Felted glass fibre scaffolds for cultivated leather

Abstract

The invention relates to the field of cell culture. The invention specifically relates to culturing of cells such as fibroblasts to obtain collagen sheets. It was found that glass fibre scaffolds can improve the characteristics of the process. The invention also relates to the collagen sheets, and to leather obtained by the tanning of such collagen sheets.

Description

[0001] Felted glass fibre scaffolds for cultivated leather

[0002] Field

[0003] The invention relates to the field of cell culture. The invention specifically relates to culturing of cells such as fibroblasts to obtain collagen sheets. It was found that glass fibre scaffolds can improve the characteristics of the process. The invention also relates to the collagen sheets, and to leather obtained by the tanning of such collagen sheets.

[0004] Background

[0005] Leather is used in many applications, including clothing, shoes, fashion accessories, upholstery, luggage, and automotive applications. It is generally made from animal hide. Historically these hides were utilized from animals hunted for sustenance, and leather was one readily accessible source of highly durable and functional fabric. With the advent of plastics, leather can be seen as a luxury good utilized mainly for fashion and luxury appeal. The current procurement of leather can be objectionable on both sustainability and ethical grounds. For instance, the carbon footprint of bovine leather can be much more significant than that of synthetic fabrics. Additionally, traditional leather requires the killing of animals, which can be seen as ethically objectionable.

[0006] Cultured leather is known in the art. It relies on cell and tissue engineering methods to create tissue that resembles sub-epidermal skin (dermis). WO2017193058 describes a method for method of preparing a cultured leather comprising culturing a plurality of layers, each layer comprising cells, wherein each layer of the plurality of layers is coupled to an adjacent layer by a nucleotide adhesion molecule. This requires specialised techniques for providing layers of cell material of sufficient thickness. No information is provided on cell seeding efficiency.

[0007] Jakab et al. (Materials Today Sustainability, 5 (2019) 100018, DOI:

[0008] 10.1016 / j.mtsust.2019.100018) describe in vitro production of collagen sheets for tanning. Their production requires the seeding of subsequent layers of cells on top of earlier layers. This requires repeated seeding of cells to achieve a collagen sheet of desired thickness, without control over the direction or boundaries of cell growth. WO2014201406 similarly requires the stacking of multiple layers of cells in successive rounds of culturing. No information is provided on cell seeding efficiency.

[0009] EP1589098 describes the use of collagen material as a scaffold for cell culture. The use of collagen waste material as a scaffold is described, which has an associated risk of contamination. Collagen sources are animal in nature, and collagen sources that are not waste material can reduce cost-effectiveness of the process.

[0010] Summary of the Invention

[0011] There is a need for improved methods for providing collagen sheets, particularly collagen sheets suitable for tanning. There is a need for improved in vitro leather. There is a need for improved collagen sheets with controlled properties. There is a need for improved scaffolds for use in cell culturing, particularly for use in the production of collagen sheets. There is a need for improved cell culturing or cell seeding methods for providing collagen sheets.

[0012] The invention provides a method for providing a collagen sheet, the method comprising the steps of: i) providing a felted glass fibre sheet; ii) placing the glass fibre sheet in a bioreactor; iii) seeding fibroblasts onto the glass fibre sheet in the bioreactor; iv) culturing the fibroblasts under conditions conducive to collagen production to form a collagen sheet. The method optionally further comprises the step of v) isolating the collagen sheet. Preferably step i) comprises autoclaving the felted glass fibre sheet. Preferably the fibroblasts are dermal fibroblasts, preferably bovine dermal fibroblasts.

[0013] In some embodiments in step ii) multiple felted glass fibre sheets are placed in the bioreactor to form a multi-layered scaffold. Preferably the glass fibres in the glass fibre sheet have an average diameter of from about 1 to about 25 pm, preferably of from about 5 to about 15 pm; and / or the fibre density in the glass fibre sheet is from about 1 to about 10 mg / cm2, preferably from about 1 .5 to about 8 mg / cm2; and / or the gap size between fibres in the glass fibre sheet is from about 10 to about 140 pm, preferably from about 25 to about 125 pm; and / or the glass fibre sheets have a thickness of about 20-2500 pm; and / or the glass fibre sheet is stitched.

[0014] In some embodiments in step iii) from about 1x104to about 1x108fibroblasts are seeded per cm2of glass fibre sheet, preferably from about 1x105to about 1x107fibroblasts per cm2, more preferably from about 5x105to about 5x106fibroblasts per cm2. Preferably the culturing in step iv) is performed in DMEM / F-12 medium that optionally comprises 2-20% serum such as 10% fetal bovine serum, and that optionally comprises 2-phospho-L-ascorbic acid. Preferably the culturing is performed for about 2 to 8 weeks, preferably 2 to 6 weeks, more preferably for about 3 to 5 weeks or for about 6 to 8 weeks.

[0015] Provided is the use of felted glass fibre sheets in the manufacture of in vitro leather. Also provided is a collagen sheet comprising felted glass fibres, preferably wherein the collagen sheet is obtainable by a method as described herein Also provided is a method for manufacturing in vitro leather, the method comprising the step of tanning a collagen sheet as described herein. Also provided is in vitro leather, or a consumer product comprising or consisting of in vitro leather, wherein the in vitro leather is obtainable by a method as described herein.

[0016] Detailed description

[0017] The inventors have surprisingly found that the use of glass fibre sheets as a scaffold for cell culture improves cell seeding and collagen production, and that these beneficial effects are particularly accessible when using felted glass fibre sheets, which can be conveniently handled and which have good flexibility. This allows the more efficient production of in vitro leather, and the resulting leather has attractive properties. Accordingly, the invention provides a method for culturing cells, the method comprising the steps of: i) providing a felted glass fibre sheet; ii) placing the glass fibre sheet in a bioreactor; iii) seeding cells, preferably fibroblasts, onto the glass fibre sheet in the bioreactor; iv) culturing the cells, preferably under conditions conducive to collagen production to form a collagen sheet.

[0018] Also provided is a method for culturing cells, the method comprising the steps of: i) providing a glass fibre sheet that has been treated with a coating agent that introduces an organic coating on the glass fibres; ii) placing the glass fibre sheet in a bioreactor; iii) seeding cells onto the glass fibre sheet in the bioreactor; iv) culturing the cells.

[0019] Also provided is a method for providing a collagen sheet, the method preferably comprising the steps of: i) providing a glass fibre sheet preferably comprising solid fibres; ii) placing the glass fibre sheet in a bioreactor; iii) seeding cells such as fibroblasts onto the glass fibre sheet in the bioreactor; iv) culturing the cells, preferably under conditions conducive to collagen production to form a collagen sheet; and optionally v) isolating the collagen sheet.

[0020] Such methods can be referred to herein as a method according to the invention. The collagen sheets produced by this method have a good collagen content, and can be tanned to produce in vitro leather of good quality. In preferred embodiments the steps are performed in numerical order. In preferred embodiments step v) is not optional. In other embodiments, step v) is not performed.

[0021] Provision of a glass fibre sheet

[0022] In step i) a glass fibre sheet is provided, and said sheet preferably comprises solid fibres. In preferred embodiments substantially all fibres of the glass fibre sheet are solid fibres. The glass fibre sheet preferably does not comprise sol-gel fibres, or fibres derived from a sol-gel process. The fibres are preferably melt-derived, wherein for example the required elements (such as silica, boron, aluminium, calcium, magnesium) are heated (1 100°C < AT < 1500°C) until molten, followed by extrusion such as pressure extrusion in single fibres. A suitable feed format can be 1 .5m x 20m (w x I). Preferred solid fibres are fused silica glass fibres. Preferably, the glass fibre comprises glass containing 5-70% SiO, 5-40% BO, and 1 -50% CaO in mass-% of oxide conversion as a glass composition. The glass fibres can comprise at least Si, B, and Ca, preferably at least Si, B, Ca, and Mg, or Si, B, Ca, and Al, even more preferably Si, B, Ca, Al, and Mg. Optionally P is also present.

[0023] In some embodiments, silica, boron, and calcium may each be present in the compositions in an amount of about 1 % to about 99%, based on the weight of the glass. In further embodiments, silica, boron, and calcium may each be present in the composition in 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 each be present in the composition in about 5 to about 10%, about 10 to about 15%, about 15 to about 20%, about 20 to about 25%, about 25 to about 30%, about 30 to about 35%, about 35 to about 40%, about 40 to about 45%, about 45 to about 50%, about 50 to about 55%, about 55 to about 60%, about 60 to about 65%, about 65 to about 70%, about 70 to about 75%, about 75 to about 80%, about 80 to about 85%, about 85 to about 90%, about 90 to about 95%, or about 95 to about 99%. Some embodiments may contain substantially each of silica, boron, calcium, aluminium, and magnesium, with only traces of other elements if any, preferably no other elements (outside of any required elements such as H or O) are present.

[0024] The glass fibres may further comprise one or more of a silicate, borosilicate, borate, or calcium, including CaO, P2O5, SiO2, and B2O3. Suitable glass types are widely known. Examples are A-glass, C-glass, D-glass, E-glass, R-glass, and S-glass. Most preferably the glass is E-glass, which is alumino-borosilicate glass preferably with less than 1 % (w / w) of alkali oxides. The glass is preferably alkali-free, which in this context refers to an alkali oxide content of less than 1 % (w / w). The glass is preferably acid / base resistant.

[0025] The form or shape of the sheet is not essential. It can be convenient to have a glass fibre sheet that is roughly the same shape as the interior space that is available in the intended bioreactor. Most glass fibre sheets can be cut to size using scissors. A skilled person can select an appropriate size. Preferred glass fibre sheets for use as scaffolds are rectangular or substantially rectangular, such as square. Preferably, the sides of the glass fibre sheet are about 0.1 -50 cm, more preferably about 1-40 cm, still more preferably about 5-30 cm, still more preferably 10-25 cm. For smaller assays, areas of 1 cm2or less can be suitable. For the eventual production of in vitro leather, areas of 100 cm2(10x10 cm) or 625 cm2(25x25 cm) or most preferably 900 cm2(30x30 cm) can be suitable. Other preferred scaffold sizes are 4x4 cm and 10x10 cm. Alternately, for use in larger bioreactors, areas of over 10000 cm2can be considered, such as 20000 cm2(100x200 cm). For sheets having areas of over 10000 cm2it is preferred that the thickness of the sheet is at least 1500 pm. As follows from the dimensions of 10x10 cm leading to a glass fibre sheet with an area of 100 cm2, area as used in this context is the measure of the entire sheet’s size on a surface.

[0026] In preferred embodiments the glass fibre sheet has an area of at least 8 cm2such as about 10 to about 20.000 cm2, preferably of at least 10 cm2such as about 15 to about 10.000 cm2, more preferably of about 100 to 1000 cm2, such as about 500 to 900 cm2. In other preferred embodiments the glass fibre sheet has an area of at least 90 cm2, more preferably at least 100 cm2, still more preferably at least 500 cm2, even more preferably at least 600 cm2, still more preferably at least 625 cm2, still more preferably at least 750 cm2, still more preferably at least 850 cm2, and even more preferably at least 900 cm2.

[0027] The glass fibre sheet can be of any suitable type. Examples of glass fibre sheets are woven fabrics, knit fabrics, unidirectional fabrics, felted glass fibre sheets, or chopped strand mats. Preferred types are chopped strand mats and felted glass fibre sheets. In some preferred embodiments the glass fibre sheet is a chopped strand mat. In some preferred embodiments the glass fibre sheet is a felted glass fibre sheet.

[0028] It was found that chopped strand mats offer an attractive spatial distribution of fibres for culturing, and accordingly in some embodiments the glass fibre sheet is preferably a chopped strand mat. In chopped strand mats glass fibres lay randomly across each other. Chopped strand mats can further comprise a binder, which in preferred embodiments is washed out of the sheet prior to cell culture. Chopped strand mats are preferably isotropic. In general, fibres in chopped strand mats are substantially of equal length. Chopped strand mats are widely available from commercial sources (see for instance the examples). Suitable commercially sourced mats are chopped strand mats, such as chopped strand mats for marine use, for roofing, or for use as battery separator, more preferably comprising GRP resin (which is a commonly used polyester resin).

[0029] For reference, chopped strand mats are often found in fibreglass that can be used for repair works, for instance on boat hulls. Such mats generally consist of glass fibres and a binder. Mats are typically processed using a hand lay-up technique, where sheets of material are placed on a mold and brushed with resin. The material conforms to different shapes when wetted, and the binder conveniently dissolves in the resin, leaving the glass fibres. After the resin cures, a hardened product remains. This makes commercially available fibreglass chopped strand mats convenient sources of glass fibre sheets for use in the invention - the non-glass component can be easily washed away and a glass fibre sheet remains. In preferred embodiments the glass fibre sheet is a washed fibreglass sheet. In preferred embodiments the glass fibre sheet essentially consists or consists of glass fibres.

[0030] It was found that felted glass fibre sheets offer good spatial distribution of fibres for culturing, while also offering an attractive material flexibility during handling, and accordingly in some embodiments the glass fibre sheet is preferably a felted glass fibre sheet. In a felted glass fibre sheet glass fibres lay randomly across each other. Glass fibres in a felted glass fibre sheets are preferably isotropic. In general, fibres in a felted glass fibre sheet are substantially of equal length. Preferably the length is about 10-200 mm, more preferably about 20-150 mm, still more preferably about 30-100 mm. Felted glass fibre sheets are widely available from commercial sources (see for instance the examples). Suitable commercially sourced felted mats can be mats for automotive use, for marine use such as shipbuilding (walls, ceilings, piping insulation), for building construction building (heat insulation), for applications in household goods (heat insulation), for applications in power plants (turbine insulation), et cetera.

[0031] Felting is a well-known technique, and a skilled person can recognise a felted mat. Various forms of felting exist. For instance, felting can be a process where one or more felting members such as a needle can be repeatedly inserted into a glass fibre sheet to promote entanglement of the fibres, resulting in a felted glass fibre sheet. In addition to this needle-felting, sheets can also be pressed, which compacts the felt and forces the fibres into closer relationship and thereby produces a more durable felt. It is highly preferred that felted glass fibre sheets do not comprise a bonding agent or binder, such as a resin.

[0032] Fibreglass sheets, particularly felted fibreglass sheets, can be reinforced by stitching. Such a sheet is a stitched sheet. Stitch-bonding is a nonwoven construction where the sheet is formed by stitching or knitting the fiberglass to form a sheet with the appearance of a knit fabric. Such a sheet can be referred to as a stitch-bonded sheet, which is a preferred stitched sheet. Stitches are preferably substantially parallel runs of stitching, more preferably parallel runs of stitching. Stitching can be performed by a sewing machine or a knitting machine. Stitching is performed using a yarn, and suitable yarns are commonly known. Polyester yarn was found to be particularly suitable. Such yarns can be referred to as stitching yarns.

[0033] In preferred embodiments the felted glass fibre sheet is a washed felted glass fibre sheet. In preferred embodiments the felted glass fibre sheet essentially consists or consists of glass fibres with optional stitching yarn. In preferred embodiments the felted glass fibre sheet is needle-felted. In preferred embodiments the felted glass fibre sheet is stitched. In preferred embodiments the felted glass fibre sheet is stitched with a polyester yarn. In preferred embodiments the felted glass fibre sheet is stitch-bonded. In preferred embodiments the felted glass fibre sheet is stitch-bonded with a polyester yarn. In preferred embodiments the felted glass fibre sheet is needle-felted and stitched. In preferred embodiments the felted glass fibre sheet is needle-felted and stitch-bonded. In preferred embodiments the felted glass fibre sheet is needle-felted and stitch-bonded with a polyester yarn.

[0034] It was found that cell seeding and proliferation was optimal for glass fibres of certain diameters, at certain density and with certain inter-fibre distances. In preferred embodiments is provided the method according to the invention, wherein the glass fibres have an average diameter of from about 1 to about 25 pm, preferably of from about 5 to about 15 pm, more preferably of from about 6 to about 14 pm; and / or the fibre density in the glass fibre sheet is from about 1 to about 30 mg / cm2, preferably from about 1 .5 to about 25 mg / cm2, more preferably from about 2 to about 20 mg / cm2; in some embodiments the density is about 20-25 mg / cm2; In some embodiments the density is about 1 .5 to about 8 mg / cm2; and / or the gap size between fibres in the glass fibre sheet is from about 10 to about 140 pm, preferably from about 25 to about 125 pm; and / or the glass fibre sheets have a thickness of about 10-3000, preferably 15-2000, more preferably 20-1000 pm; or the glass fibre sheets have a thickness of about 20-2500 pm; and / or the glass fibre sheet is stitched, preferably stich-bonded.

[0035] More preferably all four of the above apply. Glass fibre diameter is preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 pm. Glass fibre diameter is preferably at most 25, 24, 23, 22 ,21 , 20, 19, 18, 17, 16, or 15 pm. Preferably the diameter is from about 3 to about 20 pm, more preferably from about 7 to about 18 pm, still more preferably from about 10 to about 14 pm. Fibre diameter can be measured using any known technique, a suitable example is by scanning electron microscopy. The fibre diameter is preferably the radius of the cylinder that best represents the fibre - it is generally the smallest dimension of the fibre. For felted glass fibre sheets it can further be preferably that the fibre has a diameter of 4 to 16, preferably 5 to 15, more preferably 6 to 14 pm, even more preferably 7 to 13, still more preferably 8 to 12, even more preferably 9 to 11 pm. Fibre 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 / cm2. Fibre density is preferably at most 10, 9.5, 8, 7.5, 7, 6.5, 6.4, 6.3, 6.2, 6.1 , 6, 5.9, or 5.8 mg / cm2. A thicker sheet generally has a higher density in mg / cm2. Further preferred densities are about 2 to about 7 mg / cm2, or about 3.5 to about 6.5 mg / cm2. Fibre density can be determined by conventional weighing of a fragment of known dimensions. In some embodiments the density is higher, for instance at most 30 mg / cm2, such as about 15-30 mg / cm2, preferably 17-27 mg / cm2, more preferably about 20-25 mg / cm2. These values are particularly relevant for non-felted glass fibre sheets. For felted glass fibre sheets, mass per unit area is generally somewhat higher, for instance about 10-500 mg / cm2, more preferably about 30-450 mg / cm2, even more preferably about 35-300 mg / cm2.

[0036] The gap size between fibres can influence the dimensions available to cells when they are mobile, or during proliferation. It is known that the dimensions of a surrounding matrix can influence the behaviour of cells, and thus can impact collagen output. Good results were obtained with glass fibre sheets having a gap size between fibres from about 10 to about 140 pm, preferably about 15 to about 130 pm, more preferably about 20 to about 120 pm, more preferably about 30 to about 110 pm, still more preferably about 40 to about 100 pm, more preferably about 50 to about 90 pm, more preferably about 60 to about 80 pm. These values are particularly relevant for non-felted glass fibre sheets. Gap size can be determined by scanning electron microscopy.

[0037] When not felted, the glass fibre sheet preferably has a thickness of not more than 3000 pm. When thicker scaffolds are desired, multiple sheets can be placed on top of one another. For mechanical robustness, the sheets preferably do not have a thickness of less than 20 pm. The sheets can have a thickness of 30-2000 pm, preferably 40-1700 pm, preferably 45-1500 pm, more preferably 50-800 pm, still more preferably 100-750 pm, still more preferably 150-700, 200-650, 250-600, 300-550, 350-500, or most preferably about 400-450 pm. In preferred embodiments the thickness is 1500 pm. Thickness can be determined by scanning electron microscopy or by using high precision callipers. Felted glass fibre sheets can have a thickness as described above, but can also be thicker. For instance, felted glass fibre sheets can have a thickness in between 0.5-30 mm, preferably in between 1-10 mm, more preferably in between 1.5-8 mm, even more preferably in between 2-7 mm, or 2-6 mm, or 2-5 mm, or 2-4 mm, most preferably of about 2-3 mm.

[0038] Increased cell seeding and collagen content were obtained with glass fibre sheets having a coating. In preferred embodiments the glass fibre sheet has been treated with a coating agent that introduces an organic coating on the glass fibres. Coating agents are a well-known class of compounds, and coating chemistry, particularly for glass material, is an established field.

[0039] It is not preferred that an organic coating refers to a proteinaceous coating or to an opsonisation or a deposition of extracellular material. An organic coating in this context is a coating of organic moieties, preferably of small organic moieties such as short alkyl chains bearing at least one heteroatom. The coating is preferably homogeneous, as can be studied using microscopy or FTIR techniques. The coating preferably consists of only a single type of moiety. The organic coating is preferably covalently linked to the glass fibres. Covalent linkage can be confirmed using ATR techniques such as ATR-FTIR.

[0040] In preferred embodiments the organic coating comprises amines or thiols. Thiols are generally of formula -SH and can also be referred to as mercaptans. Thiols are preferably free thiols. Amines are preferably of general formula -NH2 and can also be quaternary amines as later described herein. Glass fibres coated with primary amines are particularly useful as a material that can be converted into glass fibres coated with quaternary amines.

[0041] It was found that the invention could be advantageously practiced when the coating agent is a small molecule. Such a small molecule is preferably organic and preferably has a molecular weight of no more than 1000 Dalton, more preferably of no more than 500 Dalton.

[0042] Silanization is the attachment of an organosilyl group to chemical species such as glass. Silanization often refers to conversion of a silanol-terminated surface such as glass to an alkylsiloxy-terminated surface. This conversion can introduce new chemical moieties to the treated surface, and is often used to modify the surface properties of glass. Silanizing agents often have the formula (RO)3-n(CH3)nSiR' or R'SiCh, where R is an alkyl chain, preferably a short alkyl chain such as methyl or ethyl, n is 1 or 2, and R' is an alkyl chain or a functionalized alkyl group. Preferred agents are of the formula (RO)3SiR', where R is methyl, ethyl, or propyl, preferably methyl or ethyl, and R' is -CH2(CH2)m-X, wherein m is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 , preferably 1 , 2, 3, 4, 5, 6, or 7, more preferably 1 , 2, or 3, most preferably 2, and X is -SH or -NH2 or -N+(CH3)3, preferably -SH or -NH2. In preferred embodiments the coating agent is a silanization agent, preferably a trialkoxysilane such as a triethoxysilane or a trimethoxysilane. A preferred silane is a triethoxysilane or a trimethoxysilane. A preferred aminoalkylsilane is a trimethoxysilane or a triethoxysilane, preferably a triethoxysilane. A preferred mercaptoalkylsilane is a trimethoxysilane or a trmethoxysilane, preferably a trimethoxysilane. The term silane generally refers to the -Si(RO)3 moiety as comprised in (RO)3SiR' defined above.

[0043] In further preferred embodiments the coating agent is an aminoalkylsilane such as a (3- aminopropyl)silane, a mercaptoalkylsilane such as a (3-mercaptopropyl)silane, a glycidyloxyalkylsilane such as a (3-glycidyloxypropyl)silane, a cyanoalkylsilane such as a 2- (cyanoethyl)silane, or an isocyanatoalkylsilane such as a (3-isocyanatopropyl)silane; preferably an aminoalkylsilane or a mercaptoalkylsilane; more preferably a (3-aminopropyl)silane or a (3-mercaptopropyl)silane; most preferably a (3-aminopropyl)silane such as (3-aminopropyl)triethoxysilane.

[0044] The coating agent preferably introduces thiols (-SH) or amines. Amines can be primary amines or can be further substituted amines. Preferred amines are primary amines (-NH2) and quaternary amines (-N+(alkyl)3), which are herein sometimes referred to as QA. The alkyl moieties in quaternary amines are preferably identical, and are preferably methyl, ethyl, or propyl, most preferably methyl. Quaternary amines are charged and can have any suitable counterion, preferably a physiologically acceptable counterion such as chloride. The selection of a suitable counterion is trivial and can be performed by any skilled person. Quaternary amines can be conveniently obtained from primary amines using haloalkyl. In preferred embodiments, the coating agent introduces organic amines, wherein the organic amines are subsequently quaternized, preferably using a halomethane such as iodomethane. An amine- functionalized glass fibre sheet, such as one formed by APTES treatment of glass fibre, can be contacted with a solution of haloalkyl in a suitable organic solvent, such as in a lower alcohol such as methanol, ethanol, or propanol, preferably ethanol. A convenient way of contacting is to submerge the glass fibre in the organic solvent comprising the haloalkyl. The contacting is preferably performed in the absence of light, and the contacting can be of any suitable duration, such as from 1-96 hours, or 12-84 hours, preferably 18-76 hours. After this contacting the glass fibre is preferably washed such as by using PBS, for instance washed three times.

[0045] In some embodiments in step i) a glass fibre sheet that has been treated with a coating agent that introduces an organic coating on the glass fibres is provided. In other words, in step i) a glass fibre sheet having an organic coating on the glass fibres is provided. The organic coating is introduced by a coating agent, and this can be part of step i) but this can also have occurred prior to the method. Introducing an organic coating on the glass fibres can be achieved by treating the glass fibre sheet with a coating agent. In preferred embodiments this comprises contacting the glass fibre sheet with a solution comprising 0.05-5 vol.-% of the coating agent, preferably 0.1 -1.5 vol.-%, more preferably about 0.5-1 .2 vol.-%. In general, as used herein, when a percentage is mentioned without further specification, it is intended to be vol.-%.

[0046] The solution is preferably an organic solution, more preferably a protic organic solution. In preferred embodiments the solution consists or essentially consists of the coating agent and an organic solvent. Preferred organic solvents are lower alcohols such as methanol, ethanol, propanol, butanol, and isomers thereof, preferably methanol or ethanol, most preferably ethanol. In some embodiments the solution comprises 0.05-10 vol.-% of the coating agent, preferably 0.1 -7.5 vol.- %, more preferably about 0.2-5 vol.-% , more preferably about 0.3-4 vol.-% , more preferably about 0.4-3 vol.-%, still more preferably about 0.5-2.5 vol.-%, most preferably about 0.75-1 .25 vol.-%.

[0047] Treating the glass fibre sheet with a coating agent is preferably performed in a fume hood. Preferably, the glass fibre sheet to be treated is a dry glass fibre sheet. A glass fibre sheet can be conveniently dried by dehydration in acetone, for instance at about 20 °C, for instance for about 2- 12 minutes such as 5 minutes. The acetone can be evaporated at room temperature. The glass fibre sheet is then contacted with the solution comprising the coating agent, wherein the contacting is preferably submerging of the glass fibre sheet. The contacting can be for any suitable amount of time, such as 1-120 minutes, preferably 5-60, more preferably 10-30 minutes, most preferably 15- 25 minutes such as about 20 minutes. The contacting can be at any suitable temperature such as about 20 °C. The contacting can also be at a temperature above ambient temperature, for instance at a temperature of about 40-80 °C, preferably about 50-70 °C, such as about 65 to 68 °C. After this contacting the glass fibre sheet is a coated glass fibre sheet, or a glass fibre sheet that has an organic coating. This coated glass fibre sheet can be washed, for instance washed 3 to 5 times such as 4 times, in an aqueous solution such as a buffer, for instance PBS. Optionally prior to washing or as part of the first wash, coated glass fibre sheets can be incubated in an aqueous solution such as PBS to allow unused coating agent to hydrolyse. Coated sheets can be kept in a desiccator until their further use.

[0048] The glass fibre sheet can be additionally pre-treated before cells such as fibroblasts are seeded on it. This pre-treatment can be prior to placement in the bioreactor, or it can be performed in the bioreactor. The latter is convenient when the pre-treatment comprises washing steps because a bioreactor can often conveniently be filled with or drained of various liquids. Pretreatment before placement in the bioreactor is convenient when it comprises steps such as heating the sheet, or autoclaving it. Preferred glass fibre sheets are pre-treated.

[0049] Suitable pre-treatments can comprise steps of washing, heating, drying, or prewetting. Washings can be 1 , 2, 3, 4, 5, or more washes. Washes can be performed for 1 , 5, 10, 15, 20, 25, 30 minutes or more. Washings can be performed in any suitable medium, such as water, saline buffer, or organic solvents. 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 can be mixed with water. A preferred washing medium is 70% C1 -C4 alcohol in water, more preferably ethanol. Washing medium can be decanted, drained, or aspirated, preferably aspirated.

[0050] Heating can be to 37 °C, to 50 or 55 °C, to 80 °C, to 100 °C, to 120 °C, or more. Drying can be in a vacuum or preferably under an air flow, for instance under a nitrogen flow. Drying can be performed for 15, 30, 45, 60, 90, 120, 150, 180, 210 minute or more. Drying under air flow can conveniently be performed in a flow cabinet. Drying is preferably performed when all washing steps have concluded. Autoclaving can be performed under any suitable condition, for instance using PBS (preferably at a pH of about 7-8 such as about 7.2). Good results were obtained when the glass fibre sheet was autoclaved as part of pre-treatment, accordingly in preferred embodiments step i) comprises autoclaving the glass fibre sheet, preferably comprises autoclaving the felted glass fibre sheet. Preferably this is autoclaving in PBS, more preferably at a pH of 7-8, even more preferably of about 12.

[0051] Prewetting was found to improve performance of the glass fibre sheet, particularly when prewetting was performed with a medium comprising proteins. This makes cell culture media excellent prewetting media. Prewetting is preferably performed using a cell culture medium, such as DMEM or SF medium. The medium can be complete medium (for instance DMEM with FBS such as 10% FBS and optionally with antibiotics such as Penicillin-Streptomycin-Amphotericin B, also known as P / S / A). Prewetting can be for about 0.5-48 hours, preferably about 1 -36 hours, more preferably about 2-24 hours, more preferably about 4-20 hours, more preferably about 6-18 hours, more preferably about 8-16 hours, more preferably about 10-14 hours, such as about 12 hours. Prewetting is concluded by removal of the prewetting medium, optionally followed by washing steps as described above. Prewetting is most effective when it is performed with sterile medium under sterile conditions, which can conveniently be the case when prewetting is performed after autoclaving. Improved cell seeding was observed when prewetting was performed using cell culture medium, preferably complete cell culture medium.

[0052] An examples of a pre-treatment is three washes with 70% C1 -C4 alcohol (preferably ethanol) in water, followed by drying. A more preferred example is autoclaving of the glass fibre sheet followed by three washes with 70% C1 -C4 alcohol (preferably ethanol) in water, followed by drying. These examples are preferably followed by prewetting in cell culture medium.

[0053] In step ii) the glass fibre sheet is placed in a bioreactor. This can be any bioreactor suitable for culturing cells such as fibroblasts, and a skilled person is able to select an appropriate one. Examples are culture flasks such as T-75 flasks, petri dishes, or multiwell-plates such as 6-well plates or 24-well plates or 48-well plates. It is convenient when the bioreactor can be opened to facilitate eventual isolation of the collagen sheet. When disposable bioreactors are used, such as a flask, the flask can also be cut open. In general, the bioreactor can be a cell culture dish, flask, roller, chamber (e.g., rotating chamber) or the like. Dishes are preferred, particularly glass dishes.

[0054] Bioreactors are preferably low-adhesion bioreactors, such as siliconized ones. Bioreactors are preferably sterilised before the glass fibre sheet is placed. After the sheet is placed, the scaffold inside the bioreactor can be sterilised, for instance by an ethanol wash such as 70% ethanol incubation for 10-40 minutes, preferably 20-30 minutes. Prior to seeding of cells, the bioreactor and / or the glass fibre sheet are preferably washed with saline such as with PBS.

[0055] In other embodiments the bioreactor is a perfusion bioreactor. Perfusion bioreactors are commonly known. This type of bioreactor employs a perfusion process that involves a continuous culturing method in which cells are retained in the bioreactor and typically cultured over what are often relatively long periods of time. In particular, cells are usually kept in a cell retention compartment inside of the bioreactor while culture medium is continuously exchanged. This way, fresh medium replenishes the nutrient(s) delivered to the cells while cellular waste and medium that has been depleted of nutrients are removed. The exchange is typically performed at the same rate for the introduction of fresh media and the removal of waste product(s). By continuously removing spent media and replacing it with new or reconditioned media, nutrient levels are maintained for improved growing conditions and cell waste product is removed to avoid toxicity.

[0056] In some embodiments in step ii) multiple glass fibre sheets are placed in the bioreactor to form a multi-layered scaffold. Preferably multiple felted glass fibre sheets are placed in the bioreactor to form a multi-layered scaffold. Preferably when multiple glass fibre sheets are used, the sheets are of substantially equal size, or are of equal size. In some embodiments two sheets are used. In some embodiments three sheets are used. In some embodiments, 4, 5, 6, 7, or 8 sheets are used. As discussed above, the glass fibre sheet can be placed in a bioreactor prior to any pre-treatment. Steps i) and ii) can be performed simultaneously, for instance through the provision of a bioreactor that already comprises a glass fibre sheet. In some embodiments the felted glass fibre sheet is placed in a bioreactor as described herein, and the bioreactor is sterilised before the sheet is placed. After the sheet is placed, the sheet can be sterilised inside the bioreactor, for instance by an ethanol wash such as 70% ethanol incubation for 10-40 minutes, preferably 20-30 minutes. Prior to seeding of cells, the bioreactor and / or the sheet are preferably washed with saline such as with PBS. Alternately, the sheet is placed inside the bioreactor after which both are sterilised simultaneously. Alternately, the sheet and the bioreactor are sterilised separately before the sheet is placed inside the bioreactor.

[0057] In preferred embodiments the bioreactor is sterilised prior to step iii). Sterilisation can be performed using any known suitable method, such as rinsing with organic solvents such as ethanol, or heat treatment. Heat treatment is preferred, and can comprise passing steam through the bioreactor, or autoclaving the bioreactor. Advantageously, the bioreactor can be heat treated in its entirety, either in its empty state, or while already comprising the glass fibre sheet. It was found that certain materials such as stainless steel or PEEK (polyetheretherketone) were particularly suitable for forming part or all of the bioreactor, as these materials were particularly suitable for heat treatment. Accordingly a preferred bioreactor is a bioreactor that is stable to heat treatment, such as a bioreactor that comprises or substantially consists of stainless steel or PEEK.

[0058] Seeding and culturing of cells

[0059] In steps iii) and iv), cells are cultured on the glass fibre sheet in the bioreactor. The cells excrete collagen, forming a collagen sheet suitable for the production of in vitro leather, for instance through tanning. Conventional animal hide is formed primarily of collagen, a fibrous protein. Collagen is a generic term for a family of at least 28 distinct collagen types; animal skin is typically type 1 collagen, and thus a preferred collagen is type 1 collagen. Other types of collagen do exist and may also be used in forming leather. Collagens are characterized by a repeating triplet of amino acids, -(Gly-X-Y)n-, so that approximately one-third of the amino acid residues in collagen are glycine. X is often proline and Y is often hydroxyproline. Structurally, collagen may consist of twined triple units of peptide chains of differing lengths. Different animals and thus different cells may produce different amino acid compositions of the collagen, which may result in different properties. Collagen fibre monomers may be produced from alpha-chains of about 1050 amino acids long, so that the triple helix takes the form of a rod of about 300 nm long, with a diameter of 1 .5 nm. In the production of extracellular matrix by cells such as fibroblasts, triple helix monomers may be synthesized and the monomers may self-assemble into a fibrous form. These triple helices may be held together by salt links, hydrogen bonding, hydrophobic bonding, and covalent bonding. Triple helices can be bound together in bundles called fibrils, and fibril bundles come together to create fibres. Fibres typically divide and join with each other throughout a layer of skin. Variations of the crosslinking or linking may provide strength to the material.

[0060] In step iii), cells are seeded onto the glass fibre sheet. The cells are preferably animal cells, more preferably mammalian or reptile cells, most preferably mammalian cells such as from cows (bovine), sheep, goats, equines, buffalo, pigs, and aquatic mammals such as seals, or reptile cells such as snakes and alligators. Good results were obtained using bovine cells. The cell is preferably a collagen-releasing cell, more preferably a fibroblast. A mixture of different cells may also be used. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, and it produces the structural framework (stroma) for animal tissues. Fibroblasts are the most common cells of connective tissue in animals. Preferred fibroblasts are dermal fibroblasts, more preferably bovine dermal fibroblasts.

[0061] In some preferred embodiments, cells are primary cells, preferably fibroblasts are primary fibroblasts. Cells may be derived from tissue extracts / explants, or manipulated (transgenic) cell lines, or any variation thereof. In some variations, the cell may be grown to complete confluence (e.g., 100% confluence), in which cells are inhibited from further growth but may continue to produce or be stimulated to produce and release collagen. In some variations, the cells may not be grown to complete confluence, (e.g., approximately 99% confluence, 95 % confluence, 90% confluence, 85% confluence, 80% confluence, etc.). Cells may be cultured until greater than 80% confluence, greater than 85% confluence, greater than 90% confluence, greater than 95% confluence and / or just under full (100%) confluence. Primary fibroblasts can be used after initial expansion, preferably after about 3 to 10 passages, more preferably about 4 to 9 passages, even more preferably about 5 to 8 passages, such as after 6 passages (thus using P6 cells).

[0062] Before seeding, cells can be proliferated in any suitable culturing medium. Preferred media are DMEM or SF media. Media can be complete (generally comprising 10% serum), or serum-free, or comprise 5% serum. Preferably, media for proliferation prior to seeding are serum-free or comprise 5% serum. A preferred medium for proliferation prior to seeding is 50% DMEM such as DMEM-F12 + 10% serum such as FBS + 1 % antibiotics such as P / S / A, combined with 50% serum- free proliferation media based on DMEM such as DMEM-F12, supplemented with 1 -5 such as about 2.5 mg / mL protein such as albumin, 1 -10 such as 5 pg / mL hormone such as insulin, 2-10 such as about 5.5 pg / mL iron source such as an iron binding protein such as transferrin, 2-12 such as 6.7 pg / mL trace elements such as selenium, 100-500 such as about 250 nM vitamin such as vitamin C, preferably 2-phospho-L-ascorbic acid, and 1 -100 ng / mL growth factors such as 10 ng / mL basic fibroblast growth factor (bFGF) and 50 ng / mL platelet-derived growth factor (PDGF), and 0.02-0.5 mM such as 0.1 mM amino acid such as glutamine. This serum-free proliferation medium can also be used by itself. Another preferred medium for proliferation prior to seeding is FBS medium. Another preferred medium for proliferation prior to seeding is SF medium. In any medium described herein, as an antibiotic, gentamicin can be used, for instance 50 pg / mL gentamicin.

[0063] Cells can be seeded at any suitable density. Good results were achieved with 1 x 106cells I cm2. Accordingly, cells are preferably seeded at a density from about 1x104to about 1x108cells per cm2of glass fibre sheet, preferably from about 1x105to about 1x107cells per cm2, more preferably from about 5x105to about 5x106cells per cm2. In preferred embodiments is provided the method, wherein in step iii) from about 1x104to about 1x108fibroblasts are seeded per cm2of glass fibre sheet, preferably from about 1x105to about 1x107fibroblasts per cm2, more preferably from about 5x105to about 5x106fibroblasts per cm2. In other embodiments seeding density is from about 5x105to about 1x107, most preferably from about 1x106to about 5x106cells per cm2. Alternately, the same good results were achieved with 4.5x104cells / mm3. Accordingly, cells are preferably seeded at a density from about 1x104to about 1x105cells per mm3of glass fibre sheet, preferably from about 2.5x104to about 4.5x104cells per mm3.

[0064] Other preferred seeding densities were identified using a model square scaffold having an area of 2.5 cm2(being 25 mm3of glass fibre in this screening test). Preferred densities were found to be 2.5x107cells / cm3to 1x108cells / cm3, preferably 3.5x107to 6x107cells / cm3, such as about 4.5x107cells / cm3, which gave the best results, particularly for seeding on a scaffold where only a single seeding was performed. Good results were also obtained for a larger scaffold (15 cm3), where multiple seedings such as 4 seedings were performed. Good cell density was found to be in line with the screening test described above.

[0065] Cells can be seeded from a concentrated suspension. Cells can be seeded by dropping a volume of cell suspension in the centre of the scaffold. Cells can also be seeded via multiple drops, which can be spatially distributed over the scaffold. After seeding, the cells can be allowed to attach to the glass fibre sheet prior to the addition of additional culturing medium. This can be for 1 -3 hours, preferably for 1 .5-2 hours.

[0066] Multiple seedings can be performed to improve cell density. In some embodiments 2, 3, 4, 5, or 6 seedings are performed, preferably 2, 3, 4, or 5 seedings, more preferably 3 or 4 seedings such as 4 seedings. Further seedings can be performed identically to the first seeding. Seedings are preferably separated by about 1 -4 days of culturing, more preferably by 2-3 days of culturing. This allows the original cells to attach. The ratio between attached cells and suspended cells can be referred to as the seeding efficiency. This is preferably determined 24h after seeding. During the seeding process, culturing is preferably under static conditions - not under gentle agitation. For felted glass fibre sheets as scaffolds, preferably 1 or 2 seedings are performed. In some embodiments 2 seedings are performed. In some embodiments 1 seeding is performed.

[0067] In step iv) the cells are cultured under conditions conducive to collagen production to form a collagen sheet. A skilled person knows how to culture cells such as fibroblasts. Generally, cells are cultured at 35-40 °C, preferably at about 37 °C. Generally, cells are cultured under a controlled CO2 atmosphere, such as 3-8% CO2, preferably at about 5% CO2. Preferably cells are cultured in a humidified environment. Conventional cell incubators can be used to provide such conditions. Media are preferably changed every 2-3 days. Culturing under perfusion was found to lead to attractive results, and is therefore preferred.

[0068] In preferred embodiments, the culturing is performed for about 1 to 6 or 2 to 6 weeks, preferably for about 2 to 5 weeks, more preferably for about 3 to 5 weeks. It was found that after about 35 days the collagen sheet was suitable for isolation, and accordingly culturing can be for about 30 to about 40 days. In some embodiments, the culturing is performed for about 2 to 3 weeks, which was found suitable for development purposes. Cells can be cultured in any suitable medium. When glass fibre sheets were prewetted, the cells are preferably cultured in the same medium as was used for prewetting. DMEM and SF were found to be very suitable for production of collagen, of which DMEM was preferred. Preferably the culturing in step iv) is performed in DMEM / F-12 medium that optionally comprises 2-20% serum such as 10% fetal bovine serum, and that optionally comprises 2-phospho-L-ascorbic acid. In some embodiments the culturing in step iv) is performed in medium that comprises 2-20% serum such as 10% fetal bovine serum, and that optionally comprises 2-phospho-L-ascorbic acid. In some embodiments the culturing in step iv) is performed in medium that comprises 2-phospho-L-ascorbic acid.

[0069] When medium comprises 2-20% serum, it preferably comprises 4-18%, more preferably 5- 16%, more preferably 7-14%, still more preferably 8-12%, still more preferably 9-1 1 %, most preferably 10% serum. A skilled person can select a suitable serum, examples are platelet lysate such as human platelet lysate, fetal calf serum, horse serum, and newborn calf serum. Fetal bovine serum is most preferred.

[0070] 2-phospho-L-ascorbic acid was found to promote collagen production. When medium comprises 2-phospho-L-ascorbic acid, it preferably comprises 20-2000 pM, more preferably 50- 1500 pM, still more preferably 100-1250 pM, still more preferably 150-1000 pM. Good results were obtained with 200-750 pM, for instance with 250-500 pM.

[0071] Culturing can be performed under static conditions, without agitation of the bioreactor, the glass fibre scaffold, or the cell culture medium. The culturing can also be performed under gentle agitation, such as using an orbital shaker. The orbital shaker can be set to a gentle setting, such as 2-50 rpm, preferably 5-35 rpm such as 10 rpm. Gentle agitation generally comprises a shear stress that ranges between 0.01-5 Pa or 0.1-50 dyne / cm2. In some embodiments, shear stress ranges between 0.1-3 Pa or 1-40 dyne / cm2. In some embodiments, shear stress ranges between 0.5-2 Pa or 5-30 dyne / cm2. Preferably, shear stress ranges between 1-1.5 Pa or 10-20 dyne / cm2. In some embodiments, culturing is first performed under static conditions, followed by culturing under gentle agitation. Preferably, the first 10-40%, more preferably 15-30, such as 20% of total culturing duration is performed under static conditions with the remainder under gentle agitation. Alternately, seeding is performed under static conditions, and subsequent culturing is performed under gentle agitation.

[0072] Collagen sheets and their use

[0073] In preferred embodiments the culturing of step iv) is under conditions conducive to collagen production to form a collagen sheet; and wherein the method optionally further comprises the step of: v) isolating the collagen sheet.

[0074] In step v) the collagen sheet that has formed is isolated. The sheet was found to retain its shape, while collagen sheets cultured without glass fibre sheets were found to contract when removed from the bioreactor. Thus the invention provides a means for obtaining form-fast collagen sheets by using a readily available material as a scaffold. The collagen sheet can be fixed before or after its isolation. Means for fixing a tissue are well-known, and a skilled person can select a suitable method. For instance, the collagen sheet can be fixed using formaldehyde such as 4% paraformaldehyde in water. The collagen sheet can be isolated using any suitable means, such as manually, with a spatula, with a scraper, or preferably using tweezers.

[0075] The invention provides a collagen sheet comprising glass fibre, wherein the glass fibre preferably has an organic coating. Also provided is a collagen sheet comprising felted glass fibre. This collagen sheet is preferably obtainable by a method according to the invention. Herein, the collagen sheet can comprise up to 50 wt.-% glass fibre, preferably 0.5-40 wt.-%, more preferably 1-30 wt.-%, more preferably 2-25 wt.-%, still more preferably 3-20 wt.-%, still more preferably 4-15 wt.-%, most preferably 5-10 wt.-%. These collagen sheets do not contract, or preferably maintain their dimensions for at least 1 , 2, 3, 4, 5, 6, or 7 days after formation of the sheet. In this context, maintenance of dimensions implies that the largest loss of dimension is no larger than a contraction to 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the original dimensions. Dimensions of the collagen sheet are preferably as defined for the glass fibre sheet.

[0076] Hydroxyproline content is a good measure for collagen content. In preferred embodiments the collagen sheet comprises at least 1 pg / mg hydroxyproline, more preferably at least 2, still more preferably at least 3, more preferably at least 4 pg / mg, still more preferably at least 8 pg / mg, still more preferably at least 12 pg / mg, still more preferably at least 20 pg / mg, still more preferably at least 30 pg / mg, most preferably at least 50 pg / mg. Hydroxyproline content can be assayed using known techniques, for instance by measuring absorbance in a chromogenic assay, conveniently by using commercially available kits such as described in the examples.

[0077] In vitro leather

[0078] The invention provides the use of glass fibre in the manufacture of in vitro leather, particularly the use of felted glass fibre sheets in the manufacture of in vitro leather. The use is substantially as described above, wherein the glass fibre is used as glass fibre sheets in the method according to the invention, and wherein the glass fibre preferably has an organic coating as described above, and wherein the glass fibre sheet is preferably a felted glass fibre sheet as described above. The obtained collagen sheets are then subsequently used for the provision of in vitro leather. These sheets preferably comprise glass fibre wherein the glass fibre has an organic coating as described above. These sheets preferably comprise felted glass fibre as described above. Thus the invention also provides a method for manufacturing in vitro leather, the method comprising the step of tanning a collagen sheet, wherein the collagen sheet is as defined above. Also provided is in vitro leather or a consumer product, the consumer product comprising or consisting of in vitro leather, wherein the in vitro leather is obtainable by a method as described above.

[0079] Tanning is an ancient technique that is well-known to the skilled person. It is generally understood to be the process of treating the skins of animals to produce leather. Tanning may be performed in any number of well-understood ways, including vegetable tanning (e.g., using tannin), chrome tanning (chromium salts including chromium sulfate), aldehyde tanning (using glutaraldehyde or oxazolidine compounds), syntans (synthetic tannins, using aromatic polymers), and the like. Tanning can be proceeded by preparatory stages, such as preservation, soaking (re hydrating), liming, splitting, re-liming, deliming, bating (protein proteolysis), degreasing, frizzing, bleaching, pickling, and de-pickling. Tanning can comprise adjustments in the pH to enhance the tanning, preferably lowered, more preferably to pH 2.8-3.2. Preferably, tanning is performed using a chrome-based tanning method.

[0080] Tanning can be succeeded by post-tanning treatments, also referred to as crusting, such as wetting (rehydrating), sammying (drying), thinning (splitting into thinner layers), shaving, neutralization (adjusting pH to a more neutral level), retanning, dyeing (coloring), fatliquoring, filling, stuffing, stripping, whitening, fixation of unbound chemicals, setting, conditioning, softening, buffing, and the like.

[0081] After tanning an in vitro leather is obtained. The invention thus provides in vitro leather obtainable by a method according to the invention. The in vitro leather can have a thickness of 0.01 - 0.5 mm after the tanning and dying process, preferably 0.015 - 0.4 mm, more preferably 0.2 - 0.3 mm, most preferably 0.24 mm. The in vitro leather can include a plurality of layers to reach the desired thickness, for example comprising 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 7-8 layers. The in vitro leather can be coloured or pigmented, for example using one or more colorants or pigments. It can also be patterned, for example patterned after a skin pattern of an animal.

[0082] The in vitro leather is an attractive alternative for leather derived from animal hides. Accordingly it can be used in consumer products. Examples of consumer products are clothing, such as jackets, vests, hats, dresses, pants, and gloves; footwear, such as shoes, sandals, flipflops, and boot; accessories, such as belts, suit cases, bracelets, wristwatch straps, handbags, purses, key holders, mobile phone holders, phone cases, laptop cases, spectacle cases, cases for electronics, wallets, card holders, billfolds, briefcases, eyeglass cases, luggage tags, and portfolio holders; automotive applications, such as car seat covers, steering wheel covers, dashboards, armrests, head restraints, doors, motorcycle saddles, and motorcycle handles; home and office items, such as sofas, hassocks, footrests, chair backs, armrests, leather wall hangings, mats, carpet, lamp shades, and pillows; sport items, such as sports balls, 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 items, such as heat resistant aprons and gloves, handle covers, holsters, quivers and knife sheaths; musical items, such as percussion instruments, guitar straps, instrument cases; pets accessories, such as collars, chains, muzzles, harness, saddles, bridles, leash, and pets furniture.

[0083] General Definitions

[0084] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of’ meaning that a product or a composition or a nucleic acid molecule or a peptide or polypeptide of a nucleic acid construct or vector or cell as defined herein may comprise additional component(s) than the ones specifically identified; said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value.

[0085] Whenever a parameter or a substance is discussed in the context of this invention, it is assumed that unless otherwise specified, the parameter is determined, measured, or manifested under physiological conditions. Physiological conditions are known to a person skilled in the art, and comprise aqueous solvent systems, atmospheric pressure, pH-values between 6 and 8, a temperature ranging from room temperature to about 37 °C (from about 20 °C to about 40 °C), and a suitable concentration of buffer salts or other components.

[0086] All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

[0087] Particular embodiments

[0088] A. Method for culturing cells, the method comprising the steps of: i) providing a glass fibre sheet that has preferably been treated with a coating agent that introduces an organic coating on the glass fibres; ii) placing the glass fibre sheet in a bioreactor; iii) seeding cells onto the glass fibre sheet in the bioreactor; iv) culturing the cells.

[0089] B. The method according to embodiment A, wherein the coating agent is a small molecule.

[0090] C. The method according to embodiment A or B, wherein the organic coating comprises amines or thiols.

[0091] D. The method according to any one of embodiments A-C, wherein the organic coating is covalently linked to the glass fibres.

[0092] E. The method according to any one of embodiments A-D, wherein the coating agent is a silanization agent, preferably a trialkoxysilane such as a triethoxysilane.

[0093] F. The method according to any one of embodiments A-E, wherein the coating agent is an aminoalkylsilane such as a (3-aminopropyl)silane, a mercaptoalkylsilane such as a (3- mercaptopropyl)silane, a glycidyloxyalkylsilane such as a (3-glycidyloxypropyl)silane, a cyanoalkylsilane such as a 2-(cyanoethyl)silane, or an isocyanatoalkylsilane such as a (3- isocyanatopropyl)silane; preferably an aminoalkylsilane or a mercaptoalkylsilane; more preferably a (3-aminopropyl)silane or a (3-mercaptopropyl)silane; most preferably a (3-aminopropyl)silane such as (3-aminopropyl)triethoxysilane.

[0094] G. The method according to any one of embodiments A-F, wherein the coating agent introduces organic amines, wherein the organic amines are subsequently quaternized, preferably using a halomethane such as iodomethane.

[0095] H. The method according to any one of embodiments A-G, wherein treating the glass fibre sheet with a coating agent comprises contacting the glass fibre sheet with a solution comprising 0.05-5 vol.-% of the coating agent, preferably 0.1 -1 .5 vol.-%, more preferably about 0.5-1 .2 vol.-%.

[0096] I. The method according to any one of embodiments A-H, wherein step i) comprises autoclaving the glass fibre sheet.

[0097] J. The method according to any one of embodiments A-l, wherein the cells are fibroblasts, preferably dermal fibroblasts, more preferably bovine dermal fibroblasts.

[0098] K. The method according to any one of embodiments A-J, wherein the culturing of step iv) is under conditions conducive to collagen production to form a collagen sheet; and wherein the method optionally further comprises the step of: v) isolating the collagen sheet.

[0099] L. Use of glass fibre having an organic coating in the manufacture of a collagen sheet or of in vitro leather.

[0100] M. Collagen sheet comprising glass fibre wherein the glass fibre has an organic coating.

[0101] N. Method for manufacturing in vitro leather, the method comprising the step of tanning a collagen sheet according to embodiment M.

[0102] O. In vitro leather obtainable by a method according to embodiment N.

[0103] P. Collagen sheet according to embodiment M, wherein the collagen sheet is obtainable by a method according to embodiment K.

[0104] Q. Consumer product comprising or consisting of in vitro leather according to embodiment O.

[0105] R. The method according to any one of embodiments A-K, wherein the glass fibre sheet comprises solid fibres.

[0106] S. The method according to any one of embodiments A-K or R, wherein in step ii) multiple glass fibre sheets are placed in the bioreactor to form a multi-layered scaffold.

[0107] T. The method according to any one of embodiments A-K or R or S, wherein the glass fibres have an average diameter of from about 1 to about 25 pm, preferably of from about 5 to about 15 pm; and / or the fibre density in the glass fibre sheet is from about 1 to about 30 mg / cm2, preferably from about 1 .5 to about 25 mg / cm2, more preferably from about 2 to about 20 mg / cm2; in some embodiments the density is about 20-25 mg / cm2; in some embodiments the density is about 1 .5 to about 8 mg / cm2;; and / or the gap size between fibres in the glass fibre sheet is from about 10 to about 140 pm, preferably from about 25 to about 125 pm; and / or the glass fibre sheets have a thickness of about 10-3000, preferably 15-2000, more preferably 20-1000 pm.

[0108] U. The method according to any one of embodiments A-K or R-T, wherein in step iii) from about 1x104to about 1x108cells such as fibroblasts are seeded per cm2of glass fibre sheet, preferably from about 1x105to about 1x107cells per cm2, more preferably from about 5x105to about 1x107, most preferably from about 1x106to about 5x106cells per cm2, or wherein in step iii) from about 1x104to about 1x105cells such as fibroblasts are seeded per mm3of glass fibre sheet, preferably from about 2.5x104to about 4.5x104cells per mm3.

[0109] V. The method according to any one of embodiments A-K or R-U, wherein the culturing in step iv) is performed in DMEM / F-12 medium that optionally comprises 2-20% serum such as 10% fetal bovine serum, and that optionally comprises 2-phospho-L-ascorbic acid.

[0110] W. The method according to any one of embodiments A-K or R-V, wherein the culturing is performed for about 2 to 6 weeks, preferably for about 2 to 5 weeks, more preferably for about 3 to 5 weeks.

[0111] X. The method according to any one of embodiments A-K or R-W, wherein the culturing is performed under gentle agitation, such as using an orbital shaker.

[0112] Y. The method according to any one of embodiments A-K or R-X, wherein the glass fibre sheet has an area of at least 8 cm2such as about 10 to about 20.000 cm2, preferably of at least 10 cm2such as about 15 to about 10.000 cm2, more preferably of about 100 to 1000 cm2, such as about 500 to 900 cm2.

[0113] Z. The method according to any one of embodiments A-K or R-Y, wherein the glass fibre sheet has a rectangular or substantially rectangular shape, such as a square shape.

[0114] Legend to the Figures

[0115] Fig. 1 A - Brig htfield microscopy (5X) of non-felted glass fibre sheet of example 1.1.1.

[0116] Fig. 1B - Brightfield microscopy of felted glass fibre sheet of example 1.1.1. Shown is an edge of a scaffold that was seeded with primary bovine fibroblasts twice at 45,000 cells / mm3and cultured for seven weeks.

[0117] Fig. 1C - Scanning electron microscopy of various glass fibre sheets. Magnification as indicated, scale bar for 40x is 500 pm, scale bar for 250x is 100 pm. Top two images for EsportsMJJ mat, middle two images for Utek mat, bottom two images for GuoTai mat.

[0118] Fig. 2A - Live / DEAD results for cells (from FBS-media) seeded onto scaffolds of Fig. 1 A which underwent pre-treatment 2 optionally followed by SF-medium prewetting (indicated SF) or FBS- medium prewetting (indicated FBS). (Et-H1 - Ethidium Homodimer-1).

[0119] Fig. 2B -Z-stack imaging of cells nuclei (Hoechst staining blue) and collagen (CNA35 staining red) distribution on glass fibre scaffolds after 7 weeks of culture on a scaffold as Fig. 1 B. Images show collagen staining and cell distribution across the samples. Biopsy punch diameter: 8 mm.

[0120] Fig. 3 - Seeding efficiency as determined by PicoGreen assay. Pre-treatment 1 was outperformed by all pre-treatment 2 options, indicating a beneficial effect of autoclaving.

[0121] Fig. 4A - Fluorescence microscopy shows good cell behaviour on the glass fibre scaffold. Results shown after 7 days and after 21 days.

[0122] Fig. 4B - Fluorescence microscopy shows closing of gaps within 21 days on a glass fibre scaffold that was treated with 0.1 vol.-% MPTES. Fig. 4C - Fluorescence microscopy shows closing of gaps within about 14 days on a quaternary amine (QA) glass fibre scaffold that was produced from a scaffold treated with 1 .0 vol.-% APTES.

[0123] Fig. 5 - Brightfield imaging (5X magnification) of cell culture as indicated. Gap closure is observed over time.

[0124] Fig. 6A - Z-stack imaging of cells nuclei (Hoechst staining blue) and collagen (CNA35 staining red) distribution on glass fibre scaffolds after 5-weeks of culture. Small-scale prototypes show collagen staining across the sample and high variability of cell distribution across samples. Samples from the centre or the corner of the scaffold as indicated. Low background staining is observed on blank control (data not shown). Biopsy punch diameter: 6 mm.

[0125] Fig. 6B - LIVE / DEAD staining of cultures of Fig. 6A. Viability is high. Some cell death is observed, mostly for unmodified scaffolds. Percentages indicated are vol.-%. MPTES is (3- Mercaptopropyl)trimethoxysilane. QA (quaternary amine) scaffolds appear to be most populated.

[0126] Fig. 7 - in vitro leather produced by tanning a collagen sheet cultured on a glass fibre scaffold.

[0127] Examples

[0128] Example 1 - Glass fibre sheet as scaffold for cell culture

[0129] 1.1 Materials and Methods

[0130] 1.1.1 Provision of a glass fibre sheet

[0131] Felted glass fibre sheets were procured from a commercial supplier (valmiera glass, stitch- bonded mats E-glass 600 °C). These are stitch-bonded glass fibre mats with a thermal resistance of 600 °C, made of alkali-free alumina borosilicate E-glass fibre yarns with a filament diameter of 6 to 13 mm and a length of the cut fibre of 30 to 100 mm, without any bonding agent. Polyester yarn is used for stitching. Material is commonly available with a standard width of 100 cm, thickness 4- 12 mm, mass per unit area 800 to 2900 g / m2. Used felted glass fibre sheets had a mass per unit area of 800 g / m2and a thickness of 3mm. Scaffolds were cut to 10cm by 10 cm squares and were autoclaved prior to further use as described below.

[0132] Additional scaffolds were also procured. A roll of 1x5 meter 30g chopped strand emulsion mat fibreglass was procured from a commercial supplier (FEVERWORK 1x5m 30g CSM Fiberglass Mat Fibreglass Strand Pads Fibreglass Roll Marine for GRP Resin; Brand Name: EsportsMJJ; Ean: 6953105511954; Part Number: 511954). The white mat comprised 30g Alkali-free fibreglass and was a fibre-reinforced polymer produced by pultrusion winding. The glass fiber mat is a sheet-like product made by continuous strands or surface non-directionally bonded together by chemical bonding or mechanical action. It featured fiber distribution uniformity, good tensile strength, and good flexibility. The glass type was Alkali-free, E-type, acid / base resistant, melting point: 1100°C < AT < 1500 °C. Notably the cost per 20m2was below 20€; equaling less than 0.001€ I cm2.

[0133] 25cm2of the mat was cut into 0.25cm2pieces (for 48-well plates) or 1 cm2pieces (for 24- well plates) using a ruler and scalpel and was given 1 of 2 different pre-treatments.

[0134] Pre-treatment 1 : 70% Ethanol(aq) washing (x3) for 20min per wash.

[0135] Pre-treatment 2: Autoclaving followed by pre-treatment 1 . The autoclaving of pre-treatment 2 was in x1 PBS; pH 7.2. Autoclaved material can be stored in a dessicator prior to use. The 1 cm2scaffolds were placed in ultra-low attachment (ULA) 24-MW plates (or 48-MW plates, or other as required), followed by a single 70% Ethanol(aq) wash, aspirated, then drying under constant flow in a BSL2 flow cabinet.

[0136] Optionally the pre-treatments included prewetting, in this case incubation in complete media [DMEM + 10% FBS + 1 % P / S / A] overnight once fully dried from the ethanol washes. For comparison, prewetting was also performed in SF media overnight, or in 1x PBS overnight. Prior to cell seeding the scaffolds were washed (x3) with ethanol, aspirated dry and left to air-dry under constant air flow in a flow cabinet for at least 3h.

[0137] Glass fibre sheets were analysed such as by brightfield microscopy or scanning electron microscopy. Sheets from additional suppliers (for instance Utek or GuoTai) were compared. Results for chopped strand mats are shown in Fig. 1. Good results were also obtained with other scaffold sizes, such as 4x4 cm and 10x10 cm.

[0138] 1.1.1 .1 Modification of glass fibre sheets with a coating agent

[0139] For some experiments, as indicated, sheets were functionalized using coating agents (3- Aminopropyl)triethoxysilane (APTES) or (3-Mercaptopropyl)trimethoxysilane (MPTES). APTES covalently adds amine groups while MPTES covalently adds thiol groups to surfaces. In a fume hood, glass fibre sheets were dehydrated in acetone for 5 minutes and dried at room temperature for a couple of minutes (scaffolds may turn from transparent to white). Then the sheet was submerged in an APTES or MPTES solution comprising a desired vol.-% of the functionalization agent in ethanol, wherein the sheet is incubated for 20 minutes at room temperature. Afterwards the sheets were washed 4x in PBS and dried under the flow hood. Optionally prior to washing or as part of the first wash, glass fibre sheets can be incubated in an aqueous solution such as PBS to allow unused coating agent to hydrolyse. Functionalized sheets were kept in a desiccator until their further use. Coating agents were used at 0.1 vol.-%, 0.5 vol.-%, and 1.0 vol.-%. Felted scaffolds were coated using 0.5 vol.-% MPTES.

[0140] 1 .1 .1 .2 Quaternary amination of amine-coated fibreglass

[0141] A 25cm2(5cm x 5cm) sheet of glass fibre was autoclaved at 121 ° C for 20min in x1 PBS (pH 7.2), after which it was immersed in acetone or ethanol for 10 min to precipitate out the salts from the PBS solution. It was subsequently washed (x2) with acetone or ethanol. The glass fibre sheet was then transferred to a solution of 0.1 - 1 .0 vol.-% APTES in 100% EtOH, submerged for 20 minutes, followed by a PBS washing step (x3) and rinsing with acetone or ethanol as above. The resulting amine-functionalized glass fibre sheet was then submerged in a 5.0 vol.-% solution of CH3I in 100% EtOH, protected from light, and allowed to react over 24-72h (generally bringing the reaction to completion). Then the ethanol solution was aspirated after which the quaternary amine (QA) glass fibre was washed (x3) in x1 PBS (pH 7.2). QA scaffolds can be stored in PBS or 70% EtOH(aq) until their further use. 1 .1 .2 Serum free proliferation medium

[0142] Cells (primary bovine fibroblasts) were proliferated in serum-free medium (for chopped strand mats). The medium was based on DMEM / F12 medium, supplemented with 2.5 mg / mL albumin, 5 pg / mL insulin, 5.5 pg / mL transferrin, 6.7 pg / mL selenium, 250 nM vitamin C, 10 ng / mL basic fibroblast growth factor (bFGF), 50 ng / mL platelet-derived growth factor (PDGF) and 0.1 mM glutamine. For feited scaffolds, cells were proliferated in the presence of medium.

[0143] 1 .1 .3 Petri dish preparation and scaffold sterilisation

[0144] A glass petri dish was siliconized with Sigmacote according to manufacturer instructions, and then sterilised. The glass fibre scaffold is then placed inside the petri dish inside a flow cabinet. Scaffolds were sterilized by 70% (v / v) ethanol incubation for 20-30 min. Solvent remains are washed (x1) with PBS. To dry the scaffolds, they were aspirated as much as possible with a pipette vacuum aspirator. Resulting scaffolds are sterile and ready for the cell seeding step.

[0145] 1.1.4 Seeding of scaffolds

[0146] Scaffolds (chopped strand mats) were seeded with primary bovine fibroblasts (PBF) at 1 x 106cells / cm2(250 000 cells / scaffold; cells grown in FBS medium or cells grown in SF medium; scaffold thickness about 0.1 -0.15 mm), using a 125pL drop seeding method in the centre of the scaffold. The 24-MW plates with seeded scaffolds were carefully transferred to a 5% CO2 37 °C humidified incubator and incubated there for 1.5-2h. After the cells were allowed to attach to the scaffold, 500pL complete media was added gently to the wells. Media was changed every 2-3 days; total culture duration was 7 days. All samples were done in triplicate; a representative scaffold without cells was used as blank.

[0147] Alternately, PBF cells were seeded onto the scaffold (thickness about 0.1-0.15 mm) using a drop-seeding method for 1 x 106cells / cm2; shortly 30-40|JL of cell containing media was seeded onto the scaffolds which were placed in 48MW plates. The 48MW with seeded scaffolds were carefully transferred to the 5% CO2 37° C humidified incubator for 1.5-2h. After the cells were allowed to attach to the scaffold, 500|JL media was added gently to the wells. The media used was complete media (+250|JM 2-phosphate ascorbic acid) or SF, dependent on the scaffold prewetting (matching it). Cell seeded onto PBS-rinsed scaffolds were cultured in SF. Media was changed at 24h; with total culture time of 3 days. For some analyses, culturing was done for 21 days. All samples were done in triplicate; a representative scaffold without cells was used as blank.

[0148] Felted scaffolds were seeded twice at 45.000 cells / mm3, and were cultured for 7 days in serum-containing media.

[0149] 1.1.5 Analysis of seeding efficiency

[0150] A seeding efficiency count, DNA Picogreen assay and Live / DEAD (Hoechst / Ethidium Homodimer-1) staining was done 24h post seeding. Seeding efficiency was done by using the PicoGreen assay to estimate cell content as a function of total DNA content. Briefly, representative scaffolds (2-3) per condition were sacrificed and homogenized for 2min in ice-cold Tris-buffer (pH 8) followed by manufacturer protocol. Estimation was based on 6pg of DNA content per single cell.

[0151] A cell number fold increase over 72h was also done with the DNA Picogreen assay. Foldincrease was calculated with the relative fluorescence unit (RFU) derived [DNA] at 24h compared to the RFU [DNA] at 72h = (72h #Cells) / (24h # Cells).

[0152] Live / DEAD (Hoechst / Ethidium Homodimer-1) staining was done 24h post seeding with representative samples per condition. Hoechst / Phalloidin-FITC / CNA35-Alexa Fluor™ 568 staining was done for samples on the last day and analysed by fluorescence microscopy. Scaffolds were washed with warm media and the cells fixed with 4% paraformaldehyde (PFA, pH 7.2) for 15min at room temperature or overnight at in the refrigerator. Scaffolds (chopped strand mat) were stained first with the Hoechst / CNA35, per standard protocol (For a 5 mL staining solution in media / PBS: 5 pL stock Hoechst ([Hoechst] FINAL = 1 pg / mL; 1 .6 pL stock EthD-1 ([EthD-1]FiNAL = 650 nM; Add enough staining solution to cover the sample and incubate 15min in the dark; Wash 1x with media) for 15-30min at room temperature, then washed (x3) with x1 PBS (pH 7.2). Scaffolds were then treated with permeabilization buffer (0.1 % Tergitol in PBS) for 15-30min at room temperature, washed (x3) with x1 PBS (pH 7.2), and a drop of the Phalloidin-FITC was added per well.

[0153] 1 .1 .6 Hoechst / Phalloidin / CNA35 staining

[0154] To stain the nucleus, actin, and collagen from cells, cells were fixed with PFA. Then cell walls were permeabilized with 0.5% Tween 20 in PBS and incubated for 1 h. Staining solution in PBS (in the same tube):

[0155] • Hoechst (blue): 1 pL per 1000 pL

[0156] • Phalloidin (green) 1 pL per 1000 pL

[0157] CNA35 (red) 1 pL per 100 pL

[0158] Sample was fully covered in staining solution to cover the sample, roughly 500 pL for scaffolds as described above. Samples were incubated for 1 h in the dark, wash 2x with PBS.

[0159] 1 .2 Results

[0160] 1.2.1 Properties of glass fibre sheet

[0161] For chopped strand mats, brig htfie Id microscopy revealed a sheet height of 150-200 pm as determined with a tanner’s gauge. The fibre diameter was from 5-10 pm, or 10 pm + / - 1.1 pm as determined by SEM. Other manufacturers provided sheets with fibre diameters as shown in table S1 .2.1 . Representative microscopy images are shown in Fig. 1 . Autoclaving was found to not affect the mechanical structure of the glass fibre sheet. Table S. 1.2.1 - properties of glass fibre sheets (CSM denoted chopped strand mat)

[0162] Fibres with a diameter below 5 pm were not observed. Sheets with a thickness of 0.5 mm were also used, having a density of 5.8 mg / cm2and average diameters of 14-17 pm. The glass fibre sheets were not brittle and did not transfer fibres to hands / tweezers while handling. Sheets were flexible and robust, and breakdown products were silica, boron, aluminium, calcium, and magnesium.

[0163] Felted scaffolds generally had somewhat smaller fibre diameters, and sheets of about 3 mm in thickness. Mass was about 80 mg / cm2, taking into account that mats were stitched (into stitch-bonded felted glass fibre mats) with a polyester yarn. Thermal resistance was 600 °C, fibres being made of alkali-free alumina borosilicate E-glass fibre yarns with a filament diameter of 6 to 13 mm and a length of the cut fibre of 30 to 100 mm, without any bonding agent. Felted scaffolds were found more convenient to handle (e.g. to place in bioreactors) owing to their greater flexibility.

[0164] 1 .2.1 .1 Properties of modified glass fibre sheet

[0165] Different surface modification procedures were followed by brightfield microscopy, revealing no unexpected changes to the glass fibre appearance. ATR-FTIR (attenuated total reflection - Fourier transform infrared) analysis of APTES modified scaffolds revealed, as compared to unmodified glass fibre, a reduction in the Si-O-Si bands around 900 cm'1, an appearance of N-H bending around 1550 cm'1, and an appearance of N-H stretching around 3400 cm'1, indicative of successful surface modification.

[0166] 1 .2.2 Live / Dead assays

[0167] The Live / Dead assay for all permutations of Pre-treatment 1 showed no viable cells at 24h. The Live / DEAD assay results for Pre-treatment 2 (autoclaved in x1 PBS) shows a relatively high cell death. Less cell death was observed for “no wetting” conditions than for media pre-treated scaffolds. The above was observed for cells from FBS-media as well as for cells from SF-media from spinner flasks. Fig. 2 shows microscopy images.

[0168] 1.2.3 Seeding Efficiency

[0169] Seeding efficiencies for glass fibre scaffolds of pre-treatment 2 were satisfactory, both for modified and unmodified glass fibre sheets. The best seeding conditions for all cell origins (FBS or SF media) was when scaffolds were prewetted in complete serum media or in SF media. A decrease in seeding efficiency was observed for scaffolds that were not prewetted, as well as for all conditions of pre-treatment 1 . The seeding efficiency was determined using a PicoGreen assay. Pre-treatment 2 (without prewetting) and Pre-treatment 1 (regardless of prewetting) had low seeding efficiencies for FBS grown cells, where a Live / DEAD staining showed no viable cells present on the scaffolds for Pre-treatment 1 . Seeding efficiency of serum-free cells on modified scaffolds (vol.-% indicated) appeared to be higher than for unmodified scaffolds, with standardised (unmodified = 1) values shown in the table below (n = 6). All conditions initially showed decreased cell numbers at day 3, followed by numbers increased above original seeding numbers at day 7.

[0170] Fluorescent labelling was done on day 7 to assess cell distribution, cytoskeleton, and collagen production on unmodified scaffolds (Fig. 4A), and collagen production was seen to still be ongoing at day 21 although open gaps were visible. Scaffolds treated with a coating agent resulted in good collagen layers after 21 days (Fig. 4B for 0.1 vol.-% MPTES; closed collagen layer also visible at 21 days for 0.5 vol.-% MPTES) or after 14 days (Fig. 4C for 1.0 vol.-% QA). APTES scaffolds, like unmodified scaffolds, also led to collagen layers that still showed open gaps, both at 0.1 vol.-% and 0.5 vol.-%. Both unmodified and modified scaffolds show a time-dependent qualitative increase in collagen detected. The nuclei appear to be agglomerated from Day 1 -7, followed by spreading and gap closure in what can be seen as a proliferative manner.

[0171] Overall, good results were seen for all Pre-treatment 2 scaffolds. Cells attach, proliferate, and produce collagen over at least 7 days. Cells produce collagen over at least 21 days. Surface modification led to increased cell proliferation and collagen production.

[0172] Seeding efficiency at 24 hours post seeding at 1x106cells / cm2was 1 18% for prewetting and culturing in complete media, 79% for prewetting and culturing in SF media, and 64% for prewetting in PBS and culturing in SF. Thus prewetting in cell culture medium leads to improved cell seeding efficiency

[0173] Assessed at n=6, the fold increase for cells over 72 hours revealed that culturing in complete medium led to a slight decrease of cells (73%). SF prewetted scaffolds and culturing in SF led to an increase in cell density (111 % when prewetted in SF or 144% when prewetted in PBS). There is a possibility that exposure of the cells to complete media results in a short-term burst in proliferation due to exposure to richer medium, this may lead to an overestimation at t=24h. Low seeding efficiency for the PBS-wetted scaffolds is likely related to the lack of surface passivation due to an absence of relevant biomolecules in PBS. In any case, serum-free cells from the subculture (spinner flasks) readily adhered and thrived on the fibre glass scaffold.

[0174] Seeding on felted scaffolds was comparable to seeding on chopped strand mats, if not improved. Felted scaffolds were found to achieve satisfactory cell densities after only two seeding waves, where results with chopped strand mats as presented herein were achieved after four seeding waves. As a comparative experiment, seeding efficiencies were also determined on a different glass fibre material (non-felted so-gel fibres of a fibre diameter 30pm - 70pm; gap size 150-250 pm; fibre density of about 12 mg / cm2; thickness about 450 - 700pm). Seeding was about 80% of the seeding on the scaffolds described above.

[0175] Example 2 - production of in vitro leather

[0176] 2.1 - Scaffolds, cell culture, and seeding

[0177] Glass fibre scaffolds were prepared as described above. EsportsMJJ Alkali-free, E-type, acid / base resistant, melting point: 1 100°C < AT < 1500 °C glass fibre was cut into 4x4 cm to create scaffolds. 2-layered scaffolds (dimensions: 4 cm x 4 cm size, ~0.2 pm thickness, V= 320 mm3) were autoclaved in PBS. Scaffolds were sterilised with 70%(v / v) ethanol and left to dry overnight on a siliconized glass petri dish. Felted scaffolds were cut to 10x10 cm instead.

[0178] Primary bovine fibroblasts were used. During expansion, cells were cultured with “50 / 50” media (50% DMEM-F12+10% FBS+ 1 % P / S / A combined with 50% serum-free proliferation media (see section 1 .1 .2)). Scaffolds were seeded 4 times (for chopped strand mats) or 2 times (for felted sheets) in interval of 2-3 days. For chopped strand mats, each seeding, 32x106cells were concentrated in 1 .6 mL, the cell suspension was spread on top of the scaffolds surface and incubated during 1 h before adding 25mL culture media (DMEM-F12+ 10% (v / v) FBS + 50 pg / mL gentamicin + 500 pM L-Ascorbic acid 2-phosphate sesqui magnesium salt hydrate (Vitamin C)) . For felted sheets 45.000 cells per mm3of scaffold were seeded. During the seeding process, scaffolds were cultured in static conditions for one week. After this process, petri dishes were then placed in dynamic culture using an orbital shaker at 35 RPM during the remaining 4 weeks of culture (inducing a shear stress of 0.01 -5 Pa or 0.1-50 dyne / cm2). It was found that the orbital shaker promoted collagen formation and improved overall collagen deposition. Seeding efficiency was measured as the number of cells in suspension after 24h of each seeding step. Once the last seeding step was finished, 50% culture media was changed weekly.

[0179] Cells proliferation was primarily access by brightfield imaging (5X magnification). On the last day of culture, 6 mm biopsy punches were collected, and LIVE / DEAD stained (See example 1.1.5). Remaining of the scaffolds was fixed with 4% (vZv) PFA and cut in half (halving one axis) for analysis and the other half was sent for tanning. Samples were collected and stained with Hoechst and CNA35 fluorescent dye to assess cell and collagen distribution on scaffolds, respectively (See example 1 .1 .6). Z-stack imaging (16 slices, 10 pm step) was performed using ImageExpress Pico (Molecular devices).

[0180] 2.2 - Hydroxyproline content

[0181] The amount of collagen content can be assessed based on hydroxyproline content of scaffolds. For quantitative analysis, hydroxyproline assay were performed at the end of the culture on -10 mg fixed samples versus respective blank scaffold. The protocol for a commercially available kit was followed: OH-Proline assay (Sigma-Aldrich kit #MAK357). Briefly, before starting, the kit components were allowed to warm to room temperature and mixed by vortexing prior to use. Dry sample tissue was weighed. 100pL of dFW per 10 mg of tissue was added (increments of 100 pL were used, no smaller volume than 100 pL was used, rounding up to the nearest multiple of 100) and the sample was homogenized. Homogenate was transferred to a sample container which is heat resistant and has a PTFE screwcap. 100pL of NaOH (10M) was added to the homogenate and mixed thoroughly. Samples were autoclaved at 121 °C for 1 h, and then allowed to cool to room temperature before adding 100pL HCL (10M), after which they were mixed to neutralize the homogenate. Samples were centrifuged (10 000 x g, 5min) to sediment the insoluble debris.

[0182] Standards were prepared and all samples added to a 96-well plate. For standards, 20pL of hydroxyproline (HP) standard stock solution (1 mg / mL) was diluted with 180 pL dH2<D. A working solution = 200 pL of 0.1 mg / mL. Wells of 0, 0.2, 0.4, 0.6, 0.8, and 1 .0 were used as a standard. 10 pL of sample was used per well, in duplicate or preferably triplicate. Samples can be evaluated at x100 dilution, x10 dilution, or neat. Samples and standards are dried to evaporate all the liquid.

[0183] Then assay components are vortexed to ensure mixing and dissolution of precipitates. Oxidation Reagent Mix (ORM) was prepared by mixing 6 pL Chloramine T and 94 pL oxidation buffer per sample, after which it was protected from light until use. For the assay, 100 pL ORM is added to each well of the 96-well plate. Then 50 pL of the Develop Solution is added to each well and incubate at 37 °C for 5min, followed by 50 pL of the DMAB solution to each well, after which contents are mixed (e.g. using a multichannel micropipette), followed by heating to 65 °C for 45min. Absorbance (OD560) is read on a plate reader (SpectraMax iD3).

[0184] Blank samples are used for sample correction. The standard series is plotted to determine the linear equation (y=mx; y= OD, x = hydroxyproline (HP) pg / well). The mass of HP per well (pg) for the samples is determined using the linear equation. [HP] in pg / pL:

[0185] [HP] = (BA / ) x D

[0186] Where B = mass of HP (pg); V = volume of sample added to well (pL); D = dilution factor for 96-well volume = 1 ; dilution factor for homogenate = 3 for 10mg tissue. The equation to calculate the collagen content (HP content of collagen for bovine dermis is 12-15%):

[0187] Mass of collagen (pg) = (Mass of HP pg) / 0.135

[0188] Collagen content can be normalized to the sample mass: pg Collagen / mg

[0189] Example 2.3 - cell attachment and distribution

[0190] High seeding efficiency (%) was observed, followed by a slight decrease overtime. Possibly as cells populate, spread and close gaps within the fibrous scaffold new cells might face a higher challenge to find space for attachment. This might lead to loss of cells. The following efficiencies were seen for experiments where chopped strand mats were used: 1stseeding 98%, 3rdseeding 90%, 4thseeding 98%. Brightfield observations on the 14thday culture showed no empty regions or cell aggregates across the culture. On the last day of culture, media had not altered colour towards the yellow spectrum, which indicates that pH had not lowered too much. pH was measured during culture time and never decreased bellow 6.5. Samples had an approximate thickness of 0.3 mm before and after PFA fixing. Fig. 5 shows cell distribution on the scaffold. Gaps were found to close over time, particularly for MPTES and QA scaffolds. Fig. 6A shows good distribution of living cells and an even distribution of collagen (unmodified scaffold shown, effect observed for all conditions, including felted mats that were also tested). Fig. 6B shows how QA scaffolds appear to have a higher number of cells in comparison to the other conditions, although viability is high overall. Cell death was highest on unmodified scaffolds. Tweezer manipulation revealed that collagen sheets kept the same flexibility before and after 4% (v / v) PFA fixing.

[0191] For QA-functionalized scaffolds, tested at 0.1 vol.-%, 0.5 vol.-%, and 1.0 vol.-%, the cells also proliferated on the scaffolds and had a comparative high collagen production. There appears to have been no challenge for gap closure based on nuclear- and collagen staining.

[0192] Example 2.4 - collagen content

[0193] A good collagen content was found for the sheets after culture for 14 days as described above. The collagen sheets were assessed for hydroxyproline content as described in example 2.2, and a hydroxyproline content of 4.06 pg per mg (±0.07, Mean±SD) of dry mass was found for chopped strand mats, and of about 3.65 pg per mg for feited scaffolds (hydroxyproline quantification on stitched bonded fiberglass seeded with primary bovine fibroblasts and cultured for 7 weeks in serum-containing media in static conditions, after only two seeding waves instead of four). Without a scaffold according to the invention, collagen sheets suitable for hydroxyproline assays could not be harvested. Such sheets contracted.

[0194] For collagen sheets grown on scaffolds of glass fibre sheets that had been treated with a coating agent that introduces an organic coating on the glass fibres, the collagen content was even higher. For instance, chopped strand scaffolds treated with 1 vol.-% APTES, quaternised to form a QA coating, the hydroxyproline content was 6.37 pg per mg (±0.01 , Mean±SD). Glass fibre sheets treated with 0.1 vol.-% MPTES led to an hydroxy pro line content of 6.88 pg per mg (±0.04, Mean±SD).

[0195] The below table shows collagen content standardized to collagen content of shaved tanned bull leather (0.6 mm) for various collagen sheets obtained using the methods described herein , after 21 days of culture on 0.25 cm2scaffolds (h = 200 pm). The condition that is listed under “sample” is the coating agent used on the glass fibre sheet that was used as a scaffold. MPTES in serum-free medium and QA in complete medium outperformed unmodified scaffolds in serum-free medium. APTES underperformed compared to MPTES and QA, but APTES-modified scaffolds were used to obtain QA scaffolds. Results are triplicates. Collagen formation was followed in time for unmodified scaffolds and for MPTES at both 0.1 and 0.5 vol.-%, and for these conditions the amount of collagen was shown to increase from day 7 to day 14 to day 21 .

[0196] Example 2.5 - provision of in vitro leather

[0197] Collagen sheets provided as described above were tanned following a traditional chromebased tanning method. Obtained in vitro leather samples (for chopped strand mat see Fig. 7) had thickness as shown in the table below after the tanning and dying process, as measured using a digital gauge. Tensile stress testing was performed on the tanned cultured leather pieces. Sheets of 15 cm3were also successfully tanned.

[0198] Example 2.6 - conclusion

[0199] Culture leather was obtained using a glass fibre scaffold. At the end of the culture, a high cell viability was observed. Cells and collagen were widely distributed in the scaffold and hydroxyproline content showed that scaffolds facilitated collagen secretion and deposition. The scaffolds were able to be tanned and presented good flexibility. Felted scaffolds and chopped strand scaffolds were both found to result in good hydroxyproline content.

Claims

Claims1 . Method for providing a collagen sheet, the method comprising the steps of: i) providing a felted glass fibre sheet; ii) placing the glass fibre sheet in a bioreactor; iii) seeding fibroblasts onto the glass fibre sheet in the bioreactor; iv) culturing the fibroblasts under conditions conducive to collagen production to form a collagen sheet.

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

3. The method according to claim 1 or 2, wherein step i) comprises autoclaving the felted glass fibre sheet.

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

5. The method according to any one of claims 1 -4, wherein in step ii) multiple felted glass fibre sheets are placed in the bioreactor to form a multi-layered scaffold.

6. The method according to any one of claims 1 -5, wherein the glass fibres in the glass fibre sheet have an average diameter of from about 1 to about 25 pm, preferably of from about 5 to about 15 pm; the fibre density in the glass fibre sheet is from about 1 to about 10 mg / cm2, preferably from about 1 .5 to about 8 mg / cm2; the gap size between fibres in the glass fibre sheet is from about 10 to about 140 pm, preferably from about 25 to about 125 pm; the glass fibre sheets have a thickness of about 20-2500 pm; and / or the glass fibre sheet is stitched.

7. The method according to any one of claims 1 -6, wherein in step iii) from about 1x104to about 1x108fibroblasts are seeded per cm2of glass fibre sheet, preferably from about 1x105to about 1x107fibroblasts per cm2, more preferably from about 5x105to about 5x106fibroblasts per cm2.

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

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

10. The method according to any one of claims 1 -9, wherein step i) comprises: i) providing a felted glass fibre sheet that has been treated with a coating agent that introduces an organic coating on the glass fibres.11 . The method according to claim 10, wherein the coating agent is a silanization agent.

12. The method according to any one of claims 1 -11 , wherein the glass fibre sheet that is provided in step i), or that is placed inside the bioreactor in step ii), or onto which fibroblasts are seeded in step iii), is not coated with a polymer.

13. The method according to any one of claims 1 -12, wherein the bioreactor is a perfusion bioreactor.

14. The method according to any one of claims 1 -13, wherein the culturing of step iv) comprises culturing under perfusion.

15. The method according to any one of claims 1 -14, wherein the glass fibre sheet has a rectangular or substantially rectangular shape, such as a square shape.

16. The method according to any one of claims 1 -15, wherein the glass fibre sheet has an area of at least 8 cm2such as about 10 to about 20.000 cm2, preferably of at least 10 cm2such as about 15 to about 10.000 cm2, more preferably of about 100 to 1000 cm2, such as about 500 to 900 cm2.

17. Use of felted glass fibre sheets in the manufacture of in vitro leather.

18. Collagen sheet comprising felted glass fibres, preferably wherein the collagen sheet is obtainable by a method according to any one of claims 1 -16.

19. Collagen sheet according to claim 18, wherein the collagen sheet is obtained by a method according to any one of claims 1 -16.

20. Collagen sheet according to claim 18 or 19, wherein the collagen sheet comprises felted glass fibres and stitching yarn.21 . Method for manufacturing in vitro leather, the method comprising the step of tanning a collagen sheet as described in any one of claims 18-20.

22. In vitro leather or consumer product comprising or consisting of in vitro leather, wherein the in vitro leather is obtainable by a method according to claim 21 .

23. In vitro leather or consumer product according to claim 22, wherein the in vitro leather is obtained by a method according to claim 21 .

24. In vitro leather or consumer product according to claim 22 or 23, wherein the in vitro leather comprises glass fibres.

25. Consumer product according any one of claims 22-24, wherein the consumer product is clothing, such as jackets, vests, hats, dresses, pants, and gloves; footwear, such as shoes, sandals, flipflops, and boots; an accessory, such as belts, suit cases, bracelets, wristwatch straps, handbags, purses, key holders, mobile phone holders, phone cases, laptop cases, spectacle cases, cases for electronics, wallets, card holders, billfolds, briefcases, eyeglass cases, luggage tags, and portfolio holders; for automotive applications, such as car seat covers, steering wheel covers, dashboards, armrests, head restraints, doors, motorcycle saddles, and motorcycle handles; a home or office item, such as sofas, hassocks, footrests, chair backs, armrests, leather wall hangings, mats, carpet, lamp shades, and pillows; a sports item, such as sports balls, sports footwear, shin guards, sports gloves, costumes, racing suits, masks, knee- and elbow protection, bicycle seats, boxing gloves, helmets, roller skates, and punching bags; a protective item, such as heat resistant aprons and gloves, handle covers, holsters, quivers and knife sheaths; a musical item, such as percussion instruments, guitar straps, instrument cases; a pet accessory, such as collars, chains, muzzles, harness, saddles, bridles, leash, and pets furniture.

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