Patent Application
20240425813
The field of this invention relates to devices and methods for growing cells or making cell sheets. Some examples involve substrate surface chemistry, substrate topography, or methods of processing cell sheets.
Silicone-based polymer family members, such as PDMS, have favorable features for cell culture related applications, including their transparency and ease of microscopy, non-toxic and inert nature both chemical and biological, as well as their gas permeability. On the other hand, their intrinsic hydrophobicity results in non-specific protein adsorption, conformation and denaturation, as well as inappropriate cell attachment, agglomeration, and detachment (Walsh, 2017).
Multiple methods have been proposed to enhance surface hydrophilicity and improve cell attachment of the silicone-based elastomers, such as PDMS. For example, some groups propose use of high energy plasma radiation, which is a fast method, but the created Si-hydroxyl groups are short lived and migration of molecules from the bulk of the material can neutralize its effect (Fritz, 1995).
Other groups have proposed use of covalent immobilization of extracellular matrix proteins on the surface of the PDMS as an example method. However, this method requires functionalization of the PDMS surface by salinization and addition of linkers, such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EDC/NHS), glutaraldehyde, and polymer brushes. These modifications require multi-step and complicated processes involving harsh and often toxic materials which could leave residues and the use of organic solvents that can swell PDMS and damage its surface features (Li, 2018).
As another example, some groups have proposed coating the PDMS with dopamine (Chuah, 2015). However, dopamine monomers are expensive, potentially toxic, and the dark nature of the coating interferes with microscopy. Other proposed methods of improving PDMS cell attachment 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).
Further, some groups have proposed coating PDMS through the formation and deposition of iron (III)-tannic acid networks (Lv, 2020).
Cell sheet engineering was originally introduced in 1990s using cell culture surfaces, tissue culture polystyrene in particular, grafted with N-isopropylacrylamide (PIPAAm). PIPAAm is a temperature sensitive material that allows for detachment of cells and their secreted extracellular matrix (ECM) proteins without using enzymes, such as trypsin, that digest and destroy ECM proteins. This results in an intact cellular construct in the form of a sheet by decreasing the temperature and changing the hydrophilicity of the grafted polymer (Yamada, 1990). Alternatively, other responsive surfaces have been created for this purpose by layer-by-layer deposition of cationic and anionic polyelectrolytes on indium tin oxide (ITO). The trigger for starting the delamination of cell sheets in this case is lowering the pH as these layers become unstable at low pH and will not allow cell attachment (Guillaume-Gentil, 2011).
Another cell sheet formation technique includes growing cells on a feeder layer and detaching them by using another enzyme, dispase, that digests some of the ECM proteins and not cell-cell junctions. Growing cells on an amniotic membrane as a carrier and then using them with the membrane has been proposed as well. Using surfaces sensitive to other external stimuli such as light, electrochemical polarization, ionic solution, and magnetic force has also been used for making cell sheets (Owaki, 2014). A more recent technique that does not rely on characteristics of the surface for making cell sheets uses slight changes in the pH of the environment to cause cells to show contraction and detach from the surface. This method is only applicable to cells that show syncytialization and fusion such as skeletal muscle cells (Shahin-Shamsabadi, 2020). Thus, alternative systems and methods for creating cell sheets are needed.
Example related art includes:
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U.S. Pat. No. 5,776,747A describes a method of derivatizing or adsorbing polyethylene oxide-poly (dimethylsiloxane) copolymer (PEO-PDMS) onto a surface within a bioartificial organ to inhibit cellular attachment.
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IN201721015210A describes a bio-inspired 3D-micro/nanofluidic device manufactured by a scalable fabrication process. The device comprises thin micro/nano vascularized membrane matrices made of PDMS or dimethicone.
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This specification describes apparatus and methods for growing cells and optionally for growing a cell construct such as a cell sheet.
In some examples, a substrate for growing cells or a cell construct includes an elastomeric membrane having a patterned surface and a hydrophilic agent on the surface of the elastomeric membrane. The substrate may be made by casting an elastomeric membrane on a mold having a patterned surface and attaching a hydrophilic agent to the elastomeric membrane. A method of growing a cell construct may include providing an elastomeric, patterned and hydrophilic membrane, growing cells on the membrane in layers wherein the cells produce an extra-cellular matrix (ECM), and removing at least a portion of the cell layers from the membrane. In at least some examples, cells layers may be removed from the membrane by physical force, for example scraping and/or pulling, without the use of an enzyme or a responsive surface.
In some examples the cell constructs have high ECM content. In some examples, the cell constructs have controlled cell alignment. The ECM is a three-dimensional (3D) network that, depending on the tissue, can consist of extracellular macromolecules and/or minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite, that provide structural and biochemical support to surrounding cells. In some examples, the cell sheets may be made with any adherent cell type from any species. The technique of the present invention may be simple, easy to perform, have a low-cost, or use biocompatible, non-toxic, food-grade or food-safe materials.
In some examples, the present invention allows for cell alignment, optionally in selected or sophisticated patterns. The patterns of cell alignment are induced by patterns in a membrane, optionally made using molding processes with 3D printed molds. Molds may be prepared with 3D printers and using different types of filament materials. Similar patterns can alternatively be created on a mold, for example using CNC machining or laser engraving, on different materials. It is also possible to make the patterns directly on the membrane, for example using CNC machining or laser engraving, without the need to use a patterned mold. Moreover, the present invention optionally provides for the reuse of membranes by autoclaving and/or washing with an appropriate solvent with defined polarity to induce controlled and limited swelling in the membrane that helps with the release of the absorbed elements, for example washing a PDMS membrane with isopropyl alcohol. Additionally, the present invention may provide multi-layer cell constructs, induced ECM production, or ECM crosslinking using biocompatible or food-grade materials. The method may result in sheets, optionally formed in a short time (usually≤18 days), that can be partially or completely removed from a membrane using a physical force, e.g. scraping and/or pulling, optionally without the use of an enzyme or responsive surface.
An example embodiment described herein includes a method to create a cell construct such as a cell sheet. 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 treated directly with about 10-200 mg/ml of the aqueous solution of tannic acid or about 0.1-5 mg/ml of the solution of lignin at room temperature for a time 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. Elastomeric membranes can be pre-treated with aqueous solution of sodium hydroxide with 0.1-5 molar concentrations for 12 hours up to 5 days before treatment with tannic acid or lignin solutions are performed to enhance their effect. If pre-treated with sodium hydroxide or other techniques, depending on the elastomer, lower concentrations and shorter treatment times of either tannic acid or lignin can potentially have similar effects as their higher concentrations and longer treatment times.
Next, the method includes growing cells on the treated elastomeric membrane and aligning the cells on the treated elastomeric membrane. The aligning of the cells on the treated elastomeric membrane occurs by creating patterns on a surface of the treated elastomeric membrane. In some examples, aligning the cells on the treated elastomeric membrane occurs by utilizing three-dimensional (3D) printing to create a master mold with patterns that can be replicated to the treated elastomeric membrane by casting. In some implementations, the 3D printing comprises Fused Deposition Modeling (FDM) 3D printing. Alternatively, stereolithography (SLA) based 3D printing can be used to create defined patterns similar to the ones created by FDM methods. It should be appreciated that the patterns (e.g., a parallel pattern, a circular pattern, a concentric pattern, or a more complex pattern) are used as topographical signaling cues for the cells to be aligned in certain directions.
Further, the method includes detaching the cell construct (i.e. one or more layers of cells and ECM produced by the cells) from the membrane. In some examples, detaching the cell construct creates a cell sheet, in particular a detached cell sheet. Detaching the cell construct may occur spontaneously as the cell construct grows, or may be initiated by a force such as scraping or shaking, or may be performed by one or more physical actions such as scraping, scratching, shaking or pulling. Detaching the cell construct may occur without applying a stimuli to trigger a change in a responsive surface (e.g. a surface responsive to a change in temperature, an ionic solution, pH, light, magnetic force or electrochemical polarization) attached to the membrane. Detaching the cell construct may occur without inducing contraction of the cells. Detaching the cell construct may occur without applying a chemical agent such as an enzyme.
In some examples, the method additionally includes: inducing the cells to produce a sufficient amount of extracellular matrix (ECM) by treating the cells with an ascorbate or by macromolecular crowding, for example by adding a polyethylene glycol (PEG) or carrageenan to media. The method may also include: determining that the cells are confluent and have produced the sufficient amount of ECM. The method may also include growing additional cells on the initial cell-ECM layer.
In some examples, the method additionally includes: inducing the cells to produce a sufficient amount of extracellular matrix (ECM) by treating the cells with an ascorbate or by macromolecular crowding, for example by adding a polyethylene glycol (PEG) or carrageenan to media. Optionally, the method may include cross-linking the ECM produced by the cells with significantly lower concentrations of tannic acid (less than 0.1 mg/mL) or lignin (less than 10 g/mL) for much shorter times (5-30 minutes) to create a stable structure and removing the cells and ECM of the cells from the surface of the elastomeric membrane to create the cell sheet.
In some examples, the method may include: determining that the cells are confluent and have produced the sufficient amount of ECM. The method may also include growing additional cells on the initial cell-ECM layer.
Removal of the cells and the ECM of the cells from the surface of the elastomeric membrane can occur by scraping the cells and the ECM of the cells, or a portion thereof, off of the surface of the elastomeric membrane. In some examples, the method may include washing and autoclaving the treated elastomeric membrane to reuse the treated elastomeric membrane to grow new cells and make new cell sheets.
The present invention illustrates the possibility of using natural polyphenols such as tannic acid in its solution format with proper solvents such as deionized water and their effect on the physical or chemical properties (e.g. hydrophilicity) of the elastomeric membranes in order to enhance cell attachment. Elastomeric membranes may include silicone-based materials such as PDMS or other elastomers including but not limited to poly(butylene adipate-co-terephthalate) (PBAT) and urethane rubbers. Similarly, lignin is shown in this application to alter the hydrophilicity of elastomeric membranes and may 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. A membrane, for example an elastomeric membrane, made be provided with a hydrophilic surface without adding an exogenous ECM component to the membrane.
In some examples, the steps can also be adapted for use with non-elastomeric membranes for example, hydrophilic polycarbonate, polysulfone, and polyethersulfone.
The specification also describes a polyphenol-coated substrate for growing a cell sheet. The substrate may include an elastomeric membrane and one or more polyphenols attached to the surface of the elastomeric membrane. The polyphenol may include tannic-acid. The substrate may be made without iron. For example, the tannic acid may be un-cross-linked, or not bound to iron. In some examples, the polyphenol is lignin. The elastomeric membrane may comprise a silicone, for example PDMS, or PBAT. The substrate may 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 having parallel grooves with a spacing in the range of 0.01-500 microns or 5-50 microns.
This specification also describes a method of making a polyphenol coated substrate. The method includes treating an elastomeric membrane with an aqueous solution of a plant-based polyphenol, optionally in the substantial absence of iron, metals and/or added cross-linkers. The aqueous solution may include 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 of tannic acid or up to 200 mg/mL. The aqueous solution may have a pH of 7.5 or less. Alternatively or additionally, the aqueous solution may comprise 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, for example of NaOH, before treating the elastomeric membrane with the aqueous solution of the plant-based polyphenol. The membrane may be made of a silicone, for example polydimethylsiloxane (PDMS), or PBAT. The membrane may be treated with the aqueous solution for a time period of about 6 hours to 4 days.
This specification also describes a method of making a cell sheet. The method includes creating a patterned membrane; and, growing cells on the patterned membrane in layers wherein the layers include ECM produced by the cells. A layer may be formed by adding cells to 60-90% confluence and incubating the cells to full confluence. Multiple cell layers may attach or fuse together to form a cell sheet. The patterns may be created by casting the membrane on a mold made by three-dimensional (3D) printing, wherein patterns of the mold are replicated on the membrane. The 3D printing may be Fused Deposition Modeling (FDM) 3D printing. The pattern may include grooves, for example in a parallel pattern, a circular pattern, and a concentric pattern. The cells may be induced to produce more extracellular matrix (ECM) components by treating the cells with an ascorbate, or by macromolecular crowding, for example by adding polyethylene glycol (PEG) or carrageenan to media. The ECM may be cross-linked, for example with tannic acid or lignin. The cells and ECM of the cells may be removed from the surface of the membrane as a cell sheet, for example by scraping at least a portion of the layers from the surface of the membrane. The remainder of the layers may be removed from from the membrane, for example, by scraping, pulling, shaking or spontaneous detachment. The call sheet may be rolled to form a cellular fiber. A plurality of cell sheets or cell fibers may be stacked together.
This specification also describes a patterned membrane. The membrane may have a region with parallel grooves with a spacing of 0.01 to 500 microns. The membrane may have multiple sets of groves with different spacings, for example spacings of 5-99 or 5-50 microns, spacings of 100-500 microns and/or spacings 0.01 to 5, 0.01-1 microns, or 0.1-1 microns. The membrane may comprise an elastomer, optionally with a polyphenol coating.
This specification also describes a method of making a patterned membrane. The method includes making a mold by way of fused deposition modeling 3D printing and casting a membrane on the mold. The nozzle size of the 3D printer may be 0.1-0.6 mm. The printing speed may be 1-100 mm/s. The mold may be treated with a solvent vapor before casting the membrane on the mold. A filament may be laid down in parallel lines in at least a portion of the mold. The membrane may be an elastomer.
This specification also describes a method of making a cell sheet with stimulation. The method includes growing a cells in one or more layers on a membrane and mechanically or electrically stimulating the cell layers. The membrane may be stretched in a repeated cycle, for example to provide static stimulation or dynamic stimulation. The method may include applying a fluctuating voltage to the cell layers.
This specification also includes a method of increasing ECM production. The method includes creating a membrane; growing cells on the membrane in layers, and inducing the cells to produce more extracellular matrix (ECM) components and/or crosslinking the ECM. The cells may be treated with an ascorbate. A crowding agent, for example polyethylene glycol (PEG) or carrageenan, may be added to media. The ECM produced by the cells may be crosslinked, for example with tannic acid or lignin.
This specification also describes a method of making ECM. The method includes growing cells in layers to form a cell construct and decellularizing the cell construct. The decellularized cell construct may be solubilized and DNA residues may be removed. The solubilized ECM may be dried, for example freeze dried. An ECM gel may be reconstituted from solubilized or dried ECM. The ECM may be used as a coating or scaffold for growing cells, for example growing human cells.
The apparatus elements and steps described above can be used alone or in any permutation or sub-combinations.
The preferred embodiments of the present invention will now be described with reference to the drawings. Identical elements in the various figures are identified with the same reference numerals.
Reference will now be made in detail to each embodiment of the present invention. Such embodiments are provided by way of explanation of the present invention, which is not intended to be limited thereto. In fact, those of ordinary skill in the art may appreciate upon reading the present specification and viewing the present drawings that various modifications and variations can be made thereto.
The present invention provides a method or process to create cell sheets, optionally with high extracellular matrix (ECM) content, optionally while controlling cell alignment. It should be appreciated that the cell sheets described herein can be made with any cell type from any species. The technique of the present invention is simple, easy to perform, has a low-cost, and uses non-toxic and biocompatible, optionally food-grade and food-safe, materials. Further, the present invention allows for cell alignment, optionally in sophisticated or selected patterns, using a simple molding process optionally with 3D printed molds, optionally prepared with cheap open-source 3D printers and using different types of filament materials. Alternatively, similar patterns 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 a solvent (e.g. isopropyl) washing step or a combination thereof. In some examples, the present invention provides multi-layer cell constructs and induced ECM production with optional ECM crosslinking resulting in strong sheets. The method may use biocompatible, optionally food-grade or food-safe, materials. In some examples, sheets are formed in a short process (≤18 days). In some examples, sheets can be detached partially or completely using scraping.
In some embodiments, a general method includes three steps. First, the method includes growing cells on a treated elastomeric membrane, such as PDMS or PBAT. PDMS treatment has been done with a variety of materials to make it suitable for cell culture, but these materials are usually expensive or toxic or require multistep treatments, including activation of the PDMS surface. To remedy this concern, the present invention may utilize direct treatment of PDMS or another elastomeric 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, in order to make the membrane suitable for cell attachment. It should be appreciated that both tannic acid and lignin are food safe, natural, and non-toxic materials. These treated membranes can be used for growing cells. The treated membranes can also be washed and autoclaved in order to reuse them for growing new cells. The treatment is stable and no re-treatment is required after autoclaving.
Second, the method includes aligning the cells on a surface of the treated
membrane. Cell alignment on different membranes have been performed by creating micron or nanoscale features on the surface using lithography, or by creating protein patterns on the surface of the membrane. However, these processes are expensive, lengthy, and require specialized equipment. Some of the materials used in these processes can be animal-derived and therefore lower the consistency of the process. Distinctly, the present invention utilizes a textured mold to provide surface features on a molded membrane. In some examples, the mold is made by 3D printing, optionally with cheap and open-source printers, or alternatively CNC machining or laser engraving, in order to create micron, nano, or meso-scale patterns that can be replicated to the membrane by casting. This one step pattern creation on the membrane surface is enough to control cell alignment.
Third, the method includes cell sheet formation. Cells are grown and align on the membranes. The membrane patterns also induce ECM formation. Optionally, the cells may be induced to produce higher amounts of ECM by using different growth factors and elements, such as ascorbic acid or by macromolecular crowding. Once the cells are completely confluent and have produced enough ECM, more cells are grown on this cell-ECM layer. Optionally, in order to make a more stable structure, the ECM can be crosslinked with low concentrations of tannic acid or lignin or other crosslinkers that can crosslink different ECM components such as transglutaminase. This can be repeated at least 3 times in a 2 to 3 week time span. At the end of the process, the layers (including the cells and their ECM) can be physically removed (e.g. scraped) from the membrane to make a coherent cell sheet. Other techniques for making cell sheets require either changing membrane properties to make it suitable for cell sheet detachment, digesting some of the proteins that cells need for remaining attached to the surface, or inducing cells to show contraction that forces them to lift off. Distinctly, the method described herein provides a cohesive multilayer cellular construct that can be, for example, scrapped off using an external object. Among other benefits, in at least some examples the process described herein can be used with any adherent cell type from any species.
Optionally, one or more aspects of the method may be used alone or in other combinations. For example, cell sheets may be grown on smooth (i.e. non-patterned) membranes without cell alignment. In other examples, alternative membrane materials may be used that do not require surface treatment. In other examples, membranes may be patterned by machining or other physical processes applied to a solid 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 bulk material. The membrane is non-porous to bulk liquid flow or at least capable of supporting a liquid media over the cells. Membranes may be made of various thermoplastics or thermosets. The membranes may be rigid or elastomeric. Rigid membranes can be molded, for example, from biocompatible epoxies. Elastomeric membranes can include, for example, thermoplastic elastomers based on poly (butylene adipate-co-terephthalate) (PBAT), and urethane rubbers 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. Elastomeric membranes may also be made of a silicone, for example PDMS. However, elastomeric membranes typically have limitations due to their hydrophobicity, such as inappropriate cell attachment, agglomeration, and detachment, which may be improved with surface treatment as 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.
A method of fabricating an elastomeric membrane, such as a PDMS membrane, is also described herein. It should be appreciated that PDMS comprises an elastomer base and an elastomer curing agent. Other silicone-based or non-silicone-based elastomers have two components that upon mixing will catalyze their reaction into forming elastomeric resins or rubbers. The method includes several process steps, such as: mixing the elastomer base and the elastomer curing agent or the two components of the elastomer at certain ratios (e.g., a 1:1 to 1:20 volume or weight ratios of the curing agent to the base) and casting and heating the elastomeric solution to cure the polymer and form the membrane. Methods of 3D printing may be used to print molds that are then used for casting the elastomeric solution and creating the membranes. An example 3D printing method that may be used includes Fused Deposition Modeling (FDM) 3D printing. Patterns on the surface of the 3D printed molds (which may be or include patterns inherent to the 3D printing process) may then be mirrored on the membrane. This may be leveraged to create patterns (e.g., parallel patterns) on the elastomeric membrane that can be used for aligning the cells.
Different polymeric materials can be used with different commercially available 3D printers, such as UltiMaker and Prusa, for printing the molds. Alternatively or additionally, other techniques of creating patterns such as CNC machining or laser engraving, may be used to make a mold or modify a 3D printed mold. A 3D printer may be configured or operated to create patterns with different resolutions that can have different effects on cell alignment. When 3D printers are used for printing the molds, nozzle sizes can be 0.1-1 mm, for example 0.25 mm, 0.4 mm, or 0.6 mm, and the printing speed can be 20-100 mm/s. It should be noted that smaller nozzle sizes and lower printing speeds will create smaller features that might perform better in cell alignment depending on the cell type and its sensitivity to the environmental conditions. In some examples, thermoplastic polymers, such as PETG, ABS, and PVB (polyvinyl butyral) are used in 3D printing. However, it should be appreciated that other polymers may be used that are not explicitly listed herein. Further, other methods can also be used to create molds using non-polymeric materials, such as metallic molds. The molds may be optionally treated to alter the patterns. For example, ABS molds may be treated with acetone vapor and PVB molds may be treated with isopropyl alcohol vapor to melt the surfaces of the polymeric mold and change the resolution of the 3D printed part and consequently change the size or smoothness of the patterns on the membrane. It should be appreciated that timing of the treatment defines a final pattern or lack thereof.
Independent of the material used for 3D printing the molds, 3 levels of features may be created on the surface of the mold. The meso scale patterns, larger lines seen in
Other methods such as using sandpapers with different grits can also be used to change the features on the surface of the mold after printing but they can create new extra features while eliminating the old ones. Such methods might not be as reproducible as treatment with solvent vapor.
In some examples, treatment times with the vapor phase of a solvent of a polymeric filament 3D printed mold ranged from about 0 minutes to about 30 minutes. After the vapor treatment, the molds can be heat treated at temperatures 20-60° C. for 1-12 hours in order for the solvent to evaporate from the surface of the mold and for the polymer to solidify. When the molds are ready, the elastomer solution may be cast and heated at a temperatures ranging from 25-90° C. for a time period of 1-24 hours to allow the polymer to cure. Performing the heating process at higher temperatures will result in less time required for solidifying the elastomeric resin.
In the examples shown, molds 102 have a platform 110 and a moat 112. Membrane 106 has a bottom 116 and a wall 118. In the examples shown, the wall 118 is polygonal with multiple segments. In other examples, the wall 118 may be of another shape, for example round. In
The photograph of mold 104 shows an enlargement of a region of the platform 110 of the photograph of mold 102. The platform 110 has a topographical pattern, in this example produced inherently by a process of 3D printing and optionally, as in the case of second row 110, with further modification. In at least the region of platform 110 shown in the photograph of mold 104, the pattern includes a set of generally parallel grooves 110. The photograph of membrane 108 is an enlargement of a region of the bottom 116 of the photograph of membrane 106. The upper surface of the bottom 116 of the membrane 106, 108 has a topographical pattern mirroring the topographical pattern of the platform 110 of the mold 102, 104. In at least in the region shown in the photograph of the membrane 108, the upper surface of bottom 116 of membrane 106, 108 has a set of generally parallel grooves 120. The wall 118 may be, for example 3-10 mm high, and can contain a liquid media and cells prior to attachment on the bottom 116 of membrane 106, 108. A first layer of the cells attaches, preferably temporarily, to the bottom 116 of membrane 106, 108. In the example shown, the membrane 106, 108 is square in plan view and about 30 mm wide and long. Optionally, the membrane 106, 108 may have other shapes, for example round or rectangular. The membrane 106, 108 may also have other sizes, for example a length or diameter of 10-100 mm or more.
It should be appreciated that independent of the pattern or material used for printing, the cross-section of the pattern, e.g. grooves of the pattern, is typically similar. Other methods and materials can also be used for making the molds, preferably wherein the size of the patterns (e.g. the spacing between grooves) is similar to what is created using 3D printing, or other sizes that can have similar effects on attachment and alignment of the cells or ECM production. For example, CNC machining and laser ablation of different materials may be used to make similar molds. Furthermore, the molds can be made from metallic or polymeric materials, as well.
Many elastomeric materials, including silicones and many non-silicone-based membranes, are hydrophobic materials. Hydrophobic materials are generally unsuitable for cell attachment and tend to cause cells to form aggregate clumps rather than sheets. Membrane fabrication to impart surface topography as described above may improve cell sheet formation. Further, the surface of elastomeric or other membranes are optionally modified by chemical treatment, e.g. with a hydrophobic agent, to make them more suitable for cell attachment.
In some examples, elastomeric membranes, for example silicone membranes such as PDMS or PBAT, are treated with an aqueous solution of a polyphenol. The polyphenol may be plant based, for example tannic acid or lignin. The polyphenol may be applied to the membrane without additional cross-linkers or compounding agents, for example iron.
In some examples, the treatment may include contacting the membrane with 10-200 mg/ml of tannic acid, optionally 25-100 mg/mL. The treatment may be done generally at ambient or room temperature. The contact period may be about 6 hours to 4 days, or 1-3 days. The tannic acid solution may be essentially unbuffered, for example, the aqueous solution may have a pH of 7.5 or less or 7.0 or less. Optionally, the tannic acid solution contains only tannic acid and water. The tannic acid is applied to the membrane without additional cross-linkers or compounding agents, for example no iron is present. Without intending to be limited by theory, dihydroxyphenol or truhydroxyphenol groups of the lignin may be able to adsorb to the surface of PDMS or other elastomers even without surface activation of the membranes. After optional treatment with a caustic solution, as described further below, the membrane may also have functional groups (e.g. silanol groups) available for chemical binding. However, surface activation is not required. Further, additional cross-linking of the polyphenol (e.g. with iron or polyethyleneimine, or beyond any crosslinking that may be present in the natural molecule) is not required. In some examples, a cell growth substrate is produced that has essentially only the membrane material and attached polyphenol molecules, wherein the polyphenol molecules are not bound to each other except via the membrane.
In some examples, the treatment may include contacting the membrane with a solution of about 0.1-6 mg/mL, or 1-4 mg/ml of lignin. The treatment may be done generally at ambient or room temperature. The contact period may be about 6 hours to 4 days, or 1-3 days. The tannic acid solution may be essentially unbuffered. Optionally, the tannic acid solution contains only tannic acid, water and a water-miscible solvent, for example methanol or another alcohol.
It should be appreciated that lower or higher concentrations of polyphenol can be used for this purpose as well, but the timing of the treatment should 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 one after the other.
In order to improve the attachment of tannic acid, lignin or another polyphenol to the surface of the membrane, the membrane can be pretreated with a caustic solution, for example an aqueous sodium hydroxide solution. Alternatively, the membrane may be pretreated with an oxygen, atmospheric air or carbon dioxide plasma. Optionally, the membrane is not pre-treated with either the caustic solution of plasma.
After the polyphenol treatment (i.e. with tannic acid and/or lignin), the membranes may be washed to remove residues. For example, the membranes may be washed with deionized water, optionally at least twice, to remove the residues. Then, the membranes may be used immediately for cell culture or can be stored at room temperature for a time period of a few weeks or in a refrigerator for a longer time period.
For sterilization purposes, before starting the polyphenol treatment, the membranes can be washed with about 70% ethanol and subjected to UV afterwards or they can be autoclaved. The polyphenol (i.e. tannic acid and/or lignin) solutions can be sterile-filtered using about 0.2 μm syringe filters. The treatment may be done inside a biosafety cabinet to avoid possibility of contamination.
The treated membrane is hydrophilic but not coated or functionalized with exogenous ECM components (i.e. ECM components not made by cells grown on the membrane) such as structural proteins (e.g. collagen, elastin, fibronectin and laminin), proteoglycans and glycosaminoglycans. While adding ECM components to the membrane encourages cell attachment, this form of attachment may be too strong for cell sheet detachment. Further, in at least some examples, it is desirable to avoid all use of exogenous ECM components and to instead produce cell layer or a cell sheet that contains only ECM made by the cells grown on the membrane. Similarly, in some examples the contact angle of the treated membrane is moderate, for example 40 degrees or more 55 degrees or more, since a lower contact angle may result in a cell construct that is more difficult to separate from the membrane.
It should be appreciated that 3D printing using FDM has a very low resolution and creates meso-scale patterns (up to a few hundred microns) as well as other micron and nano-scale patterns. Although these patterns can be considered as artifacts, they can be used as topographical signaling cues for cells that are much smaller to be aligned in certain directions, as shown in
As described herein, a “cell sheet” is a scaffold-free or self-assembled sheet-like construct including cells and the ECM that they have secreted, or a portion of such a construct. Cell attachment to each other and to their ECM is preserved in the cell sheet and therefore the cells sheet can be maintained independent of a membrane or surface to adhere to at least for a period of time. Since the ECM is preserved in each sheet, if multiple sheets are stacked on top of each other, they can bind and fuse to one another or to tissues or organs.
Further, elastomeric or other membranes, with or without patterns, and optionally treated with lignin and/or tannic acid, can be used for sheet formation in a simple process. The membrane provides a proper surface for cell adherence and cells form strong attachments to the membrane at the beginning. Over time, cells proliferate and start adhering to each other and form strong cell-cell junctions. They will also secrete their own ECM, which they will then adhere to.
As cells become more confluent and establish strong attachments to each other or their ECM, and as more layers are added as described below, the attachment between the cells and the membrane weakens. Once the attachment of cells to each other and their ECM becomes stronger than their attachment to the membrane, cells and their ECM can be detached, or detachment can be initiated, by a scraping method. It is also possible to make a cell sheet by scratching the edges of the membrane and shaking it to help cells delaminate as a sheet by applying shear force. It should be appreciated that the sheets are also strong enough to be grabbed and pulled off of the surface using a tweezer or similar means. Once pulled off from the surface, cells will maintain their alignment due to the high density of the cells and high ECM content in the sheets that prevents them from changing their alignment for a period of time. Optionally, the membrane can be scraped to release edges of cell sheets, followed by a) pulling the cell sheet edges to form or release the rest of the cell sheet, b) waiting for a period of time, for example 0.2 to 2.0 hours and then scrapping or pulling to form or release the rest of the cell sheet, and/or c) waiting optionally while continuing to incubate the cells wherein the rest of the cell sheet may be formed or released spontaneously. Separating the lower layer of the cells from the membrane may cause a nearly instantaneous change in the bottom surface of the separated cells of the lower layer towards a detached sheet structure, and may trigger further changes in any cells that are still attached to the membrane. With an elastomeric membrane, scraping the membrane may also deform parts of the membrane that are not directly scraped, thus causing or helping with detachment of cells attached to parts of the membrane that are not directly scraped. A cell sheet may include a complete construct fully released from a membrane, or an edge or other portion of a construct that has been released from a membrane while another part of the construct remains attached to the membrane.
A cell sheet may have one or more layers. Once the cells cover an entire surface of the membrane and produce ECM, it is possible to add more cells in order to grow on the initial layer of cells and create a multilayer construct. This can be performed multiple times to make a thicker construct, but if it is done more than a certain number of times, depending on the cell types and species up to 10 times, the attachment between cells of different layers becomes much stronger than attachment of the first layer to the membrane, which could result in spontaneous detachment of the cells from the membrane. While spontaneous detachment may be useful, particularly if instigated by scraping, scratching or shaking, uncontrolled spontaneous detachment of the cell layers may be followed by excessive shrinkage that may rapidly turn the sheet into an agglomerate.
Further, it is possible to induce cells to secrete more ECM components by treating them with components, such as ascorbate family members, or by creating macromolecular crowding by addition of elements such as high molecular weight PEG or carrageenan with proper concentrations to the media. It is also possible to crosslink the ECM secreted by cells prior to addition of more cells to increase stability of the cellular constructs. Lower concentrations of tannic acid or lignin (e.g. less than 0.1 mg/mL tannic acid or less than 10 μg/mL lignin) for short amounts of time in serum-free media to avoid agglomeration of media components can be used for crosslinking ECM components. The timing of treatment can be in a range from about 5 minutes to about 100 minutes depending on the crosslinker type and its concentration prior to addition of the 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 with handling the sheets and moving them from one vessel to another after delamination. Additionally, the present invention contemplates use of multiple cell types either in each layer or different cell types on different layers. For example,
In an exemplary method, on Day 1, growth media is added to a depth of about 3-5 mm over the membrane. Enough cells are added to over the membrane to about 60-90% or 75-85% confluence. The cells are incubated, for example at 37 C or the physiological temperature of the cell animal, for 2 days. On Day 3, the cells will have grown to form a completely confluent first (single cell) layer. The growth media is refreshed, i.e. removed (e.g. by aspiration) and replaced with fresh media. In the case of stem cells, the fresh media may be a differentiation media. For stem cells, differentiation and fusing to adjacent cells typically happen together. For other cell types that adhere (rather than fuse) to each other, the fresh media may be more growth media.
On Day 4, a similar number of cells as were added on Day 1 are added onto the first layer of cells. On Day 5, the media is refreshed, with either new growth media or new differentiation media for stem cells. On Day 6, a second layer has formed and a similar number of cells as were added on Day 1 are added on the second layer of cells. On Day 7 the media is refreshed, with either new growth media or new differentiation media for stem cells. On Day 8, a third layer has formed and a similar number of cells as were added on Day 1 are added on the third layer of cells. On Day 9 the media is refreshed, with either new growth media or new differentiation media for stem cells. On Day 10, a fourth cell layer has formed. In this example, the cell sheet will have four layers. Optionally, more or less, i.e. 2 or 3 or 5 or more, layers may be produced. On Day 11, and every two days thereafter, the media is refreshed again. The cells continue to produce ECM and the cell layers becomes more robust. On Day 18, an edge of the cell layers may be scraped from the membrane, and the cell layers may be pulled away from the membrane. The cells transform quickly after they are detached from the membrane and a cell sheet is formed. This example describes a typical process but the schedule of cell and media addition or various other steps may be varied.
Once a cell sheet is delaminated from the membrane, the cells and their ECM are removed. Although the cell sheet is removed, the membrane could allow unspecific penetration of certain smaller molecules to its bulk, which renders the membrane opaque. It is possible to first remove most of these components by submerging the membrane in deionized water and autoclaving the membrane, for example for a time period of about twenty minutes. To further remove such residues, after autoclaving is performed, the membrane may be submerged in isopropyl or other non-polar solvents that can cause swelling in elastomeric membranes, for example for a time period of about 10-60 minutes. The solvent will swell the membrane and the rest of these residues may be removed. At this point, the membrane can be used for cell culture again without repeating the treatment with tannic acid and/or lignin.
As an illustrative example,
The membranes, for example tannic acid/lignin treated elastomeric membranes with or without patterning, can be used for growing different cell types from different tissues of different species, for both primary and immortalized cells.
Specifically, according to
In some examples, the cell sheets can be formed or remodeled into cell constructs of other configurations. For example, cell sheets can be rolled on themselves, and preserved ECM will result in formation of a coherent cellular structure that cannot be unrolled. Multiple cellular fibers 902 made out of rolled sheets can be stacked on top of each other, and they will also form firm attachment to each other, as shown in
The present invention also contemplates creating cellular fibers with different cell types and stacking them. Examples of this application include creating marbling for cultivated meat by using skeletal muscle, adipose, and connective tissue derived cells, or creating more physiologically relevant models of different tissues and organs by recreating cellular crosstalk. Sheets stacked on top of each other without first rolling them, will also adhere to each other and form a coherent structure. An example of this includes making sheets of hepatocytes and stacking them to make liver-like tissue for either in vitro modeling or making foie gras as food.
Cells constantly receive dynamic cues in in vivo conditions. These cues include electrical and/or mechanical stimulation. Recreating such cues in the in vitro conditions can be used to guide cellular behavior including cell alignment, ECM production, and even differentiation path of the stem cells to different cell types. It is possible to apply mechanical stimulation to the cells grown on an elastomeric membrane by stretching or compressing the membrane that is flexible. Electrodes can also be inserted inside the bulk of the membrane close to the surface or above the surface to apply electrical stimulation to the cells.
Different parameters can be controlled to deliver appropriate mechanical or electrical stimulation to the cells depending on the cell type, species, or the specific application. For mechanical stimulation, cells can be exposed to static or dynamic stimulation. In static stimulation, the elastomeric membrane can be stretched for 1-20%. This stretched condition can be maintained for 10-100 minutes or longer time before the elastomeric membrane is returned to its original resting size. After a certain amount of time, a similar or different stretching condition for similar or different amount of time can be implemented. In dynamic stimulation, important parameters are amplitude and frequency of the stimulation as well as the active time. The amplitude of the deformation/stretching can be 1-20% or more and the frequency can be 0.1-10 Hz or more. The stimulation can be performed for 1-10 minutes or more followed by 1-10 minutes or more in rest mode before the same or different stimulation is applied. Similar static or dynamic electrical stimuli can be applied by controlling the voltage (0.1-5V), frequency (1 mHz to 20 Hz or more), as well as duty cycle.
The patterns created on the surface of the membrane 1102 of
Patterning Different Cell Types culture media 1104 of
After the sheets are formed and removed from the membrane, since there is a high content of ECM present, the sheets can be decellularized using mild detergents such as Triton X-100, SDS, or Tween 20, at room temperature for a short time or on ice for longer times. Decellularization can be performed with 0.1-1% V/V or wt/V solutions of each of the mentioned detergents or a combination of them at room temperature or at 4° C. The decellularization can be performed for 1-48 hours depending on the temperature or concentration of the detergent. The solvent for the detergents can be either deionized water or a buffer such as phosphate buffered saline (PBS). It is also possible to treat the sheets with one detergent solution, wash them with deionized water or PBS, and continue the treatment with a fresh batch of similar or different detergent. Using fresh detergent solution can further improve the decellularization process.
After decellularization, a fibrous sheet of rich ECM will remain that can be used for 3D cell culture in vitro or in vivo regenerative medicine and tissue engineering applications. The ECM sheets can also be treated with DNase and RNases to remove any DNA and RNA residues. By using different cell types or treating them with different types of media, the composition of ECM can be controlled. Membrane surface patterns, as well as external stimuli including electrical and mechanical stimuli, can be used to align the ECM components in certain directions, as described herein. Further crosslinking of the ECM proteins, before or after decellularization can also be performed. The directionality of ECM will be preserved during decellularization and can guide cellular behavior after re-cellularization.
The decellularized ECM can also be solubilized to create 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, decellularized sheets can be submerged in such solutions and kept on ice for 24-48 hours. Every 6-12 hours, sonication with 1-10 Hz frequency for 2-5 seconds can be applied to disrupt the ECM and further ease the dissolution of the ECM molecules in the buffer. The sonication should be implemented while the samples are kept on ice to avoid denaturation of ECM proteins due to the generated heat. Similarly, treating the decellularized sheets with enzymes such as Pronase, Dispase, or different collagenases (such as collagenase type I, II, III, and IV) or their combinations. Depending on the origin of the cells as well as the conditions of ECM production, different concentrations of these enzymes can be used including 0.1 to 2 mg/mL, 0.5 and 5 U/mL, or 0.1 to 5 mg/mL. The timing of the treatment varies between 12 hours and up to 4 days.
Sonication and homogenization can be performed to help with solubilization. It should be appreciated that these processes are performed on ice. After solubilization, the solution is filtered using dialysis filters to remove the enzymes or ionic solution. It can then be lyophilized and solubilized again in the deionized water to achieve the desired concentration. This solution can then be kept refrigerated or frozen until use. The potential applications for this include treatment of surfaces that are not suitable for cell attachment to render them cell adhesive or 3D culture systems using “cell-derived ECM” as the main matrix. This solution is temperature sensitive and can be gelled quickly in a few minutes by increasing the temperature to a temperature of about 37° C.
In this experiment, rabbit myoblasts are differentiated to skeletal muscle cells and used to form a cell sheet with aligned cells, following these steps:
The descriptions of the various embodiments of the present invention have been presented for purposes of illustration, but are not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The terminology used herein was chosen to best explain the principles of the embodiments, the practical application or technical improvement over technologies found in the marketplace, or to enable others or ordinary skill in the art to understand the embodiments disclosed herein.
When introducing elements of the present disclosure or the embodiments thereof, the articles “a,” “an,” and “the” are intended to mean that there are one or more of the elements. 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 there may be additional elements other than the listed elements.
Although this invention has been described with a certain degree of particularity, it is to be understood that the present disclosure has been made only by way of illustration and that numerous changes in the details of construction and arrangement of parts may be resorted to without departing from the spirit and the scope of the invention.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
Chakrabarty, T., et al., “Bioinspired tannic acid-copper complexes as selective coating for nanofiltration membranes,” Separation and Purification Technology, 2017. 184:188-94.
Chuah, Y. J., et al., “Simple surface engineering of polydimethylsiloxane with polydopamine for stabilized mesenchymal stem cell adhesion and multipotency,” Scientific Reports, 2015. 5 (1): 18162.
Fritz, J. L. and M. J. Owen, “Hydrophobic Recovery of Plasma-Treated Polydimethylsiloxane,” The Journal of Adhesion, 1995. 54 (1-4): 33-45.
Guillaume-Gentil, O., et al., “pH-controlled recovery of placenta-derived mesenchymal stem cell sheets,” Biomaterials, 2011. 32 (19): 4376-84 .
Li, Q., et al., “Polydopamine-collagen complex to enhance the biocompatibility of polydimethylsiloxane substrates for sustaining long-term culture of L929 fibroblasts and tendon stem cells,” Journal of Biomedical Materials Research Part A, 2018. 106 (2): 408-18.
Lv, X., et al., “A one-step tannic acid coating to improve cell adhesion and proliferation on polydimethylsiloxane,” New Journal of Chemistry, 2020. 44 (35): 15140-47.
Owaki, T., et al., “Cell sheet engineering for regenerative medicine: current challenges and strategies,” Biotechnol J, 2014. 9 (7): 904-14.
Shahin-Shamsabadi, A. and P.R. Selvaganapathy, “TT-SACS: pH Induced Self-Assembled Cell Sheets Without the Need for Modified Surfaces,” ACS Biomater Sci Eng, 2020. 6 (9): 5346-56.
Sharma, V., et al., “Surface characterization of plasma-treated and PEG-grafted PDMS for micro fluidic applications,” Vacuum, 2007. 81 (9): 1094-1100.
Walsh, D. I., et al., “Enabling Microfluidics: from Clean Rooms to Makerspaces,” Trends in Biotechnology, 2017. 35 (5): 383-92.
Wu, M.-H., “Simple poly (dimethylsiloxane) surface modification to control cell adhesion,” Surface and Interface Analysis, 2009. 41 (1): 11-16.
Yamada, N., et al., “Thermo-responsive polymeric surfaces; control of attachment and detachment of cultured cells,” Die Makromolekulare Chemie, Rapid Communications, 1990. 11 (11): 571-76.
This application claims priority from: U.S. Provisional Application Ser. No. 63/504,774, filed May 29, 2023; U.S. application Ser. No. 17/882,693, filed Aug. 8, 2022; and, U.S. application Ser. No. 17/838,284, filed Jun. 13, 2022. For the United States of America, this application claims the benefit of U.S. Provisional Application Ser. No. 63/504,774, and this application is a continuation-in-part of U.S. application Ser. No. 17/882,693, filed Aug. 8, 2022, which is a continuation of U.S. application Ser. No. 17/838,284, filed Jun. 13, 2022. Application Ser. Nos. 63/504,774; 17/882,693; and 17/838,284 are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2023/050779 | 6/7/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63504774 | May 2023 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17838284 | Jun 2022 | US |
| Child | 17882693 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17882693 | Aug 2022 | US |
| Child | 18694213 | US | |
| Parent | 17838284 | Jun 2022 | US |
| Child | PCT/CA2023/050779 | WO |
