Apparatus and method for producing a cell sheet

Abstract

A system and method for growing cells or cell sheets are described. The method can use biocompatible or food-safe materials. The method can enable cell orientation in an optionally selected pattern that can be created by casting a membrane onto a 3D printed mold. The method can include surface treatment of the elastomeric membrane. The method can enable reuse of the membrane by an autoclaving process and / or a washing process. The method can create a multi-layer cell sheet having an extracellular matrix (ECM) created by the cells, which is detached from the membrane. Optionally, the cells can be separated from the ECM.

Description

Technical Field

[0001] This application claims priority from U.S. Provisional Patent Application No. 63 / 504,774, filed May 29, 2023; U.S. Patent Application No. 17 / 882,693, filed Aug. 8, 2022; and U.S. Patent Application No. 17 / 838,284, filed Jun. 13, 2022. In the United States of America, this application claims the benefit of U.S. Provisional Patent Application No. 63 / 504,774, and this application is a divisional of U.S. Patent Application No. 17 / 882,693, which is a continuation of U.S. Patent Application No. 17 / 838,284, filed Jun. 13, 2022. Patent Applications Nos. 63 / 504,774, 17 / 882,693, and 17 / 838,284 are hereby incorporated by reference herein.

[0002] The field of the present invention relates to devices and methods for growing cells or for making cell sheets. Some examples involve substrate surface chemistry, substrate topography, or methods of processing cell sheets.

Background Art

[0003] Members of the silicone-based polymer family, such as PDMS, have characteristics advantageous for cell culture applications, such as their transparency and ease of microscopic observation, non-toxic and inert properties both chemically and biologically, and their gas permeability. On the other hand, their inherent hydrophobicity results in non-specific protein adsorption, conformation and denaturation, and inappropriate cell adhesion, aggregation, and detachment (Walsh, 2017).

[0004] Multiple methods have been proposed to increase the surface hydrophilicity of silicone-based elastomers, such as PDMS, and improve cell adhesion. For example, some groups have proposed the use of high-energy plasma radiation, which is a rapid method, but the created Si-hydroxyl groups are short-lived, and the movement of molecules from the bulk of the material can neutralize its effect (Fritz, 1995).

[0005] As another example, some groups have proposed the use of covalent immobilization of extracellular matrix proteins on the surface of PDMS. However, this method requires functionalization of the PDMS surface by salinization and the addition of linkers such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N-hydroxysuccinimide (EDC / NHS), glutaraldehyde, and polymer brushes. These modifications require a multi-step and complex process that includes the use of harsh and often toxic materials that can leave residues, and organic solvents that can swell the PDMS and damage its surface features (Li, 2018).

[0006] As another example, some groups have proposed coating PDMS with dopamine (Chuah, 2015). However, dopamine monomers are expensive and potentially toxic, and the dark nature of the coating interferes with microscopic observation. Other proposed methods for improving cell attachment to PDMS include grafting with polyethylene glycol or PEG after plasma treatment (Sharma, 2007) and surface modification with Pluronic® F68, poly-L-lysine, and fibronectin (Wu, 2009).

[0007] Furthermore, some groups have proposed coating PDMS by the formation and deposition of iron(III)-tannic acid networks (Lv, 2020).

[0008] Cell sheet engineering was first introduced in the 1990s using a tissue culture polystyrene grafted with N-isopropylacrylamide (PIPAAm) on the cell culture surface. PIPAAm is a temperature-sensitive material that enables the detachment of cells and their secreted extracellular matrix (ECM) proteins without using enzymes such as trypsin that digest and destroy ECM proteins. Thus, by lowering the temperature and changing the hydrophilicity of the grafted polymer, an intact cell construct in the form of a sheet is brought about (Yamada, 1990). Alternatively, other responsive surfaces for this purpose have been created by layer-by-layer deposition of cationic or anionic polyelectrolytes on indium tin oxide (ITO). These layers become unstable at low pH and prevent cell attachment, so the trigger for initiating delamination of the cell sheet in this case is a decrease in pH (Guillaume-Gentil, 2011).

[0009] Another cell sheet formation technique involves growing cells on a feeder layer and detaching the cells using another enzyme dispase that digests some ECM proteins rather than cell-cell junctions. It has also been proposed to grow cells on amniotic membranes and use them together with the membranes. The use of surfaces sensitive to other external stimuli such as light, electrochemical polarization, ionic solutions, and magnetic forces has also been employed in the production of cell sheets (Owaki, 2014). In a recent technique that does not depend on the properties of the surface for the production of cell sheets, a slight change in the environmental pH is used to shrink the cells and detach them from the surface. This method is applicable only to cells that show syncytialization and fusion, such as skeletal muscle cells (Shahin-Shamsabadi, 2020). Therefore, alternative systems and methods for creating cell sheets are needed.

[0010] Exemplary related technologies include the following: U.S. Patent Application Publication No. 20210180012A1 describes a method for manufacturing a concavo-convex gel surface with defined curvature (C-D) or defined shape (S-D) for use in cell and tissue culture and other surface and interface applications, and provides a method for using C-D or S-D concavo-convex surfaces with various curvatures to direct cell attachment, spreading, and migration.

[0011] U.S. Patent No. 5776747A describes a method for derivatizing or adsorbing a polyethylene oxide-poly(dimethylsiloxane) copolymer (PEO-PDMS) onto the surface within a bioartificial organ to inhibit cell attachment.

[0012] Korean Patent No. 102215710B1 describes a multilayer foam sheet of acrylonitrile-butadiene-styrene (ABS) resin, a thermoplastic resin having excellent mechanical strength and low-temperature impact resistance, a method for manufacturing the same, and a method for manufacturing a three-dimensional (3D) molded article using the sheet.

[0013] Indian Patent No. 201721015210A describes a bio-inspired 3D-micro / nano fluidic device manufactured by a scalable manufacturing process. The device includes a thin membrane matrix with micro / nano blood vessels made of PDMS or dimethicone.

[0014] International Publication No. 2018058135A1 describes a photocurable poly(siloxane) formulation for fabricating a 3D-printed PDMS structure by stereolithography, a method for 3D printing by stereolithography for fabricating a PDMS structure, and a PDMS structure 3D-printed by stereolithography.

[0015] Chinese Patent No. 105504759B describes an ABS composite material used for 3D printing.

[0016] Chinese Patent No. 106046700A describes a method for preparing 3D printing materials from PETG plastic and plant fibers. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] U.S. Patent Application Publication No. 2021 / 0180012 [Patent Document 2] U.S. Patent No. 5,776,747 [Patent Document 3] Korean Patent No. 10-2215710 [Patent Document 4] Indian Patent Application Publication No. 201721015210 [Patent Document 5] International Publication No. 2018 / 058135 [Patent Document 6] Chinese Patent No. 105504759 [Patent Document 7] Chinese Patent Application Publication No. 106046700 [Non-patent literature]

[0018] [Non-Patent Document 1] Walsh, 2017 [Non-patent document 2] Fritz, 1995 [Non-patent document 3] Li, 2018 [Non-patent document 4] Chuah, 2015 [Non-Patent Document 5] Sharma, 2007 [Non-patent document 6] Wu, 2009 [Non-Patent Document 7] Level 2020 [Non-patent document 8] Yamada, 1990 [Non-Patent Document 9] Guillaume-Gentil, 2011 [Non-Patent Document 10] Owaki, 2014 [Non-Patent Document 11] Shahin-Shamsabadi, 2020 [Summary of the Invention] [Problems to be Solved by the Invention]

[0019] This specification describes an apparatus and method for growing cells, and optionally for growing cell constructs such as cell sheets, for growing cells. [Means for Solving the Problems]

[0020] In some examples, the substrate for growing cells or cell constructs includes an elastomeric membrane having a patterned surface and a hydrophilic agent on the surface of the elastomeric membrane. The substrate can be manufactured by casting the elastomeric membrane on a mold having a patterned surface and attaching the hydrophilic agent to the elastomeric membrane. The method for growing a cell construct can include the steps of preparing a patterned hydrophilic membrane of elastomer, growing cells in layers on the membrane, wherein the cells produce an extracellular matrix (ECM), and removing at least a part of the cell layer from the membrane. In at least some examples, the cell layer may be removed from the membrane by physical forces, such as scraping and / or pulling, without using an enzyme or a responsive surface.

[0021] In some examples, the cell construct has a high ECM content. In some examples, the cell construct has a controlled cell alignment. The ECM is a three-dimensional (3D) network depending on the tissue and can consist of extracellular polymers and / or minerals such as collagen, enzymes, glycoproteins, and hydroxyapatite that provide structural and biochemical support around the cells. In some examples, the cell sheet may be made of any adherent cell type from any species. The techniques of the present invention are simple, easy to perform, can have low cost, or can use biocompatible, non-toxic, food-grade, or food-safe materials.

[0022] In some examples, the present invention enables cell alignment in an optionally selected or refined pattern. The pattern of cell alignment is induced by a pattern of a membrane made using a molding process with an optionally 3D printed mold. The mold can be prepared using a 3D printer with different types of filament materials. Similar patterns can alternatively be created with the mold, for example, using CNC machining or laser engraving on different materials. Also, for example, it is possible to directly create a pattern on the membrane using CNC machining or laser engraving without the need to use a patterned mold. Further, the present invention optionally provides for membrane reuse by autoclaving and / or washing with a suitable solvent having a defined polarity to induce controlled, limited swelling in the membrane to assist in the release of absorbed elements, for example, washing a PDMS membrane with isopropyl alcohol. Further, the present invention may provide for multi-layer cell constructs, induced ECM production, or ECM crosslinking using biocompatible or food-grade materials. The method can optionally result in a sheet formed in a short time (usually within 18 days) and can optionally be partially or completely removed from the membrane by physical forces such as scraping and / or pulling without using enzymes or responsive surfaces.

[0023] The exemplary embodiments described herein include a method for creating cell constructs such as cell sheets. The method may include process steps such as treating an elastomeric membrane with an aqueous solution (e.g., an aqueous solution of tannic acid and / or an aqueous solution of lignin). In some examples, the elastomeric membrane is directly treated at room temperature with an aqueous solution of tannic acid at about 10 - 200 mg / mL or a solution of lignin at about 0.1 - 5 mg / mL for a period of about 6 hours to 4 days. In some examples, the method also includes treating the surface of the treated elastomeric membrane with a solution (e.g., a sodium hydroxide solution in deionized water) or plasma. The elastomeric membranes can be pretreated with an aqueous sodium hydroxide solution at a concentration of 0.1 - 5 molar for up to 5 days, up to 12 hours prior to the treatment with the solution of tannic acid or lignin to enhance their effects. When pretreated with sodium hydroxide or other means, depending on the elastomer, lower concentrations and shorter treatment times of tannic acid or lignin may have effects similar to their higher concentrations and longer treatment times.

[0024] Next, the method includes the steps of growing cells on the treated elastomer membrane and imparting orientation to the cells on the treated elastomer membrane. The imparting of cell orientation on the treated elastomer membrane is performed by creating a pattern on the surface of the treated elastomer membrane. In some examples, imparting orientation to cells on the treated elastomer membrane is performed by using 3D printing to create a master mold having a pattern that can be replicated onto the elastomer membrane treated by casting. In some implementations, 3D printing includes fused deposition modeling (FDM) 3D printing. Alternatively, stereolithography (SLA)-based 3D printing can be used to create a defined pattern similar to that created by the FDM method. It will be understood that the pattern (e.g., parallel pattern, circular pattern, concentric circular pattern, or a more complex pattern) is used as a topographical signal transduction cue for orienting cells in a particular direction.

[0025] Furthermore, the method includes the step of detaching the cell construct (i.e., one or more layers of cells and the ECM produced by the cells) from the membrane. In some examples, detaching the cell construct creates a cell sheet, particularly a detached cell sheet. Detaching the cell construct can occur spontaneously when the cell construct grows, or can be initiated by a force such as scraping or vibration, or can be performed by one or more physical actions such as scraping, scratching, vibrating, or pulling. Detaching the cell construct can be performed without applying a stimulus to cause a change in a responsive surface attached to the membrane (e.g., a surface that responds to changes in temperature, ionic solution, pH, light, magnetic force, or electrochemical polarization). Detaching the cell construct can be performed without inducing cell contraction. Detaching the cell construct can be performed without applying a chemical agent such as an enzyme.

[0026] In some examples, the method further includes inducing the cells to produce a sufficient amount of extracellular matrix (ECM) by treating the cells with ascorbate or by polymer crowding, such as by adding polyethylene glycol (PEG) or carrageenan to the culture medium. The method may also include determining that the cells are in a confluent state and producing a sufficient amount of ECM. The method may also include growing additional cells on the initial cell-ECM layer.

[0027] In some examples, the method further includes inducing the cells to produce a sufficient amount of extracellular matrix (ECM) by treating the cells with ascorbate or by polymer crowding, such as by adding polyethylene glycol (PEG) or carrageenan to the culture medium. Optionally, the method includes crosslinking the ECM produced by the cells with a very low concentration of tannic acid (less than 0.1 mg / mL) or lignin (less than 10 μg / mL) for a very short time (5 - 30 minutes) to create a stable structure, and removing the cells and the ECM of the cells from the surface of the elastomer membrane to form a cell sheet.

[0028] In some examples, the method may include determining that the cells are in a confluent state and have produced a sufficient amount of ECM. The method may also include growing additional cells on the initial cell-ECM layer.

[0029] The removal of the cells and the ECM of the cells from the surface of the elastomer membrane can be performed by peeling the cells and the ECM of the cells, or a part thereof, from the surface of the elastomer membrane. In some examples, the method may include washing the treated elastomer membrane, autoclaving it, growing new cells, and reusing the treated elastomer membrane to produce a new cell sheet.

[0030] The present invention exemplifies the possibility of using natural polyphenols such as tannic acid in solution form using a suitable solvent such as deionized water, and the effect on the physical or chemical properties (e.g., hydrophilicity) of these elastomer membranes in order to enhance cell attachment. The elastomer membrane may include silicone-based materials such as PDMS or other elastomers including, but not limited to, poly(butylene adipate-co-terephthalate) (PBAT) and urethane rubber. Similarly, lignin has been shown in the present application to be able to modify the hydrophilicity of the elastomer membrane and can be applied using solvents such as deionized water and methanol. The effects of these treatments are stable and can withstand autoclaving and washing steps with organic solvents such as isopropyl that can cause elastomer swelling. Membranes, e.g., elastomer membranes, are made to have a hydrophilic surface without adding exogenous ECM components to the membrane.

[0031] In some examples, these steps can also be adapted for use with non-elastomer membranes such as hydrophilic polycarbonate, polysulfone, and polyethersulfone.

[0032] This specification also describes a polyphenol-coated substrate for growing cell sheets. The substrate may include an elastomer membrane and one or more polyphenols attached to the surface of the elastomer membrane. The polyphenol may include tannic acid. The substrate can be made without containing iron. For example, tannic acid may be non-crosslinkable or may not be bound to iron. In some examples, the polyphenol is lignin. The elastomer membrane may include silicone such as PDMS or PBAT. The substrate can have a contact angle that is at least 20, 40, or 55 degrees and at most 70, 75, 80, 85, or 90 degrees. The substrate may have a portion with parallel grooves having a spacing in the range of from 0.01 to 500 microns or from 5 to 50 microns.

[0033] This specification also describes a method for producing a polyphenol-coated substrate. The method includes treating an elastomeric film with an aqueous solution of a plant-derived polyphenol, optionally in the substantial absence of iron, metal, and / or an added crosslinking agent. The aqueous solution may contain tannic acid, for example, at least 10 mg / ml of tannic acid, at least 25 mg / mL of tannic acid, or at least 50 mg / mL of tannic acid, optionally up to 100 mg / mL or up to 200 mg / mL of tannic acid. The aqueous solution may have a pH of 7.5 or less. Alternatively or additionally, the aqueous solution may contain lignin, for example, at least 0.1 mg / ml of lignin, at least 1 mg / mL of lignin, or at least 2 mg / mL of lignin, optionally up to 4 mg / mL or up to 6 mg / mL of lignin. The substrate may be treated with a basic solution, such as a basic solution of NaOH, before treating the elastomeric film with the aqueous solution of the plant-derived polyphenol. The film may be made of silicone, such as polydimethylsiloxane (PDMS) or PBAT. The film may be treated with the aqueous solution for a period of about 6 hours to 4 days.

[0034] This specification also describes a method of producing a cell sheet. The method includes creating a patterned membrane and growing cells layer by layer on the patterned membrane, where the layer contains extracellular matrix (ECM) produced by the cells. The layer can be formed by adding cells to a confluency of 60 - 90% and incubating the cells to full confluency. Multiple cell layers can adhere or fuse to each other to form a cell sheet. The pattern can be created by casting a membrane on a mold produced by three-dimensional (3D) printing, and the pattern of the mold is replicated on the membrane. The 3D printing can be, for example, fused deposition modeling (FDM) 3D printing. The pattern may include grooves, for example, in a parallel pattern, circular pattern, and concentric circle pattern. Cells can be induced to produce more extracellular matrix (ECM) components by treating the cells with ascorbate or by polymer crowding, for example, by adding polyethylene glycol (PEG) or carrageenan to the culture medium. The ECM can be cross-linked, for example, with tannic acid or lignin. The cells and the ECM of the cells can be removed from the surface of the membrane as a cell sheet, for example, by peeling off at least a part of the layer from the surface of the membrane. The remaining part of the layer can be removed from the membrane, for example, by scraping, pulling, vibrating, or spontaneous detachment. The cell sheet can be rolled to form cell fibers. Multiple cell sheets or cell fibers can be stacked together.

[0035] This specification also describes a patterned membrane. The membrane can have a region with parallel grooves having a spacing between 0.01 and 500 microns. The membrane may have multiple sets of grooves with different spacings, for example, a spacing between 5 and 99 or 5 and 50 microns, a spacing between 100 and 500 microns, and / or a spacing between 0.01 and 5, 0.01 and 1 micron, or 0.1 and 1 micron. The membrane may optionally include an elastomer with a polyphenol coating.

[0036] This specification also describes a method of fabricating a patterned membrane. The method includes fabricating a mold through thermally dissolving lamination type 3D printing and casting a membrane on the mold. The nozzle size of the 3D printer can be 0.1 - 0.6 mm. The printing speed can be 1 - 100 mm / second. The mold can be treated with solvent vapor before forming the membrane on the mold. The filaments can be arranged in parallel lines on at least a part of the mold. The membrane can be an elastomer.

[0037] This specification also describes a method of fabricating a cell sheet using stimuli. The method includes growing cells in one or more layers on a membrane and mechanically or electrically stimulating the cell layer. The membrane can be stretched in repeated cycles, for example, to provide static or dynamic stimuli. The method can include applying a varying voltage to the cell layer.

[0038] This specification also includes a method of increasing ECM production. The method includes creating a membrane, growing cells in layers on the membrane, and inducing the cells to produce more extracellular matrix (ECM) components and / or crosslinking the ECM. The cells may be treated with ascorbate. A crowding agent, for example, polyethylene glycol (PEG) or carrageenan, may be added to the culture medium. The ECM produced by the cells can be crosslinked with, for example, tannic acid or lignin.

[0039] This specification also describes a method of fabricating ECM. The method includes growing cells in layers to form a cell construct and decellularizing the cell construct. The decellularized cell construct can be solubilized and DNA residues can be removed. The solubilized ECM can be dried, for example, lyophilized. The ECM gel can be reconstituted from the solubilized or dried ECM. The ECM can be used as a coating or scaffold for growing cells, for example, for growing human cells.

[0040] The devices, elements, and processes described above can be used alone or in any order or combination of components.

Brief Description of the Drawings

[0041]

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DETAILED DESCRIPTION OF THE INVENTION

[0042] Preferred embodiments of the present invention will now be described with reference to the drawings. Like elements in the various figures are identified by like reference numerals.

[0043] Reference will now be made in detail to each embodiment of the invention. Such embodiments are provided as an illustration of the invention and are not intended to limit the invention. Indeed, those skilled in the art will appreciate that various modifications and variations can be made upon reading this specification and viewing the drawings herein.

[0044] The first exemplary process The present invention provides a method or process for creating cell sheets, optionally having a high extracellular matrix (ECM) content and optionally controlling cell orientation. It should be understood that the cell sheets described herein can be made from any cell type from any species. The technology of the present invention is simple, easy to implement, has a low cost, uses non-toxic and biocompatible materials that are optionally food grade and safe as food. Further, the present invention optionally uses a simple molding process using an inexpensive open-source 3D printer and optionally a 3D printed mold prepared using different types of filament materials to enable cell orientation in an optionally refined or selected pattern. Alternatively, a similar pattern can be created on the mold using CNC machining or laser engraving on different materials. In some examples, the present invention provides for the reuse of membranes, for example, by autoclaving, or by a solvent (e.g., isopropyl) washing step, or by a combination thereof. In some examples, the present invention provides multi-layer cell constructs, resulting in strong sheets by induced ECM production with optional ECM cross-linking. The method can use biocompatible, optionally food grade, or food-safe materials. In some examples, the sheets are formed in a short period (18 days or less). In some examples, the sheets can be partially or completely detached using scraping.

[0045] In some embodiments, the general method comprises three steps. First, the method involves growing cells on a processed elastomer membrane such as PDMS or PBAT. The treatment of PDMS has been carried out with various materials to make it suitable for cell culture, but these materials are usually expensive or toxic, or require a multi-step process involving the activation of the PDMS surface. To address this concern, the present invention may utilize the direct treatment of PDMS or another elastomer membrane with a polyphenol solution. In some examples, the polyphenol solution is a tannic acid solution in water or a lignin solution in water and / or methanol or another alcohol to make the membrane suitable for cell attachment. It should be understood that both tannic acid and lignin are safe for food, natural, and non-toxic materials. These treated membranes can be used to grow cells. The treated membranes can also be washed and autoclaved for reuse in growing new cells. This treatment is stable and does not require re-treatment after autoclaving.

[0046] Second, the method involves imparting cell orientation on the surface of the treated membrane. Cell orientation on different membranes has been achieved by using lithography to create micron- or nanoscale features on the surface or by creating protein patterns on the surface of the membrane. However, these processes are expensive, time-consuming, and require special equipment. Some of the materials used in these processes may be of animal origin, thus reducing the consistency of the process. Clearly, the present invention utilizes a textured mold to provide surface features on the formed membrane. In some examples, the mold is optionally made by 3D printing using an inexpensive, open-source printer or by CNC machining or laser engraving to create micron, nano, or mesoscale patterns that can be replicated onto the membrane by molding. The one-step patterning on the membrane surface is sufficient to control cell orientation.

[0047] Thirdly, the method involves the formation of a cell sheet. Cells are grown on a membrane and given an orientation. The membrane pattern also induces ECM formation. Optionally, the cells can be induced to produce a greater amount of ECM by using different growth factors and elements such as ascorbic acid, or by polymer crowding. Once the cells are completely confluent and produce sufficient ECM, more cells are grown on this cell-ECM layer. Optionally, to create a more stable structure, the ECM can be crosslinked with a low concentration of tannic acid or lignin, or other crosslinking agents such as transglutaminase, which are other ECM components. This can be repeated at least three times over a period of two to three weeks. At the end of the process, the layer (including the cells and their ECM) can be physically removed (e.g., peeled off) from the membrane to create a coherent cell sheet. Other techniques for creating a cell sheet involve modifying the membrane properties to make it suitable for cell sheet detachment, digesting some of the proteins necessary for the cells to remain attached to the surface, or inducing the cells to show contraction in order to force them to lift up. Clearly, the method described herein provides, for example, a cohesive multi-layer cell construct that can be peeled off using an external object. Among other advantages, in at least some examples, the process described herein can use any adherent cell type from any species.

[0048] Optionally, one or more aspects of the method may be used alone or in other combinations. For example, the cell sheet may be grown on a smooth (i.e., unpatterned) membrane without cell orientation. In other examples, alternative membrane materials that do not require surface treatment may be used. In other examples, the membrane may be patterned by machining or other physical processes applied to solid membranes.

[0049] Manufacture of the membrane As used herein, the term "membrane" refers to a substrate suitable for supporting a cell sheet. Porosity is optional, but the membrane is typically a bulky material. The membrane is non-porous so that it can flow a large amount of liquid or at least support a liquid medium over the entire cell. The membrane may be made from various thermoplastic or thermosetting materials. The membrane may be rigid or elastomeric. The rigid membrane may be molded, for example, from a biocompatible epoxy resin. The elastomeric membrane may include, for example, a thermoplastic elastomer based on poly(butylene adipate-co-terephthalate) (PBAT), and a urethane rubber formed by reacting a polyol with an isocyanate. Optionally, commercially available materials from Smooth-On, Polytek, and Reynolds Advanced Materials can be used for cell culture-related applications. The elastomeric membrane may also be made of silicone, for example PDMS. However, elastomeric membranes typically have limitations due to their hydrophobicity, such as inappropriate cell attachment, aggregation, and detachment, which can be improved by the surface treatments described herein. PDMS is a preferred material and is used as an exemplary membrane material in many examples herein, but other materials may be used.

[0050] Methods for manufacturing an elastomeric membrane such as a PDMS membrane are also described herein. It should be understood that PDMS comprises an elastomer base and an elastomer curing agent. Other silicone-based or non-silicone-based elastomers contain two components that catalyze their reactions to form an elastomeric resin or rubber upon mixing. The method includes mixing an elastomer base and an elastomer curing agent, or two components of an elastomer, in a specific ratio (e.g., a volume or weight ratio of curing agent to base of 1:1 to 1:20), and several process steps such as casting and heating an elastomer solution to cure the polymer and form a membrane. A mold may be printed using a 3D printing method and then used for casting an elastomer solution to create a membrane. Exemplary 3D printing methods that may be used include fused deposition modeling (FDM) 3D printing. Subsequently, a pattern on the surface of the 3D printed mold (if present or included, a pattern specific to the 3D printing process) can be reflected onto the membrane. This can be utilized to create a pattern (e.g., a parallel pattern) on the elastomeric membrane that can be used to orient cells.

[0051] Different polymer materials can be used with various commercially available 3D printers such as Ultimaker and Prusa for printing molds. Alternatively or additionally, other techniques for creating patterns such as CNC machining or laser engraving may be used to fabricate the mold or to modify a 3D printed mold. The 3D printer may be configured or operated to create patterns with different resolutions that can have various effects on cell orientation. When the 3D printer is used to print the mold, the nozzle size can be from 0.1 to 1 mm, for example, 0.25 mm, 0.4 mm, or 0.6 mm, and the printing speed can be from 20 to 100 mm / second. It should be noted that smaller nozzle sizes and lower printing speeds can create smaller features that may exhibit better performance in cell orientation depending on the cell type and its environmental conditions. In some examples, thermoplastic polymers such as PETG, ABS, and PVB (polyvinyl butyral) are used in 3D printing. However, it should be understood that other polymers not explicitly listed herein may also be used. Additionally, other methods may be used to create the mold using non-polymeric materials such as molds. The mold may optionally be treated to change the pattern. For example, to melt the surface of a polymer mold and change the resolution of the 3D printed part, and as a result, change the size or smoothness of the pattern on the membrane, the ABS mold may be treated with acetone vapor and the PVB mold may be treated with isopropyl alcohol vapor. Understand that the timing of the treatment defines the final pattern or its absence.

[0052] Regardless of the material used to 3D print the mold, three levels of features can be created on the surface of the mold. The wider lines of the mesoscale pattern seen in FIGS. 13 and 14 can have a spacing between 100 and 500 μm. These patterns can have a certain moderate effect on cell orientation. The thinner lines with a spacing of 5 to 50 μm of the micron-scale pattern are particularly useful for imparting alignment to cells. Features smaller than 1 μm of the nanoscale pattern create a surface roughness that induces cells to secrete a greater amount of ECM components. Together, these features create a 3D-like environment for cell growth and function and create a basal morphology, changing cell behavior compared to in vivo conditions, compared to a surface that does not have complete two-dimensional features. Using a larger size of the printer nozzle or faster printing will shift the size of the features to larger ones within the aforementioned range.

[0053] Other methods such as using sandpaper with various grits can also be used to modify the features on the surface of the mold after printing, but they may create new additional features while removing the old ones. Such methods may not be as reproducible as treatment with solvent vapor.

[0054] In some examples, the treatment time with the vapor phase of the solvent for a 3D printed mold of polymer filament was in the range of about 0 minutes to about 30 minutes. After vapor treatment, the mold can be heat treated for 1 to 12 hours in the range of 20 to 60 °C to evaporate the solvent from the surface of the mold and solidify the polymer. Once the mold is prepared, the elastomer solution is poured in and heated for a period of 1 to 24 hours at a temperature in the range of 25 to 90 °C to cure the polymer. By performing the heating process at a high temperature, the required time is consequently shortened to solidify the elastomer resin.

[0055] Figure 1 shows images of ABS molds with and without acetone vapor treatment, and PDMS membranes formed using the ABS molds, according to at least some embodiments disclosed herein. Specifically, Figure 1 shows a first row 112 associated with ABS molds 102, 104 without 30 minutes of acetone vapor treatment and PDMS membranes 106, 108 formed using the ABS molds 102, 104, and a second row 110 associated with ABS molds 102, 104 with 30 minutes of acetone vapor treatment and PDMS membranes 106, 108 formed using the ABS molds 102, 104.

[0056] In the example shown, mold 102 has a platform 110 and a moat 112. Membrane 106 has a bottom 116 and a wall 118. In the example shown, wall 118 is a polygon having a plurality of segments. In other examples, wall 118 may have another shape, such as circular. In Figure 1, membrane 106 is removed from mold 102 and inverted. The upper surface of bottom 116 is formed by resin cast on platform 110. Wall 118 is formed by resin cast in moat 112.

[0057] The photograph of the mold 104 shows an enlarged view of the area of the platform 110 in the photograph of the mold 102. The platform 110, in this example, is essentially produced by a 3D printing process with a topographical pattern, and optionally, as in the case of the second row 110, is further modified. In at least the area of the platform 110 shown in the photograph of the mold 104, the pattern includes a series of generally parallel grooves 110. The photograph of the membrane 108 is an enlarged view of the area of the bottom 116 in the photograph of the membrane 106. The upper surface of the bottom 116 of the membranes 106, 108 has a topographical pattern that reflects the topographical pattern of the platform 110 of the molds 102, 104. In at least the area shown in the photograph of the membrane 108, the upper surface of the bottom 116 of the membranes 106, 108 has a series of generally parallel grooves 120. The wall portion 118 may be, for example, 3 to 10 mm in height and can contain a liquid medium and cells prior to attachment on the bottom 116 of the membranes 106, 108. The first layer of cells preferably temporarily attaches to the bottom 116 of the membranes 106, 108. In the example shown, the membranes 106, 108 are square in plan view and the width and length are about 30 mm. Optionally, the membranes 106, 108 may have other shapes, such as circular or rectangular. The membranes 106, 108 may also have other sizes, such as lengths or diameters of 10 to 100 nm or more.

[0058] Figure 2 displays an image showing the effect of materials (e.g., ABS202, ABS204 with acetone vapor treatment, or PETG206) used for 3D printing of molds according to at least some embodiments disclosed herein. The present invention also contemplates creating patterns such as circular or concentric patterns by controlling the pattern in which materials, such as those shown in Figure 3, and in particular those not explicitly listed or shown herein, are deposited during the 3D printing process.

[0059] Regardless of the pattern or material used for printing, it should be understood that the cross-section of the pattern, for example, the grooves of the pattern, are typically similar. It is also possible to use other methods and materials to fabricate the mold, preferably using sizes of patterns (e.g., the spacing between grooves) that are similar to those created using 3D printing, or other sizes that can produce similar effects on cell attachment and orientation or ECM production. For example, similar molds may be fabricated using CNC machining and laser ablation of other materials. Furthermore, the mold can be fabricated from metal or polymer materials as well.

[0060] Treatment of the surface of the elastomeric membrane for cell attachment Many elastomeric materials, including silicone and many non-silicone-based membranes, are hydrophobic materials. Hydrophobic materials are generally inappropriate for cell attachment and tend to cause cells to form aggregates rather than sheets. The manufacture of membranes for imparting surface topographies as described above can improve cell sheet formation. Furthermore, the surface of the elastomer or other membrane can optionally be modified by chemical treatment, for example, using a hydrophobizing agent, to make them more suitable for cell attachment.

[0061] In some examples, elastomeric membranes, silicone, membranes such as PDMS or PBAT, are treated with an aqueous solution of polyphenol. The polyphenol may be of plant origin, for example, tannic acid or lignin. The polyphenol may be applied to the membrane without additional cross-linking agents or additives (e.g., iron).

[0062] In some examples, the treatment can include contacting the membrane with tannic acid at 10 - 200 mg / mL, optionally 25 - 100 mg / mL. The treatment can generally be carried out at ambient temperature or room temperature. The contact period can be about 6 hours to 4 days, or 1 - 3 days. The tannic acid solution is not essentially buffered, for example, the aqueous solution can have a pH of 7.5 or less, or 7.0 or less. Optionally, the tannic acid solution contains only tannic acid and water. Tannic acid is applied to the membrane without additional cross-linking agents or compounding agents, for example, in the absence of iron. While not intending to be bound by theory, the dihydroxyphenol groups or trihydroxyphenol groups of lignin may be able to adsorb onto the surface of PDMS or other elastomers without surface activation of the membrane. As further described below, after optional treatment with a caustic solution, the membrane can also have functional groups available for chemical bonding (e.g., silanol groups). However, surface activation is not necessary. Furthermore, additional cross-linking of the polyphenol (e.g., by iron or polyethyleneimine, or beyond cross-linking that may be present in natural molecules) is not necessary. In some examples, a cell growth surface is produced that essentially has only the membrane material and the attached polyphenol molecules, and the polyphenol molecules are not bonded to each other except through the membrane.

[0063] In some examples, the treatment can include contacting the membrane with a solution of lignin at about 0.1 - 6 mg / mL, or 1 - 4 mg / mL. The treatment can generally be carried out at ambient temperature or room temperature. The contact period can be about 6 hours to 4 days, or 1 - 3 days. The tannic acid solution may not be essentially buffered. Optionally, the tannic acid solution contains only tannic acid, water, and a water-miscible solvent, such as methanol or another alcohol.

[0064] For this purpose, polyphenols of lower or higher concentrations can also be used, but it should be understood that the timing of the treatment has to be adjusted accordingly. In some embodiments, a solution containing both tannic acid and lignin can be used, or different solutions containing tannic acid and lignin can be used individually and alternately.

[0065] To improve the adhesion of tannic acid, lignin or another polyphenol to the surface of the membrane, the membrane can be pretreated with a caustic solution, such as an aqueous sodium hydroxide solution. Alternatively, the membrane may be pretreated with oxygen, air or carbon dioxide plasma. Optionally, the membrane is not pretreated with any plasma caustic solution.

[0066] After the polyphenol treatment (i.e., using tannic acid and / or lignin), the membrane can be washed to remove the residue. For example, the membrane can be washed with deionized water, optionally at least twice, to remove the residue. The membrane can then be used immediately for cell culture or stored at room temperature for a period of weeks or in a refrigerator for a long period.

[0067] For sterilization purposes, before starting the polyphenol treatment, the membranes can be washed with approximately 70% ethanol and then be subjected to UV radiation, or they can be autoclaved. The polyphenol (tannic acid and / or lignin) solution can be sterilization filtered using a syringe filter of approximately 0.2 μm. To avoid the possibility of contamination, the treatment may be carried out in a biosafety cabinet.

[0068] FIG. 4 shows an image depicting the effect of treatment on the improvement of cell adhesion among the PDMS membrane 404, the treated PDMS membrane 406, and cells grown in the control group 402 (e.g., commercially available cell cultures treated with polystyrene) according to at least some embodiments disclosed herein. As shown in FIG. 4, the untreated PDMS membrane 404 does not allow for proper cell adhesion, and the cells will either form aggregates or clumps or die. Here, the membrane has no pattern and thus no orientation is imparted to the cells. Optionally, a smooth membrane can also be used to create a cell sheet, but there is no intentional cell orientation.

[0069] Figure 12 shows the effect of concentration and time as measured by their contact angles in various examples of treatment with tannic acid or lignin on the hydrophobicity of the PDMS membrane. The PDMS membranes were not pretreated with, for example, caustic solution or plasma to activate their surfaces. Three concentrations of aqueous solutions of tannic acid (25, 50, and 100 mg / mL) and lignin (1, 2, and 4 mg / mL) were used, either for 24-hour treatment or 72-hour treatment. In each case, three 1×1 cm pieces of the PDMS membrane were cut out and treated at room temperature. Contact angle measurements were performed by taking photographs of 10 μL water droplets on each membrane at magnifications of 50 - 1000 times using a portable digital microscope. The photographs were analyzed using the Contact Angle Plugin in ImageJ. For both tannic acid and lignin, increasing the concentration and time of treatment decreased the contact angle. In the cell growth experiment, cell sheets were grown using membranes having contact angles in the range of about 60 - 87 degrees. However, better results were obtained at lower contact angles closer to 60 degrees than at contact angles closer to 87 degrees. Furthermore, the literature suggests that contact angles reduced to 55 degrees and further to 20 degrees may be useful. Thus, the concentration and time of treatment are optionally selected to result in a contact angle of 87 degrees or less, 85 degrees or less, 80 degrees or less, 75 degrees or less, or 70 degrees or less. The concentration and time of treatment are optionally selected to result in a contact angle of 20 degrees or more, 40 degrees or more, 55 degrees or more, or 60 degrees or more. The preferred contact angle may also vary depending on the cell type or the surface topography of the membrane. Treatment with a 50 mg / mL solution of tannic acid for 72 hours using various cell types and PDMS membranes with smooth or textured structures produced high-quality cell sheets.

[0070] The treated membrane is hydrophilic but is not coated or functionalized with exogenous ECM components such as structural proteins (e.g., collagen, elastin, fibronectin, and laminin), proteoglycans, and glycosaminoglycans (i.e., ECM components not produced by cells grown on the membrane). Adding ECM components to the membrane promotes cell attachment, but the form of this attachment may be too strong for detachment of the cell sheet. Further, in at least some examples, it is desirable to avoid using any exogenous ECM components and instead generate a cell layer or cell sheet that contains only ECM produced by cells grown on the membrane. Similarly, in some examples, the contact angle of the treated membrane is moderate, e.g., 40 degrees or more or 55 degrees or more, as a lower contact angle can result in a cell construct that is more difficult to separate from the membrane.

[0071] Cell orientation 3D printing using FDM has a very low resolution and it should be understood that it creates mesoscale patterns (up to several hundred microns) as well as other micron and nanoscale patterns. These patterns can be considered artifacts, but as shown in FIG. 5, they can be used as topographical signaling cues for very small cells because they are oriented in a certain direction. Specifically, FIG. 5 shows parallel pattern 502, circular pattern 504, concentric circular pattern 506, and pattern shades of the mold 508 treated with acetone vapor. By controlling the printing pattern at various locations of the mold, a membrane having different patterns at various locations can be created and used to pattern cells. This example is for skeletal muscle tissue, where all cells need to be properly differentiated and oriented to form muscle fibers that align with each other. As an illustrative example, rabbit primary myoblasts are used for demonstration purposes in FIG. 5. In the case of a mold treated with ABS, treatment with acetone vapor for a certain period of time (e.g., 5 to 30 minutes or more) results in reducing the patterns on the mold completely or to an extent that cells cannot recognize them. As a result, the cells still show proper attachment but do not have any orientation.

[0072] Cell sheet formation As described herein, a "cell sheet" is a scaffold-free or self-organizing sheet-like construct that includes cells and the ECM secreted by the cells, or a part of such a construct. Cell adhesion to each other and cell adhesion to their ECM are preserved in the cell sheet, and thus the cell sheet can be maintained independently of a membrane or surface and adhere for at least a certain period of time. Since the ECM is preserved in each sheet, when multiple sheets are stacked on top of each other, they can bind, fuse with each other, or fuse with a tissue or organ.

[0073] Furthermore, an elastomer or other membrane, optionally treated with lignin and / or tannic acid, with or without a pattern, can be used for sheet formation in a single process. The membrane provides a surface suitable for cell adhesion, and the cells first form a strong adhesion to the membrane. Over time, the cells proliferate, begin to adhere to each other, and form strong cell-cell junctions. They also secrete their own ECM and then adhere to that ECM.

[0074] As the cells become more confluent and establish strong adhesions to each other or to their ECM, and as more layers are added as described below, the adhesion between the cells and the membrane weakens. When the adhesion of the cells to each other and to their ECM becomes stronger than their adhesion to the membrane, the cells and their ECM can be detached or detachment can be initiated by the scraping method. It is also possible to scratch the edge of the membrane, vibrate it to create a cell sheet, and apply shear force to assist in cell detachment as a sheet. It should also be understood that tweezers or similar means are used to grasp the sheet with sufficient strength to pull it away from the surface. When pulled away from the surface, the cells will maintain their orientation because the cell density is high, the ECM content in the sheet is high, and this prevents the cells from changing their orientation over a certain period of time. Optionally, the membrane can be peeled off to release the edge of the cell sheet, and subsequently, a) pull the edge of the cell sheet to form or release the rest of the cell sheet, b) wait for a certain period, e.g., 0.2 - 2.0 hours and then peel or pull to form or release the rest of the cell sheet, and / or c) optionally wait while continuing to incubate the cells, where the rest of the cell sheet can be formed or released naturally. When separating the lower layer of cells from the membrane, the bottom surface of the separated lower layer of cells may change almost instantaneously towards the detached sheet structure, which may cause further changes to the cells still attached to the membrane. Using an elastomeric membrane may also deform the part of the membrane that is not directly scraped off, thus causing or assisting in the detachment of the cells attached to the part of the membrane that is not directly scraped off. The cell sheet may include a completed construct completely released from the membrane, or the edge or other part of a construct released from the membrane with another part of the construct remaining attached to the membrane.

[0075] The cell sheet may have one or more layers. When cells cover the entire surface of the membrane and produce ECM, it is possible to grow them on top of the first layer of cells and add more cells to create a multi-layered construct. This can be done multiple times to produce thicker constructs, but if it is done more than a certain number of times depending on the cell type and species, up to 10 times maximum, the adhesion between cells of different layers becomes much stronger than the adhesion to the membrane of the first layer, which can lead to spontaneous detachment of the cells from the membrane. Spontaneous detachment can be useful, especially when caused by peeling, scratching, or vibrating, but uncontrolled spontaneous detachment of the cell layer can be followed by excessive contraction and the sheet may rapidly turn into aggregates.

[0076] Furthermore, by treating the cells with components such as ascorbate family members, or by adding elements such as high molecular weight PEG or carrageenan to the medium at appropriate concentrations, or by creating polymer crowding, it is possible to induce the cells to secrete more ECM components. To enhance the stability of the cell construct, it is also possible to crosslink the ECM secreted by the cells before adding more cells. To avoid aggregation of the medium components, low concentrations of tannic acid or lignin in serum-free medium (e.g., less than 0.1 mg / mL of tannic acid or less than 10 μg / mL of lignin) can be used to crosslink the ECM components in a short time. The timing of the treatment can be in the range of about 5 minutes to about 100 minutes, depending on the type of crosslinking agent and its concentration before adding new cells. Multiple cell layers as well as more ECM content and crosslinking can result in a sheet with better mechanical integrity that can help in handling the sheet and moving them from one container to another after delamination. Furthermore, the present invention contemplates the use of either multiple cell types in each layer or different cell types on different layers. For example, FIG. 6 shows images depicting different steps of sheet formation using a PDMS membrane and the preserved cell orientation in a stand-alone cell sheet according to at least some of the embodiments disclosed herein.

[0077] In an exemplary method, on day 1, a growth medium is added on top of the membrane at a depth of about 3 - 5 mm. A sufficient number of cells are added on top of the membrane at a confluence density of about 60 - 90% or 75 - 85%. The cells are incubated, for example, at 37°C or the physiological temperature of animal cells for 2 days. On day 3, the cells will have grown to form a first (single cell) layer that is completely confluent. The growth medium is refreshed, i.e., removed (e.g., by aspiration) and replaced with fresh medium. In the case of stem cells, the fresh medium can be a differentiation medium. For stem cells, differentiation and fusion to adjacent cells typically occur together. For other cell types that adhere to each other (as opposed to fusing), the fresh medium can be more growth medium.

[0078] On day 4, the same number of cells as added on day 1 are added to the first layer of cells. On day 5, the medium is refreshed with either fresh growth medium or, for stem cells, fresh differentiation medium. On day 6, a second layer is formed and the same number of cells as added on day 1 are added on top of the second layer of cells. On day 7, it is refreshed with either fresh growth medium or, for stem cells, fresh differentiation medium. On day 8, a third layer is formed and the same number of cells as added on day 1 are added on top of the third layer of cells. On day 9, the medium is refreshed with either fresh growth medium or, for stem cells, fresh differentiation medium. On day 10, a fourth cell layer is formed. In this example, the cell sheet will have four layers. Optionally, more or fewer layers, i.e., 2 or 3 or 5 or more layers, may be created. On day 11 and then every 2 days thereafter, the medium is refreshed again. The cells continue to produce ECM and the cell layer becomes stronger. On day 18, the edges of the cell layer can be peeled off the membrane and the cell layer can be detached from the membrane. The cells change rapidly after they are detached from the membrane and a cell sheet is formed. This example illustrates a typical process, but the schedule of cell and medium addition, or various other steps, may vary.

[0079] Reuse of the mold When the cell sheet is delaminated from the membrane, the cells and their ECM are removed. The cell sheet is removed, but the membrane allows non-specific penetration of certain small molecules into its bulk, making the membrane opaque. By immersing the membrane in deionized water and autoclaving the membrane, for example, over a period of about 20 minutes, the initial removal of these components is enabled. To further remove such residues, after the autoclaving process, the membrane may be immersed in isopropyl or other non-polar solvents that can cause swelling of the elastomeric membrane for a period of, for example, about 10 - 60 minutes. The solvent swells the membrane and the remaining of these residues can be removed. At this point, the membrane can be reused for cell culture without repeating the treatment with tannic acid and / or lignin.

[0080] As an exemplary example, FIG. 7 shows an image of a PDMS membrane used for cell culture and reused three times, in rounds 1 702, round 2 704, and round 3 706, according to at least some embodiments disclosed herein. There are no apparent differences during cell attachment, proliferation, differentiation, or orientation. Further, rabbit myoblasts were grown on the PDMS membrane for a period of about 8 days in each round (e.g., round 1 702, round 2 704, and round 3 706) and differentiated into myofibers.

[0081] Cell type Membranes, e.g., tannic acid / lignin-treated elastomer membranes with or without patterning, can be used to grow different cell types from different tissues of different species for both primary and immortalized cells. FIG. 8 shows images of rabbit primary myoblasts 802, bovine primary myoblasts 804, bovine primary fibroblasts 806, and human HepG2 hepatocytes 808 at low confluence density 812 and high confluence density 810, according to at least some embodiments disclosed herein. Different stem cells, such as adult stem cells, embryonic stem cells, or induced pluripotent stem cells, can also be used in this process. Stem cells can also be differentiated into target cells while being grown and maintained on these membranes by switching the culture medium from a growth medium to an appropriate differentiation medium. As described herein, "confluence density" is the percentage of the area covered by adherent cells. This measurement is routinely used to monitor cell growth and expansion during cell culture experiments. This is useful in determining the optimal timing for process interventions such as cell harvesting, passage, and drug treatment, or for cell differentiation.

[0082] Specifically, according to FIG. 8, myoblasts having an elongated spindle shape from both rabbit and bovine sources (e.g., rabbit primary myoblasts 802 and bovine primary myoblasts 804) showed an orientation following the membrane pattern, while bovine fibroblasts (e.g., bovine primary fibroblasts 806) maintained their flattened shape. Hepatocytes having a circular shape (e.g., human HepG2 hepatocytes 808) also maintained their shape independent of the membrane pattern.

[0083] Assembly of the sheet In some examples, the cell sheet can be formed or modified into other configured cell constructs. For example, the cell sheet can be rolled onto them, and the preserved ECM will result in the formation of an adhered cell structure that cannot be spread. A plurality of cell fibers 902 made from the rolled sheet can be stacked on top of each other, and they will also form a firm adhesion to each other as shown in FIG. 9. When skeletal muscle cells are used, rolling can be performed in a direction that results in muscle cells aligned in the direction of the larger fibers. This functional unit can be used in different applications such as for in vitro modeling of skeletal muscle tissue for drug discovery or disease modeling, development of cultured meat, or as a building block for biofabrication of skeletal muscle tissue for regenerative medicine applications.

[0084] The present invention also contemplates creating cell fibers with different cell types and laminating them. Examples of this application include creating marbling in cultured meat using cells derived from skeletal muscle, fat, and connective tissue, or creating a more physiologically relevant model of different tissues and organs by reproducing cell crosstalk. Sheets stacked on top of each other without first rolling them will also adhere to each other and form an adhered structure. This example includes creating a sheet of hepatocytes, stacking them to create a liver-like tissue for in vitro modeling or creating foie gras as a food.

[0085] Bioreactor for dynamic environment Cells are constantly receiving dynamic cues in in vivo conditions. These cues include electrical and / or mechanical stimuli. Reproducing such cues in in vitro conditions can be used to induce cell behaviors such as cell orientation, ECM production, and further differentiation pathways of stem cells into different cell types. By stretching or compressing a flexible membrane, it is possible to apply mechanical stimuli to cells grown on an elastomer membrane. Electrodes can also be inserted close to the surface or into the bulk of the membrane above the surface to apply electrical stimuli to the cells. Figure 10 shows a scenario of a first mechanical stimulus 1002 and a scenario of a second electrical stimulus 1004. For the mechanical stimulus, the membrane 106 is attached to a linear actuator 1006. The linear actuator 1006 is adapted to compress or stretch the membrane 106. For the electrical stimulus, the electrode 1008 is cast into the membrane 106. The electrode 1008 is attached to a power source 1010 adapted to provide voltage and / or current to the electrode 1008.

[0086] Depending on the cell type, species, or specific application, different parameters can be controlled to deliver appropriate mechanical or electrical stimuli to the cells. For mechanical stimuli, the cells can be exposed to static or dynamic stimuli. In static stimuli, the elastomer membrane can be stretched by 1 - 20%. This stretched state can be maintained for 10 - 100 minutes or longer, after which the elastomer membrane returns to its original resting size. After a certain time, similar or different stretching conditions can be performed for similar or different times. In dynamic stimuli, the important parameters are the amplitude and frequency of the stimulus and the active time. The amplitude of deformation / stretching can be 1 - 20% or more, and the frequency can be 0.1 - 10 Hz or more. The stimulus is applied for 1 - 10 minutes or more, followed by a resting mode of 1 - 10 minutes or more, after which the same or different stimuli are applied. Similar static or dynamic electrical stimuli can be applied by controlling the voltage (0.1 - 5 V), frequency (1 mHz - 20 Hz or more), and duty cycle.

[0087] Patterning of Different Cell Types The pattern created on the surface of membrane 1102 of FIG. 11 creates grooves 120 having a concave shape. The depth of these grooves is not much larger than the size of the cells 1106 themselves (the size of the cells 1106 is highly exaggerated in the central panel of FIG. 11), and they can further cover the entire surface uniformly, but this feature can be used to pattern different cell types on the membrane 1102 or to impart orientation. Optionally, each of these concave grooves 120 can be filled with a small amount of the cell culture medium 1104 of FIG. 11 having a particular cell type in such a way that the solutions in adjacent grooves 120 do not contact each other. After a while (e.g., after about 30 minutes to about 60 minutes), the cells adhere to the surface of the grooves 120, and excess medium 1104 can be added to cover the entire surface. This can be used to position different cell types at different locations on each membrane 1102. For example, by patterning fat and muscle cells, it is possible to create marbling for cultured meat applications.

[0088] Decellularized Cell Sheet as a Fibrous Scaffold After the sheet is formed and removed from the membrane, since a high content of ECM is present, the sheet can be decellularized using a mild detergent such as Triton X-100, SDS, or Tween20 at room temperature for a short time or on ice for a longer time. Decellularization can be carried out at room temperature or at 4°C using a 0.1 - 1% V / V or wt / V solution of each of the above-mentioned detergents or a combination thereof. Decellularization can be carried out for 1 - 48 hours depending on the temperature or concentration of the detergent. The solvent for the detergent can be either deionized water or a buffer such as phosphate buffered saline (PBS). It is also possible to treat the sheet with one detergent solution, wash them with deionized water or PBS, and continue the treatment with a fresh batch of the same or different detergents. Using a fresh detergent solution can further improve the decellularization process.

[0089] After decellularization, an ECM-rich fibrous sheet remains, which can be used for 3D cell culture in in vitro or in vivo regenerative medicine and tissue engineering applications. The ECM sheet can also be treated with DNase and RNase to remove any DNA and RNA residues. The composition of the ECM can be controlled by using different cell types or treating them with different types of media. External stimuli, including membrane surface patterning, as well as electrical and mechanical stimuli, can be used to orient ECM components in a specific direction, as described herein. Further cross-linking of ECM proteins can also be performed before or after decellularization. The directionality of the ECM is preserved during decellularization and can induce cell behavior after recellularization.

[0090] Solubilized ECM The decellularized ECM can also be solubilized to create a liquid ECM that can be used for surface treatment or 3D cell culture. The decellularized ECM can be solubilized using a high ionic strength solution such as 3-4 molar urea solution in deionized water or PBS. For this purpose, the decellularized sheet can be immersed in such a solution and held on ice for 24-48 hours. Ultrasonic treatment at a frequency of 1-10 Hz for 2-5 seconds every 6-12 hours can be applied to disrupt the ECM and further facilitate the dissolution of ECM molecules in the buffer. The ultrasonic treatment should be performed while holding the sample on ice to avoid denaturation of ECM proteins due to heat generation. Similarly, the decellularized sheet is treated with enzymes such as pronase, dispase, or various collagenases (such as collagenase types I, II, III, and IV) or combinations thereof. Depending on the origin of the cells and the conditions of ECM production, different concentrations of these enzymes can be used, such as 0.1-2 mg / mL, 0.5 and 5 U / mL, or 0.1-5 mg / mL. The timing of the treatment varies from 12 hours to a maximum of 4 days. Ultrasonic treatment and homogenization may be performed to assist solubilization. It should be understood that these processes are performed on ice. After solubilization, the solution is filtered using a dialysis filter to remove the enzyme or ionic solution. It is then lyophilized, re-solubilized in deionized water to achieve the desired concentration. This solution can then be stored refrigerated or frozen until use. Potential uses for this include treating surfaces that are not suitable for cell attachment to make them cell adhesive or 3D culture systems using "cell-derived ECM" as the main matrix. This solution is temperature-sensitive and can rapidly gel within minutes by raising the temperature to about 37°C.

Example

[0091] [Example 1]: Formation of a cell sheet using rabbit myoblasts on a patterned membrane fabricated using an orientation-imparted ABS mold In this experiment, rabbit myoblasts were used to differentiate into skeletal muscle cells to form a cell sheet with oriented cells, and these steps were followed: 1. The master mold was 3D printed using an Original Prusa i3 MK3S+ and ABS. The nozzle size was 0.4 mm, and the printing speed was 60 mm / s. 2. PDMS SYLGARD184 was used for membrane fabrication using a 10:1 ratio of base and curing agent. Curing was carried out at 60 °C for 4 hours. The membrane was 3 × 3 cm in size. 3. After sterilizing the membrane with 70% ethanol, it was treated with 3 mL of a sterile aqueous solution of tannic acid at a concentration of 50 mg / mL for 72 hours, and then it was washed with deionized water to remove any trace of tannic acid. 4. Rabbit myoblasts (Sigma-Aldrich, RB150-05) were grown in a 10 cm Petri dish in their growth medium (Sigma-Aldrich, RB151-500) to a confluence density of 80%. They were lysed using trypsin, and 5×10 5 of them were added to the membrane in the same growth medium. This was considered day 1 of the sheet formation process, and the cell number would result in a confluence density of 80% when the cells adhered to the membrane. 5. On day 3 of the experiment, the medium was switched to the cell differentiation medium (Sigma-Aldrich, 151D-250) with sufficient L-ascorbate-2-phosphate added to achieve a concentration of 100 μg / mL. This medium was refreshed every 2 days until the experiment ended. 6. On days 4, 6, and 8, the same number of cells (5×10 5 ) were directly added to the differentiation medium on top of the membrane. The newly added cells immediately adhered on top of the previous layer of cells and would differentiate into skeletal muscle cells. 7. On day 18, the cells and their ECM could be peeled off as a sheet using a cell scraper (Sigma-Aldrich, CLS3010-100EA).

[0092] [Example 2]: Formation of cell sheets by bovine fibroblasts on a minimally patterned membrane fabricated using a PVB mold treated with mechanical stimulation 1. The master mold is 3D printed using an Original Prusa i3 MK3S+ and PVB. The nozzle size is 0.4 mm and the printing speed is 60 mm / second. 2. The mold is treated with isopropyl alcohol for 45 minutes and then left overnight at room temperature (25 °C) to evaporate, solidify the polymer, and eliminate the pattern. 3. Similar to the experiment before using PDMS, a membrane is generated by treating with a 1 mg / mL solution of lignin in a 1:1 mixture of deionized water and methanol for 72 hours. 4. After washing, 5×10 5 primary bovine fibroblasts grown in their growth medium (DMEM + 10% FBS) are added to the membrane in the same medium. 5. On day 3, the medium is refreshed with the same growth medium supplemented with sufficient L-ascorbate-2-phosphate to achieve a concentration of 200 μg / mL, and this is refreshed every two days. 6. The same number of cells (5×10 5 cells) are added on days 4, 6, 8, and 10. 7. Mechanical stimulation is applied to the membrane by continuously stretching the membrane from day 4 to day 12 at an amplitude of 10% and a frequency of 1 Hz. 8. On day 14, the cells and their ECM are peeled off to form a sheet.

[0093] [Example 3]: Use of decellularized ECM as a coating for the culture of human induced pluripotent stem cells 1. The rabbit cell sheets formed in the first experiment are used here. Four sheets are washed with PBS to remove the medium residue. Then, they are immersed in 10 mL of 0.2% Triton X-100 in deionized water and held on ice overnight. 2. The Triton X-100 in deionized water is refreshed after 24 hours. 3. Forty-eight hours after treatment with the decellularization solution, the sheet is centrifuged at 300 g for 5 minutes and then washed three times with PBS to remove any detergent residues. 4. The decellularized sheet is then immersed in a 4 M solution of urea in ice-cold deionized water. The solution is sonicated at a frequency of 1 Hz every 12 hours while being held on ice at 2-second intervals. 5. After 48 hours, the ECM is completely dissolved in the solution. This is centrifuged again at 10,000 g for 10 minutes to remove any floating components. 6. The solution is then dialyzed for 48 hours using a 1 kDa dialysis filter against deionized water. 7. The final solution is then frozen at -80 °C for 2 hours and then lyophilized to form a lyophilized powder. 8. The lyophilized decellularized ECM is then reconstituted in 1 mL of PBS containing 0.1 M acetic acid. 9. A non-tissue culture-treated 6-well plate is then treated overnight at room temperature with 2 mL of a 10-fold dilution of the solubilized cell-derived ECM in PBS. 10. This treated well plate is then used to grow human induced pluripotent stem cells (iPSCs) (Stem Cell Technologies, 200-0511). These cells typically require coating with vitronectin, laminin, or Matrigel™ on both tissue culture-treated well plates and non-tissue culture-treated well plates. The cell-derived ECM solution is an alternative to these animal-derived or recombinantly produced ECM solutions.

[0094] [Example 4]: Formation of a cell sheet by bovine myoblasts on a patterned membrane made of an Ecoflex 00-30 film on an ABS mold 9. The master mold is 3D printed using an Original Prusa i3 MK3S+ and ABS. The nozzle size is 0.4 mm and the printing speed is 60 mm / s. 10. Similar to the previous experiment, a membrane is fabricated using Ecoflex 00-30 (PBAT) instead of PDMS. Components A and B of the resin are mixed at a volume ratio of 1:1 and cast on an ABS mold. The curing of the resin is carried out at room temperature for 4 hours. The solidified membrane is white and opaque, and cannot be examined under a microscope. Tannic acid treatment is carried out for 72 hours with a 50 mg / mL solution as described herein. 11. After washing, 1×10 6 primary bovine fibroblasts grown in their growth medium (DMEM + 17% FBS, 2% fetal bovine serum, and 1% egg yolk extract factor) are added to the membranes in the same medium. 12. On day 3, the medium is refreshed to a differentiation medium (DMEM + 2% fetal bovine serum and 10 ng / mL FGF) with 100 μg / mL L-ascorbate-2-phosphate added thereto, and it is refreshed every two days. 13. The same number of cells (7.5×10 5 cells) are added on days 4, 6, and 8. 14. On day 18, the cells and their ECM are stripped off to form a sheet.

[0095] The descriptions of the various embodiments of the present invention have been presented for purposes of illustration, but are not intended to be an exhaustive or limiting listing of the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the above-described embodiments. The terms used herein have been selected to best explain the principles of the embodiments, the practical application or technical improvement to technologies found in the market, or to enable others or those skilled in the art to understand the embodiments disclosed herein.

[0096] When introducing an element or an embodiment of the present disclosure, the articles "a," "an," and "the" are intended to mean that one or more of the elements are present. Similarly, the adjective "another," when used to introduce an element, is intended to mean one or more elements. The terms "including" and "having" are intended to be inclusive such that additional elements may exist in addition to the recited elements.

[0097] Although the present invention has been described with a certain degree of particularity, it should be understood that the present disclosure has been made by way of example only, and that numerous changes in the details of the structure and arrangement of parts may be used without departing from the spirit and scope of the present invention.

[0098] [Table 1] TIFF2025524312000003.tif147169

Claims

1. A substrate for growing cells or cell sheets, comprising: an elastomeric membrane having a patterned surface; and a hydrophilic agent on the surface of the elastomeric membrane.

2. The substrate according to claim 1, wherein the hydrophilic agent is not an exogenous extracellular matrix (ECM) component.

3. The substrate according to claim 1 or 2, wherein the patterned surface is formed on the elastomeric membrane.

4. The substrate according to any one of claims 1 to 3, wherein the hydrophilic agent is a polyphenol.

5. The substrate according to any one of claims 1 to 4, wherein the hydrophilic agent is tannic acid or lignin.

6. The substrate according to any one of claims 1 to 5, wherein the hydrophilic agent is tannic acid and the tannic acid is not bound to iron.

7. The substrate according to any one of claims 1 to 6, wherein the elastomeric membrane contains silicone, such as PDMS or PBAT.

8. The substrate according to any one of claims 1 to 7, having a contact angle of at least 40 degrees and at most 85 degrees.

9. The substrate according to any one of claims 1 to 8, wherein the patterned surface includes a portion having parallel grooves spaced at a distance in the range of 0.01 to 500 microns or 5 to 50 microns.

10. A method for producing a substrate for growing cells or cell sheets, comprising: casting an elastomeric membrane onto a mold having a patterned surface; and attaching a hydrophilic agent to the elastomeric membrane.

11. The method according to claim 10, wherein the hydrophilic agent is not an exogenous extracellular matrix (ECM) component.

12. The method according to claim 10 or 11, wherein the mold is produced by fused deposition modeling (FDM) 3D printing.

13. The method according to any one of claims 10 to 12, wherein the treatment step includes contacting the elastomeric membrane with an aqueous solution of polyphenol, and the aqueous solution is substantially free of iron.

14. The method according to any one of claims 10 to 13, wherein the treatment step includes contacting the elastomeric membrane with an aqueous solution having 10 to 200 mg / ml of tannic acid and a pH of 7.5 or less.

15. The method according to any one of claims 10 to 14, wherein the treatment step includes contacting the elastomeric membrane with an aqueous solution having 0.1 to 6 mg / mL of lignin.

16. The method according to any one of claims 10 to 15, wherein the membrane comprises silicone, such as PDMS or PBAT.

17. A method for creating a cell sheet, comprising: providing an elastomeric membrane having a hydrophilic and patterned surface; growing cells in layers on the membrane, wherein the cells produce an extracellular matrix (ECM); removing at least a portion of the layer from the membrane.

18. The method according to claim 17, wherein the removing step comprises applying a physical force to the cells and / or spontaneous separation of the cells from the membrane.

19. The step of growing cells in layers on the membrane comprises: adding a first set of cells onto the membrane to a confluence density of 60 to 90%; incubating the first set of cells to a confluence density of at least 95% to create a first cell layer; adding a second set of cells onto the first cell layer; incubating the second set of cells to create a second cell layer; whereby the cells of the first and second cell layers produce an extracellular matrix and the cells of the second cell layer attach to or fuse with the cells of the first cell layer.

20. The method according to any one of claims 17 to 19, comprising creating a patterned surface by casting the membrane onto a patterned mold.

21. The method according to any one of claims 17 to 20, wherein the elastomeric membrane comprises silicone, such as PDMS or PBAT.

22. The method according to any one of claims 17 to 21, comprising a hydrophilic agent attached to the surface of the elastomeric membrane.

23. The method according to claim 22, wherein the hydrophilic agent is not an exogenous extracellular matrix (ECM) component.

24. The method according to claim 22 or 23, wherein the hydrophilic agent comprises polyphenol.

25. The method according to any one of claims 17 to 24, wherein the removing step consists of peeling off at least a portion of the layer from the membrane.

26. A method for creating a cell sheet, comprising: treating a silicone-based membrane with an aqueous solution, wherein the silicone-based membrane comprises a polydimethylsiloxane (PDMS) elastomer; growing cells on the treated membrane; [[ID=3X]]imparting orientation to the cells on the treated membrane. It should be noted that there seems to be a typo in the original text where "31" is written as "3X" in the last line. I have translated it as it is, but it might need to be corrected in the original content. A method comprising the step of creating a cell sheet. **Claim 27** The method according to claim 26, wherein the aqueous solution is selected from the group consisting of an aqueous solution of tannic acid, an aqueous solution of tannic acid optionally free of iron, an aqueous solution of tannic acid having a pH of 7.5 or less, and an aqueous solution of lignin. **Claim 28** The method according to claim 27, wherein the membrane is directly treated with an aqueous solution of tannic acid at about 10 to 200 mg / mL, for example, about 50 mg / mL, or with an aqueous solution of lignin at about 0.1 to 6 mg / mL, for example, about 1 mg / mL, optionally at room temperature, for a period of about 6 hours to 4 days, for example, about 3 days. **Claim 29** The method according to any one of claims 26 to 28, further comprising the step of treating the surface of the membrane with oxygen or air plasma to improve the wettability of the surface of the membrane. **Claim 30** The method according to any one of claims 26 to 29, further comprising the step of infiltrating a solution into the surface of the treated membrane to improve the wettability of the surface of the membrane. **Claim 31** The method according to claim 30, wherein the solution comprises a sodium hydroxide solution, for example, a sodium hydroxide solution in deionized water. **Claim 32** The method according to any one of claims 26 to 31, wherein the step of imparting orientation to cells on the treated membrane is performed by creating a pattern on the surface of the membrane treated via lithography. **Claim 33** The method according to any one of claims 26 to 31, wherein the step of imparting orientation to cells on the treated membrane is performed by creating a protein pattern on the surface of the treated membrane. **Claim 34** The method according to any one of claims 26 to 31, wherein the step of imparting orientation to cells on the treated membrane is performed by creating a pattern that can be replicated by casting on the membrane treated by 3D printing using 3D printing. **Claim 35** The method according to claim 34, wherein the 3D printing includes fused deposition modeling (FDM) 3D printing. **Claim 36** The method according to any one of claims 32 to 35, wherein the pattern is used as a topographical signal transduction cue for smaller cells to impart orientation in a specific direction. **Claim 37** The method according to any one of claims 32 to 36, wherein each pattern of the pattern is selected from the group consisting of a parallel pattern, a circular pattern, and a concentric circular pattern. **Claim 38** The method according to any one of claims 26 to 37, further comprising the step of inducing the cells to produce a sufficient amount of extracellular matrix (ECM) by treating the cells with ascorbate.

39. The method according to any one of claims 26 to 38, further comprising the step of inducing the cells to produce a sufficient amount of extracellular matrix (ECM) by adding polyethylene glycol (PEG) or carrageenan to the medium.

40. The method according to any one of claims 26 to 39, further comprising the step of determining that the cells are in a confluent state and have produced a predetermined amount of ECM.

41. The method according to any one of claims 26 to 40, further comprising the step of growing additional cells on the cell-ECM layer.

42. The method according to any one of claims 26 to 41, further comprising the step of crosslinking a predetermined amount of ECM produced by the cells with tannic acid or lignin to create a stable structure.

43. The method according to any one of claims 26 to 42, further comprising the step of removing the cells and the ECM of the cells from the surface of the membrane to create a cell sheet.

44. The method according to claim 43, wherein the step of removing the cells and the ECM of the cells from the surface of the membrane is performed by peeling off the cells and the ECM of the cells from the surface of the membrane.

45. The method according to any one of claims 26 to 44, further comprising the step of washing and / or autoclaving the treated membrane and reusing the treated membrane for growing new cells.

46. A substrate for growing cells, an elastomer membrane, and one or more polyphenols attached to the surface of the elastomer membrane.

47. The substrate according to claim 46, wherein the one or more polyphenols contain tannic acid.

48. The substrate according to claim 47, which does not contain iron.

49. The substrate according to claim 47, wherein the tannic acid is not crosslinked or the tannic acid is not bound to iron.

50. The substrate according to any one of claims 46 to 49, wherein the one or more polyphenols contain lignin.

51. The substrate according to any one of claims 46 to 50, wherein the elastomer membrane contains silicone, such as PDMS or PBAT.

52. The substrate according to any one of claims 46 to 51, having a contact angle of at least 20, 40, or 55 degrees and a maximum of 70, 75, 80, 85, or 90 degrees.

53. The substrate according to any one of claims 46 to 52, comprising a portion having parallel grooves with an interval in the range of 0.01 to 500 microns, for example, 5 to 50 microns.

54. A method for treating a substrate for growing cells, comprising the step of treating an elastomeric membrane with an aqueous solution of one or more polyphenols, optionally in the substantial absence of iron, and optionally without pre-activating the surface of the elastomeric membrane.

55. The method according to claim 54, wherein the aqueous solution contains tannic acid.

56. The method according to claim 55, wherein the aqueous solution contains at least 10 mg / ml of tannic acid, at least 25 mg / mL of tannic acid, or at least 50 mg / mL of tannic acid, and optionally up to 100 mg / mL or up to 200 mg / mL of tannic acid.

57. The method according to claim 55 or 56, wherein the aqueous solution has a pH of 7.5 or less.

58. The method according to claim 55, wherein the aqueous solution contains lignin.

59. The method according to claim 58, wherein the aqueous solution contains at least 0.1 mg / ml of lignin, at least 1 mg / ml of lignin, or at least 2 mg / ml of lignin, and optionally up to 4 mg / ml or up to 6 mg / ml of lignin.

60. The method according to any one of claims 54 to 59, comprising treating the substrate with a basic solution, such as NaOH, before treating the elastomeric membrane with the aqueous solution.

61. The method according to any one of claims 54 to 60, wherein the membrane contains silicone, such as polydimethylsiloxane (PDMS) or PBAT.

62. The method according to any one of claims 54 to 61, wherein the membrane is treated with the aqueous solution for a period of about 6 hours to 4 days.

63. A method for growing cells or creating a cell sheet, comprising the step of providing a membrane having a region with a pattern having an interval in the range of 0.01 to 500 microns, the step of growing cells in one or more layers on the patterned membrane, wherein the layer contains an extracellular matrix (ECM) produced by the cells, and the step of removing one or more layers from the membrane, optionally including the step of applying a physical force to the cells.

64. The method according to claim 63, wherein the layer is formed by adding cells to a confluence density of 60-90% and incubating the cells to a substantially complete confluence density.

65. The method according to claim 63 or 64, wherein the cell layer adheres or fuses together.

66. The method according to any one of claims 63-65, wherein the pattern is created by casting a membrane on a mold produced by three-dimensional (3D) printing, and the pattern of the mold is replicated on the membrane.

67. The method according to claim 66, wherein the 3D printing includes fused deposition modeling (FDM) 3D printing.

68. The method according to any one of claims 63-67, wherein the pattern includes grooves and / or is selected from the group consisting of parallel patterns, circular patterns, and concentric circular patterns.

69. The method according to any one of claims 63-68, further comprising the step of inducing the cells to produce more extracellular matrix (ECM) components by treating the cells with ascorbate or by polymer crowding, for example, by adding polyethylene glycol (PEG) or carrageenan to the culture medium.

70. The method according to any one of claims 63-69, further comprising the step of crosslinking the ECM produced by the cells, for example, with tannic acid or lignin.

71. The method according to any one of claims 63-70, comprising the step of removing at least a part of one or more layers from the surface of the membrane as a cell sheet.

72. The method according to claim 71, wherein the removal of at least a part of one or more layers from the surface of the membrane includes the step of peeling off at least a part of one or more layers from the surface of the membrane.

73. The method according to claim 71 or 72, further comprising the step of rolling the cell sheet to form cell fibers.

74. The method according to any one of claims 71-73, further comprising the step of stacking a plurality of cell sheets or cell fibers.

75. A substrate for cell culture, comprising a membrane, the membrane including a region having parallel grooves with a spacing of 0.01-500 microns.

76. The substrate according to claim 75, wherein the region includes parallel grooves having a spacing of 5-50 microns.

77. The substrate according to claim 75 or 76, wherein the region further comprises parallel grooves having one or more intervals selected from the group consisting of a) 5 to 99 or 5 to 50 microns, b) 100 to 500 microns, and c) 0.01 to 5, 0.01 to 1 micron, or 0.1 to 1 micron.

78. The substrate according to any one of claims 75 to 77, wherein the film comprises an elastomer, such as PDMS or PBAT.

79. The substrate according to any one of claims 75 to 78, wherein the film comprises polyphenol.

80. A method for producing a substrate for cell culture, comprising: producing a mold by thermally dissolving and laminating 3D printing; and casting a film on the mold.

81. The method according to claim 80, wherein the nozzle size is 0.1 to 0.6 mm.

82. The method according to claim 80 or 81, wherein the printing speed is 1 to 100 mm / second.

83. The method according to any one of claims 80 to 82, further comprising treating the mold with solvent vapor before casting the film on the mold.

84. The method according to any one of claims 80 to 83, wherein the filaments are arranged in parallel lines in at least a part of the mold.

85. The method according to any one of claims 80 to 84, wherein the film is an elastomer.

86. A method for growing cells or producing a cell sheet, comprising: growing one or more layers of cells on a film; and mechanically or electrically stimulating the cells.

87. The method according to claim 86, further comprising stretching the film in repeated cycles.

88. The method according to claim 86, comprising static stimulation or dynamic stimulation.

89. The method according to claim 88, further comprising applying a varying voltage.

90. A method for growing cells or producing a cell sheet, comprising: producing a film; growing cells in layers on the film; and inducing the cells to produce more extracellular matrix (ECM) components and / or crosslinking the ECM.

91. The method according to claim 90, further comprising treating the cells with ascorbate.

92. The method according to claim 90 or 91, further comprising adding a crowding agent, such as polyethylene glycol (PEG) or carrageenan, to the medium.

93. The method according to any one of claims 90 to 92, comprising the step of crosslinking the ECM produced by cells with, for example, tannic acid or lignin.

94. A method for producing ECM, comprising: a step of growing cells to form a cell sheet, the cell sheet containing ECM produced by the cells; and a step of decellularizing the cell sheet.

95. The method according to claim 94, further comprising the step of solubilizing the decellularized cell sheet.

96. The method according to claim 94 or 95, further comprising drying the solubilized ECM, for example, by lyophilization.

97. The method according to any one of claims 94 to 96, comprising the step of removing DNA residues.

98. The method according to any one of claims 94 to 97, further comprising the step of reconstituting the solubilized or dried ECM into a gel.

99. The method according to any one of claims 94 to 98, comprising the step of using the ECM as a coating or scaffold for growing cells, for example, for growing human cells.

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