Substrate for cell culture comprising modified prolamins

Abstract

A substrate for culturing cells that comprises a modified prolamin having a net positive charge is provided. The modified prolamin may be a prolamin that has been modified through amidation or esterification, such as the addition of a methyl-ester group or an ethanolamine group. The modified prolamin is animal-free may be food-grade and / or may be free of cross-linkers. The modified prolamin may be in a coating that can be applied to a surface for culturing adherent cells, such as a microcarrier (e.g., beads) or well plates. Substrates coated with the modified prolamin are able to support cell expansion and can be used for food-grade applications such as cultured meat.

Description

Attorney Docket No. : SP24-283 SUBSTRATE FOR CELL CULTURE COMPRISING MODIFIED PROLAMINS CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S.Provisional Application Serial No. 63 / 719,387 filed on November 12, 2024, the content of which is relied upon and incorporated herein by reference in its entirety.FIELD

[0002] The present disclosure relates to adherent cell production (including for cultivated meat and stem cells), and substrates for cell production.BACKGROUND

[0003] Some cell types are anchorage dependent. With anchorage dependent cells, the cells must adhere to a surface to be able to survive and grow (expand in number). In vitro, anchorage dependent cells can be grown in several ways, but microcarriers can be used to culture large amounts of cells within a small footprint. Microcarriers are tiny support matrices for anchorage dependent cells to adhere to and allow for the cells to expand in number. However, when the anchorage dependent cells used are for cultivated (cultured) meat, using microcarriers becomes complicated because of difficulties removing them after the meat cells have been grown and because of the need for the cells to be food-grade. Currently available microcarriers are either not food-grade and / or have non-food-grade coatings or other components with them, and they have difficulties being removed from downstream processes. A need exists for low cost microcarriers that are simple to produce, that are food grade, and yet still promote sufficient levels of cell attachment and expansion.SUMMARY

[0004] Aspect 1. A coating composition, comprising a modified prolamin having a net positive charge throughout a pH range of pH 6-8. The modified prolamin is an amidated or ester ified prolamin, and the coating is free from animal components.Attorney Docket No. : SP24-283

[0005] Aspect 2. The coating composition of aspect 1, wherein the coating is free of crosslinkers.

[0006] Aspect 3. The coating composition of any one of aspects 1 or 2, wherein the prolamin is gliadin, hordein, secalin, zein, kafirin, and avenin, or combinations thereof.

[0007] Aspect 4. The coating composition of aspect 3, wherein the prolamin is zein.

[0008] Aspect 5. The coating composition of any one of aspects 1-4, wherein the coating composition is food-grade.

[0009] Aspect 6. The coating composition of any one of aspects 1-5, wherein the amidated prolamin has either an amide modification or an ethanolamide modification, and wherein the esterified prolamin is a methyl-ester modification.

[0010] Aspect 7. A cell culture surface, comprising a surface for culturing adherent cells and a coating on the surface. The coating on the surface comprises a modified prolamin having a net positive charge when at pH 7.5. The modified prolamin is an amidated or esterified prolamin.

[0011] Aspect 8. The cell culturing surface of aspect 7, wherein the coating is free from crosslinkers and is free from animal components.

[0012] Aspect 9. The cell culturing surface of any one of aspects 7 or 8, wherein the modified prolamin comprises zein.

[0013] Aspect 10. The cell culturing surface of any one of aspects 7-9, wherein the amidated prolamin has either an amide modification or an ethanolamide modification, and the esterified prolamin has a methyl-ester modification.

[0014] Aspect 11. The cell culturing surface of any one of aspects 7-10, wherein the modified prolamin is present in the coating in a percent by weight between 0.01 wt.% and 5 wt.% of the coating.Attorney Docket No. : SP24-283

[0015] Aspect 12. The cell culturing surface of any one of aspects 7-11, wherein the modified prolamin has a surface density on the cell culturing surface of between 0.1 pg / cm2and 100 pg / cm2.

[0016] Aspect 13. The cell culturing surface of any one of aspects 7-12, wherein the surface is a microcarrier.

[0017] Aspect 14. The cell culturing surface of aspect 13, wherein the microcarrier is a bead for culturing adherent cells.

[0018] Aspect 15. The cell culturing surface of any one of aspects 13-14, wherein the microcarrier is a dissolvable microcarrier.

[0019] Aspect 16. The cell culturing surface of any one of aspects 13-15, wherein the modified prolamin is present on the microcarrier in an amount between 10 pg modified prolamin / mg dry microcarrier and 100 pg modified prolamin / mg dry microcarrier.

[0020] Aspect 17. The cell culturing surface of any one of aspects 7-16, wherein the prolamin is selected from the group consisting of gliadin, hordein, secalin, zein, kafirin, and avenin, or combinations thereof.

[0021] Aspect 18. The cell culturing surface of any one of aspects 7-17, wherein the cell culturing surface is a sanitized cell culturing surface.

[0022] Aspect 19. A method of culturing cells, comprising the steps of providing a cell culturing surface, providing adherent cells to the cell culturing surface, and culturing the adherent cells on the cell culturing surface. The cell culturing surface comprises a surface for culturing adherent cells, and a coating on the surface. The coating on the surface comprises a modified prolamin having a net positive charge at pH 7.5, and the modified prolamin is an amidated or esterified prolamin.

[0023] Aspect 20. The method of aspect 19, wherein the modified prolamin comprises a methyl-ester modification, an ethanolamide modification, or an amidated modification.Attorney Docket No. : SP24-283

[0024] Aspect 21. The method of any one of aspects 19-20, wherein the modified prolamin comprises zein.

[0025] Aspect 22. The method of any one of aspects 19-21 , wherein the cell culture surface is sterilized.

[0026] Aspect 23. The method of any one of aspects 19-22, wherein the surface for culturing adherent cell comprises a bead.

[0027] Aspect 24. The method of aspect 23, wherein the bead is dissolvable.

[0028] Aspect 25. The method of any one of aspects 23 or 24, wherein the cell culturing surface comprises modified prolamin at an amount of between 10 pg modified prolamin / mg dry beads and 100 pg modified prolamin / mg dry beads.

[0029] Aspect 26. The method of any one of aspects 19-25, wherein the cell culturing surface comprises a surface density of modified prolamin of between 0.1 pg / cm2and 100 pg / cm2.

[0030] Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0031] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide an overview or framework to understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s), and together with the description serve to explain principles and operation of the various embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following is a description of the figures in the accompanying drawings, given purely by way of non-limiting example. The figures are not necessarily to scale, and certainAttorney Docket No. : SP24-283 features and certain views of the figures may be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

[0033] FIG. 1 is a Fourier Transform Infrared Spectroscopy (FUR) spectrograph of an unmodified prolamin (“Zein” in the figure) compared to a methyl-ester modified prolamin (“Zein-ME” in the figure), according to some aspects of the present disclosure.

[0034] FIG. 2 is a line graph showing the zeta potential of an unmodified prolamin (“raw Zein” in the legend) compared to an esterified zein (methyl-ester modified zein, “Zein methyl ester” in the legend) from pH 4 to pH 9, according to some aspects of the present disclosure.

[0035] FIGS. 3A-3C are images from an optical microscope in fluorescent mode at lOx magnification of calcein AM stained C2C12 cells grown on an ultra-low attachment (ULA) well plate (FIG. 3C), a tissue culture treated (TCT) well plate (FIG. 3B), and on an ultra-low attachment (ULA) well plate coated with an esterified prolamin (methyl-ester modified zein) (FIG. 3A), according to some aspects of the present disclosure.

[0036] FIG. 4A is an image from an optical microscope in fluorescent mode at 4x magnification of calcein AM stained C2C12 cells following incubation with uncoated PGA beads, according to some aspects of the present disclosure.

[0037] FIG. 4B is an image from an optical microscope in fluorescent mode at 4x magnification of calcein AM stained C2C12 cells following incubation with polygalacturonic acid (PGA) beads coated with 0.2 wt.% methyl-ester modified zein, according to some aspects of the present disclosure.

[0038] FIG. 4C is an image from an optical microscope in fluorescent mode at 4x magnification of calcein AM stained C2C12 cells following incubation with PGA beads coated with 0. lwt.% methyl-ester modified zein, according to some aspects of the present disclosure.

[0039] FIG. 4D is an image from an optical microscope in fluorescent mode at 4x magnification of calcein AM stained C2C12 cells following incubation with PGA beads coated with 0.05 wt.% methyl-ester modified zein, according to some aspects of the present disclosure.Attorney Docket No. : SP24-283

[0040] FIG. 4E is an image from an optical microscope in fluorescent mode at 4x magnification of calcein AM stained C2C12 cells following incubation with PGA beads coated with 0.025 wt.% methyl-ester modified zein, according to some aspects of the present disclosure.

[0041] FIG. 5A is an image from an optical microscope using white light and set at 4x magnification of calcein AM stained C2C12 cells following incubation with uncoated PGA beads, according to some aspects of the present disclosure.

[0042] FIG. 5B is an image from an optical microscope using white light and set at 4x magnification of calcein AM stained C2C12 cells following incubation with PGA beads coated with 0.2 wt.% methyl-ester modified zein, according to some aspects of the present disclosure.

[0043] FIG. 5C is an image from an optical microscope using white light and set at 4x magnification of calcein AM stained C2C12 cells following incubation with PGA beads coated with 0. lwt.% methyl-ester modified zein, according to some aspects of the present disclosure.

[0044] FIG. 5D is an image from an optical microscope using white light and set at 4x magnification of calcein AM stained C2C12 cells following incubation with PGA beads coated with 0.05 wt.% methyl-ester modified zein, according to some aspects of the present disclosure.

[0045] FIG. 5E is an image from an optical microscope using white light and set at 4x magnification of calcein AM stained C2C12 cells following incubation with PGA beads coated with 0.025 wt.% methyl-ester modified zein, according to some aspects of the present disclosure.

[0046] FIG. 6 is an image from an optical microscope using white light and set at lOx magnification of calcein AM stained C2C12 cells following incubation with PGA beads coated with 0.2 wt.% methyl-ester modified zein, according to some aspects of the present disclosure. The coated beads were sanitized with ethanol prior to addition of C2C12 cells.

[0047] FIG. 7 is an image from an optical microscope using white light and set at lOx magnification of MRC5 cells post-incubation with PGA beads coated with 0.025 wt.% amidated zein, according to some aspects of the present disclosure.

[0048] FIG. 8 shows images from an optical microscope in using white light of C2C12 cells cultured on PGA beads coated with methyl-ester zein both before (left photograph, at lOxAttorney Docket No. : SP24-283 magnification) and after (right photograph, at 20xmagnification) dissolving the PGA beads with collagenase and EDTA, according to some aspects of the present disclosure.

[0049] FIG. 9 is a line graph of the zeta potential for an unmodified prolamin (“raw Zein” in the legend) compared to an amidated prolamin (“ZeinAM” in the legend), according to some aspects of the present disclosure.

[0050] FIG. 10A is an image from an optical microscope using white light and set at 4x magnification of C2C12 cells following incubation on an ultra-low attachment well plate coated with 0.2 wt.% esterified prolamin (methyl-ester modified zein), according to some aspects of the present disclosure.

[0051] FIG. 10B is an image from an optical microscope using white light and set at 4x magnification of C2C12 cells following incubation on an uncoated ultra-low attachment well plate, according to some aspects of the present disclosure.

[0052] FIG. 10C is an image from an optical microscope using white light and set at lOx magnification of C2C12 cells following incubation on an ultra-low attachment well plate coated with 0.2 wt.% esterified prolamin (methyl-ester modified zein), according to some aspects of the present disclosure.

[0053] FIG. 10D is an image from an optical microscope using white light and set at lOx magnification of C2C12 cells following incubation on an uncoated ultra-low attachment well plate, according to some aspects of the present disclosure.

[0054] FIG. 11 is a line graph of the zeta potential for an unmodified prolamin (“raw Zein” in the legend) compared to an ethanolamide modified prolamin (“ZeinEA” in the legend), according to some aspects of the present disclosure.DETAILED DESCRIPTION

[0055] The various aspects and embodiments will now be fully described herein. These aspects and embodiments may, however, be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided so the disclosure will be thorough and complete, and will fully convey the scope of the present subject matter to those skilled in theAttorney Docket No. : SP24-283 art. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0056] Modifications of the disclosure will occur to those skilled in the art and to those who make or use the disclosure. Therefore, it is understood that the embodiments shown in the drawings and described above are merely for illustrative purposes and not intended to limit the scope of the disclosure, which is defined by the following claims, as interpreted according to the principles of patent law, including the doctrine of equivalents.A. Definitions

[0057] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, particular methods and materials are now described.

[0058] As used herein the terms “the,” “a,” or “an,” mean “at least one,” and should not be limited to “only one” unless explicitly indicated to the contrary. Thus, for example, reference to “a component” includes embodiments having two or more such components unless the context clearly indicates otherwise.

[0059] Unless otherwise stated, the use of individual numerical values are stated as approximations as though the values were preceded by the word “about” or “approximately.” Similarly, the numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about” or “approximately.” In this manner, variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. As used herein, the terms “about” and “approximately” when referring to a numerical value shall have their plain and ordinary meanings to a person of ordinary skill in the art to which the disclosed subject matter is most closely related or the art relevant to the range or element at issue. The amount of broadening from the strict numerical boundary depends upon many factors. For example, some of the factors which may be considered includeAttorney Docket No. : SP24-283 the criticality of the element and / or the effect a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those of skill in the art. As used herein, the use of differing amounts of significant digits for different numerical values is not meant to limit how the use of the words “about” or “approximately” will serve to broaden a particular numerical value or range. Thus, as a general matter, “about” or “approximately” broaden the numerical value. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values plus the broadening of the range afforded by the use of the term “about” or “approximately.” Consequently, recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein.

[0060] As used herein, the term “and / or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing components A, B, and / or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0061] As used herein, “have,” “having,” “include,” “including,” “comprise,” “comprising” or the like are used in their open ended sense, and generally mean “including, but not limited to.”

[0062] “Optional” or “optionally” means that the subsequently described element, component or circumstance may or may not occur, so that the description includes instances where the element, component, or circumstance occurs and instances where it does not.

[0063] In this document, relational terms, such as first and second, top and bottom, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions.Attorney Docket No. : SP24-283

[0064] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.B. Introduction

[0065] Anchorage dependent cells are cultured on a substrate as this type of cell needs to attach to a substrate to survive and grow in numbers (expansion). An important use of certain types of anchorage dependent cells is in the generation of cultivated meat, also known as cultured meat. Cultured meat is genuine animal meat (including seafood) that is produced by cultivating animal cells directly. This production method eliminates the need to raise and farm animals for food, eliminates animal slaughtering for food, and has a positive impact on the environment by reducing greenhouse-gas emissions and water consumption involved in farming and processing animals for food.

[0066] Microcarriers are one way to grow cells for cultured meat. However, most microcarriers are non-dissolvable and so need to be removed from the cultured cells with downstream processing. This is inefficient because of the extra processing steps that are required to remove the microcarriers from cell culture. Further, microcarriers (or parts of them) can be internalized by cells and can contaminate the final meat product. It is advantageous if the microcarriers are dissolvable, meaning they act as a temporary substrate in the form of microcarriers during the phase of cell attachment and expansion but can then be degraded or dissolved thereafter. Alternatively, it is advantageous if the microcarriers stay embedded in the final meat product but are edible because this decreases the number of processing steps that need to be performed.

[0067] With any of non-dissolvable, dissolvable, or edible microcarriers, it is critical that cells be able to adequately adhere to the microcarrier and be able to proliferate to expand the numbers of cells being cultured. Currently available microcarriers suffer from not being edible, not being suitable for use with edible materials, not adequately performing (adherence and growth are stunted) or being difficult to dissolve. With edible microcarriers, the microcarriers do not perform adequately and they have a high rigidity, which imparts un undesirable crunch in the final meat product. There is therefore a need for a low cost, edible microcarrier composed of food-gradeAttorney Docket No. : SP24-283 ingredients that promote cell attachment and expansion at levels that allow for commercially viability, and further that are easy to generate and use.

[0068] The present disclosure has discovered a solution to the problem of non-edible substrates that can promote cell binding and attachment by creating a novel substrate in the form of a coating comprising water insoluble modified prolamin exhibiting a net positive charge at physiological pH after modification. The novel substrate does not use crosslinkers, cultures anchorage dependent cells, is food grade, is free of animal components, and can be coated onto food grade microcarriers.

[0069] Additional features and advantages will be set forth in the detailed description which follows and will be apparent to those skilled in the art from the description or recognized by practicing the embodiments as described in the following description, together with the claims and appended drawings.C. Modified Prolamin Substrate

[0070] Adherent cell culture requires a surface for culturing cells as the cells must attach to the surface in order to proliferate. The surface for culturing cells may comprise a substrate that aids in cell attachment and / or proliferation. This substrate for adherent cell attachment and growth comprises a modified prolamin. In some embodiments, the substrate for adherent cell attachment and growth comprises at least two modified prolamins. Prolamins are storage proteins from plants that have a high content of hydrophobic amino acids (for example, proline, alanine and leucine). In one embodiment, the prolamin is a cereal prolamin. A cereal prolamin is an endosperm storage protein of a cereal grain. In some embodiments, the prolamin is gliadin (wheat), hordein (barley), secalin (rye), zein (com), kafirin (sorghum), or avenin (oats). In one specific embodiment, the prolamin is zein. However, it should be understood that any other prolamin beyond those listed that are known to those of ordinary skill in the art may be used. Because of their hydrophobic nature, prolamins are generally insoluble in water. In some embodiments, the prolamin is waterinsoluble.

[0071] The prolamin for the substrate is a modified prolamin. A modified prolamin is a prolamin that has been altered to have a net positive charge (a positive zeta potential) atAttorney Docket No. : SP24-283 physiological pH (i.e., pH 6-8) after the modification. Modifications that alter a prolamin to have a net positive charge at physiological pH include amidation and esterification, among other modifications known to those of ordinary skill in the art. In some embodiments, the modification to form the modified prolamin is amidation, esterification, or a combination thereof.

[0072] Amidation is a chemical reaction in which carboxyl groups on the prolamin protein are converted into amide groups. For example, amino acids in the prolamin protein such as aspartic acid and / or glutamic acid have carboxyl group side chains and when reacted with an amino compound such as ammonia or alkylamines (among other known to those of ordinary skill in the art) the carboxyl groups in those amino acids are converted to amide groups. If ammonia is the reactant, aspartic acid is converted to asparagine and glutamic acid is converted to glutamine, converting a negative charge into a neutral charge. The decrease in negative charges in the modified prolamin protein results in a prolamin with a net positive charge.

[0073] In some embodiments, the reactant for amidation is ammonia or an ammonium salt, an alkylamine, an aminoalcohol, or a combination thereof. Alkylamines used for the reactant may include those comprising 2-18 carbons. Aminoalchohols may include ethanolamine, propanolamine, butanolamine, N-methyl diethanolamine, and dimethylethanolamine, among others known to those of ordinary skill in the art. In one embodiment, the reactant for amidation is ammonia. In a preferred embodiment, the reactant for amidation is food grade. Food grade reactants for amidation include food grade ammonia, among others known to those of ordinary skill in the art.

[0074] Esterification is a chemical reaction in which carboxyl groups or amide groups in the prolamin’s amino acid sidechains are converted to alkyl ester groups. For example, amino acids in the prolamin protein such as aspartic acid and / or glutamic acid have carboxyl side chains and when reacted with an alcohol (for example an alcohol that the prolamin may be solubilized in) in acidic conditions, the carboxyl groups in those amino acids will be converted to alkyl ester groups. Likewise, amino acids such as glutamine and asparagine having an amide group in their side chain convert the amide into an alkyl ester group when exposed to an alcohol. For example, if methanol is the reactant, the carboxyl groups of the side chains of glutamate and aspartate are converted from a carboxylic acid group to a group having a methyl-ester. Similarly, methanol will convertAttorney Docket No. : SP24-283 the amide groups of glutamine and asparagine side chains from an amide group to a group having a methyl-ester. These changes in the esterified prolamin protein result in a prolamin protein having a net positive charge.

[0075] In some embodiments, the modified prolamin is generated with an esterification reaction, the esterification reaction comprising an alcohol reactant. In some embodiments, the alcohol reactant is a primary, secondary, or tertiary alcohol. In some embodiments, the alcohol reactant is methanol, ethanol, n-propanol, isopropanol, butanol, pentanol, hexanol, octanol, decanol, or a combination thereof. In one embodiment, the alcohol reactant is methanol, ethanol, or a combination thereof. In a preferred embodiment, the alcohol reactant is an edible or food grade alcohol. In one embodiment, the alcohol reactant is food grade and is ethanol, a sugar alcohol, polypropylene glycol, or a combination thereof. Sugar alcohols that are edible include xylitol, sorbitol, maltitol, mannitol, lactitol, isomalt, erythritol, and hydrogenated starch hydrolysates.

[0076] In some embodiments, the esterification reaction further comprises an acid catalyst. In such embodiments, the acid catalyst may be an organic acid or an inorganic acid. Any suitable organic acid known to those of ordinary skill in the art may be used, including but not limited to paratoluene sulfonic acid. Alternatively, any suitable inorganic acid known to those of ordinary skill in the art may be used, including but not limited to sulfuric acid, hydrochloric acid, and hydrochloride gas. In some embodiments, the acid catalyst is paratoluene sulfonic acid, sulfuric acid, or hydrochloric acid. In some embodiments, the acid catalyst is a food-grade acid catalyst. Food-grade acid catalysts include hydrochloric acid and sulfuric acid, among others known to those of ordinary skill in the art.

[0077] In some embodiments, the modified prolamin may be packaged in a vessel as part of a kit. In such embodiments, the kit includes a vessel that has the modified prolamin. In some further embodiments, the modified prolamin in the vessel may be in dry form (e.g., in precipitated or lyophilized form). Alternatively, the modified prolamin in the vessel may be solubilized in an alcohol or hydroalcoholic solution. In some embodiments, the alcohol or hydroalcoholic solution includes ethanol or methanol.

[0078] The vessel or kit may further include an article that has a cell culture surface. The cell culture surface is a surface for adherent cells to be cultured. The cell culture surface may be aAttorney Docket No. : SP24-283 treated or untreated cell culture surface. Treated cell culture surfaces include surfaces that are coated or surface treated to encourage cell attachment (for example, coatings that comprise cell attachment peptides or plasma treated polymer surfaces) or to prevent cell attachment (for example, ultra-low attachment coatings that are hydrophilic and neutrally charged), as known to those of ordinary skill in the art. The article may be any container, culture plate, well plate, or other vessel that can be used to culture adherent cells known to those of ordinary skill in the art.D. Methods of Making Modified Prolamins

[0079] Methods of making certain modified prolamins will now be described.Esterification Modification

[0080] A modified prolamin that is an esterified prolamin may have a methyl-ester modification. To produce a modified prolamin wherein the modification is a methyl-ester, the prolamin is combined with paratolulene sulfonic acid (“PTSA”) and methanol, with sulfuric acid and methanol, or methanol and a Lewis acid known to those of ordinary skill of the art, such as scandium (III) triflate. In some embodiments, the methyl-ester prolamin is generated from combining prolamin with methanol and either PTSA or sulfuric acid. The ratio of prolamin to PTSA, sulfuric acid, or Lewis acid in the mixture may be between 0.5:20 and 20:0.5, between 0.5:15 and 15:0.5, between 0.5:10 and 10:0.5, or between 0.5:5 and 0.5:15. In some embodiments the mixture is left at rest to react. In some embodiments, the mixture is agitated to react. In some embodiments the mixture is both left at rest and agitated to react. In some embodiments, the reaction may be carried out at a temperature between 4 °C and 80 °C, between 4 °C and 70 °C between 4 °C and 60 °C, between 4 °C and 50 °C, between 4 °C and 40 °C, between 10 °C and 80 °C, between 20°C and 80 °C, between 30 °C and 80 °C, between 40 °C and 80 °C, between 50 °C and 80 °C, between 60 °C and 80 °C, between 70 °C and 80 °C, between 15 °C and 70 °C, between 15 °C and 60 °C, between 15 °C and 50 °C, between 15 °C and 40 °C, or at any range or value between 4 °C and 80 °C. In some embodiments, the time for reaction may be between 5 minutes and 14 days.

[0081] Alternatively, the modified prolamin that is an esterified prolamin may have an ethyl-ester modification. In this modification, the prolamin is combined with PTSA and ethanol,Attorney Docket No. : SP24-283 with sulfuric acid and ethanol, or ethanol and a Lewis acid known to those of ordinary skill of the art, such as scandium(III) triflate. In some embodiments, the ethyl-ester prolamin is generated from combining prolamin with ethanol and either PTS A or sulfuric acid. The ratio of prolamin to PTS A, sulfuric acid, or Lewis acid in the mixture, the temperature for the reaction, the length of time for the reaction, and style of mixing (at rest and / or agitation) is the same for the ethyl-ester modification as described above for the methyl-ester modification.

[0082] After the reaction has been completed, the esterified prolamin having a methylester or ethyl-ester modification may be recovered by any method known to those of ordinary skill in the art. In one embodiment, the esterified prolamin having a methyl-ester or ethyl-ester modification is recovered from the mixture by precipitation using the addition of water to the mixture. The supernatant may then be decanted and optionally, the precipitate may be washed with a basic buffer such as sodium hydroxide and / or water. In another embodiment, the esterified prolamin having a methyl-ester or ethyl-ester modification is recovered by dialysis with an alcohol or hydroalcoholic solution. In some embodiments, the alcohol is ethanol or methanol. In some embodiments, the hydroalcoholic solution is a mixture of alcohol and water at a ratio between 30:70 alcohol to water and 95:5 alcohol to water. In one specific embodiment, the hydroalcoholic solution is a mixture of alcohol and water at a ratio between 80:20 alcohol to water and 60:40 alcohol to water. For any embodiment, the esterified prolamin having a methyl-ester or ethyl-ester modification may be dried prior to use as a coating material.

[0083] In some embodiments, the yield of esterified prolamin having a methyl-ester or ethyl-ester modification from the starting amount of unmodified prolamin used is between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 30% and 90%, between 30% and 80%, between 30% and 70%, between 30% and 60%, between 40% and 60%, between 40% and 70%, between 40% and 80%, or at any range or value between 30% and 100% yield.Amidation Modification

[0084] To produce a modified prolamin wherein the modification results in an amidated prolamin, raw prolamin may be dissolved in an alcohol or hydroalcoholic solution and then mixed with l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (“EDC”), dicyclohexyl carbodiimideAttorney Docket No. : SP24-283 (“DCC”), or carbonyldiimidazole (“CDI”). This mixture creates an activated prolamin. The ratio of the EDC, DCC, or CDI in the mixture to carboxylic acid groups on the raw prolamin may be at a ratio between 1:1 and 50:1. For example, the ratio of EDC, DCC, or CDI in the mixture to carboxylic acid groups on the prolamin may be 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1 :45, or 1 : 50, or at any ratio therebetween. In one specific embodiment, the mixture comprises raw prolamin, an alcohol or hydroalcoholic solution, and EDC.

[0085] Regarding the alcohol in the mixture to create an activated prolamin, the alcohol can be any alcohol known to those of ordinary skill in the art to dissolve zein, including methanol, ethanol, and isopropanol. In one embodiment, the alcohol is ethanol or isopropanol. In another embodiment, the hydroalcoholic solution is a mixture of alcohol and water at a ratio between 30:70 and 95:5. In one specific embodiment, the hydroalcoholic solution is a mixture of alcohol and water at a ratio between 80:20 alcohol to water and 60:40 alcohol to water. In a further specific embodiment, the alcohol in the hydroalcoholic solution is ethanol or isopropanol.

[0086] In some embodiments, the mixture to generate an activated prolamin further includes N-hydroxysuccinimide (“NHS”). In mixtures comprising NHS, the ratio of EDC, DCC, or CDI in the mixture to NHS may be at a ratio between 0.5:1 and 1:4. For example, the ratio of EDC, DCC, or CDI in the mixture to NHS may be at a ratio of 0.5: 1, 1:1, 1:2, 1:3, or 1 :4, or at any ratio therebetween. In one specific embodiment, the mixture comprises raw prolamin, an alcohol or hydroalcoholic solution, EDC, and NHS, and the ratio of NHS to EDC in the mixture is between 0.5:1 and 1:2.

[0087] Once the activated prolamin has been created, the activated prolamin is then reacted with a salt comprising an ammonium salt or an ethanolamine salt to generate an ami dated prolamin. When an ammonium salt is used, the modified prolamin comprises an amide modification, whereas when an ethanolamine salt is used, the modified prolamin comprises an ethanolamide modification. The mixture comprising the activated prolamin may be agitated before the ammonium salt or ethanolamine salt is added to create the reaction mixture. The reaction mixture may then left at rest to react. The reaction mixture may instead be agitated to react. Alternatively, the reaction mixture may be both left at rest and agitated during the reaction time. In some embodiments, the reaction mixture may be carried out at a temperature between 4 °C and 80 °C,Attorney Docket No. : SP24-283 between 4 °C and 70 °C between 4 °C and 60 °C, between 4 °C and 50 °C, between 4 °C and 40 °C, between 10 °C and 80 °C, between 20°C and 80 °C, between 30 °C and 80 °C, between 40 °C and 80 °C, between 50 °C and 80 °C, between 60 °C and 80 °C, between 70 °C and 80 °C, between 15 °C and 70 °C, between 15 °C and 60 °C, between 15 °C and 50 °C, between 15 °C and 40 °C, or at any range or value between 4 °C and 80 °C. In some embodiments, the time for reaction may be between 5 minutes and 14 days.

[0088] In some aspects of the disclosure, the modified prolamin is an amidated prolamin comprising an amide modification. To produce the amidated prolamin with a primary amide modification, the activated prolamin mixture above is reacted with ammonia or an ammonium salt. Ammonium salts include ammonium chloride, ammonium carbonate, ammonium bicarbonate, and ammonium sulfate, among others. The ammonium salt may be food grade. Food grade ammonium salts include but are not limited to ammonium bicarbonate, ammonium carbonate, and ammonium chloride. In one embodiment, the ammonium salt is ammonium chloride. In another embodiment, the ammonium salt is food grade ammonium chloride. The ratio of ammonia or ammonium salt to activated prolamin may be at a ratio between 0.5:100 and 5:100, or at any value or in any range therebetween. For example, the ratio of ammonia or ammonium salt is between 1:100 and 5:100, between 2:100 and 5:100, between 3:100 and 5:100, between 4:100 and 5:100, between 0.5:100 and 4:100, between 0.5:100 and 3:100, between 0.5:100 and 2:100, or between 0.5:100 and 1:100.

[0089] In other aspects of the disclosure, the modified prolamin is an amidated prolamin comprising an ethanolamide modification. To produce the amidated prolamin with an ethanolamide modification, the activated prolamin mixture described above is reacted with ethanolamine or an ethanolamine salt. In one embodiment, the ethanolamine salt is ethanolamine chloride. The ratio of ethanolamine or ethanolamine salt to activated prolamin may be at a ratio between 0.5:100 and 5:100, or at any value or in any range therebetween. For example, the ratio of ethanolamine or ethanolamine salt is between 1:100 and 5:100, between 2:100 and 5:100, between 3:100 and 5:100, between 4:100 and 5:100, between 0.5:100 and 4:100, between 0.5:100 and 3:100, between 0.5:100 and 2:100, or between 0.5:100 and 1:100.

[0090] After an amidation reaction has been completed, the amidated prolamin may be recovered by any method known to those of ordinary skill in the art. For example, the amidatedAttorney Docket No. : SP24-283 prolamin is recovered by precipitation with water. The amidated prolamin precipitate may then be dried. As another example, the amidated prolamin is recovered from the mixture by dialysis followed by freeze drying. The dialysis solution may be an alcohol or a hydroalcoholic solution. In some embodiments, the alcohol may be ethanol or methanol. For example, the hydroalcoholic solution may be a mixture of alcohol and water at a ratio between 30:70 alcohol to water and 95:5 alcohol to water. In one specific embodiment, the hydroalcoholic solution is a mixture of alcohol and water at a ratio between 80:20 alcohol to water and 60:40 alcohol to water. The yield of amidated prolamin from the starting amount of prolamin used may be between 30% and 100%, between 40% and 100%, between 50% and 100%, between 60% and 100%, between 70% and 100%, between 30% and 90%, between 30% and 80%, between 30% and 70%, between 30% and 60%, between 40% and 60%, between 40% and 70%, between 40% and 80%, or at any range or value between 30% and 100% yield.Characteristics of Modified Prolamins

[0091] For any of the modified prolamins of the present disclosure, the modified prolamin has a net positive charge (a positive zeta potential) at physiological pHs. The net positive charge of a prolamin or modified prolamin can be determined by testing a solution comprising the prolamin or modified prolamin with a zeta analyzer, such as the Zetasizer Nano ZS analyzer from Malvern Panalytical Ltd. A zeta analysis determines the zeta potential of a particle in a liquid, emulsion, or suspension. The zeta potential is a measure of the electrical potential at the particleliquid interface. When the zeta potential is greater than zero millivolts (mV), the particle has a net positive charge. Prolamin is a type of particle that can be analyzed using a zeta analysis. The modified prolamins of the present disclosure that have a positive zeta potential at a particular pH have a net positive charge at that pH.

[0092] For example, the modified prolamin may have a net positive charge at a pH between pH 4-9, between pH 5-9, between pH 6-9, between pH 7-9, between pH 6-8, or at any range or value between pH 4-9. The modified prolamin may have a net positive charge at pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH 4.5, pH 4.5, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7,Attorney Docket No. : SP24-283 pH 7.8, pH 7.9, pH 8.0, pH, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, or pH 9.0, or in any range or at any value between pH 4.0 and pH 9.0. In one embodiment, the modified prolamin has a net positive charge at pHs 6.5-8.0. In one specific embodiment, the modified prolamin has a net positive charge at pH 7.5.E. Modified Prolamin Coatings

[0093] The modified prolamin can be formed into a coating solution that can be applied to a surface to create a coating on the surface that promotes adherent cell attachment and growth (expansion). The coating solution comprises a modified prolamin and an alcohol. Any of the modified prolamins described above may be used. In some embodiments, the modified prolamin in the coating solution may be present in a percent by weight between 0.01 wt.% and 5 wt.%, between 0.01 wt.% and 1 wt.%, between 0.01 wt.% and 0.5 wt.%, between 0.01 wt.% and 0.1 wt.%, between 0.01 wt.% and 0.05 wt.%, between 0.01 wt.% and 0.025 wt.%, or between 0.015 wt.% and 0.5 wt.%, modified prolamin. In one specific embodiment, the modified prolamin in the coating is present in the coating solution in a percent by weight between 0.01 wt.% and 1 wt.%.

[0094] In some embodiments, the alcohol may be ethanol, methanol, n-propanol, or isopropanol. In some embodiments, the alcohol is food grade. In one specific embodiment, the food grade alcohol is ethanol or isopropanol. The alcohol in the coating may be pure alcohol (i.e., highest purity of the alcohol that is commercially available, such as 95% ethanol), or it may be a hydroalcoholic alcohol. If the alcohol is a hydroalcoholic alcohol, the ratio of alcohol to water may be between 60:40 and 95:5, between 70:30 and 95:5, between 80:20 and 95:5, or between 90:10 and 95:5. In one embodiment, the alcohol is a hydroalcoholic alcohol and the ratio of alcohol to water is between 70:30 and 90: 10.

[0095] The modified prolamin coating solution may further include a buffer. Any buffer known to those of ordinary skill in the art may be used, including 2-(N-morpholino)ethanesulfonic acid (“MES”). Other non-limiting examples include Tris-HCl and HEPES. The ratio of alcohol to buffer may be between 60:40 and 95:5, between 70:30 and 95:5, between 80:20 and 95:5, or between 90: 10 and 95:5. In one embodiment, the coating solution further includes a buffer with a ratio of alcohol to buffer between 60:40 and 95:5. In another embodiment, the coating solution further includes a buffer with a ratio of alcohol to buffer between 70:30 and 90: 10.Attorney Docket No. : SP24-283

[0096] The modified prolamin coating solution and the modified prolamin coating on the cell culture surface are free from animal components (i. e. , animal -free). This means that the coating solution and the modified prolamin coating on the cell culture surface does not include animal-derived materials in the coating solution or coating itself. Having an animal-free coating solution and the modified prolamin on the cell culture surface is particularly useful for fields where animal-derived materials can contaminate the process or product, such as in stem cell research areas including cell therapy, regenerative medicine, and vaccine production, and in drug discovery and testing areas including cancer research, genetics research, and molecular biology research. It should be understood that the modified prolamin coating can be used for application like microcarriers for cultured meat, but it may instead be used for any other application that cultures adherent cells. Likewise, the coating can be used in applications seeking to use animal-free coatings or crosslinker-free coatings that promote adherent cell attachment and growth.

[0097] Further, the modified prolamin coating solution and the modified prolamin coating on the cell culture surface may be free of crosslinkers. Crosslinkers are molecules that chemically link together one or more target components in a solution. Most crosslinkers are toxic and so cannot be used for food grade products. Crosslinking also requires extra processing steps and so may be undesirable both for time efficiency of the process and from a cost perspective.

[0098] In some embodiments, the modified prolamin coating solution or the modified prolamin coating on the cell culture surface is free from both animal components and crosslinkers. In other embodiments, the modified prolamin coating solution and the modified prolamin coating on the cell culture surface is free from both animal components and crosslinkers.F. Modified Prolamin Coated Surfaces

[0099] The modified prolamin coating solution can be applied to any surface that can be used to culture adherent cells, known to those of ordinary skill in the art. Suitable surfaces include but are not limited to, coverslips, slides, culture dishes, culture plates, roller bottles, well plates having any number of wells between 1 and 10,000, flasks, microcarriers, and multilayer flasks. The cell culture surface may be made from materials known to those of ordinary skill in the art, including but not limited to dextran, glass, polystyrene, polyethylene, polypropylene, gelatin, polygalacturonic acid, collagen, chitin, alginate, cellulose, raw prolamins, and derivatives thereof.Attorney Docket No. : SP24-283 The cell culture surface may be a treated surface or an untreated surface. Treated surfaces may include surfaces that improve cell attachment such as tissue culture treated surfaces and Corning® CellBIND® surfaces among others known to those of ordinary skill in the art. Treated surfaces may also include surfaces that reduce cell attachment, such as ultra-low attachment (“ULA”) surfaces using covalently bound hydrogels that are hydrophilic and neutrally charged, among others known to those of ordinary skill in the art.

[0100] In some aspects of the disclosure, the cell culture surface is a microcarrier. A microcarrier is a small support matrix that allows adherent cells to attach and supports growth (expansion) of the cells. In some embodiments, the microcarrier is porous. In some embodiments, the microcarrier is between 90 pm and 400 pm in diameter or at its widest point. In one specific embodiment, the microcarrier is between 100 pm and 300 pm in diameter or at its widest point. In one embodiment, the microcarrier is a bead. Commercially available beads for adherent cell culture may be used, or beads may be generated by processes such as those stated in US. Pat. App. No.2018 / 0179489, incorporated herein by reference. In some embodiments, the microcarrier is dissolvable. In some embodiments, the microcarrier is a food grade microcarrier.

[0101] To coat the cell culture surface, a modified prolamin coating described above is applied to the cell culture surface and is left to adsorb on the surface. The time the modified prolamin coating is left to adsorb on the surface may be for a time between 5 seconds and 48 hours (with or without agitation). In some embodiments, the cell culture surface is then washed one or more times with a rinse agent. The rinse agent may be water or a buffer. The cell culture surface may then be washed between 1 and 10 times with water or a buffer with a volume approximately equivalent to the volume used to coat the cell culture surface. The coated cell culture surface may further be dried before use.

[0102] The cell culture surface may have a surface density of the modified prolamin (either with the amidation modification or the esterification modification) of between 0.1 pg / cm2and 100 pg / cm2, or at any value or range between 0.1 pg / cm2and 100 pg / cm2. The surface density of the modified prolamin on the cell culture surface refers to the amount of modified prolamin per coated surface area. The surface density of prolamin on the coated surface is determined by a bicinchoninic acid (“BCA”) assay, using assay kits such as a Micro BC Assay Kit (Interchim, Cat.Attorney Docket No. : SP24-283 No. UP75760A). BCA assays create a coloring with proteins (like prolamin) that allow a determination of protein amounts with a U V / VIS spectrophotometer. In one embodiment, the cell culture surface has a surface density of the modified prolamin of between 0.1 pg / cm2and 90 pg / cm2, between 0.1 pg / cm2and 80 pg / cm2, between 0.1 pg / cm2and 70 pg / cm2, between 0.1 pg / cm2and 60 pg / cm2, between 0.1 pg / cm2and 50 pg / cm2, between 0.1 pg / cm2and 40 pg / cm2, 0.1 pg / cm2and 30 pg / cm2, 0.1 pg / cm2and 20 pg / cm2, 0.1 pg / cm2and 10 pg / cm2, 0.5 pg / cm2and 100 pg / cm2, 1 pg / cm2and 100 pg / cm2, 10 pg / cm2and 100 pg / cm2, 20 pg / cm2and 100 pg / cm2, 30 pg / cm2and 100 pg / cm2, 40 pg / cm2and 100 pg / cm2, between 50 pg / cm2and 100 pg / cm2, between 60 pg / cm2and 100 pg / cm2, between 70 pg / cm2and 100 pg / cm2, between 80 pg / cm2and 100 pg / cm2, between 90 pg / cm2and 100 pg / cm2, between 5 pg / cm2and 90 pg / cm2, between 1 pg / cm2and 50 pg / cm2, or between 10 pg / cm2and 40 pg / cm2.

[0103] In embodiments where the cell culture surface is a bead, the amount of modified prolamin adsorbed onto the cell culture surface is between 10 pg prolamin / mg dry beads and 300 pg prolamin / mg dry beads, between 10 pg prolamin / mg dry beads and 250 pg prolamin / mg dry beads, between 10 pg prolamin / mg dry beads and 200 pg prolamin / mg dry beads, between 10 pg prolamin / mg dry beads and 150 pg prolamin / mg dry beads, between 10 pg prolamin / mg dry beads and 100 pg prolamin / mg dry beads between 20 pg prolamin / mg dry beads and 90 pg prolamin / mg dry beads, between 30 pg prolamin / mg dry beads and 70 pg prolamin / mg dry beads, between 40 pg prolamin / mg dry beads and 60 pg prolamin / mg dry beads, between 10 pg prolamin / mg dry beads and 60 pg prolamin / mg dry beads, between 40 pg prolamin / mg dry beads and 100 pg prolamin / mg dry beads, or in any range or value between 10 pg prolamin / mg dry beads and 300 pg prolamin / mg dry beads. The amount of prolamin per mg dry beads can be determined using a BCA assay as described above.

[0104] Adherent cells may be cultured on the coated cell culture surface. Adherent cells attach to the modified prolamin coated surface and may be expanded (grown) on the modified prolamin coated surface to produce large amounts of cultured cells. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more than 90% of the cells added to the culture surface attach to the modified prolamin coated cell culture surface. In one specific embodiment, at least 50% of the cells added to the culture surface attach to the1Attorney Docket No. : SP24-283 modified prolamin coated cell culture surface. In some embodiments, the adherent cells grown on the cell culture surface expand at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, or at any range or value between 2-fold and 1000-fold over the number of cells added to the cell culture surface. In one specific embodiment, the adherent cells grown on the cell culture surface expand at least 5-fold over the number of cells added to the cell culture surface.

[0105] In some embodiments, cultured cells may be harvested. Methods of harvesting adherent cells grown on cell culture surfaces are known to those of ordinary skill in the art. In embodiments where cultured cells are on dissolvable microcarriers, the cells may be harvested by dissolving the microcarriers. Dissolvable microcarriers are dissolved by adding an appropriate proteolytic enzyme to the cell culture, which disrupts bonds in the crosslinkers in the microcarrier, causing the microcarrier to dissolve. Dissolvable microcarriers and how to dissolve them are known to those of ordinary skill in the art. In embodiments where cultured cells are on the surfaces of a vessel such as culture plate, a slide, a flask, in wells of a well plate, and the like, the cells may be harvested by adding a dissociation reagent that disrupts the attachment of the cells to the cell culturing surface. Dissociation reagents include proteolytic enzymes such as trypsin, collagenase, dispase, Accutase®, among others known to those of ordinary skill in the art. Other dissociation reagents include non-enzymatic solutions with chelators, such as Corning® Cell Recovery Solution and Corning® Cellstripper® solution.EXAMPLES

[0106] The embodiments described herein will be further clarified by the following examples.Example 1 - Raw ZeinRaw Zein Coatins

[0107] To create a 0.2 wt. % raw zein hydroalcoholic coating, 100 mg of raw zein (Sigma Aldrich, Cat. No. Z3625) was dissolved in 50 mL of ethanol / 2-(N-morpholino) ethanesulfonic acid (“MES”) in a 70:30 ratio by volume while being stirred.Attorney Docket No. : SP24-283

[0108] To create microcarrier beads, calcium cross-linked polygalacturonic acid (PGA) beads were prepared using a method disclosed in commonly-assigned U.S. Patent Application No.2018 / 0179489, incorporated by reference herein in its entirety, and employing a laminar jet break up system from Nisco. The calcium crosslinked PGA microcarrier had a mean diameter of 250 pm. The calcium cross-linked PGA beads were then equilibrated for 10 minutes in MES buffer pH 5.5. The MES buffer was created by dissolving 0.39 g MES hydrate in 195 mL ultra purified water and the pH was adjusted to 5.5 by adding IN NaOH. Ultra purified water was then added until the solution was 200 mL in total.

[0109] To coat the raw zein coating mixture on the microcarrier beads, 12 mL of the 0.2 wt.% raw zein coating mixture was added to 2 mL of PGA beads (volume of the bead bed) in a 15 mL plastic centrifuge tube. The tube was agitated for 2 hours at room temperature on a lab roller. Then the beads were washed three times with 10 mL ultra purified water. The resulting beads were colorless and exhibited high transparency.Raw Zein Cell Testins

[0110] To evaluate myoblast cell attachment on the raw zein coated beads, C2C12 cells (from passage 6) in Roswell Park Memorial Institute (“RPMI”) medium supplemented with 10% fetal bovine serum (“PBS”), PenStrep (100 U / mL final concentration) and GlutaMAX™ (Gibco) (lOOx concentration, 5 mL added to 500 mL RPMI medium) were seeded onto a monolayer of the raw zein coated PGA beads that had been placed in wells of a 24-well ultra-low attachment (“ULA”) well plate. The density of cells added was 100K cells per well. The beads were observed both one day and three days after seeding using an 1X73 Inverted Microscope (Olympus) in brightfield mode. As shown in FIG. 10B (4x magnification) and FIG. 10D (lOx magnification), no cell attachment occurred with the raw zein coated beads three days after seeding.Example 2 - Zein-methyl-esterZein Methyl-Ester Synthesis

[0111] A modified zein with a methyl-ester modification (“Zein-Me”) was created as a substrate by the following procedure. 1 g of zein (Sigma Aldrich, Cat. No. Z3625) and 10 g paratolulene sulfonic acid (“PTS A”) was dissolved in 50 mL of 99.8% methanol. The mixture wasAttorney Docket No. : SP24-283 covered and left at room temperature for 120 hours. The mixture was then refluxed for 2 hours at 65°C. Next, 150 mL of ultra-pure water was added to the mixture to precipitate the Zein-Me. The mixture was allowed to rest until the Zein-Me precipitate settled on the bottom of the vessel. The supernatant liquid was decanted and 50 mL of aqueous IN NaOH was added the vessel containing the precipitate to neutralize any residual acid. The precipitate was then isolated by centrifugation, washed with a small quantity of cold ultra-pure water and then dried in vacuum for 24 h at 60 °C. A total of 0.55 g of Zein-Me was recovered after the drying step (equivalent to a 55% yield).

[0112] The Zein-Me was solubilized in aqueous ethanol 70:30 (i.e., 70% of the total volume was ethanol, and 30% of the total volume was ultra-pure water) for further analysis. A Fourier Transform Infrared (“FTIR”) spectroscopy analysis was performed using a (Nicolet IS 50 FT-IR, Thermo Fisher Scientific, Inc.) spectrometer on both Zein-Me and on the raw zein (unmodified) material to investigate esterification with the Zein-Me. A comparison of the unmodified zein with the Zein-Me in an FTIR analysis is shown in FIG. 1. New peaks appeared at 1740 cm'1and 1173 cm'1in the Zein-Me sample, which were assigned to the C=O and C-0 stretching vibrations respectively of methyl ester groups, indicative of esterification. Also, the reduction in the area of the NH stretch mode centered at 3295 cm'1between the Zein-Me and the unmodified zein samples is indicative of the esterification. Further, the intensities of the primary amide components at 3203 cm'1and 3450 cm'1between the Zein-Me and unmodified zein are also reduced, which is also indicative of esterification.

[0113] A zeta potential analysis was also performed on the Zein-Me and unmodified zein, using a zeta potential analyzer (Zetasizer Nano ZS, Malvern Panalytical, Ltd.). The zeta potential of the Zein-Me was evaluated at a concentration of 20 mg / ml Ethanol / MES 70:30 w / v at different pH values ranging from pH 4 to pH 9. The results, shown in FIG. 2, indicate that Zein-Me has a positive zeta potential throughout the entire range of pH 4-9, whereas the zeta potential of the raw zein drops when the pH is increased over pH 4 and then reaches zero charge near pH 7.Zein-Me Coatins on Cell Culture Plates

[0114] The Zein-Me substrate was used to improve cell attachment on a 2D surface. The 2D surface used were well bottoms of an ultra-low attachment (“ULA”) well plate comprising 24 wells. The well bottoms were coated with 30 pl of a 0.05 wt.% Zein-Me solution in methanol. TheAttorney Docket No. : SP24-283 coating was left to dry overnight at 37 °C, then rinsed with ultra -pure water several times leading to a hydrophobic surface that beaded water. The coated surface was tested for cell adhesion using C2C12 cells, with a cell amount of 25,000 cells (C2C12 cells from passage no. 10) per well in Iscove's Modified Dulbecco's Medium (“IMDM’). The attachment of C2C12 cells was also evaluated in parallel on Tissue Culture Treated (“TCT”) and ULA plates without Zein-Me coating as reference. After letting the cells attach to the plates, they were stained with calcein AM and observed on a 1X73 Inverted Microscope (Olympus) in fluorescence mode at lOx magnification. The resultant images are shown in FIG. 3. As shown in FIG. 3, the C2C12 cells attach and spread well on the Zein-Me coated plates (FIG. 3A), and the adhesion is comparable to the adhesion on standard TCT plates (FIG. 3B), whereas no adhesion was present on the ULA plates without Zein-Me coating (FIG. 3C).Zein-Me Coatins on Microcarriers

[0115] Calcium cross-linked PGA beads (a type of microcarrier) were prepared as described in Example 1. The microcarriers had mean diameter of 250 pm. To create a 0.2 wt. % Zein-Me hydroalcoholic coating, 100 mg of precipitated and washed Zein-Me was dissolved in 50 mL of ethanol / MES in a 70:30 ratio by volume while being stirred. Then, 12 mL of the Zein-Me solution was added to 2 mL of PGA beads (volume of the bead bed) in a 15 mL plastic centrifuge tube. The tube was agitated for 2 hours at room temperature on a lab roller. Then the beads were washed three times with 10 mL ultra-pure water. The resulting beads were colorless and exhibited high transparency. Coatings with 0.025 wt.%, 0.05 wt.%, and 0.1 wt.% Zein-Me were likewise created, but using 400 mL, 200 mL, and 100 mL ethanol / MES in a 70:30 ratio by volume respectively.Zein-Me Coatins Cell Testins

[0116] To evaluate myoblast cell attachment on the Zein-Me coated beads, C2C12 cells (from passage 6) in RPMI medium supplemented with 10% FBS, PenStrep (100 U / mL final concentration) and GlutaMAX™ (lOOx concentration, 5 mL added to 500 mL RPMI medium) were seeded onto a monolayer of the Zein-Me coated PGA beads that had been placed in wells of a 24- well ULA well plate. The number of cells added was 100,000 cells per well. The beads were observed both one day and four days after seeding with an 1X73 Inverted Microscope (Olympus)Attorney Docket No. : SP24-283 in brightfield mode. As shown in FIG. 10A (4x magnification) and FIG. IOC (lOx magnification), the cells attach well and spread well on Zein-Me coated PGA beads.

[0117] Four days after seeding, the Zein-Me coated PGA beads were stained with calcein AM staining and then observed with a 1X73 Inverted Microscope (Olympus) in fluorescent mode, using fluorescent light. Images of the calcein AM stained cells at 4x magnification under fluorescent light with the uncoated beads and the Zein-Me coated beads at 0.025 wt.%, 0.05 wt.%, 0.1 wt.%, and 0.2 wt.% Zein-Me coated beads are shown in FIGS. 4A-4E. No C2C12 cell adhesion is present on uncoated PGA beads (FIG. 4A). All of the Zein-Me coated PGA beads promoted cell attachment and growth as C2C12 cells are present all around the 0.025 wt.% Zein-Me coated beads (FIG. 4E), the 0.05 wt.% Zein-Me coated beads (FIG. 4D), the 0.1 wt.% Zein-Me coated beads (FIG. 4C), and the 0.2 wt.% Zein-Me coated beads (FIG. 4B).

[0118] Likewise, images of the calcein AM stained cells at 4x magnification under white light with the uncoated beads and the Zein-Me coated beads at 0.025 wt.%, 0.05 wt.%, 0.1 wt.%, and 0.2 wt.% Zein-Me coated beads are shown in FIGS. 5A-5E. Again, no C2C12 cell adhesion is present on uncoated PGA beads (FIG. 5A). All of the Zein-Me coated PGA beads promoted cell attachment and growth as C2C12 cells are present all around the 0.025 wt.% Zein-Me coated beads (FIG. 5E), the 0.05 wt.% Zein-Me coated beads (FIG. 5D), the 0.1 wt.% Zein-Me coated beads (FIG. 5C), and the 0.2 wt.% Zein-Me coated beads (FIG. 5B).Zein-Me Coatins Sanitization

[0119] PGA beads coated with 0.2 wt.% Zein-Me were sanitized for 15 minutes in 70% ethanol and then rinsed with ultrapure water three times and then rinsed once with cell culture medium. The sanitized coated beads were then seeded with C2C12 cells as described above and then left for 4 days for cell attachment and growth to occur. The cells were then stained with calcein AM staining and observed in a 1X73 Inverted Microscope (Olympus) in brightfield mode at lOx magnification. It was expected that the ethanol would degrade the Zein-Me and would remove the coating from the beads due to the solubility of the Zein-Me in ethanol, which would result in the beads being unable to support cell attachment and growth. However, unexpectedly, as shown in FIG. 6, the sterilized coated beads were resistant to ethanol sanitization and still promoted C2C12 cell attachment and growth.Attorney Docket No. : SP24-283 Harvest of Cells from Zein-Me Coated Microcarriers

[0120] C2C12 cells that had been grown on Zein-Me coated beads as described above were tested for harvesting capabilities from the coated, cultured beads. To harvest the cells, the coated beads with the attached and grown C2C12 cells on them (left-most photo in FIG. 8, at lOx magnification) were incubated with 3.5 mg / mL collagenase IV and 5 mM EDTA for 2 min. at room temperature, followed by stirring the mixture at 37 °C for 5 min. The mixture was then imaged on an 1X73 Inverted Microscope (Olympus) in brightfield mode at 20x magnification, which showed the harvest was successful (right-most photo in FIG. 8). The image of the mixture showed a suspension of single cells post-harvest (right-most photo of FIG. 8).Example 3 - Amidated-ZeinAmidated-Zein Synthesis

[0121] A modified zein with an amidated modification (“Zein-Am”) was created as a substrate by the following procedure. A 10 wt.% raw zein solution in 70:30 v / v ethanol / water was prepared by dissolving 500 mg of raw zein in 5 mb of 70:30 v / v ethanol / water. Separately, 760 mg EDC and 114 mg N-hydroxysuccinimide (NHS) were dissolved in 20 mL of 70:30 v / v ethanol / water to create an EDC / NHS solution. Eight mL of the EDC / NHS solution was added to 4 mL of the 10 wt.% raw zein solution and then stirred for 30 minutes at room temperature to convert the carboxylic acid groups on the zein into NHS esters. Next, 0.8 mL of ammonium chloride solution (prepared by dissolving 4 mg NH4CI in 1 mL ultra-pure water) was added to the activated-Zein solution. The solution was vigorously shaken and left to react overnight under stirring at room temperature. To purify the Zein-Am, the crude reaction mixture was then dialyzed for 24 hrs. against 1 liter of 70:30 v / v ethanol / water using a regenerated cellulose membrane tubing, with 6-8 kDa cut-off (CelluSep®). The solution was exchanged 4 times over the 24 hours. The dialyzed solution was then transferred in a glass flask and freeze-dried.

[0122] A zeta potential analysis was performed on the Zein-Am and unmodified zein, using a zeta potential analyzer (Zetasizer Nano ZS, Malvern Panalytical, Ltd.). The zeta potential of the Zein-Am was evaluated at a concentration of 20 mg / ml ethanol / MES 70:30 w / v at different pH values ranging from pH 4 to pH 9. The results, shown in FIG. 9, indicate that Zein-Am isAttorney Docket No. : SP24-283 positively charged throughout the entire range of pH 4-9, whereas the zeta of the raw zein drops when the pH is increased over pH 4 and then reaches zero charge near pH 7.Zein-Am Coatins on Microcarriers

[0123] Calcium cross-linked PGA beads (a type of microcarrier) were prepared as described in Example 1. The microcarriers had a mean diameter of 250 pm. To create a 0.025 wt. % Zein-Am hydroalcoholic coating, 100 mg of precipitated and washed Zein-Am was dissolved in 400 mL of ethanol / MES in a 70:30 ratio by volume while being stirred. Then, 12 mL of the Zein-Me solution was added to 2 mL of PGA beads (volume of the bead bed) in a 15 mL plastic centrifuge tube. The tube was agitated for 2 hours at room temperature on a lab roller. Then the beads were washed three times with 10 mL ultra-pure water. The resulting beads were colorless and exhibited high transparency.

[0124] A Bicinchoninic Acid assay (“BCA”) (Micro BC Assay Kit, Interchim, Cat. No. UP75760A) on the Zein-Am coated beads was performed. The BCA assay determined that about 50 pg zein-Am / mg dry PGA was adsorbed.Zein-Am Coatins Cell Testins

[0125] As a first pass to evaluate cell attachment and growth on the Zein-Am coated beads, Vero cells (cells derived from African green monkey kidneys) in IMDM medium supplemented with 10% PBS, PenStrep (100 U / mL final concentration) and GlutaMAX™ (lOOx concentration, 5 mL added to 500 mL medium) were seeded onto a monolayer of the Zein-Am coated PGA beads that had been placed in wells of a 24-well ULA well plate. The number of cells added was 100,000 cells per well. The beads were observed one day after seeding with an optical microscope. As shown in FIG. 7 (4x magnification), the Vero cells attach and spread well on Zein-Am coated PGA beads.

[0126] Next, the Zein-Am coated beads were tested with MRC5 cells (fibroblast cell line isolated from human lung) to determine applicability to dynamic cell cultures. The MRC5 cells in IMDM medium supplemented with 10% FBS, PenStrep (100 U / mL final concentration), and GlutaMAX™ (5 mL of lOOx concentration in 500 mL of medium) were seeded onto a monolayer of the Zein-Am coated PGA beads that had been placed in a 125 mL disposable spinner flaskAttorney Docket No. : SP24-283 (Corning®). The MRC5 cells on Zein- Am coated PGA beads also attached and spread well on the beads. The cells were cultured for seven days and achieved a 9-fold expansion in cell numbers.Example 4 - Ethanolamide-ZeinEthanolamide-Zein Synthesis

[0127] To create ethanolamide-zein (“Zein-EtAm”), the protocol to create purified Zein-Am that was described in Example 3 was repeated except ethanolamine hydrochloride was used instead of NH4CI. Zein-EtAm coated PGA beads were also created as described in Example 3.

[0128] A zeta potential analysis was performed on the Zein-EtAm and unmodified zein, using a zeta potential analyzer (Zetasizer Nano ZS, Malvern Panalytical, Ltd.). The zeta potential of the Zein-EtAm was evaluated at a concentration of 20 mg / ml Ethanol / MES 70:30 w / v at different pH values ranging from pH 4 to pH 9. The results, shown in FIG. 11, indicate that Zein-EtAm is positively charged throughout the entire range of pH 4-9, whereas the zeta of the raw zein drops when the pH is increased over pH 4 and then reaches zero charge near pH 7.Zein-EtAm Coatins on Microcarriers

[0129] Calcium cross-linked PGA beads (a type of microcarrier) were prepared as described in Example 1 and a coating solution with Zein-EtAm was applied on the beads as described in Example 3.Zein-EtAm Coatins Cell Testins

[0130] To evaluate cell attachment and growth on the Zein-EtAm coated beads, the cell testing protocol from Example 3 for C2C12 cells was followed except that Zein-EtAm coated beads were used. The C2C12 cells attached and spread well on the Zein-EtAm coated PGA beads.

[0131] While the present disclosure includes a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the present disclosure.

Claims

Attorney Docket No. : SP24-283CLAIMSWhat Is Claimed Is:

1. A coating composition, comprising:a modified prolamin having a net positive charge throughout a pH range of pH 6-8; wherein the modified prolamin is an amidated or esterified prolamin, andwherein the coating is free from animal components.

2. The coating composition of claim 1, wherein the coating is free of crosslinkers.

3. The coating composition of any one of claims 1-2, wherein the prolamin is selected from the group consisting of gliadin, hordein, secalin, zein, kafirin, and avenin, or combinations thereof.

4. The coating composition of claim 3, wherein the prolamin is zein.

5. The coating composition of any one of claims 1-4, wherein the coating composition is food-grade.

6. The coating composition of any one of claims 1-5, wherein the amidated prolamin has either an amide modification or an ethanolamide modification, and wherein the esterified prolamin is a methyl-ester modification.

7. A cell culture surface, comprising:a surface for culturing adherent cells; anda coating on the surface comprising a modified prolamin having a net positive charge at pH 7.5;wherein the modified prolamin is an amidated or esterified prolamin.

8. The cell culturing surface of claim 7, wherein the coating is free from crosslinkers and is free from animal components.Attorney Docket No. : SP24-283 9. The cell culturing surface of any one of claims 7-8, wherein the modified prolamin comprises zein.

10. The cell culturing surface of any one of claims 7-9, wherein the amidated prolamin has either an amide modification or an ethanolamide modification, and the esterified prolamin has a methyl-ester modification.

11. The cell culturing surface of any one of claims 7-10, wherein the modified prolamin is present in the coating in a percent by weight between 0.01 wt.% and 5 wt.% of the coating.

12. The cell culturing surface of any one of claims 7-11, wherein the modified prolamin has a surface density on the cell culturing surface of between 0.1 pg / cm2and 100 pg / cm2.

13. The cell culturing surface of any one of claims 7-12, wherein the surface is a microcarrier.

14. The cell culturing surface of claim 13, wherein the microcarrier is a bead for culturing adherent cells.

15. The cell culturing surface of any one of claims 13-14, wherein the microcarrier is a dissolvable microcarrier.

16. The cell culturing surface of any one of claims 13-15, wherein the modified prolamin is present on the microcarrier in an amount between 10 pg modified prolamin / mg dry microcarrier and 100 pg modified prolamin / mg dry microcarrier.

17. The cell culturing surface of any one of claims 7-16, wherein the prolamin is selected from the group consisting of gliadin, hordein, secalin, zein, kafirin, and avenin, or combinations thereof.

18. The cell culturing surface of any one of claims 7-17, wherein the cell culturing surface is a sanitized cell culturing surface.

19. A method of culturing cells, comprising the steps of:providing a cell culturing surface, wherein the cell culturing surface comprises:Attorney Docket No. : SP24-283 a surface for culturing adherent cells, anda coating on the surface comprising a modified prolamin having a net positive charge at pH 7.5, wherein the modified prolamin is an amidated or esterified prolamin; providing adherent cells to the cell culturing surface;culturing the adherent cells on the cell culturing surface.

20. The method of claim 19, wherein the modified prolamin comprises a methyl-ester modification, an ethanolamide modification, or an amidated modification.

21. The method of any one of claims 19-20, wherein the modified prolamin comprises zein.

22. The method of any one of claims 19-21, wherein the cell culture surface is sterilized.

23. The method of any one of claims 19-22, wherein the surface for culturing adherent cell comprises a bead.

24. The method of claim 23, wherein the bead is dissolvable.

25. The method of any one of claims 23-24, wherein the cell culturing surface comprises modified prolamin at an amount of between 10 pg modified prolamin / mg dry beads and 100 pg modified prolamin / mg dry beads.

26. The method of any one of claims 19-25, wherein the cell culturing surface comprises a surface density of modified prolamin of between 0.1 pg / cm2and 100 pg / cm2.

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