperature before adding 100 μL (10M) of HCl, and subsequently mixed to neutralize the homogenate. The sample was centrifuged (10000 × g, 5 min), and insoluble debris settled.

[0083] Standard materials were prepared, and all samples were added to a 96-well plate. For the standard material, 20 μL of hydroxyproline (HP) standard stock solution (1 mg / mL) was diluted with 180 μL of dH2O. Standard solution = 200 μL of 0.1 mg / mL solution. Wells 0, 0.2, 0.4, 0.6, 0.8, and 1.0 were used as standards. 10 μL of sample per well was used in duplicate (2-well), preferably in 3-well duplicates. Samples could be evaluated at 100-fold dilution, 10-fold dilution, or undiluted. The samples and standard materials were dried, and all liquids were evaporated.

[0084] Next, the assay components were vortexed to ensure thorough mixing and dissolution of the precipitate. For each sample, 6 μL of chloramine T and 94 μL of oxidation buffer were mixed to prepare an Oxidation Reagent Mix (ORM), which was then protected from light until use. For the assay, 100 μL of ORM was added to each well of a 96-well plate. Then, 50 μL of Develop Solution was added to each well and incubated at 37°C for 5 minutes, followed by the addition of 50 μL of DMAB solution to each well, after which the contents were mixed (e.g., using a multichannel micropipette), and then heated at 65°C for 45 minutes. Absorbance (OD560) was read using a plate reader (SpectraMax iD3).

[0085] A blank sample is used for sample correction. A standard series is plotted, and a linear equation (y=mx; y=OD, ×=hydroxyproline (HP) μg / well) is determined. The mass (μg) of HP in the sample per well is determined using this linear equation. [HP] is expressed in μg / μL: [HP] = (B / V) × D In the above formula, B = mass of HP (μg); V = volume of sample added to the well (μL); D = dilution ratio relative to the 96-well volume = 1; homogenate dilution ratio = 3 per 10 mg of tissue. Formula for calculating collagen content (the collagen HP content of bovine dermis is 12-15%): Collagen mass (μg) = (HP mass μg) / 0.135 The collagen content can be normalized to the mass sample: collagen μg / mg.

[0086] Example 2.3 - Cell attachment and distribution High seeding efficiency was observed, which then decreased slightly. Perhaps, as cells aggregate and spread, and gaps within the fibrous scaffold close, new cells may face a greater challenge in finding space for attachment. This can lead to cell loss. The following efficiencies were observed: 98% for the first seeding, 90% for the third seeding, and 98% for the fourth seeding. Bright-field observation of the culture on day 14 showed no empty areas or cell aggregates throughout the culture. On the final day of culture, the medium did not change color against the yellow spectrum, indicating that the pH had not become too low. pH was measured during the culture period and never dropped below 6.5. The samples had an approximate thickness of 0.3 mm before and after PFA fixation. Figure 5 shows the cell distribution on the scaffold. The gaps were found to close over time. Figure 6 shows the good distribution of viable cells and the uniform distribution of collagen. Tweezers manipulation revealed that the collagen sheet maintained the same flexibility before and after 4% (v / v) PFA fixation.

[0087] Example 2.4 - Collagen Content Regarding the cultured sheets described above, a good collagen content was found. The collagen sheets were evaluated for hydroxyproline content as described in Example 2.2, and a dry weight hydroxyproline content of 4.06 μg / mg (±0.07, mean ±SD) was found. When the scaffold according to the present invention was not used, it was not possible to obtain collagen sheets suitable for the hydroxyproline assay. Such sheets shrunk. When a silk (modified with RGD peptide)-based scaffold was used, a hydroxyproline content slightly below 75% was achieved compared to comparative experiments using a glass fiber scaffold.

[0088] Example 2.5 - Provision of leather in vitro As described above, the provided collagen sheets were tanned according to a conventional chromium-based tanning method. The resulting in vitro leather samples (see Figure 7) had a thickness of 0.24 ± 0.05 mm after the tanning and drying process, as measured using a digital gauge. Results are shown as mean ± SD. Tensile stress tests were performed on the tanned cultured leather pieces.

[0089] Example 2.6 - Conclusion Cultured leather was obtained using a glass fiber scaffold. High cell viability was confirmed at the end of the culture. Cells and collagen were widely distributed in the scaffold, and the hydroxyproline content indicated that the scaffold promoted collagen secretion and deposition. The scaffold was tannable and exhibited good flexibility.

Claims

1. A method for providing collagen sheets: i) A process of providing a glass fiber sheet containing solid fibers; ii) The step of placing the glass fiber sheet inside the bioreactor; iii) A step of seeding fibroblasts onto the glass fiber sheet in the bioreactor; iv) A step of culturing the fibroblasts under conditions that facilitate collagen production to form a collagen sheet. Methods that include...

2. v) The method according to claim 1, further comprising the step of isolating the collagen sheet.

3. The method according to claim 1 or 2, wherein step i) includes pressurizing the glass fiber sheet.

4. The method according to any one of claims 1 to 3, wherein the fibroblasts are dermal fibroblasts, preferably bovine dermal fibroblasts.

5. The method according to any one of claims 1 to 4, wherein in step ii), a plurality of glass fiber sheets are placed in the bioreactor to form a multilayer scaffold.

6. - The glass fibers have an average diameter of about 1 to about 25 μm, preferably about 5 to about 15 μm; - The fiber density in the glass fiber sheet is approximately 1 to approximately 10 mg / cm³. ２ Preferably about 1.5 to about 8 mg / cm³ ２ And; - The gap size between fibers in the glass fiber sheet is about 10 to about 140 μm, preferably about 25 to about 125 μm; and / or - The glass fiber sheet has a thickness of approximately 20 to 2500 μm. The method according to any one of claims 1 to 5.

7. In step iii), about 1×10 ４ to about 1×10 ８ fibroblasts / cm ２ are seeded onto a glass fiber sheet, preferably about 1×10 ５ to about 1×10 ７ fibroblasts / cm ２ , more preferably about 5×10 ５ to about 5×10 ６ fibroblasts / cm ２ The method according to any one of claims 1 to 6, wherein the fibroblasts are seeded.

8. The method according to any one of claims 1 to 7, wherein the culture in step iv) is carried out in a DMEM / F-12 medium which optionally contains 2-20% serum, such as 10% fetal bovine serum, and optionally contains 2-phospho-L-ascorbic acid.

9. The method according to any one of claims 1 to 8, wherein the culture is carried out for about 2 to 8 weeks, preferably 2 to 6 weeks, more preferably 3 to 5 weeks or about 6 to 8 weeks.

10. The method according to any one of claims 1 to 9, wherein the culture is carried out under gentle stirring, for example using an orbital shaker.

11. The method according to any one of claims 1 to 10, wherein the glass fiber sheet is a chopped strand mat.

12. The use of glass fibers in the production of leather in vitro.

13. The glass fibers have an average diameter of approximately 1 to approximately 25 μm; and The fiber density in the glass fiber sheet is approximately 1 to approximately 10 mg / cm³. ２ And; The gap size between fibers in the glass fiber sheet is approximately 10 to approximately 140 μm; and The glass fiber sheet has a thickness of approximately 20 to 2500 μm. The use described in claim 12.

14. Collagen sheet containing glass fibers.

15. The collagen sheet according to claim 14, wherein the collagen sheet can be obtained by the method described in any one of claims 1 to 11.

16. The collagen sheet according to claim 14 or 15, wherein the glass fibers have an average diameter of about 1 to about 25 μm, and / or the gap size between the glass fibers is about 10 to about 140 μm.

17. The collagen sheet according to any one of claims 14 to 16, wherein the glass fibers are intersected and randomly overlapped.

18. A method for producing in vivo leather, comprising the step of tanning a collagen sheet according to any one of claims 14 to 17.

19. Ex vivo leather obtained by the method described in claim 18.

20. A consumer product comprising or consisting of the ex vivo leather described in claim 19.

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