WO2025244838A1 - Improved collagen coatings - Google Patents

Abstract

Functional collagen coating compositions that bind to a surface for culturing adherent cells are provided, where the adherent cells can attach to the surface through the collagen in the coating, even after sterilization. The collagen coatings compositions comprise collagen and a buffer and comprise a pH between a 3.5 pH and 5.5 pH. Methods to make surfaces comprising a uniform collagen coating from the collagen coating compositions are also provided. The method includes providing a surface for the collagen coating, adding to the surface a collagen coating composition, and removing the excess collagen coating from the surface. The method may further include a sterilization step after the collagen coated surface has been formed.

Description

IMPROVED COLLAGEN COATINGS

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. 63/650,169 filed on May 21, 2024, the content of which is relied upon and incorporated herein by reference in its entirety their entireties.

BACKGROUND

Field

[0002] The present specification generally relates to compositions for collagen-based coatings and methods of making and using same.

Technical Background

[0003] Collagen is biocompatible with human tissue, regardless of its source. This property makes collagen a gold standard for incorporation into tissue engineering. Collagen can form different types of structures depending on its environment, including individual collagen proteins, triple-helices, fibrils, networks, binding anchors, and composites with inorganic molecules, which further enhances a wide integration of collagen as the primary choice of biomaterials in tissue engineering and cell therapy. One application of collagen in tissue engineering and cell therapy is in a coating, which facilitates mammalian cell attachment during adherent cell culture. However, existing commercial practices for coatings with collagen are sub-optimal due to the amounts of collagen coating needed to achieve the appropriate density of collagen for the tissue engineering and cell therapy systems, particularly for large applications. Further, the existing practices for coatings with collagen render them unable to be sterilized due to a loss of collagen function upon sterilization. Therefore, in many applications for collagen coatings, existing practices require coatings with collagen to be performed aseptically, which is problematic for applications using these collagen coatings. Not only does the requirement of aseptic application significantly increase the risk of contamination by introducing a significant number of steps for which contamination may occur, it also causes problems to be able to sufficiently dry the coatings. Accordingly, a continuing need exists for a collagen composition that can be used in large-scale applications and that can be sterilized. SUMMARY

[0004] The present disclosure provides improved collagen compositions and methods of making and using such compositions fortissue engineering and cell therapy systems.

[0005] In one aspect of the present disclosure, a cell culture device having at least a portion of its surface with a collagen coating is provided. The cell culture device comprises a surface and a collagen coating on at least a portion of the surface. The collagen coating comprises a collagen and the surface density of collagen on the coated surface portion is between 0.4 pg/cm² and 2.0 pg/cm². The collagen coated surface is configured to attach adherent cells. In some embodiments of this aspect, the collagen in the collagen coating is type I collagen. In some embodiments, the type I collagen may be from bovine skin or rat tail. For any of the embodiments of this aspect, the collagen coating may further comprise a buffer in the coating and the buffer may have a pKa of between pKa 3.8 and 5.2. The buffer in the coating may instead have a pKa of between pKa 4.0 and 5.0. For any of the embodiments of this aspect having a buffer, the buffer in the coating may be citric acid, acetic acid, or a combination thereof. For any of the embodiments with this aspect having a buffer, the buffer in the coating may comprise an acid that has at least one carboxylic acid group or its carboxylate ion. In other embodiments, the buffer in the coating may comprise an acid that comprises one, two, or three carboxylic acid groups, their carboxylate ions, or a combination of carboxylic acid groups and carboxylate ions. For any of the embodiments with this aspect, the pH of the collagen coating may be between pH 3.7 and pH 5.5. In other embodiments, the pH of the collagen coating may be between pH 4.4 and pH 5.0. For any of the embodiments with this aspect, the collagen coating may be substantially uniform on a millimeter scale. In some embodiments, the collagen on the collagen coated surface may be substantially uniform on a micrometer scale. In one specific embodiment, the collagen on the collagen coated surface is substantially uniform on a millimeter scale and a micrometer scale.

[0006] In one specific embodiment of this aspect, the collagen is bovine skin type I collagen, and the collagen surface density is in a range of 0.7 pg/cm² to 1.6 pg/cm². In another specific embodiment of this aspect, the collagen is rat tail tendon type I collagen, and the collagen surface density is in a range of 0.6 pg/cm² to 1.1 pg/cm².

[0007] For any of the above embodiments of this aspect, the collagen coated surface may be a high frequency irradiated collagen coated surface. In some embodiments of this aspect, the high frequency irradiation may be at a dosage between 10 kGy and 50 kGy. In one specific embodiment, the high frequency irradiation is gamma irradiation at a dosage between 10 kGy and 50 kGy.

[0008] For any of the above embodiments of this aspect, the collagen coated surface may have at least 50% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test. In some embodiments, wherein the collagen coated surface may have at least 75% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

[0009] In a second aspect of the present disclosure, a method for forming a collagen coated cell culture device surface is provided. The method comprises the steps of providing a surface, adding a collagen coating to at least a portion of the surface, and removing coating excess from the collagen coated surface. The collagen coating comprises collagen, a buffer, and a pH between pH 3.5 and pH 5.5. After coating excess has been removed from the coated the surface, the coated surface has a coating efficiency of at least 2%. In some embodiments of this aspect, after coating excess has been removed from the collagen coated surface, the collagen coated surface has a coating efficiency of at least 5%. In some embodiments, after coating excess has been removed from the collagen coated surface, the collagen coated surface has a coating efficiency of at least 15%.

[0010] For any of the embodiments of this aspect, the pH of the collagen coating may be between pH 3.7 and pH 5.5. For any of the embodiments of this aspect, the collagen may be type I collagen. In one specific embodiment, the type I collagen may be from bovine skin, rat tail, or a combination thereof. For any of the embodiments of this aspect, the buffer may have a pKa of between pH 4.0 and pH 5.0. For any of the embodiments of this aspect, the buffer may comprise an acid that comprises one, two, or three carboxylic acid groups, their carboxylate ions, or a combination of carboxylic acid groups and carboxylate ions.

[0011] In some embodiments of this aspect, the collagen on the collagen coated surface is substantially uniform on a millimeter scale. In some embodiments of this aspect, the collagen on the collagen coated surface is substantially uniform on a micrometer scale.

[0012] In some embodiments of this aspect, the method may comprise a further step of washing the collagen coated surface after the step of removing coating excess from the collagen coated surface. In one further embodiment, the method may also comprise the step of drying the collagen coated surface after the step of washing the collagen coated surface. [0013] For any of the embodiments of this aspect, the method may further comprise a step of sterilizing the collagen coated surface with high energy irradiation at a dosage between 10 kGy and 50 kGy. In one specific embodiment for any of the embodiments of this aspect, the method may comprise a step of sterilizing the collagen coated surface with gamma irradiation at a dosage between 10 kGy and 50 kGy.

[0014] For any of the embodiments of this aspect, the coated surface may have at least 50% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test. For any of the embodiments of this aspect, the collagen coated surface may have at least 75% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

[0015] In a third aspect of the present disclosure, a cell culture device is provided. The cell culture device comprises a surface and a collagen coating on at least a portion of the surface. The collagen coating comprises collagen, a buffer, and the coating has a pH between pH 3.6 and 5.6. The collagen coated surface comprises a collagen surface density on the at least a portion of the surface that as measured by a bicinchoninic acid assay is between 1.2 and 20 times greater than a collagen surface density of an identically collagen coated surface comprising a collagen composition comprising a pH between pH 2.0 and 3.4. In some embodiments, the collagen in the collagen coating is type I collagen. In some embodiments, the type I collagen is from bovine skin, rat tail, or a combination thereof.

[0016] It is to be understood that both the foregoing general description and the following detailed description describe various embodiments and are intended to provide an overview or framework for understanding the nature and character of the claimed subject matter. The accompanying drawings are included to provide a further understanding of the various embodiments, and are incorporated into and constitute a part of this specification. The drawings illustrate the various embodiments described herein, and together with the description serve to explain the principles and operations of the claimed subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A is an optical image at lOx magnification of HT1080 cells grown on a tissue culture treated well plate coated with a collagen coating having 0.1% acetic acid (pH 3.4) and 50 pg/mL rat tail collagen. [0018] FIG. IB is an optical image at lOx magnification of HT1080 cells grown on a tissue culture treated well plate without any collagen coating.

[0019] FIG. 2 is a bar graph of collagen surface density measurements from collagen coated well plates with rat tail collagen coating compositions at different pH values and different buffer solutions.

[0020] FIG. 3 is a series of photographs of collagen coated well plates after colloidal gold staining, where the wells were coated with rat tail collagen coating compositions at different pH values and different buffer solutions.

[0021] FIG. 4 is a series of scanning electron micrograph images with a 200 pm scale bar of tissue culture treated wells coated with rat tail collagen coating compositions in citric acid buffer at different pH values.

[0022] FIG. 5 is a bar graph of collagen surface density measurements from collagen coated well plates with rat tail collagen coating compositions in acetic acid or acetate at different pH values.

[0023] FIG. 6 is a series of photographs of collagen coated well plates after colloidal gold staining, where the wells were coated with rat tail collagen coating compositions in acetic acid or its ion, acetate, at different pH values.

[0024] FIG. 7 is a bar graph of collagen surface density measurements from collagen coated well plates with rat tail collagen coating compositions in citric acid buffer at pH 4.7, where the coating was equilibrated on the well surface for different lengths of time.

[0025] FIG. 8 is a series of photographs of collagen coated well plates after colloidal gold staining, where the wells were coated with rat tail collagen coating compositions in citric acid buffer at pH 4.7, where the coating was equilibrated on the well surface for different lengths of time.

[0026] FIG. 9 is a bar graph of collagen surface density measurements from collagen coated well plates with rat tail collagen coating compositions in citric acid buffer at pH 4.7 or 0.1% acetic acid (pH 3.4), comparing the effect of different collagen concentrations in the coating solutions that were equilibrated on the well surface for 4 hours. Also included in the comparison was one collagen concentration that was equilibrated on the well surface for 1 hour instead of 4 hours.

[0027] FIG. 10 is a series of photographs of collagen coated well plates after colloidal gold staining, comparing the effect of different collagen concentrations in coating solutions that were equilibrated on the well surface for 4 hours. Also included in the comparison was one collagen concentration that was equilibrated on the well surface for 1 hour instead of 4 hours.

[0028] FIG. 11A is a bar graph of collagen surface density measurements from collagen coated well plates with rat tail collagen coating compositions at different pHs in either citric acid buffer or acetic acid buffer, comparing the effect of well surface treatment prior to coating (uncoated versus tissue culture treated well plates) on the collagen coating surface density.

[0029] FIG. 11B is a bar graph of collagen surface density measurements from collagen coated well plates with rat tail collagen coating compositions at different pHs in either citric acid buffer or acetic acid buffer, to see the effect of CellBIND® treated well plates on collagen coating surface density.

[0030] FIG. 12 is a series of optical images of collagen coated well plates after colloidal gold staining, depicting the effect no surface treatment (untreated) on well plates has on coatings with rat tail collagen in either citric acid buffer or acetic acid buffer at varying coating solution pHs.

[0031] FIG. 13 is a series of optical images of collagen coated well plates after colloidal gold staining, depicting the effect that tissue culture pre-treatment on the surface well plates has on coatings with rat tail collagen in either citric acid buffer or acetic acid buffer at varying coating solution pHs.

[0032] FIG. 14 is a series of optical images of collagen coated well plates after colloidal gold staining, depicting the effect that CellBIND® pre-treatment on the surface well plates has on coatings with rat tail collagen in either citric acid buffer or acetic acid buffer at varying coating solution pHs.

[0033] FIG. 15 is a series of optical images at lOx magnification of HT1080 cells grown on tissue culture treated plates with no collagen coating (negative control), on tissue culture treated plates with collagen coating having rat tail collagen at a concentration of 50 pg/mL in 0.1% acetic acid (pH 3.4) (positive control), on tissue culture treated plates with collagen coating having rat tail collagen at a concentration of 50 pg/mL in citric acid at pH 3.7, or on tissue culture treated plates with collagen coating having rat tail collagen at a concentration of 50 pg/mL in citric acid at pH 4.7.

[0034] FIG. 16 is a series of optical images at lOx magnification of HT1080 cells grown on tissue culture treated plates coated with 50 pg/mL rat tail collagen in 0.1% acetic acid (pH 3.4), 5 pg/mL rat tail collagen in citric acid buffer at pH 4.7, 10 pg/mL rat tail collagen type I in citric acid buffer at pH 4.7, 25 pg/mL rat tail collagen in citric acid buffer at pH 4.7, and 50 pg/mL rat tail collagen in citric acid buffer at pH 4.7. The coated plates were not irradiated before the HT1080 cells were grown in the wells.

[0035] FIG. 17 is a series of optical images at lOx magnification of HT1080 cells grown on tissue culture treated plates coated with 50 pg/mL rat tail collagen in 0.1% acetic acid (pH 3.4), 5 pg/mL rat tail collagen in citric acid buffer at pH 4.7, 10 pg/mL rat tail collagen in citric acid buffer at pH 4.7, 25 pg/mL rat tail collagen in citric acid buffer at pH 4.7, and 50 pg/mL rat tail collagen in citric acid buffer at pH 4.7. The coated plates were subjected to gamma irradiated and then equilibrated before the HT1080 cells were grown in the wells of the plates.

[0036] FIG. 18 is a bar graph of collagen surface density measurements of collagen from collagen coated well plates with bovine skin collagen coating compositions coated on tissue culture treated well plates at different pHs in either citric acid buffer or hydrochloric acid buffer.

[0037] FIG. 19 is a series of photographs of collagen coated well plates after colloidal gold staining, the coatings being at different pHs. The coating compositions shown are bovine skin collagen in either citric acid buffer (pH 3.7, pH 4.7, pH 5.5) or 0.0 IN HC1 (pH 2.0).

[0038] FIG. 20 is a series of scanning electron micrographs with a 200 pm scale bar of collagen coated well plates at different pHs. The collagen compositions shown are bovine skin collagen in citric acid buffer at pH 3.7, pH 4.7, or pH 5.5. [0039] FIG. 21 is a bar graph of collagen surface density measurements from collagen coated well plates with bovine skin collagen coating compositions at different collagen concentrations in the coatings, in either citric acid buffer or hydrochloric acid buffer, and at different lengths of time to equilibrate the coating on the well surface during the coating process.

[0040] FIG. 22 is a series of photographs of collagen coated well plates after colloidal gold staining, the coatings being at different concentrations of bovine skin collagen in either citric acid buffer or hydrochloric acid buffer, and at different lengths of time to equilibrate the coating on the well surface during the coating process.

[0041] FIG. 23 are optical images at lOx magnification of HT1080 cells grown on collagen coated wells that were not irradiated (top row) or that were irradiated before the cells were grown on them (bottom row). The top row of images is of HT1080 cells grown on collagen coated well plates without gamma irradiation having coating compositions with a collagen concentration of 50 mg/mL bovine skin collagen in either 0.01 N HC1 (pH 2.0), citric acid at pH 3.7, or citric acid at pH 4.7. The bottom row of images is of HT1080 cells grown on the collagen coated well plates that were gamma irradiated prior to the cell assay, with the same collagen compositions as the top row.

[0042] FIG. 24 are optical images at lOx magnification of HT1080 cells grown on either collagen coated wells or uncoated wells (labeled “TCT”), at different concentrations of bovine skin collagen in the coating composition in either citric acid buffer or hydrochloric acid. No gamma irradiation was performed on the coated well plates.

[0043] FIG. 25 are optical images at lOx magnification of HT1080 cells grown on either collagen coated wells or uncoated wells (labeled “TCT”), at different concentrations of bovine skin collagen in the coating composition in either citric acid buffer or hydrochloric acid. The well plates were gamma irradiated before the HT1080 cells were grown in the plates.

DETAILED DESCRIPTION

[0044] 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 the art. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0045] 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

[0046] 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 invention, particular methods and materials are now described.

[0047] 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 include 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.

[0048] 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.

[0049] As used herein, “has,” “have,” “having,” “include,” “including,” “comprise,” “comprising” or the like are used in their open ended sense, and generally mean “including, but not limited to.”

[0050] “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.

[0051] 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.

[0052] 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.

[0053] 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

[0054] Collagen is a fibrous structural protein and the main component of connective tissues. It is an important component of certain coatings used to facilitate attachment of cells during adherent cell cultures, among other uses for collagen. Collagen coatings are conventionally very acidic in nature (pH 3.4 or less), as more acidic pHs have better cell binding and cell response than more neutral pHs. As such, standard protocols for collagen compositions for coatings use very acidic pHs. In applications where a gel including collagen is used, a neutral pH is used because a pH of about 7.7 is needed to form gels with collagen. However, at more neutral pHs, the collagen aggregates and is unsuitable for coatings.

[0055] In commercial applications such as tissue culture plates and other tissue-related culturing equipment and surfaces, collagen coatings are an important component for successfully growing adherent cell cultures. However, collagen coated surfaces have been unable to be sterilized by irradiation as irradiation causes a loss of functionality of the collagen. When collagen is no longer functional, the cells will no longer attach to the surface and so will not proliferate.

[0056] As described further below in the present disclosure, it has been unexpectedly found that coatings with a pH around the isoelectric point of collagen provide more than ten times better efficiency with the coating process than conventional acidic and neutral pHs while maintaining a uniformly coated surface. It has also been unexpectedly found that the below- described collagen coated surfaces are able to be sterilized with irradiation and yet still maintain collagen functionality.

Collagen Coating Compositions

[0057] Surfaces that adherent cells can attach to are required for adherent cell culturing techniques. Surfaces made from polyolefins or glass require some sort of surface treatment for culturing adherent cells. One such surface treatment is a coating that improves the attachment of adherent cells to the surface for growing the cells, such as a coating that comprises collagen. Typically, adherent cell cultures are grown in roller bottles, well plates, flasks, on microcarriers, and on other types of devices that have at least one surface of the device that cells can adhere to. [0058] Several types of collagen exist, the most common being Types I, II, III, IV, and V. Type I collagen is the most prolific type of collagen. At physiological pHs, collagen forms large fibers that are found primarily in connective tissues in vertebrates.

[0059] Commercial indications for collagen use either acidic buffer solutions or neutral buffer solutions depending on the application. Standard protocols for coating collagen onto cell culture devices solubilize collagen in buffers at very acidic pHs (pH 3.4 or less), whereas standard protocols for forming collagen hydrogels use collagen in buffers at neutral pH (e.g., pH 7.0-8.0). However, as disclosed herein, it has been unexpectedly found that coating collagen onto cell culture devices using pHs of about pH 3.5 -5.5 instead of either very acidic or more neutral pHs results in the highest amount of collagen coating the cell culture device uniformly and can generate a functionalized collagen coating after sterilization with irradiation (e.g., gamma, e-beam, and x-rays).

[0060] In one aspect of the present disclosure, a collagen coating composition is provided. The collagen coating composition comprises collagen and a buffer. In some embodiments, the collagen is a type I collagen. In some embodiments, the type I collagen is from rat or bovine. However, it should be understood that any type I collagen can be used known to those skilled in the art, including but not limited to type I collagen from human placenta, human skin, rabbit skin, bovine tendon, bovine skin, and rat tail.

[0061] Buffers are aqueous solutions that are resistant to pH change and so their pH can be adjusted to specified levels. In some embodiments, the collagen coating composition comprises a buffer with a pKa between pKa 3.8 and pKa 5.2, between pKa 3.9 and pKa 5. 1, or between pKa 4.0 and pKa 5.0, or at any range or value between pKa 3.8 and pKa 5.2. In some embodiments, the buffer comprises citric acid, acetic acid, hydrochloric acid, oxalic acid, maleic acid, malonic acid, fumaric acid, ascorbic acid, succinic acid, adipic acid, glutaric acid, or tartaric acid, isoniazid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, hyaluronic acid, polygalacturonic acid, or a combination thereof. In one specific embodiment, the buffer comprises citric acid, acetic acid, hydrochloric acid, or a combination thereof. In another specific embodiment, the buffer comprises citric acid. In some embodiments the buffer comprises an acid having at least one carboxylic acid group or its carboxylate ion, at least two carboxylic acid groups or their carboxylate ions or a combination thereof, at least three carboxylic acid groups or their carboxylate ions or a combination thereof, or more than three carboxylic acid groups or their carboxylate ions or a combination thereof. In one specific embodiment, the buffer comprises an acid having one, two, or three carboxylic acid groups or their carboxylate ions or a combination of carboxylic acid groups and carboxylate ions.

[0062] In some embodiments, the collagen coating composition comprises a buffer whose pH can be adjusted to a pH less than pH 5.5. In some embodiments, the pH of the collagen coating composition may be between pH 2.0 and pH 7.0. In some embodiments the pH of the collagen coating composition is less than pH 5.5 and at least pH 2.0. In one embodiment, the pH of the collagen coating composition is less than pH 5.5 and at least pH

2.5, less than pH 5.5 and at least pH 3.0, less than pH 5.5 and at least pH 3.5, less than pH 5.5 and at least pH 4.0, less than pH 5.5, and at least pH 4.5, or less than pH 5.5 and at least pH 5.0. In another embodiment, the pH of the collagen coating composition is less than pH 5.0 and at least pH 2.0, less than pH 4.5 and at least pH 2.0, less than pH 4.0 and at least pH 2.0, less than pH 3.5 and at least pH 2.0, less than pH 3.0 and at least pH 2.0, or less than pH 2.5 and at least pH 2.0. In yet another embodiment, the pH of the collagen coating composition is less than pH 5.4 and at least pH 2.5, less than pH 5.3 and at least pH 3.0, less than pH 5.3 and at least pH 3.5, less than pH 5.3 and at least pH 3.6, less than pH 5.3, and at least pH 3.7, less than pH 5.3 and at least pH 3.8, less than pH 5.3 and at least pH 3.9, less than pH 5.3 and at least pH 3.9, less than pH 5.3 and at least pH 4.0, less than pH 5.3 and at least pH 4.1, less than pH 5.3 and at least pH 4.2, less than pH 5.3 and at least pH 4.3, less than pH 5.2 and at least pH 4.4, less than pH 5.1 and at least pH 4.5, less than pH 5.0 and at least pH 4.5, less than pH 4.9 and at least pH 4.6, or less than pH 4.8 and at least pH 4.6. In one embodiment, the collagen coating composition has a pH of less than pH 5.0 and at least pH 4.0. In one embodiment, the collagen coating composition has a pH of about pH 4.0, pH 4.1, pH 4.2, pH 4.3, pH 4.4, pH

4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, or about pH 5.0, or any value therebetween.

[0063] The concentration of the collagen in the collagen coating composition is between 1 pg/mL and 100 pg/mL. or any value therebetween. In one embodiment, the concentration in the collagen coating composition is between 1 pg/mL and 95 pg/mL, between 1 pg/mL and 90 pg/mL, between 1 pg/mL and 85 pg/mL, between 1 pg/mL and 80 pg/mL, between 1 pg/mL and 75 pg/mL, between 1 pg/mL and 70 pg/mL, between 1 pg/mL and 65 pg/mL, between 1 pg/mL and 60 pg/mL, between 1 pg/mL and 55 pg/mL, between 1 pg/mL and 50 pg/mL, between 1 pg/mL and 45 pg/mL, between 1 pg/mL and 40 pg/mL, between 1 pg/mL and 35 pg/mL, between 1 pg/mL and 30 pg/mL, between 1 pg/mL and 25 pg/mL, between 1 pg/mL and 20 pg/mL, between 1 pg/mL and 15 pg/mL. or between 1 pg/mL and 10 pg/mL. In another embodiment, the concentration in the collagen coating composition is between 5 pg/mL and 100 pg/mL, between 10 pg/mL and 100 pg/mL, between 15 pg/mL and 100 pg/mL, between 20 pg/mL and 100 pg/mL, between 25 pg/mL and 100 pg/mL, between 30 pg/mL and 100 pg/mL, between 35 pg/mL and 100 pg/mL, between 40 pg/mL and 100 pg/mL, between 45 pg/mL and 100 pg/mL, between 50 pg/mL and 100 pg/mL, between 55 pg/mL and 100 pg/mL, between 60 pg/mL and 100 pg/mL, between 65 pg/mL and 100 pg/mL, between 70 pg/mL and 100 pg/mL, between 75 pg/mL and 100 pg/mL, between 80 pg/mL and 100 pg/mL, between 85 pg/mL and 100 pg/mL, between 90 pg/mL and 100 pg/mL, or between 95 pg/mL and 100 pg/mL. In yet another embodiment, the concentration of collagen in the collagen coating composition is between 3 pg/mL and 75 pg/mL, between 3 pg/mL and 50 pg/mL, between 3 pg/mL and 30 pg/mL, between 3 pg/mL and 15 pg/mL, between 5 pg/mL and 50 pg/mL, between 40 pg/mL and 60 pg/mL, between 45 pg/mL and 55 pg/mL, or at any value or range between 3 pg/mL and 75 pg/mL. In one specific embodiment, the concentration of collagen in the collagen coating composition is about 5 pg/mL, about 10 pg/mL, about 25 pg/mL, or about 50 pg/mL.

Methods of Making Collagen Coated Cell Culturing Devices

[0064] According to other aspects of the present disclosure, a method is provided for forming a collagen coated cell culture device surface, which may include providing a surface for adhering adherent cells, adding to the surface a coating composition comprising collagen and a buffer, and removing coating excess from the coated surface.

[0065] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order, nor that with any apparatus specific orientations be required. Accordingly, where a method claim does not actually recite an order to be followed by its steps, or that any apparatus claim does not actually recite an order or orientation to individual components, or it is not otherwise specifically stated in the claims or description that the steps are to be limited to a specific order, or that a specific order or orientation to components of an apparatus is not recited, it is in no way intended that an order or orientation be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps, operational flow, order of components, or orientation of components; plain meaning derived from grammatical organization or punctuation, and; the number or type of embodiments described in the specification.

[0066] To generate a collagen coated cell culturing device, a surface is provided that the collagen coating will adhere to. Such surfaces include thermoplastic polymers such as polystyrene, polypropylene, polyethylene, polyethylene terephthalate, polycarbonate, polyvinyl chloride, nylon, polyether ether ketone. Other types of surfaces include glass or ceramics. However, these are merely exemplary, and any thermoplastic polymer or other compound known to those of ordinary skill in the art may be used. In some embodiments, prior to any application of a collagen coating to the surface, the surface may be treated physically or chemically to promote attachment of adherent cells. Exposing the surface to a plasma gas (such as with tissue culture treated surfaces) or to microwave plasma (such as for a CellBind® surface) are some examples, but any other surface modifications for tissue culture known to those of ordinary skill in the art may be used.

[0067] A collagen coating composition can be applied to a provided surface to create a collagen coated surface. In some embodiments, the loading concentration of collagen for the coated surface is between 0.1 pg of collagen per 1 cm² surface area to be coated (i.e., 0.1 pg/cm²) and between 20 pg of collagen per 1 cm² surface area to be coated (i.e., 20 pg/cm²), or in any range or at any value between 0. 1 pg/cm² and 20 pg/cm². In some embodiments, the loading concentration of collagen for the surface area to be coated is between 0.1 pg/cm² and 15 pg/cm², between 0.1 pg/cm² and 10 pg/cm², or between 0.1 pg/cm² and 5 pg/cm². In some embodiments, the loading concentration of collagen for the surface area to be coated is between 0.5 pg/cm² and 20 pg/cm², between 1 pg/cm² and 20 pg/cm², between 5 pg/cm² and 20 pg/cm², between 10 pg/cm² and 20 pg/cm², or between 15 pg/cm² and 20 pg/cm². In some embodiments, the loading concentration of collagen for the surface area to be coated is between 3 pg/cm² and 17 pg/cm², between 5 pg/cm² and 15 pg/cm², between 7 pg/cm² and 13 pg/cm², or between 9 pg/cm² and 11 pg/cm². In one specific embodiment, the loading concentration of collagen for the surface area to be coated is about 0.1 pg/cm², about 0.2 pg/cm², about 0.3 pg/cm², about 0.4 pg/cm², about 0.5 pg/cm², about 0.6 pg/cm², about 0.7 pg/cm², about 0.8 pg/cm², about 0.9 pg/cm², about 1 pg/cm², about 2 pg/cm², about 3 pg/cm², about 4 pg/cm², about 5 pg/cm², about 6 pg/cm², about 7 pg/cm², about 8 pg/cm², about 9 pg/cm², about 10 pg/cm², about 11 pg/cm², about 12 pg/cm², about 13 pg/cm², about 14 pg/cm², about 15 pg/cm², about 16 pg/cm², about 17 pg/cm², about 18 pg/cm². about 19 pg/cm², about 20 pg/cm², or any value therebetween.

[0068] In some embodiments, the collagen coating composition may be added in liquid form to a surface until the liquid covers the surface to be coated. In other embodiments, the surface may be submerged in a vessel containing the liquid collagen coating composition. However, it should be understood that any technique of applying a liquid to a surface may be used. In some embodiments, the liquid may contact the surface for between 5 minutes and 48 hours. In one embodiment, the collagen coating composition may contact the surface for between 10 minutes and 48 hours, between 15 minutes and 48 hours, between 30 minutes and 48 hours, between 45 minutes and 48 hours, between 1 hour and 48 hours, between 2 hours and 48 hours, between 4 hours and 48 hours, between 6 hours and 48 hours, between 8 hours and 48 hours, between 10 hours and 48 hours, between 15 hours and 48 hours, between 20 hours and 48 hours, or between 25 hours and 48 hours, between 5 minutes and 24 hours, between 10 minutes and 22 hours, between 15 minutes and 20 hours, between 30 minutes and 18 hours, between 1 hour and 16 hours, or between 2 hours and 14 hours, or between 3 hours and 12 hours, or any other range or value between 5 minutes and 48 hours. In one specific embodiment, the collagen coating composition may contact the surface for between 30 minutes and 18 hours. In another specific embodiment, the collagen coating composition may contact the surface for about 1 hour, about 2 hours, about 4 hours, or about 16 hours.

[0069] In some embodiments, the temperature at which the contacting of the collagen coating composition with the surface may occur at a temperature between 2 °C and 45 °C. In one embodiment, the temperature at which the contacting of the collagen coating composition with the surface may occur at a temperature between 2 °C and 37 °C, between 4 °C and 37 °C, between 8 °C and 18 °C, between 18 °C and 37 °C, between 25 °C and 45 °C, between 2 °C and 10 °C, or any range or value between 2 °C and 45 °C.

[0070] Any coating excess from the collagen coated surface may be removed by any technique that removes liquid from a surface, such as decanting. In some embodiments, removing coating excess may be performed by tilting the surface an angle sufficient to allow the liquid on the surface to flow off the surface. In other embodiments, removing coating excess may be performed by removing a surface immersed in the liquid from the liquid. In yet other embodiments, removing coating excess may be performed by aspirating the liquid from the surface. In some embodiments, removing coating excess may be performed by blotting the liquid from the surface. However, it should be understood that any technique of removing the coating excess from the surface may be used known to those of ordinary skill in the art.

[0071] In some embodiments, the collagen coated surface may be washed with deionized water or a buffer. In one specific embodiment the collagen coated surface is washed with deionized water. In some embodiments, wash residual or wash excess may be removed from the coated surface after washing the coated surface.

[0072] In some embodiments, the collagen coated surface may be dried. In some embodiments, drying the collagen coated surface may occur for a time between 5 minutes and 48 hours. In one embodiment, drying the collagen coated surface may occur for a time between 10 minutes and 36 hours, between 15 minutes and 24 hours, between 30 minutes and 24 hours, between 45 minutes and 24 hours, between 1 hour and 24 hours, between 5 hours and 24 hours, between 10 hours and 24 hours, between 5 minutes and 24 hours, between 5 minutes and 20 hours, between 5 minutes and 16 hours, between 5 minutes and 12 hours, or in any range or any value between 5 minutes and 48 hours. In some embodiments, drying of the collagen coated surface may occur at a temperature of between 2 °C and 45 °C. In one embodiment, drying of the collagen coated surface may occur at a temperature between 2 °C and 37 °C, between 4 °C and 37 °C, between 8 °C and 18 °C, between 18 °C and 37 °C, between 25 °C and 45 °C, between 2 °C and 10 °C, or any range or value between 2 °C and 45 °C. In one specific embodiment, a collagen coated surface is dried at a temperature between 10 °C and 37 °C for a time between 4 hours and 24 hours.

[0073] The coating efficiency of the collagen on the collagen coated surface can be determined taking the surface density of collagen on the collagen coated surface and dividing it by the total collagen amount per total surface area coated. The total collagen amount can be determined by multiplying the collagen concentration of the coating by the volume of coating added. The following formula may be used (with “ug” being equivalent to micrograms (pg)):

[0074] In some embodiments, the coating efficiency of the collagen on the coated surface is at least 2%. In some embodiments, the coating efficiency of collagen on the coated surface is at least 5%, at least 10%, at 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or more than 70%. In some embodiments, the coating efficiency of collagen on the coated surface is between 2% and 100%, between 5% and 65%, between 8% and 60%, between 10% and 60%, between 15% and 60%, between 20% and 60%, between 25% and 60%, between 30% and 60%, between 35% and 60%, or at any range or value between 2%-100%.

[0075] One functionality of collagen is to promote binding of adherent cells to the surface. Functionality of a collagen coated surface can be determined by an HT1080 cell attachment test. The HT1080 cell attachment test is now described. HT1080 cells are a commercially available fibrosarcoma cell line from human epithelial cells originally derived from a patient’s cancerous fibroblast cells. A total of 2 x 10⁴ cells/cm² of surface for cell attachment (or coated surface if a coated surface is employed) are used in a volume of 0.2 mb of serum free Dulbecco's Modified Eagle Medium (“DMEM”) per cm² of surface to be coated. The liquid containing the cells is dispensed onto the surface (or coated surface if employed). The cells are equilibrated for 1 hour at 37 °C to allow time for the cells to attach to the surface (or coated surface if employed). The cells are then observed with a phase contrast optical microscope (at lOx resolution) to determine the morphology of the cells on the surface and to determine what percent of the cells exhibit attachment characteristics. When a collagen coated surface has functionality, adherent cells bind to the surface and adopt an elongated shape (or more elongated shape) along the surface. In particular, upon visual observation with phase contrast optical imaging, cells that have attached appear to have spread in size around the periphery of the cells and lose optical brightness compared to unattached cells (for example, the cells in FIG. 1A have over 95% of the cells showing spread). When a collagen coated surface does not have functionality, adherent cells are unable to attach properly to adopt an elongated shape or spread along the surface (for example, the cells in FIG. IB do not properly spread) and so they are unable to proliferate, which leads to cell death. Cells that do not attach appear rounder and smaller in size than attached cells, and upon visual observation with phase contrast optical imaging, these cells have notable optical brightness on the periphery of the cell. One such visual observation device that can be used to determine cell morphology and the percent of adherent cells attached to the surface is a Vi-CELL cell counter and analyzer (Beckman Coulter).

[0076] In some embodiments, a collagen coated surface of the present disclosure has at least 50% functionality, at least 55% functionality, at least 60% functionality, at least 65% functionality, at least 70% functionality, at least 75% functionality, at least 80% functionality, at least 85% functionality, at least 90% functionality, at least 95% functionality, or more than 95% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test. In some embodiments, a collagen coated surface of the present disclosure has between 50% and 100% functionality, between 60% and 100% functionality, between 70% and 100% functionality, between 80% and 100% functionality, between 90% and 100% functionality, or any range or value between 50% and 100% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

[0077] Collagen coated surfaces that have been sterilized with high frequency irradiation are currently unavailable because high frequency irradiation (gamma, e-beam, or x- rays) destroys the functionality of the collagen with currently available collagen coating compositions. The loss of functionality prevents adherent cells from correctly binding and being able to proliferate. However, collagen coating compositions of the present disclosure are functional even after sterilization with high frequency irradiation. In some embodiments, the collagen coated surface may be sterilized using high frequency irradiation once the collagen coated surface has been dried. As used herein, “high frequency” irradiation refers to radiation having frequencies of at least 1 x 10¹⁷ Hz. In some embodiments, the collagen coated surface is sterilized with gamma irradiation, e-beam irradiation, or x-ray irradiation. In one specific embodiment, the collagen coated surface is sterilized with gamma irradiation or e-beam irradiation.

[0078] Sterilization occurs after imparting a radiation dose of ionizing radiation during the high frequency irradiation. The radiation dose used for the irradiation is the dosage. In some embodiments, the dosage for irradiation (gamma, x-ray, or e-beam) is between 10 kGy and and 50 kGy, or any range or value between these two endpoints. In one embodiment, the dosage for irradiation is between 10 kGy and 45 kGy, between 10 kGy and 40 kGy, between 10 kGy and 35 kGy, between 10 kGy and 30 kGy, between 10 kGy and 25 kGy, between 10 kGy and 25 kGy, between 10 kGy and 20 kGy. In one specific embodiment, the dosage for irradiation is between 10 kGy and 30 kGy.

[0079] In some embodiments, a high frequency irradiated collagen coated surface comprising a collagen coating composition described herein has at least 50% functionality, at least 55% functionality, at least 60% functionality, at least 65% functionality, at least 70% functionality, at least 75% functionality, at least 80% functionality, at least 85% functionality, at least 90% functionality, at least 95% functionality, or more than 95% functionality with observed HT1080 cells as measured by a HT1080 cell attachment test. In another embodiment, a high frequency irradiated collagen coated surface comprising a collagen coating composition described herein has between 50% and 100% functionality, between 60% and 100% functionality, between 70% and 100% functionality, between 80% and 100% functionality, between 80% and 100% functionality, between 90% and 100% functionality, or any range or value between 50% and 100% functionality with observed HT1080 cells as measured by a HT1080 cell attachment test.

Collagen Coated Cell Culturing Devices

[0080] In one aspect of the present disclosure, the collagen coating composition may be added to the surface of cell culturing devices to assist in attachment of adherent cells. Cell culturing devices include multi-well plates (such as single well, 3-well, 6-well, 12-well, 24 well, 48 well, 96 well, and 384-well plates), culture dishes, culture flasks, cover slips, slides, permeable supports, and microcarriers, among other devices known to those of ordinary skill in art.

[0081] The adherent cells may be any cell type that is an adherent cell. Non-limiting examples of adherent cell types include brain endothelial cells, heart cardiomyocytes, hepatic stellate cells, cardiac muscle cells, neuronal cells, bronchial epithelial cells, ovarian surface epithelial cells, glioma cells, urothelial carcinoma cells, insulinoma cells, papillary thyroid carcinoma cells, oligodendroglioma cells, chondrocyte cells, R-Spondin-1 -Expressing 293T cells, mast cells, mammary tumor cells, fibrosarcoma cells, microglial cells, and oral squamous carcinoma cells. These cell types are exemplary, any adherent cell type known to those of ordinary skill in the art may be used.

[0082] Once a collagen coating composition has been applied to the surface of a cell culturing device, the coated surface of the device has a surface density and uniformity associated with it. The surface density of the collagen coated surface refers to the amount of collagen per coated surface area. The surface density of the coated surface is determined by a bicinchoninic acid (“BCA”) assay, such as the Sigma- Aldrich® QuantiPro™ BCA or BCA1 assay kits. BCA assays create a purple-blue coloring with proteins (like collagen) that allow a determination of protein amounts with a UV/VIS spectrophotometer. In some embodiments, the surface density of collagen in the coated cell culture device (sterilized or unsterilized) is between 0.4 pg/cm² and 2.0 pg/cm², or at any value or in any range therebetween. In some embodiments, the surface density of collagen in the coated cell culture device (sterilized or unsterilized) is between 0.4 pg/cm² and 1.6 pg/cm², between 0.7 pg/cm² and 1.6 pg/cm², between 0.9 pg/cm² and 1.6 pg/cm², between 0.6 pg/cm² and 1.1 pg/cm², between 0.6 pg/cm² and 1.4 pg/cm², between 0.7 pg/cm² and 1.8 pg/cm², or between 0.7 pg/cm² and 1.8 pg/cm². In one embodiment, the surface density of collagen in the coated cell culture device is about 0.4 pg/cm², about 0.5 pg/cm², about 0.6 pg/cm², about 0.7 pg/cm², about 0.8 pg/cm², about

0.9 pg/cm², about 1.0 pg/cm², about 1.1 pg/cm², about 1.1 pg/cm², about 1.2 pg/cm², about

1.3 pg/cm², about 1.4 pg/cm², about 1.5 pg/cm², about 1.6 pg/cm², about 1.7 pg/cm², about

1.8 pg/cm², about 1.9 pg/cm², or about 2.0 pg/cm², or any value therebetween.

[0083] The coated surface (sterilized or unsterilized) may be substantially uniform across a coated surface area of the device. As used herein, “uniformity” in terms of the collagen coating on a surface refers to the distribution of collagen across a coated surface area. Uniformity of a collagen coated surface can be determined on a millimeter scale by colloidal gold staining followed by visualization with the naked eye on an illuminated white background. Uniformity of a collagen coated surface can be determined on a micrometer scale by imaging colloidal gold stained surfaces in a scanning electron microscope image with a 200 pm scale bar. In some embodiments, the collagen coated surface (sterilized or unsterilized) is uniform across at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99%. In some embodiments, the collagen coated surface (sterilized or unsterilized) is substantially uniform, meaning the coating is uniform across at least about 75% on a cell culture surface. The uniformity across the collagen coated surface may be on the millimeter scale, the micrometer scale, or both the millimeter scale and micrometer scale.

[0084] In some embodiments, the surface density of the collagen on a collagen coated surface with a composition comprising a pH of about pH 4.7 is greater than the same coating composition with a pH more acidic than pH 4.7 coated the same way. In one embodiment, the density of collagen on a collagen coated surface with a collagen composition comprising a pH of about pH 4.7 is greater than the same collagen composition comprising pH of about pH 3.7 coated the same way. In another specific embodiment, the surface density of collagen on a collagen coated surface with a collagen composition comprising a pH of about pH 4.7 is between 1 .3 times and 5 times greater than the same collagen composition comprising a pH of about pH of about pH 3.7 coated the same way. In one specific embodiment, the surface density of collagen on a collagen coated surface with a collagen composition comprising a pH of about pH 4.7 is at least 1.3 times greater than the same collagen composition comprising a pH of about pH 3.7 coated the same way. In another specific embodiment, the surface density of collagen on a collagen coated surface with a rat tail or bovine skin collagen composition comprising a pH of about pH 4.7 is between 1.3 times and 5 times greater than the same collagen composition comprising a pH of about pH 3.7 coated the same way.

[0085] In some embodiments, a collagen composition with a pH between pH 3.6 and 5.5 has a collagen surface density between 1.2 and 20 times greater than the collagen surface density of a collagen composition with a pH between pH 2.0 and 3.4, where the collagen surface density is measured by a BCA assay. In some embodiments, a collagen composition with a pH between pH 3.6 and 5.5 has a collagen surface density between 1.2 and 20 times greater than the collagen surface density of a collagen composition with a pH of about 2.0, where the collagen surface density is measured by a BCA assay. In one specific embodiment, the collagen in the collagen composition with a pH of about 2.0 is bovine skin collagen type I. In some embodiments, a collagen composition with a pH between pH 3.6 and 5.5 has a collagen surface density between 1.2 and 20 times greater than the collagen surface density of a collagen composition with a pH of about 3.4, where the collagen surface density is measured by a BCA assay. In one specific embodiment, the collagen in the collagen composition with a pH of about 3.4 is rat tail collagen type I. For any of these embodiments, the pH between pH 3.6 and 5.5 may be any range or value between pH 2.6 and pH 5.5. Likewise, for any of these embodiments, the pH between pH 2.0 and 3.4 may be any range or value between pH 2.6 and 5.5.

EXAMPLES

[0086] The embodiments described herein will be further clarified by the following examples.

Example 1

Positive and Negative Controls

[0087] Positive and negative controls for HT1080 cell morphology and functionality were generated. A tissue-culture treated (“TCT”) 6-well multi -well plate (Costar®) was used for the negative control. A TCT 6-well multi-well plate (Costar®) was coated with a collagen composition made from rat tail collagen type 1 in 0.1% acetic acid buffer (pH 3.4) having a collagen concentration of 50 pg/mL was used a positive control. To each well, 2 mL of the coating composition was added with a loading concentration of collagen of 10 pg/cnr. The coating was equilibrated in the wells for 4 hours at room temperature. Excess coating liquid was then removed by aspiration with a pipette tip and the wells were rinsed with deionized water. The wells were then dried overnight at room temperature. Each well in both the positive and negative controls had bottom surface area of 9.6 cm². For each plate (both positive and negative controls), 2 mL of Dulbecco’s Modified Eagle Medium (“DMEM”) and 2 x 10⁵ HT1080 cells (ATCC®) per well were added to each well. The cells were equilibrated for 1 hour at 37 °C and then visually imaged with phase contrast optical imaging in a Vi-CELL cell counter and analyzer (Beckman Coulter®) at lOx magnification. The TCT wells (negative control) did not show functionality, as shown in FIG. 1A. The wells in the positive control showed over 95% functionality, as shown in FIG. IB.

Example 2

Coating Compositions

[0088] Collagen coating compositions with rat tail tendon collagen type I (“RTC”) were formed by mixing the collagen in different aqueous buffer solutions. Aqueous buffers tested included acetic acid (pH 3.4, pH 3.8), citric acid (pH 3.7, pH 4.7, pH 5.5, pH 6.4), and Dulbecco’s Phosphate Buffered Saline (“DPBS”) (pH 7.4). Each coating composition had a collagen concentration of 50 pg/mL. The buffers used for the collagen compositions and the pHs that were tested are listed in Table 1 below.

Coatins Well Plates

[0089] Each of the coating compositions was coated onto three wells of TCT 6-well multi-well plates (Costar®) for culturing adherent cells. To coat the wells, 2 mL of collagen coating composition at a collagen concentration of 50 pg/mL was added to each well to create a loading concentration of 10 pg/cm² in each well. Each well had a bottom surface area of 9.6 cm². To each well, 2 mL of the coating composition was added with a loading concentration of collagen of 10 pg/cm². The coating was equilibrated in the wells for 4 hours at room temperature. Excess coating liquid was then removed by aspiration with a pipette tip and the wells were rinsed with deionized water. The wells were then dried overnight at room temperature.

Analysis of Coated Well Plates

[0090] The coated well plates were then tested for the surface density of the collagen on the wells and for the uniformity of the collagen on the wells. Surface density of the collagen was measured using a QuantiPro™ bicinchoninic acid (BCA) assay (Sigma-Aldrich, Cat. No. QPBCA), which detects low concentrations of proteins (e.g., collagen) with a UV/VIS spectrophotometer measuring absorbance at 562 nm wavelength. A titration standard using RTC in known amounts was conducted for the BCA assay. To conduct the BCA assay with the coating compositions, 1 mb of BCA reagent and ImL of deionized water were added to the coated wells and the wells were equilibrated for 2 hours at 37 °C. The equilibrated reagent was then aspirated out of the wells with a pipette and measured with a UV/VIS spectrometer to determine the surface density of the collagen in the coated wells. Table 1 below summarizes the results of the QuantiPro™ BCA assays for each of the coating compositions. Table 1 also summarizes the coating efficiency for each composition, which was calculated by taking the average of the BCA assays for each composition and dividing it by the total concentration of collagen loaded per cm² for each well. FIG. 2 graphically shows the collagen surface density results of the QuanitPro™ BCA assays for the rat tail tendon collagen type I coating compositions.

[0091] Table 1

[0092] The coated plates were also tested for uniformity on the millimeter scale using colloidal gold staining. TCT 6-well multi -well plates (Costar®) were coated with the coating compositions described in Table 1 above. After drying, 2 mL colloidal gold stain (BioRad®) was added to each well and the wells were equilibrated overnight at room temperature °C. Next, the colloidal staining excess was removed and the wells were rinsed with deionized water. The plates were then left to dry for 2-4 hours. The wells were then visually observed on an illuminated white background.

[0093] Optical images of the colloidal staining tests for the RTC coating compositions are shown in FIG. 3. In FIG. 3, areas of the images for colloidal staining that are darker indicate higher densities of colloidal staining. FIG. 3 shows low colloidal staining density of rat tail tendon collagen at both low (very acidic) and high (more neutral) pHs and significantly higher colloidal gold staining density at pHs closer to pH 4.7. The collagen coatings in citric acid buffer were substantially uniform at pHs tested that were less than pH 5.5, and less uniform staining at pHs tested at pH 5.5 and above. The densest colloidal staining occurred at the coating composition comprising a pH 4.7.

[0094] The colloidal gold stained coated plates were also visually observed with a scanning electron microscope with a scale bar of 200 pm to determine the uniformity of the collagen coating on a micron scale. FIG. 4 shows micrographic images of the RTC coating compositions in citric acid buffers at different tested pHs. The images confirm that the citric collagen coatings are uniform on a micron scale at pH 3.7 and pH 4.7 but show mottling at pH

5.5 and 6.4, indicating the coating is less uniform on a micron scale at these higher pHs.

Example 3

Coatins Compositions and Coated Well Plates

[0095] A collagen coating composition with RTC at different pHs of acetate (pH 3.7, pH 4.5, pH 5.5) were formed by mixing the collagen in an aqueous buffer solution. Each coating composition had a collagen concentration of 50 pg/mL. These were compared to RTC coating compositions with 0.1% acetic acid (pH 3.4) and 0.01% acetic acid (pH 3.8) that were described in Example 2. The buffers used for the collagen compositions and the pHs that were tested and compared are listed in Table 2 below. Each of the collagen coating compositions at different pHs of acetate was coated onto three wells of TCT 6-well multi -well plates (Costar®) using the same process as described in Example 2.

Analysis of Coated Well Plates

[0096] The coated well plates were then tested for surface density of the collagen on the wells, coating efficiency, and uniformity of the collagen on a millimeter scale using the same processes described in Example 2. Table 2 below summarizes the results of the QuantiPro™ BCA assays for each of the coating compositions along with the coating efficiencies. FIG. 5 graphically shows the collagen surface density results of the QuanitPro™ BCA assays for the rat tail tendon collagen type I coating compositions.

[0097] Table 2

[0098] The uniformity on the millimeter scale of the coatings was determined using the same process for colloidal gold staining as described in Example 2. Optical images of the colloidal staining tests for the RTC acetate coating compositions are shown in FIG. 6, along with the comparison results of the RTC acetic acid coating compositions from Example 2. In FIG. 6, areas of the images for colloidal staining that are darker indicate higher densities of colloidal staining. FIG. 6 shows low colloidal staining density of RTC with acetic acid and higher staining with acetate. It also shows significantly higher colloidal gold staining density at pH 4.7 and pH 5.5. The collagen coatings in acetic acid and acetate buffers were substantially uniform on a millimeter scale at the pHs tested.

Example 4

Coating Compositions and Coated Well Plates

[0099] A collagen coating composition with RTC and citric acid at pH 4.7 was formed. The RTC collagen concentration in the composition was 5 pg/mL. The coating composition was coated onto sets of three wells in TCT 6-well multi-well plates (Costar®) using 2 mL of the coating composition per well. The loading concentration of collagen was 1 pg/cm². The coating was equilibrated in the wells at room temperature for either 1 hour, 2 hours, 4 hours, or overnight, as described below in Table 3. Excess coating liquid was then removed by aspiration with a pipette tip and the wells were rinsed with deionized water. The wells were then dried overnight at room temperature.

Analysis of Coated Well Plates [00100] The coated well plates were then tested for the surface density of the collagen on the wells, coating efficiency, and uniformity of the collagen on a millimeter scale using the same processes described in Example 2. Table 3 below summarizes the results of the QuantiPro™ BCA assays for each of the coating compositions along with the coating efficiencies. FIG. 7 graphically shows the collagen surface density results of the QuanitPro™ BCA assays for the rat tail tendon collagen type I coating compositions.

[00101] Table 3

[00102] The uniformity on the millimeter scale of the coatings was determined using the same process described in Example 2. Optical images of the colloidal staining tests for the citric acid pH 4.7 coating compositions at different coating lengths of time are shown in FIG. 8. The amount of staining increased as the coating equilibration times increased.

Example 5

Coating Compositions and Coated Well Plates

[00103] Collagen coating compositions with RTC and citric acid at pH 4.7 were formed that had varying RTC collagen concentrations. Concentrations of collagen in the coatings made were 50 pg/mL. 25 pg/mL. 10 pg/mL, and 5 pg/mL. The coating compositions were coated onto sets of three wells in TCT 6-well multi-well plates (Costar®) using 2 mb of the coating composition per well. The loading concentrations of collagen were 10 pg/cm², 5 pg/cm², 2 pg/cm², and 1 pg/cm² respectively. The coatings were equilibrated in the wells at room temperature for 4 hours. One additional set of well plates was coated with the 50 pg/mL composition but was only equilibrated for 1 hour. Excess coating liquid was then removed by aspiration with a pipette tip and the wells were rinsed with deionized water. The wells were then dried overnight at room temperature. These compositions were then compared to the coating composition using 50 pg/mL collagen in 0.1% acetic acid (pH 3.4) from Example 1, as is shown below in Table 4.

Analysis of Coated Well Plates

[00104] The coated well plates were then tested for the surface density of the collagen on the wells, coating efficiency, and uniformity of the collagen on a millimeter scale using the same processes described in Example 2. Table 4 below summarizes the results of the QuantiPro™ BCA assays for each of the coating compositions along with the coating efficiencies. FIG. 9 graphically shows the collagen surface density results of the QuanitPro™ BCA assays for the rat tail tendon collagen type I coating compositions.

[00105] Table 4

[00106] The uniformity on the millimeter scale of the coatings was determined using the same process described in Example 2. Optical images of the colloidal staining tests for the citric acid pH 4.7 coating compositions at different coating lengths of time are shown in FIG. 10. The amount of staining increased as the coating equilibration times increased. The staining was significantly increased for all concentrations of collagen in citric acid at pH 4.7 as compared to the 50 pg/mL collagen concentration in 0.1% acetic acid at pH 3.4.

Example 6

Coatins Compositions and Coated Well Plates [00107] The impact of different surface treatments on well plates prior to coating with collagen coating compositions comprising RTC was investigated. RTC was dissolved in either 0.1% acetic acid (pH 3.4), 0.01% acetic acid (pH 3.8), citric acid at pH 4.7, or citric acid at pH 3.7. The concentration of collagen in each of the coatings was 50 pg/mL. The coating compositions were coated onto sets of three wells in either untreated 6-well multi-well plates (Costar®), in TCT 6-well multi-well plates (Costar®), or in CellBIND® 6-well multi -well plates (Coming®). TCT well plates use a lower frequency plasma gas treatment compared to the CellBIND® well places, which causes the CellBIND® plate surfaces to be different from the TCT plate surfaces. The untreated and TCT wells had a bottom surface area of 9.6 cm², and the CellBIND® wells had a bottom surface area of 9.5 cm². Regardless of surface treatment type, each well received 2 mb of the coating composition. The loading concentration of collagen for each well was 10 pg/cm². The coatings were equilibrated in the wells at room temperature for 1 hour. Excess coating liquid was then removed by aspiration with a pipette tip and the wells were rinsed with deionized water. The wells were then dried overnight at room temperature.

Analysis of Coated Well Plates

[00108] The coated well plates were then tested for the surface density of the collagen on the wells, coating efficiency, and uniformity of the collagen on a millimeter scale using the same processes described in Example 2. Table 4 below summarizes the results of the QuantiPro™ BCA assays for each of the coating compositions along with the coating efficiencies. FIG. HA graphically shows the collagen surface density results of the QuanitPro™ BCA assays for the rat tail tendon collagen type I coating compositions using untreated or tissue culture treated well plates as described in Table 4. FIG. 11B graphically shows the collagen surface density results of the QuanitPro™ BCA assays for the rat tail tendon collagen type I coating compositions using CellBIND® treated well plates as described in Table 4. [00109] Table 4

[00110] The uniformity on the millimeter scale of the coatings was determined using the same process described in Example 2. Optical images of the colloidal staining tests for the untreated multi-well plates with different coating compositions are shown in FIG. 12. The untreated plates did not show uniform staining for any coating composition. Optical images of the colloidal staining tests for the TCT multi-well plates with different coating compositions are shown in FIG. 13. The TCT treated plates were substantially uniformity with acetic acid collagen coating at both pH 3.4 and pH 3.8, and with citric acid collagen coating at pH 4.7. The TCT treated plate with citric acid collagen coating at pH 6.4 was not uniform. Optical images of the colloidal staining tests for the CellBIND® multi-well plates with different coating compositions are shown in FIG. 14. The CellBIND® treated plates showed some uniformity with all coating compositions and pHs (and more uniformity than the untreated plates), but the uniformity was not as high as the TCT treated plates.

Example 7

Coatins Compositions and Coated Well Plates [00111] Collagen coating compositions with RTC and citric acid at either pH 3.7 or pH 4.7 were formed. Concentrations of collagen in the coatings made were 50 pg/mL. The coating compositions were coated onto sets of three wells in TCT 6-well multi-well plates (Costar®) using 2 mL of the coating composition per well. The loading concentration of collagen in each of the compositions was 10 pg/cm². The coatings were equilibrated in the wells at room temperature for 4 hours. Excess coating liquid was then removed by aspiration with a pipette tip and the wells were rinsed with deionized water. The wells were then dried overnight at room temperature.

Analysis of Coated Well Plates

[00112] The coated well plates were tested for functionality using the same process described in Example 1. The tested HT1080 cells were visually imaged with phase contrast optical imaging in a Vi-CELL cell counter and analyzer (Beckman Coulter®) at lOx magnification. The citric acid collagen coating compositions at both pH 3.7 and pH 4.7 showed at least 95% functionality, as shown in the bottom row of images in FIG. 15. These images were compared to the images of the positive and negative controls from Example 1 (shown for comparison in the top row of images in FIG. 15). The comparison in FIG. 15 shows the collagen coating compositions with citric acid have functionality like the positive control (0.1% acetic acid) and that uncoated plates (labeled “TCT” in FIG. 15) have little or no functionality.

Example 8

Coatins Compositions and Coated Well Plates

[00113] Collagen coating compositions with RTC in either citric acid at pH 4.7 with concentrations of collagen in the coatings of either 5 pg/m L. 10 pg/mL. 25 pg/mL or 50 pg/mL. The coating compositions were coated onto sets of three wells in TCT 6-well multi-well plates (Costar®) using 2 mL of the coating composition per well. The loading concentration of collagen in each of the compositions were 1 pg/cm², 2 pg/cm², 5 pg/cm², and 10 pg/cm², respectively. The coatings were equilibrated in the wells at room temperature for 4 hours. Excess coating liquid was then removed by aspiration with a pipette tip and the wells were rinsed with deionized water. The wells were then dried overnight at room temperature. This entire process was repeated to generate a second set of coated well plates. One set of the well plates was then irradiated with gamma irradiation set to a targeted dosage of 10-25 kGy and then left to equilibrate at room temperature for 7 days. The same irradiation process was also performed on well plates coated with the composition from Example 1 having 50 pg/mL RTC in 0. 1% acetic acid (pH 3.4)

Analysis of Coated Well Plates

[00114] The set of non-irradiated coated plates and the set of irradiated coated well plates were tested for functionality using the same process described in Example 1. For each set of plates, the tested HT1080 cells were visually imaged with phase contrast optical imaging in a Vi-CELL cell counter and analyzer (Beckman Coulter®) at lOx magnification. Images of the cells from the non-irradiated set of coating compositions are shown in FIG. 16, while images from the irradiated set of coating compositions are shown in FIG. 17. In FIG. 16, the image for the acetic acid composition is included from Example 1 for comparison. As seen in FIGS. 16-17, the compositions with 0.1% acetic acid and 50 pg/mL RTC show functionality of at least 95% before gamma irradiation, but less than 30% functionality after gamma irradiation. These figures show similar results for the composition with citric acid at pH 4.7 and 5 pg/mL RTC. Regarding the composition with citric acid at pH 4.7 and 10 pg/mL RTC, these figures show a functionality of at least 95% before gamma irradiation, and at least 75% functionality after gamma radiation. The functionality of the coating with 10 pg/mL RTC is significantly greater than the functionality of the coating with 5 pg/mL RTC. For the coating compositions with citric acid at pH 4.7 and either 25 pg/mL RTC or 50 pg/mL RTC, the figures show at least 95% functionality before gamma irradiation, and at least 90% functionality after gamma irradiation.

Example 9

Coatins Compositions

[00115] Collagen coating compositions with bovine skin collagen type I (“BC”) were formed by mixing the collagen in an aqueous buffer solution. Aqueous buffers tested included hydrochloric acid (“HQ”) (0.01 N for pH 2.0) and citric acid at pH 3.7, pH 4.7, and pH 5.5. Each coating composition had a final collagen concentration of 50 pg/mL. The ingredients used for the collagen compositions and the pHs that were tested in the present in the following Examples are listed in Table 6 below. The 0.01 N HC1 (pH 2.0) composition was used a positive control. Coatins Well Plates

[00116] Each coating composition was coated onto three wells of TCT 6-well multi-well plates (Costar®) for culturing adherent cells. To coat the wells, 2 mL of collagen coating composition at a collagen concentration of 50 pg/mL was added to each well to create a loading concentration of 10 pg/cnr in each well. Each well had a bottom surface area of 9.6 cm². To each well, 2 mL of the coating composition was added with a loading concentration of collagen of 10 pg/cm². The coating was equilibrated in the wells for 4 hours at room temperature. Excess coating liquid was then removed by aspiration with a pipette tip and the wells were rinsed with deionized water. The wells were then dried overnight at room temperature.

Analysis of Coated Well Plates

[00117] The coated well plates were then tested for the surface density of the bovine collagen on the wells and for the uniformity of the collagen on the wells with the BCA assay and colloidal gold staining assays described in Example 2. For the BCA assay that measures the surface density, a titration standard using the bovine collagen in known amounts was conducted for use with the BCA assay. The remainder of the procedure for the BCA assay was the same as described in Example 2. Table 6 below summarizes the results of the BCA assays for each of the bovine collagen coating compositions. Table 6 also summarizes the coating efficiency for each composition, which was calculated as described in Example 2. FIG. 18 graphically shows the bovine collagen surface density results from the BCA assays.

[00118] Table 6

[00119] The coated plates were also tested for uniformity on the millimeter scale using colloidal gold staining. TCT 6-well multi -well plates (Costar®) were coated with the coating compositions described in Table 1 above. After drying, 2 mL colloidal gold stain (BioRad®) was added to each well and the wells were equilibrated overnight at room temperature °C. Next, the colloidal staining excess was removed and the wells were rinsed with deionized water. The plates were then left to dry for 2-4 hours. The wells were then visually observed on an illuminated white background.

[00120] Optical images of the colloidal staining tests for the bovine coating compositions are shown in FIG. 19. In FIG. 19, areas of the images for colloidal staining that are darker indicate higher densities of colloidal staining. The colloidal staining results were consistent with the surface density results. FIG. 19 shows low colloidal staining density of bovine collagen in 0.01 N HC1 at pH 2.0 and higher staining with the citric acid compositions. With the citric acid compositions, the composition at pH 4.7 showed more colloidal staining than the composition at pH 3.7, which showed more staining than the composition at pH 5.5. All of the bovine collagen compositions tested appeared substantially uniform.

[00121] The colloidal gold stained coated plates were also visually observed with a scanning electron microscope with a scale bar at 200 pm to determine the uniformity of the collagen coating on amicron scale. FIG. 20 shows micrographic images of the bovine collagen coating compositions for the citric acid compositions (pH 3.7, pH 4.7, and pH 5.5). The images show substantially uniform micrographs on the micrometer scale for pH 3.7 and pH 4.7 but show a mottled appearance for pH 5.5, indicating the coating is less uniform on the micrometer scale at higher pHs.

Example 11

Coatins Compositions and Coated Well Plates

[00122] Collagen coating compositions with bovine skin collagen type I and either 0.01 N HC1 (pH 2.0) or citric acid at pH 3.7 or pH 4.7 were formed. Concentrations of collagen in the coatings were 50 pg/mL for the 0.01 N HC1 composition, 50 pg/mL for the citric acid at pH 3.7, and either 5 pg/mL, 10 pg/mL, 25 pg/mL or 50 pg/mL for the citric acid at pH 4.7 compositions. The coating compositions were coated onto sets of three wells in TCT 6-well multi-well plates (Costar®) using 2 mb of the coating composition per well. The loading concentration of collagen in each of the 50 pg/mL compositions was 10 pg/cm². The loading concentrations for the 5 pg/mL, 10 pg/mL, and 25 pg/mL collagen coatings was 1 pg/mL, 2 pg/mL, and 5 pg/mL respectively. The coatings were equilibrated in the wells at room temperature for 4 hours. One additional set of wells having 50 pg/mL bovine collagen in citric acid at pH 4.7 was equilibrated only for 1 hour. Excess coating liquid was then removed by aspiration with a pipette tip and the wells were rinsed with deionized water. The wells were then dried overnight at room temperature. This entire process was repeated to generate a second set of coated well plates for all of the plates except the one that equilibrated for only 1 hour. One set of the well plates (except the one that equilibrated for only 1 hr) was then irradiated with gamma irradiation set to a target dosage of 10-25 kGy and then left to equilibrate at room temperature for 7 days.

Analysis of Coated Well Plates

[00123] The non-irradiated coated well plates were then tested for the surface density of the collagen on the wells, coating efficiency, and uniformity of the collagen on a millimeter scale using the same processes described in Example 2. Table 7 below summarizes the results of the QuantiPro™ BCA assays for each of the coating compositions along with the coating efficiencies. FIG. 21 graphically shows the collagen surface density results of the QuanitPro™ BCA assays for the bovine collagen coating compositions.

[00124] Table 7

[00125] The uniformity on the millimeter scale of the non-irradiated collagen coatings (except for the citric acid at pH 3.7 composition) was determined using the same process described in Example 2. Optical images of the colloidal staining tests for the coating compositions described in Table 7 (except for the citric acid at pH 3.7 composition) above are shown in FIG. 22. Among the collagen compositions with citric acid at pH 4.7, the amount of staining increased as the collagen concentrations of the coating compositions increased. The staining for the 50 pg/mL collagen composition that was equilibrated for 1 hour showed similar staining to the same composition stained for 4 hours. All the compositions showed semiuniform staining on the millimeter scale, and the compositions with citric acid were more uniform than the composition with 0.01 N HC1.

[00126] Non-irradiated and irradiated coated well plates with the bovine collagen coating equilibrated for four hours were tested for functionality using the same HT1080 cell assay described in Example 1. An HT1080 cell assay with a non-irradiated, uncoated tissue culture treated plate (Costar®) was also performed as a negative control. For each set of plates (coated and non-coated) the tested HT1080 cells were visually imaged with phase contrast optical imaging in a Vi-CELL cell counter and analyzer (Beckman Coulter®) at lOx magnification. In FIG. 23, optical images at lOx magnification of the 50 pg/mL collagen concentration in 0.01 N HC1 (pH 2.0) with and without gamma irradiation are compared to the 50 pg/mL collagen concentration coatings in citric acid pH 3.7 and pH 4.7, also with and without gamma irradiation. In the non-irradiated samples, the citric acid collagen coating compositions at both pH 3.7 and pH 4.7 showed at least 95% functionality as did the 0.01 N HC1 collagen coating composition, as shown in the top row of FIG. 23. In the irradiated samples, the citric acid collagen coating at pH 3.7 showed at least 95% functionality, the citric acid collagen coating at pH 4.7 showed at least 90% functionality, and the 0.01 N HC1 (pH 2.0) showed less than 25% functionality, as shown in the bottom row of FIG. 23.

[00127] Optical images at lOx magnification of the cells from the non-irradiated set of coating compositions that were either uncoated (negative control), coated with 50 pg/mL collagen in 0.01 NHC1 (pH 2.0), or citric acid at pH 4.7 (having 5 pg/mL, 10 pg/mL, 25 pg/mL, or 50 pg/mL collagen concentrations in the coatings) are shown in FIG. 24, while images from the irradiated set of those coating compositions are shown in FIG. 25. As shown in FIG. 24, the uncoated TCT plate (uncoated negative control) had almost no functionality. As shown in FIGS. 24-25, coating compositions with 0.01 N HC1 (pH 2.0) and 50 pg/mL bovine collagen show functionality of at least 95% before gamma irradiation, but less than 25% functionality after gamma irradiation. As shown in FIGS. 24-25, coating compositions with citric acid and 5 pg/mL bovine collagen show functionality of at least 90% before gamma irradiation, but less than 30% functionality after gamma irradiation. Regarding the composition with citric acid at pH 4.7 and 10 pg/mL bovine collagen, these figures show a functionality of at least 95% before gamma irradiation, and at least 75% functionality after gamma radiation. The functionality of the coating with 10 pg/mL RTC is significantly greater than the functionality of the coating with 5 pg/mL RTC. For the coating compositions with citric acid at pH 4.7 and either 25 pg/mL bovine collagen or 50 pg/mL bovine collagen, the figures show at least 95% functionality before gamma irradiation, and at least 90% functionality after gamma irradiation.

[00128] It will be apparent to those skilled in the art that various modifications and variations can be made to the embodiments described herein without departing from the spirit and scope of the claimed subject matter. Thus, it is intended that the specification cover the modifications and variations of the various embodiments described herein provided such modification and variations come within the scope of the appended claims and their equivalents. The below aspects of the present disclosure are exemplary only.

[00129] Aspect 1. A cell culture device, comprising a surface and a collagen coating on at least a portion of the surface, wherein the collagen coating comprises collagen, wherein the collagen coating comprises a surface density of collagen on the at least a portion of the surface of between 0.4 pg/cm² and 2.0 pg/cm², and wherein the collagen coated surface is configured to attach adherent cells.

[00130] Aspect 2. The cell culture device of Aspect 1, wherein the collagen in the collagen coating is type I collagen.

[00131] Aspect 3. The cell culture device of Aspect 2, wherein the type I collagen is from bovine skin or rat tail.

[00132] Aspect 4. The cell culture device of any of Aspects 1-3, wherein the collagen coating further comprises a buffer and the buffer comprises apKa of between pKa 3.8 and 5.2.

[00133] Aspect 5. The cell culture device of Aspect 4, wherein the buffer comprises a pKa of between pKa 4.0 and 5.0.

[00134] Aspect 6. The cell culture device of any of Aspects 4-5, wherein the buffer is citric acid, acetic acid, or a combination thereof. [00135] Aspect 7. The cell culture device of any of Aspects 4-6, wherein the buffer comprises an acid and the acid comprises at least one carboxylic acid group or its carboxylate ion.

[00136] Aspect 8. The cell culture device of any of Aspects 4-6, wherein the buffer comprises an acid that comprises one, two, or three carboxylic acid groups, their carboxylate ions, or a combination of carboxylic acid groups and carboxylate ions.

[00137] Aspect 9. The cell culture device of Aspect 1, wherein the collagen is bovine skin type I collagen, and the collagen surface density is in a range of 0.7 pg/cm² to 1 .6 pg/cm².

[00138] Aspect 10. The cell culture device of Aspect 1, therein the collagen is rat tail tendon type I collagen, the buffer is citric acid, and the collagen surface density is in a range of 0.6 pg/cm² to 1.1 pg/cm².

[00139] Aspect 11. The cell culture device of any of Aspects 1-10, wherein the pH of the collagen coating is between pH 3.5 and pH 5.5.

[00140] Aspect 12. The cell culture device of any of Aspects 1-10, wherein the pH of the collagen coating is between pH 4.4 and pH 5.0.

[00141] Aspect 13. The cell culture device of Aspect 1, wherein the collagen coating is substantially uniform on a millimeter scale.

[00142] Aspect 14. The cell culture device of Aspect 1, wherein the collagen coating is substantially uniform on a micrometer scale.

[00143] Aspect 15. The cell culture device of Aspect 1, wherein the collagen coating is substantially uniform on a millimeter scale and a micrometer scale.

[00144] Aspect 16. The cell culture device of any of Aspects 1-15, wherein the collagen coated surface is a high frequency irradiated collagen coated surface.

[00145] Aspect 17. The cell culture device of Aspect 16, wherein the high frequency irradiation is at a dosage between 10 kGy and 50 kGy.

[00146] Aspect 18. The cell culture device of Aspect 16, wherein the high frequency irradiation is gamma irradiation at a dosage between 10 kGy and 50 kGy. [00147] Aspect 19. The cell culture device of any of Aspects 1-18, wherein the collagen coated surface has at least 50% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

[00148] Aspect 20. The cell culture device of Aspect 19, wherein the collagen coated surface has at least 75% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

[00149] Aspect 21. A method for forming a collagen coated cell culture device surface, comprising the steps of (1) providing a surface, (2) adding a collagen coating to at least a portion of the surface, wherein the collagen coating comprises collagen, a buffer, and a pH between pH 3.5 and pH 5.5, and (3) removing coating excess from the collagen coated surface, wherein after removing the coating excess from the collagen coated surface, the collagen coated surface has at least 2% coating efficiency.

[00150] Aspect 22. The method of Aspect 21, wherein after removing the coating excess from the collagen coated surface, the coated surface has at least a 5% coating efficiency.

[00151] Aspect 23. The method of Aspect 21 , wherein after removing the coating excess from the collagen coated surface, the coated surface has at least a 15% coating efficiency.

[00152] Aspect 24. The method of any of Aspects 21-23, wherein the pH of the coating is between pH 3.7 and pH 5.5.

[00153] Aspect 25. The method of any of Aspects 21 -24, wherein the collagen is type I collagen.

[00154] Aspect 26. The method of Aspect 25, wherein the type I collagen is from bovine skin, rat tail tendon, or a combination thereof.

[00155] Aspect 27. The method of any of Aspects 21-26, wherein the buffer has a pKa of between pH 4.0 and pH 5.0.

[00156] Aspect 28. The method of any of Aspects 21-27, wherein the buffer comprises an acid that comprises one, two, or three carboxylic acid groups, their carboxylate ions, or a combination of carboxylic acid groups and carboxylate ions. [00157] Aspect 29. The method of Aspect 21, wherein the coating is substantially uniform on a millimeter scale.

[00158] Aspect 30. The method of Aspect 21, wherein the coating is substantially uniform on a micrometer scale.

[00159] Aspect 31 . The method of Aspect 21 , further comprising a step of washing the collagen coated surface after the step of removing coating excess from the collagen coated surface.

[00160] Aspect 32. The method of Aspect 31, further comprising a step of drying the collagen coated surface after the step of washing the collagen coated surface.

[00161] Aspect 33. The method of any of Aspects 21-32, further comprising a step of sterilizing the collagen coated surface with high energy irradiation at a dosage between 10 kGy and 50 kGy.

[00162] Aspect 34. The method of any of Aspects 21-32, further comprising a step of sterilizing the collagen coated surface with gamma irradiation at a dosage between 10 kGy and 50 kGy.

[00163] Aspect 35. The method of any of Aspects 21-34, wherein the collagen coated surface has at least 50% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

[00164] Aspect 36. The method of any of Aspects 21-34, wherein the collagen coated surface has at least 75% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

[00165] Aspect 37. A cell culture device, comprising a surface and a collagen coating on at least a portion of the surface, wherein the collagen coating comprises collagen and a buffer, and wherein the collagen coated surface comprises a collagen surface density on the at least a portion of the surface that as measured by a bicinchoninic acid assay is between 1.2 and 20 times greater than a collagen surface density of an identically collagen coated surface comprising a collagen composition comprising a pH between pH 2.0 and 3.4. [00166] Aspect 38. The cell culture device of Aspect 37, wherein the collagen in the collagen coating is type I collagen.

[00167] Aspect 39. The cell culture device of Aspect 38, wherein the type I collagen is from bovine skin, rat tail, or a combination thereof.

Claims

1. A cell culture device, comprising: a surface; and a collagen coating on at least a portion of the surface; wherein the collagen coating comprises collagen; wherein the collagen coating comprises a surface density of collagen on the at least a portion of the surface of between 0.4 pg/cm² and 2.0 pg/cm²; and wherein the collagen coated surface is configured to attach adherent cells.

2. The cell culture device of claim 1, wherein the collagen in the collagen coating is type I collagen.

3. The cell culture device of claim 2, wherein the type I collagen is from bovine skin or rat tail.

4. The cell culture device of any of claims 1-3, wherein the collagen coating further comprises a buffer and the buffer comprises a pKa of between pKa 3.8 and 5.2.

5. The cell culture device of claim 4, wherein the buffer comprises a pKa of between pKa 4.0 and 5.0.

6. The cell culture device of any of claims 4-5, wherein the buffer is citric acid, acetic acid, or a combination thereof.

7. The cell culture device of any of claims 4-6, wherein the buffer comprises an acid and the acid comprises at least one carboxylic acid group or its carboxylate ion.

8. The cell culture device of any of claims 4-6, wherein the buffer comprises an acid that comprises one, two, or three carboxylic acid groups, their carboxylate ions, or a combination of carboxylic acid groups and carboxylate ions.

9. The cell culture device of claim 1, wherein the collagen is bovine skin type I collagen, and the collagen surface density is in a range of 0.7 pg/cm² to 1.6 pg/cm².

10. The cell culture device of claim 1, therein the collagen is rat tail tendon type I collagen, the buffer is citric acid, and the collagen surface density is in a range of 0.6 pg/cnr to 1.1 g/cm².

11. The cell culture device of any of claims 1-10, wherein the pH of the collagen coating is between pH 3.5 and pH 5.5.

12. The cell culture device of any of claims 1-10, wherein the pH of the collagen coating is between pH 4.4 and pH 5.0.

13. The cell culture device of claim 1, wherein the collagen on the collagen coated surface is substantially uniform on a millimeter scale.

14. The cell culture device of claim 1, wherein the collagen on the collagen coated surface is substantially uniform on a micrometer scale.

15. The cell culture device of claim 1, wherein the collagen on the collagen coated surface is substantially uniform on a millimeter scale and a micrometer scale.

16. The cell culture device of any of claims 1-15, wherein the collagen coated surface is a high frequency irradiated collagen coated surface.

17. The cell culture device of claim 16, wherein the high frequency irradiation is at a dosage between 10 kGy and 50 kGy.

18. The cell culture device of claim 16, wherein the high frequency irradiation is gamma irradiation at a dosage between 10 kGy and 50 kGy.

19. The cell culture device of any of claims 1-18, wherein the collagen coated surface has at least 50% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

20. The cell culture device of claim 19, wherein the collagen coated surface has at least 75% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

21. A method for forming a collagen coated cell culture device surface, comprising the steps of: providing a surface; adding a collagen coating to at least a portion of the surface, wherein the collagen coating comprises collagen, a buffer, and a pH between pH 3.5 and pH 5.5; and removing coating excess from the collagen coated surface; wherein after removing the coating excess from the collagen coated surface, the collagen coated surface has at least 2% coating efficiency.

22. The method of claim 21, wherein after removing the coating excess from the collagen coated surface, the collagen coated surface has at least a 5% coating efficiency.

23. The method of claim 21, wherein after removing the coating excess from the collagen coated surface, the collagen coated surface has at least a 15% coating efficiency.

24. The method of any of claims 21-23, wherein the pH of the collagen coating is between pH 3.7 and pH 5.5.

25. The method of any of claims 21-24, wherein the collagen is type I collagen.

26. The method of claim 25, wherein the type I collagen is from bovine skin, rat tail tendon, or a combination thereof.

27. The method of any of claims 21-26, wherein the buffer has a pKa of between pH 4.0 and pH 5.0.

28. The method of any of claims 21 -27, wherein the buffer comprises an acid that comprises one, two, or three carboxylic acid groups, their carboxylate ions, or a combination of carboxylic acid groups and carboxylate ions.

29. The method of claim 21, wherein the collagen coated surface is substantially uniform on a millimeter scale across the collagen coated surface.

30. The method of claim 21, wherein the collagen coated surface is substantially uniform on a micrometer scale.

31. The method of claim 21, further comprising a step of washing the collagen coated surface after the step of removing coating excess from the collagen coated surface.

32. The method of claim 31, further comprising a step of drying the collagen coated surface after the step of washing the collagen coated surface.

33. The method of any of claims 21-32, further comprising a step of sterilizing the collagen coated surface with high energy irradiation at a dosage between 10 kGy and 50 kGy.

34. The method of any of claims 21-32, further comprising a step of sterilizing the collagen coated surface with gamma irradiation at a dosage between 10 kGy and 50 kGy.

35. The method of any of claims 21-34, wherein the collagen coated surface has at least 50% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

36. The method of any of claims 21-34, wherein the collagen coated surface has at least 75% functionality of observed HT1080 cells as measured by a HT1080 cell attachment test.

37. A cell culture device, comprising: a surface; and a collagen coating on at least a portion of the surface; wherein the collagen coating comprises collagen and a buffer; wherein the collagen coating comprises a pH between pH 3.6 and pH 5.6; and wherein the collagen coated surface comprises a collagen surface density on the at least a portion of the surface that as measured by a bicinchoninic acid assay is between 1.2 and 20 times greater than a collagen surface density of an identically collagen coated surface comprising a collagen composition comprising a pH between pH 2.0 and 3.4.

38. The cell culture device of claim 37, wherein the collagen in the collagen coating is type

I collagen.

39. The cell culture device of claim 38, wherein the type I collagen is from bovine skin, rat tail, or a combination thereof.