Cell culture surfaces and vessels and methods of making and using same

Abstract

The present disclosure relates to coated surfaces suitable for cell culture, methods of making such surfaces, and methods of culturing adherent cells on such surfaces. One aspect of the present disclosure is a surface suitable for cell culture comprising an amorphous hydrogenated carbon coating. Another aspect of the present disclosure is a method of making a surface suitable for cell culture comprising an amorphous hydrogenated carbon coating. Another aspect of the present disclosure is a method of culturing adherent cells, the method comprising incubating a surface comprising an amorphous hydrogenated carbon coating with adherent cells and a growth medium.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 384,466, filed November 21, 2022, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to coatings for cell culture devices. More particularly, the present disclosure relates to coated surfaces suitable for cell culture, methods of making such surfaces, and methods of culturing adherent cells on such surfaces. [Background technology]

[0003] In vitro cell culture is a complex process in which cells are grown under controlled conditions, generally outside their natural environment, but under conditions sufficiently similar to in vivo conditions to allow proliferation and, in the case of adherent cells, adhesion to a growth surface. These are typically cells derived from multicellular eukaryotes, particularly cultured animal cells. However, cells derived from plants, fungi, and insects, as well as microorganisms including viruses, bacteria, and protists, can also be cultured.

[0004] In vitro cell cultures can provide materials not only for research but also for a variety of applications in pharmacology, physiology, and toxicology. Cell cultures may also be desirable for use in bioprocessing and cell therapy.

[0005] In a typical cell culture process, cells can be grown and maintained in a cell incubator at an appropriate temperature and gas mixture. Mammalian cells are typically incubated at 37°C with a pH maintained at 7.2–7.4. pH can be controlled using a bicarbonate buffer system in the culture medium, along with an incubator atmosphere of approximately 5–7% carbon dioxide by volume. Carbon dioxide reacts with water to form carbonic acid, which then interacts with bicarbonate ions in the culture medium to form a buffer system capable of maintaining pH near physiological levels. Oxygen is essential for cellular metabolism and growth of many desirable cell types. Culture conditions can vary from cell type to cell type, and changes in conditions for a particular cell type can result in the expression of different phenotypes. For example, bicarbonate-based buffers can be replaced with monosodium and disodium phosphate buffers or trisodium phosphate buffers, chloride and ammonia buffers, lactate buffers, or organic buffers such as HEPES.

[0006] Commercially available cell culture vessels in the form of cell culture bags are the traditional format used for cell culture. Cell culture bags have the advantage of being disposable, which reduces preparation and cleaning time. Furthermore, cell culture bags can be pre-sterilized, are inexpensive, easy to use, and require minimal space for storage and use. Disposability also helps reduce the risk of contamination to the cell culture and the environment.

[0007] Cell culture bags often have a fluoropolymer interior surface. Fluoropolymer surfaces are highly advantageous because they can have very low amounts of leachable organic material, which can be important in many cell culture applications. Fluoropolymer surfaces also resist protein adsorption. Fluoropolymer surfaces are further advantageous from the standpoint of manufacturability and sterilization.

[0008] However, fluoropolymer surfaces generally have low surface energy, and therefore it may be difficult for adherent cells to adhere to such surfaces, which may make cell culture systems (e.g., cell culture devices) based on fluoropolymer surfaces less useful for culturing adherent cells.

[0009] Therefore, there remains a need for surfaces suitable for cell culture, particularly for adherent cells. Summary of the Invention

[0010] Thus, one aspect of the present disclosure is a surface suitable for cell culture. a substrate having a fluoropolymer surface; an amorphous hydrogenated carbon coating disposed on a fluoropolymer surface of a substrate, the amorphous hydrogenated carbon coating having a first thickness zone proximate to and extending from the fluoropolymer surface and a second thickness zone distal to the fluoropolymer surface and at a surface of the amorphous hydrogenated carbon coating, the amorphous hydrogenated carbon coating having a higher combined concentration of oxygen and nitrogen in the second thickness zone than in the first thickness zone.

[0011] Another aspect of the present disclosure is a method of making a surface suitable for cell culture, e.g., as described elsewhere herein. The method comprises: depositing an amorphous hydrogenated carbon coating on a fluoropolymer surface of a surface substrate, said depositing comprising: depositing a first thickness of the amorphous hydrogenated carbon coating by chemical vapor deposition (e.g., plasma-enhanced chemical vapor deposition (PECVD)) using a combination of a hydrocarbon gas (e.g., ethylene or propylene) and CO2 in a first ratio of hydrocarbon gas to CO2 on a first zone of the amorphous hydrogenated carbon coating, the first zone being adjacent to and extending from the fluoropolymer surface; depositing a second thickness of the amorphous hydrogenated carbon coating on a second zone of the amorphous hydrogenated carbon coating by chemical vapor deposition (e.g., PECVD) using a combination of a hydrocarbon gas (e.g., ethylene or propylene) and at least one of CO2 and NH3, the second zone being distal from the fluoropolymer surface and at a surface of the amorphous hydrogenated carbon coating, the amorphous hydrogenated carbon coating having a higher combined concentration of oxygen and nitrogen in the second zone than in the first zone of the amorphous hydrogenated carbon coating.

[0012] Another aspect of the present disclosure is a method of culturing adherent cells, comprising incubating a surface as described herein with adherent cells and growth medium.

[0013] In various embodiments of the surfaces and methods described herein, the fluoropolymer surface is an activated fluoropolymer surface. For example, in some embodiments, the fluoropolymer surface is activated by plasma treatment (e.g., with ammonia plasma or CO2 plasma) or by corona treatment, such as C treatment.

[0014] Other aspects of the present disclosure will become apparent based on the description provided herein. [Brief explanation of the drawings]

[0015] [Figure 1] 1 is a schematic cross-sectional view of a surface suitable for cell culture applications, according to one embodiment of the present disclosure. [Figure 2] FIG. 1 is a schematic cross-sectional view of a surface suitable for cell culture applications, according to another embodiment of the present disclosure. [Figure 3] 1A and 1B are a schematic plan view and a schematic cross-sectional view of a cell culture vessel of the present invention. [Figure 4] FIG. 1 shows the elemental percentages of oxygen and nitrogen on the surfaces described in Examples 1-8. [Figure 5] FIG. 1 shows the water contact angles of the surfaces described in Examples 1 to 8. [Figure 6] FIG. 1 shows exemplary thicknesses of coatings disposed on surfaces described in Examples 3-8. [Figure 7] FIG. 1 shows cell attachment and proliferation of hMSCs on coated surfaces described in Examples 5 and 8 compared to non-activated fluoropolymer surfaces, activated fluoropolymer surfaces, and commercial TCPS surfaces. [Figure 8] FIG. 1 shows cell adhesion and proliferation of monocyte-derived dendritic cells on coated surfaces described in Examples 5 and 8 compared to non-activated fluoropolymer surfaces, activated fluoropolymer surfaces, and commercial TCPS surfaces, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present inventors have focused on various methods that can be used to treat fluoropolymer surfaces to provide improved adhesion for adherent cells. Treating fluoropolymer surfaces with plasma treatment can increase their hydrophilicity (e.g., as measured by the surface contact angle), which can promote cell adhesion and proliferation. However, simply increasing the hydrophilicity of the surface of a cell culture device may not be sufficient to provide a desirable surface for culturing adherent cells.

[0017] Cell culture devices may be coated with pro-adhesive proteins such as laminin, fibronectin, or collagen. These proteins are generally obtained from animals or humans and need to be purified and characterized for clinical application. However, the purification and characterization process can be expensive and time-consuming, which ultimately increases the cost of the cell culture device.

[0018] The present inventors note that cell culture surfaces with a relatively high concentration of nitrogen and / or oxygen functional groups (e.g., amine and / or carboxylic acid functional groups) on the surface can help enhance cell adhesion and proliferation. For example, cell culture devices with amine and / or carboxylic acid functional groups can generally exhibit better cell culture behavior in serum-free cell culture than cell culture devices without such functional groups. However, because amines and carboxylic acids can also react with oxygen and cause surface reorganization, the amount of these functional groups on the surface can decrease over time, thereby reducing the surface functionality of the cell culture device.

[0019] The present inventors have determined that a desirable cell culture system for adherent cell culture can be provided by coating a fluoropolymer layer with a coating that provides nitrogen and / or oxygen functional groups on its surface. Plasma polymerization can be used to provide such a coating in the form of an amorphous coating. However, the present inventors have determined that if a homogeneous coating with a desirable high degree of nitrogen and / or oxygen functional groups is provided on the surface, such a coating may be unstable during autoclaving procedures typically used to sterilize surfaces for cell culture. The present inventors have determined that a coating with fewer nitrogen and / or oxygen functional groups proximal to the fluoropolymer surface and more nitrogen and / or oxygen functional groups distal to the fluoropolymer surface and at the surface of the coating, e.g., a non-uniform coating, can provide a system that not only has the desirable properties of a cell culture coating surface, but also provides a coating that is robust enough to withstand autoclaving. The inventors have noted that the coatings described herein are sterilizable (e.g., by autoclaving), protein-free and relatively simple and inexpensive to manufacture, while being capable of being functionalized to provide a surface particularly suitable for adherent cell culture.

[0020] Thus, one aspect of the present disclosure is a surface suitable for cell culture applications. The surface may be, for example, the surface of a cell culture device (e.g., a cell culture bag or a cell culture tube). In various embodiments, the surface may be the interior surface of a cell culture device. The surface includes a substrate having a fluoropolymer surface. The surface also includes an amorphous hydrogenated carbon coating disposed on the fluoropolymer surface of the substrate. The amorphous hydrogenated carbon coating has a first thickness zone proximate to and extending from the fluoropolymer surface and a second thickness zone distal to the fluoropolymer surface and at the surface of the amorphous hydrogenated carbon coating. In particular, the amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in the second thickness zone than in the first thickness zone. As used herein, the term "concentration" means an elemental percentage based on atomic weight.

[0021] One embodiment of a surface according to the present disclosure is shown in the schematic cross-sectional view of Figure 1. In this embodiment, a surface 100 for cell culture applications includes a substrate 110 having a fluoropolymer surface 112. The surface 100 further includes an amorphous hydrogenated carbon coating 120 disposed on the fluoropolymer surface 112 of the substrate 110. The amorphous hydrogenated carbon coating 120 has a first thickness zone 122 proximate to and extending from the fluoropolymer surface 112 and a second thickness zone 124 distal to the fluoropolymer surface 112 and at a surface 126 of the amorphous hydrogenated carbon coating 120. The amorphous hydrogenated carbon coating 120 has a higher combined oxygen and nitrogen concentration in the second thickness zone 124 than in the first thickness zone 122. Notably, the embodiment of Figure 1 shows that the first thickness zone 122 is continuous with the second thickness zone 124.

[0022] Various fluoropolymers can be used to provide the fluoropolymer surface. In various desirable embodiments as described elsewhere herein, the fluoropolymer surface is a fluorinated ethylene propylene (FEP) surface. Other fluoropolymers can be used instead. For example, in various embodiments as described elsewhere herein, the fluoropolymer surface is a polytetrafluoroethylene (PTFE) surface, a perfluoroalkoxy (PFA) surface, an ethylene tetrafluoroethylene (ETFE) surface, a polyvinylidene fluoride (PVDF) surface, a polychlorotrifluoroethylene (PCTFE) surface, an ethylene chlorotrifluoroethylene (ECTFE) surface, an ethylene fluorinated ethylene propylene (EFEP) surface, a perfluoropolyether (PFPE) surface, a modified polytetrafluoroethylene (TFM) surface, a polyvinyl fluoride surface, or a combination of any two or more thereof.

[0023] In various desirable embodiments, as described elsewhere herein, the fluoropolymer surface is the surface of a fluoropolymer-silicone laminate film. The inventors note that silicone-fluoropolymer laminates can provide a good balance of physical properties, high oxygen and carbon dioxide (CO2) permeability, and low organic leachability. The silicone-fluoropolymer can be, for example, a silicone-FEP laminate providing an FEP fluoropolymer surface. Silicone-fluoropolymer laminates are described in U.S. Pat. No. 9,926,524, which is incorporated herein by reference in its entirety. Cell culture bags based on silicone-fluoropolymer laminates are commercially available from Saint-Gobain Performance Polymer Products Corporation under the trade name VueLife®.

[0024] As one of skill in the art will appreciate, it may be desirable for a cell culture system to have a certain degree of oxygen and CO2 permeability to allow the cell culture medium to absorb oxygen from the atmosphere and desorb CO2 through the surface of the cell culture into the atmosphere.

[0025] Thus, in various desirable embodiments as described elsewhere herein, the fluoropolymer surfaces as described herein have a fluoropolymer content of at least 1500 cc / m 2 -day-atm, e.g., at least 1800cc / m 2 -day-atm, or 1500-20000cc / m 2 -day-atm, or 1800-16000cc / m 2 The surface of the film has an oxygen permeability in the range of 10-1500-atm. The oxygen permeability was measured using a MOCON OxTran 220 O2TR Analyzer according to ASTM D3985 using the following test conditions: temperature: 23°C, test gas: 10% O2 in N2, humidity: 0% relative humidity on both sides of the film, carrier gas flow rate: 20 sccm N2, test area: 5 cm 2 ,Test cycle: 15 min.

[0026] In various desirable embodiments as described elsewhere herein, the fluoropolymer surfaces as described herein have a fluoropolymer content of at least 3500 cc / m 2 -day-atm, e.g., 4000cc / m 2 -day-atm, or 3500-25000cc / m 2 -Sun-ATM, 4000~23000cc / m 2 The film surface has a CO2 permeability in the range of 10-15 atm. The CO2 permeability was measured using a MOCON Permatran-C 441 CO2TR Analyzer according to ASTM F2476 under the following test conditions: temperature: 23°C, test gas: 100% CO2, humidity: 0% relative humidity on both sides of the film, carrier gas flow rate: 50 sccm N2, test area: 5 cm 2 ,Test cycle: 30 min.

[0027] In various desirable embodiments as described elsewhere herein, the fluoropolymer surfaces as described herein have a coating density of 0.65 to 1 g / m 2 -day-atm range, e.g., 0.72-0.94 g / m 2 The surface of the film has a water vapor transmission rate (WVTR) in the range of 10-150-150 atm. The WVTR was measured using a MOCON Permatran W700 Water Vapor Analyzer according to ASTM F1249 using the following test conditions: temperature: 23°C, humidity: 100% relative humidity on the test gas side and 0% relative humidity on the detector side of the film, carrier gas flow rate: 10 sccm N2, test area: 50 cm 2 ,Test cycle: 60 min.

[0028] The inventors have noted that the adhesion of amorphous hydrogenated carbon coatings to fluoropolymer surfaces can be improved by using an activated fluoropolymer surface, e.g., a treated fluoropolymer surface. Accordingly, in various desirable embodiments, as described elsewhere herein, the fluoropolymer surface is an activated fluoropolymer surface. The activated fluoropolymer surface provides oxygen and / or nitrogen functional groups. Without being bound by theory, the inventors speculate that such functional groups provide a reactive surface upon which an amorphous film can be grown, e.g., by plasma polymerization. The amorphous film can be covalently bonded to the activated fluoropolymer surface.

[0029] Various processing techniques can be used to provide an activated fluoropolymer surface. For example, plasma treatment, such as treatment with ammonia (NH3) plasma, CO2 plasma, or a combination of NH3 and CO2 plasma, can be used. The inventors have determined that treatment of a fluoropolymer surface with NH3 plasma can better improve the hydrophilicity and biocompatibility of the fluoropolymer surface than treatment with CO2 plasma, thereby better promoting cell adhesion and proliferation on the cell culture surface. In other embodiments, the plasma can be oxygen plasma, nitrogen plasma, or a combination of oxygen and nitrogen plasma. The inventors, without being bound by theory, speculate that plasma treatment can not only add nitrogen and / or oxygen functional groups to the fluoropolymer surface, but also remove some fluorine from the fluoropolymer surface.

[0030] In other embodiments, the fluoropolymer surface can be activated (e.g., treated) by a corona treatment, such as a C treatment. As used herein, corona treatment, also referred to as corona discharge, is a plasma treatment of a surface in an atmosphere containing an organic gas. The organic gas can be, for example, ketone- or alcohol-based. In various embodiments, the alcohol contains four or fewer carbon atoms. In one embodiment, the organic gas is acetone. In one embodiment, the organic gas is mixed with an inert gas, such as nitrogen. The acetone / nitrogen atmosphere improves adhesion of the fluoropolymer layer to layers with which it directly contacts. In one embodiment, the treatment improves adhesion of the fluoropolymer layer to the polymer layer. In an exemplary embodiment, the treatment includes a C treatment of a C-treatable fluoropolymer. C treatment is further described in U.S. Patent Nos. 6,726,979 and 8,559,100, each of which is incorporated herein by reference in its entirety.

[0031] In various embodiments as described elsewhere herein, the activated fluoropolymer surface has an oxygen concentration of less than 5 atomic % and a nitrogen concentration of less than 10 atomic %. As used herein, atomic % is determined by X-ray photoelectron spectroscopy; therefore, atomic percentages based on all atoms except hydrogen, which one skilled in the art will understand are generally not appreciable by XPS.

[0032] In various embodiments as described elsewhere herein, the activated fluoropolymer surface can desirably have a reduced water contact angle. As one skilled in the art will appreciate, surfaces with higher amounts of nitrogen and / or oxygen functional groups generally have lower water contact angles. Thus, the water contact angle can be used to measure the degree of activation of the fluoropolymer surface. In various embodiments as described elsewhere herein, the activated fluoropolymer surface has a water contact angle in the range of 60° to 120°.

[0033] As described above, an amorphous hydrogenated carbon coating is disposed on the fluoropolymer surface (e.g., activated fluoropolymer surface) of the substrate. An amorphous hydrogenated carbon coating, as used herein, is a coating that has significant hydrogen and carbon content and is substantially amorphous in nature. As described in detail below, such a coating can be produced by the polymerization of a polymerizable hydrocarbon (e.g., ethylene or propylene) under chemical vapor deposition conditions, for example, by plasma enhanced chemical vapor deposition (PECVD).

[0034] Those skilled in the art will be able to determine the desired thickness of the amorphous hydrogenated carbon coating based on the disclosure herein. In various desired embodiments, as described elsewhere herein, the amorphous hydrogenated carbon coating has a thickness in the range of 10 to 200 nm, e.g., 10 to 100 nm, or 10 to 75 nm, or 10 to 50 nm, or 15 to 200 nm, or 15 to 100 nm, or 15 to 75 nm, or 15 to 50 nm, or 20 to 200 nm, or 20 to 100 nm, or 20 to 75 nm, or 20 to 50 nm. However, other thicknesses can also be used.

[0035] The amorphous hydrogenated carbon coatings described herein, in various embodiments, have little, if any, graphitic properties. In various desirable embodiments as described elsewhere herein, the amorphous hydrogenated carbon coatings have a hydrogen-to-carbon ratio of at least 1, e.g., at least 1.2, or at least 1.4. The hydrogen-to-carbon ratio can be measured as described in P.-L. Girard-Lauriault et al., "Chemical Characterization of Nitrogen-Rich Plasma-Polymer Films Deposited in Dielectric Barrier Discharges at Atmospheric Pressure," Plasma Process. Polym., 5, 631-44 (2008), which is incorporated herein by reference in its entirety.

[0036] As discussed above, it may be advantageous to have a coating with a lower amount of nitrogen and / or oxygen functional groups proximal to the fluoropolymer surface and a higher amount of nitrogen and / or oxygen functional groups distal to the fluoropolymer surface and at the surface of the coating. This can provide a more highly crosslinked layer at the interface with the fluoropolymer substrate, thus providing a stable polymer backbone and, in turn, increasing the stability of the coating. Thus, in the embodiment of FIG. 1 , the amorphous hydrogenated carbon coating 120 has a higher hydrocarbon concentration in the first thickness zone 122 than in the second thickness zone 124, forming a more crosslinked layer in the first thickness zone 122. In various desirable embodiments, as described elsewhere herein, the amorphous hydrogenated carbon coating has a combined oxygen and nitrogen concentration in the first thickness zone that is at least 3 atomic % lower, e.g., at least 4 atomic % lower, or at least 5 atomic % lower, or at least 6 atomic % lower, or at least 7 atomic % lower, than the combined oxygen and nitrogen concentration in the second thickness zone. However, in many embodiments, the amorphous hydrogenated carbon coating has a combined oxygen and nitrogen concentration in the first thickness zone of at least 5 atomic %, such as at least 10 atomic %, or at least 15 atomic %.

[0037] Those skilled in the art will be able to determine the desired thickness of the first thickness zone of the amorphous hydrogenated carbon coating based on the disclosure herein. In various desired embodiments, as described elsewhere herein, the first thickness zone of the amorphous hydrogenated carbon coating has a thickness in the range of 8 to 190 nm, e.g., 8 to 100 nm, or 8 to 75 nm, or 8 to 50 nm, or 15 to 190 nm, or 15 to 100 nm, or 15 to 75 nm, or 15 to 50 nm, or 20 to 190 nm, or 20 to 100 nm, or 20 to 75 nm, or 20 to 50 nm. However, other thicknesses can also be used.

[0038] Additionally, as discussed above, it may be advantageous to have a coating with a high amount of nitrogen and / or oxygen functional groups at the surface of the coating, distal to the fluoropolymer surface. Thus, the coating can provide a more highly functionalized layer at the surface of the coating. The inventors have determined that a more functionalized layer of the coating, e.g., the presence of carboxylic acid and / or amine functional groups, can enhance the functionality of the coating, particularly with respect to adhesion of adherent cells. Thus, in the embodiment of FIG. 1 , the amorphous hydrogenated carbon coating 120 can include a higher oxygen and / or nitrogen concentration in the second thickness zone 124 than in the first thickness zone 122, thus providing a more functionalized layer in the second thickness zone 124. In various desirable embodiments, as described elsewhere herein, the amorphous hydrogenated carbon coating has a combined oxygen and nitrogen concentration in the second thickness zone of at least 10 atomic %, e.g., at least 15 atomic %, or at least 20 atomic %.

[0039] Those skilled in the art will be able to determine the desired thickness of the second thickness zone of the amorphous hydrogenated carbon coating based on the disclosure herein. In various desired embodiments, as described elsewhere herein, the second thickness zone of the amorphous hydrogenated carbon coating has a thickness in the range of 2 to 100 nm, e.g., 2 to 50 nm, 2 to 35 nm, 2 to 20 nm, 5 to 100 nm, 5 to 50 nm, 5 to 35 nm, 5 to 20 nm, 5 to 10 nm, 10 to 100 nm, 10 to 50 nm, 10 to 35 nm, 10 to 20 nm, 20 to 100 nm, or 20 to 50 nm. However, other thicknesses can also be used.

[0040] In various desirable embodiments, as described elsewhere herein, the amorphous hydrogenated carbon coating comprises oxygen proximate the fluoropolymer surface, e.g., in a first thickness zone. Accordingly, in various embodiments, the amorphous hydrogenated carbon coating in the first thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and oxygen (i.e., subtract hydrogen from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating can comprise oxygen at the surface of the coating distal from the fluoropolymer surface, e.g., in a second thickness zone. Accordingly, in various such embodiments, the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and oxygen (i.e., subtract hydrogen from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating can comprise nitrogen at the surface of the coating distal from the fluoropolymer surface, e.g., in a second thickness zone. Thus, in various such embodiments, the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and nitrogen (i.e., subtracting hydrogen from the analysis as described above). Also, in various such embodiments, the amorphous hydrogenated carbon coating can include nitrogen and oxygen at the surface of the coating distal from the fluoropolymer surface, e.g., in the second thickness zone. Thus, in various such embodiments, the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon, nitrogen, and oxygen (i.e., subtracting hydrogen from the analysis as described above).

[0041] In various desirable embodiments, as described elsewhere herein, the amorphous hydrogenated carbon coating comprises oxygen and nitrogen proximate the fluoropolymer surface, e.g., in a first thickness zone. Accordingly, in various embodiments, the amorphous hydrogenated carbon coating in the first thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon, nitrogen, and oxygen (i.e., subtract hydrogen from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating can comprise oxygen at the surface of the coating distal from the fluoropolymer surface, e.g., in a second thickness zone. Accordingly, in various such embodiments, the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and oxygen (i.e., subtract hydrogen from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating can comprise nitrogen at the surface of the coating distal from the fluoropolymer surface, e.g., in the second thickness zone. Thus, in various such embodiments, the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and nitrogen (i.e., subtracting hydrogen from the analysis as described above). Also, in various such embodiments, the amorphous hydrogenated carbon coating can include nitrogen and oxygen at the surface of the coating distal from the fluoropolymer surface, e.g., in the second thickness zone. Thus, in various such embodiments, the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon, nitrogen, and oxygen (i.e., subtracting hydrogen from the analysis as described above).

[0042] In various desirable embodiments, as described elsewhere herein, the amorphous hydrogenated carbon coating comprises nitrogen proximate the fluoropolymer surface, e.g., in a first thickness zone. Accordingly, in various embodiments, the amorphous hydrogenated carbon coating in the first thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and nitrogen (i.e., subtract hydrogen from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating can comprise oxygen at the surface of the coating distal from the fluoropolymer surface, e.g., in a second thickness zone. Accordingly, in various such embodiments, the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and oxygen (i.e., subtract hydrogen from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating can comprise nitrogen at the surface of the coating distal from the fluoropolymer surface, e.g., in the second thickness zone. Thus, in various such embodiments, the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and nitrogen (i.e., subtracting hydrogen from the analysis as described above). Also, in various such embodiments, the amorphous hydrogenated carbon coating can include nitrogen and oxygen at the surface of the coating distal from the fluoropolymer surface, e.g., in the second thickness zone. Thus, in various such embodiments, the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon, nitrogen, and oxygen (i.e., subtracting hydrogen from the analysis as described above).

[0043] In various embodiments, the first thickness zone of the amorphous hydrogenated carbon coating is contiguous with the second thickness zone of the amorphous hydrogenated carbon coating, for example, in the embodiment of Figure 1, the first thickness zone 122 is contiguous with the second thickness zone 124.

[0044] However, in other embodiments, one or more additional thickness zones of the amorphous hydrogenated carbon coating are disposed between the first thickness zone of the amorphous hydrogenated carbon coating and the second thickness zone of the amorphous hydrogenated carbon coating. For example, in various embodiments, the one or more additional thickness zones are third thickness zones disposed between and contiguous with the first thickness zone and the second thickness zone. One such embodiment of a surface according to the present disclosure is shown in the schematic cross-sectional view of FIG. 2. In this embodiment, a surface 200 for cell culture applications includes a substrate 210 having a fluoropolymer surface 212. Surface 200 further includes an amorphous hydrogenated carbon coating 220 disposed on fluoropolymer surface 212 of substrate 210. Amorphous hydrogenated carbon coating 220 has a first thickness zone 222 proximate to and extending from fluoropolymer surface 212 and a second thickness zone 224 distal to fluoropolymer surface 212 and at a surface 226 of amorphous hydrogenated carbon coating 220. The amorphous hydrogenated carbon coating 220 has a higher total concentration of oxygen and nitrogen in the second thickness zone 224 than in the first thickness zone 222. Notably, in this embodiment, the third thickness zone 228 is disposed between and contiguous with the first thickness zone 222 and the second thickness zone 224.

[0045] One or more thickness zones disposed between the first and second zones can have different compositions. In particular, in some embodiments, the third thickness zone can include oxygen and / or nitrogen, and the amorphous hydrogenated carbon coating can have a higher combined concentration of oxygen and nitrogen in the third thickness zone than in the first thickness zone.

[0046] The surfaces described herein can be used in a variety of cell culture systems. For example, in various embodiments as described elsewhere herein, the surface is the interior surface of a cell culture vessel. Various cell culture vessels, particularly those made from fluoropolymer materials, can be adapted to the surfaces of the present disclosure, for example, by depositing an amorphous hydrogenated carbon coating thereon. For example, in various desirable embodiments, the cell culture vessel is a cell culture bag. One such embodiment is shown in the schematic top view (top of the figure) and schematic cross-sectional view (bottom of the figure) of FIG. 3. Here, cell culture bag 350 is formed by two polymer films 355 having fluoropolymer surfaces, laminated together at their edges 358. Surface 300 as described herein is provided on the interior surface of the cell culture bag. In some embodiments, the edges of the laminate may be free of a coating so as not to interfere with lamination. Many of the fluoropolymers described above can be adapted for use in such bags. Various fluoropolymer materials described above, including fluoropolymer-silicone laminates, can be adapted for use in such cell culture bags. Of course, other cell culture vessels, for example in the form of flasks, vials, tubes, and tubing, can be adapted for use with the surfaces described herein.

[0047] Another aspect of the present disclosure is a method of making a surface suitable for cell culture applications, such as a surface as described herein. The method includes depositing an amorphous hydrogenated carbon coating on a fluoropolymer surface of a substrate. The deposition includes depositing a first thickness of the amorphous hydrogenated carbon coating by chemical vapor deposition (e.g., PECVD) using a combination of a hydrocarbon gas (e.g., ethylene or propylene) and one or more oxygen- and / or nitrogen-containing plasma reactive gases (e.g., CO2 and / or NH3) in a first ratio of hydrocarbon gas to oxygen- and / or nitrogen-containing plasma reactive gas in a first zone of the amorphous hydrogenated carbon coating. The first zone is adjacent to and extends from the fluoropolymer surface. The deposition further includes depositing a second thickness of the amorphous hydrogenated carbon coating in a second zone of the amorphous hydrogenated carbon coating by chemical vapor deposition (e.g., PECVD) using a combination of a hydrocarbon gas (e.g., ethylene or propylene) and one or more oxygen- and / or nitrogen-containing plasma reactive gases (e.g., CO and / or NH) at a second ratio of hydrocarbon gas to oxygen- and / or nitrogen-containing plasma reactive gas (e.g., CO and / or NH) that is less than the first ratio of hydrocarbon gas to oxygen- and / or nitrogen-containing plasma reactive gas. The second zone is distal from the fluoropolymer surface and at the surface of the amorphous hydrogenated carbon coating. In particular, the deposited amorphous hydrogenated carbon coating has a higher combined concentration of oxygen and nitrogen in the second zone than in the first zone.

[0048] The first and second thicknesses of the amorphous hydrogenated carbon coating can be deposited without a layer in between, such that the first zone provides a continuous surface with the second zone, as described with respect to Figure 1. In other embodiments, one or more thicknesses of the amorphous hydrogenated carbon coating can be deposited between the first and second thicknesses, as described above with respect to Figure 2. For example, in various embodiments as described elsewhere herein, the invention can further include, prior to depositing the second thickness of the amorphous hydrogenated carbon coating in the second zone of the amorphous hydrogenated carbon coating, depositing a third thickness of the amorphous hydrogenated carbon coating in the third zone of the amorphous hydrogenated carbon coating by chemical vapor deposition (e.g., PECVD) using a combination of a hydrocarbon gas (e.g., ethylene or propylene) and one or more oxygen- and / or nitrogen-containing plasma reactive gases (e.g., CO and / or NH) in a third ratio of hydrocarbon gas to oxygen- and / or nitrogen-containing plasma reactive gas that is less than the first ratio of hydrocarbon gas to oxygen- and / or nitrogen-containing plasma reactive gas. In this embodiment, after depositing a second thickness of the amorphous hydrogenated carbon coating in the second zone of the amorphous hydrogenated carbon coating as described herein, the third zone is between and contiguous with the first zone of the amorphous hydrogenated carbon coating and the second zone of the amorphous hydrogenated carbon coating. In particular, the amorphous hydrogenated carbon coating may have a higher combined concentration of oxygen and nitrogen in the third zone than in the first zone of the amorphous hydrogenated carbon coating.

[0049] Those skilled in the art can adapt conventional techniques for depositing amorphous hydrogenated carbon for use in the methods described herein. Plasma enhanced chemical vapor deposition (PECVD) is a particularly useful technique. As used herein, the hydrocarbon gas or plasma reactive gas need not be a gas under ambient conditions, but rather, be a gas under the chemical vapor deposition conditions used.

[0050] A variety of hydrocarbon gases can be used. For example, chemical vapor deposition of alkenes such as propylene or ethylene or 1,3-butadiene can be used to provide amorphous hydrogenated carbon coatings. However, other hydrocarbon gases such as alkynes (e.g., acetylene, methylacetylene) and alkanes (e.g., methane, ethane, propane) can be used.

[0051] Similarly, various oxygen- and / or nitrogen-containing gases can be used as long as they are reactive under the plasma conditions. Various oxygen-containing gases, such as alcohols (e.g., methanol, ethanol), ketones (e.g., acetone), carboxylic acids (e.g., formic acid), aldehydes (e.g., formaldehyde, acetaldehyde), and ethers (e.g., dimethyl ether), can be used to provide oxygen content to the deposited material. Carbon dioxide is a particularly desirable oxygen-containing gas. Oxygen gas can be used, but at higher concentrations, it may act as an etchant. Similarly, various nitrogen-containing gases, such as amines (e.g., methylamine, ethylamine, allylamine), imines (e.g., methanimine), and nitriles (e.g., acetonitrile), can be used to provide nitrogen content to the deposited material. Ammonia is a particularly desirable nitrogen-containing gas. Some plasma-reactive compounds can contain both oxygen and nitrogen. For example, nitrogen oxides and amides (e.g., formamide, acetamide).

[0052] By varying the relative amounts of hydrocarbon gas, oxygen-containing gas, and nitrogen-containing gas, one skilled in the art can vary the oxygen and / or nitrogen concentrations among various zones of the coating to provide the various zones described herein. For example, the mass ratio of hydrocarbon gas to oxygen-containing gas (e.g., ethylene:CO) can range from 0.5 to 20 in some embodiments, and the mass ratio of hydrocarbon gas to nitrogen-containing gas (e.g., ethylene:NH) can range from 0.1 to 3 in some embodiments. It may be desirable to use a higher ratio of hydrocarbon gas to oxygen-containing gas and / or a higher ratio of hydrocarbon gas to nitrogen-containing gas in the deposition of a first zone than in the deposition of a second zone to provide a more highly crosslinked layer in the first zone and a more highly functionalized layer in the second zone.

[0053] It should be noted that the variation in oxygen and nitrogen content can be continuous, and the concentrations need not be uniform within any zone as described herein.

[0054] As discussed above, it may be desirable for the coating to be performed on an activated fluoropolymer surface. As discussed above, an activated fluoropolymer surface can improve adhesion of the coating to the fluoropolymer surface. Thus, in various embodiments of the methods described herein, the fluoropolymer surface of the substrate on which the amorphous hydrogenated carbon coating is deposited is an activated fluoropolymer surface, for example, as described above. In various embodiments, the method further includes activating the fluoropolymer surface. The fluoropolymer surface can be activated by a plasma treatment, such as treatment with an NH3 plasma, a CO2 plasma, or a combination of NH3 and CO2. In other embodiments, the plasma is an oxygen plasma, a nitrogen plasma, or a combination of oxygen and nitrogen. In other embodiments, the fluoropolymer surface can be activated by a corona treatment, such as a C treatment. In various embodiments, as described elsewhere herein, the activated fluoropolymer surface has an oxygen concentration of less than 5% and a nitrogen concentration of less than 10%. In various embodiments, as described elsewhere herein, the activated fluoropolymer surface can have a water contact angle in the range of 60° to 120°.

[0055] Another aspect of the present disclosure is a method of culturing adherent cells, comprising incubating a surface as described herein with adherent cells and growth medium. For example, the surface may be, for example, a surface of a cell culture device (e.g., a cell culture bag, a cell culture tube, a cell culture vial, or cell culture tubing).

[0056] A variety of adherent cells can be cultured using the surfaces of the present disclosure. For example, in one embodiment, the cells are stem cells, e.g., mesenchymal stromal cells (hMSCs). In another embodiment, the cells are dendritic cells, e.g., monocyte-derived dendritic cells. Those skilled in the art will identify other cell types suitable for growth.

[0057] Those skilled in the art will identify suitable growth media for use in culturing adherent cells. In various desirable embodiments, as described elsewhere herein, the growth medium is a basal medium. In various embodiments, the growth medium comprises a buffer (e.g., sodium bicarbonate), glutamine, and growth factors. In various desirable embodiments, as described elsewhere herein, the growth medium has a pH in the range of 7.2 to 7.4.

[0058] Those skilled in the art will be able to identify appropriate incubation conditions for use in the proliferation methods described herein. For example, in various desirable embodiments, such as those described elsewhere herein, the incubation temperature for culturing adherent cells is in the range of 35-39°C (e.g., 37°C).

[0059] In particular, the surfaces and containers described herein can be sterilized prior to use in cell culture. For example, sterilization can be performed by autoclaving. It is a significant advantage that the various surfaces described herein can be sterilized by autoclaving prior to use while still retaining significant functionality. [Example]

[0060] The following examples serve to illustrate various preferred embodiments and aspects of the present disclosure and should not be construed as limiting its scope.

[0061] Example 1. Activation of fluoropolymer surfaces using CO2 plasma This example describes the activation of a fluoropolymer surface using CO2 plasma. First, a fluoropolymer surface, here a fluorinated ethylene propylene (FEP) polymer surface, is cleaned with 70% ethanol in an ultrasonic bath sonicator for 30 minutes. The fluoropolymer surface is then rinsed with reverse osmosis water and dried overnight under vacuum. The cleaned fluoropolymer surface is placed on an electrode and loaded into a PECVD reactor. PECVD reactors are generally described in M. Buddhadasa & P.-L. Girard-Lauriault, "Plasma copolymerization of ethylene, 1,3-butadiene, and ammonia mixtures: amine content and water stability," Thin Solid Films, 591, 76-85 (2015), which is incorporated herein by reference in its entirety. The surface is treated with CO2 plasma for 45 seconds at a pressure of 13.3 Pa, a radio frequency power of 60 W, and a CO2 flow rate of approximately 20 standard cubic centimeters per minute (sccm). The activated fluoropolymer surface is sealed in a low-oxygen permeable bag in a glove box under argon and stored at −40° C. In this example, the activated fluoropolymer surface was removed from the PECVD reactor and stored, but one skilled in the art will understand that activation and subsequent coating deposition can be performed sequentially within the PECVD reactor.

[0062] Example 2. Activation of fluoropolymer surfaces using ammonia (NH3) plasma This example describes the activation (e.g., treatment) of a fluoropolymer surface using NH3 plasma. First, a fluoropolymer surface, here a fluorinated ethylene propylene (FEP) polymer surface, is cleaned with 70% ethanol in an ultrasonic bath sonicator for 30 minutes. The fluoropolymer surface is then rinsed with reverse osmosis water and dried overnight under vacuum. The cleaned fluoropolymer surface is placed on an electrode and loaded into a PECVD reactor. The surface is treated with NH3 plasma for 45 seconds at a pressure of 13.3 Pa, a radio frequency power of 60 W, and an NH3 flow rate of approximately 15 sccm. The activated fluoropolymer surface is sealed in a low-oxygen permeable bag in a glove box under argon and stored at -40°C. In this example, the activated fluoropolymer surface was removed from the PECVD reactor and stored, but those skilled in the art will understand that activation and subsequent coating deposition can be performed sequentially within the PECVD reactor.

[0063] Example 3. Deposition of a coating with an oxygen-containing cross-linked layer onto an activated fluoropolymer surface This example describes the deposition of an amorphous hydrogenated carbon coating having an oxygen-containing crosslinked layer on an activated fluoropolymer surface as described herein. Specifically, the oxygen-rich crosslinked layer is deposited on the activated fluoropolymer surface by PECVD using a mixture of ethylene (C2H4) at a flow rate of about 5 sccm and CO2 at a flow rate of about 40 sccm under a pressure of 80 Pa and an RF power of 20 W for 10 minutes. The coated activated fluoropolymer surface is sealed in a low-oxygen-permeable bag in a glove box under argon and stored at -40°C.

[0064] Example 4. Deposition of a coating with an oxygen-containing functional layer onto an activated fluoropolymer surface This example describes the deposition of a coating having an oxygen-containing functional layer on an activated fluoropolymer surface as described herein. Specifically, the oxygen-containing functional layer is deposited on the activated fluoropolymer surface by PECVD using a mixture of ethylene (C2H4) at a flow rate of approximately 5 to 2.5 sccm (linearly ramped over 10 minutes) and CO2 at a flow rate of approximately 40 sccm under 80 Pa pressure and 20 W RF power for 10 minutes. The coated activated fluoropolymer surface is sealed in a low-oxygen-permeable bag in a glove box under argon and stored at -40°C.

[0065] Example 5. Deposition of a coating having an oxygen-containing crosslinked layer and an oxygen-containing functional layer onto an activated fluoropolymer surface This example describes the deposition of a coating having an oxygen-containing crosslinked layer and an oxygen-containing functional layer on an activated fluoropolymer surface as described herein. First, the oxygen-containing crosslinked layer is deposited on the activated fluoropolymer surface by PECVD using a mixture of ethylene (C2H4) at a flow rate of approximately 5 sccm and CO2 at a flow rate of approximately 40 sccm at a pressure of 80 Pa and an RF power of 20 W for 5 minutes. Then, the oxygen-containing functional layer is deposited on the oxygen-rich crosslinked layer by PECVD using a mixture of ethylene (C2H4) at a flow rate ranging from approximately 5 to 2.5 sccm (linearly ramped over 1.5 minutes) and CO2 at a flow rate of approximately 40 sccm at a pressure of 80 Pa and an RF power of 20 W for 1.5 minutes. The coated activated fluoropolymer surface is sealed in a low-oxygen-permeable bag in a glove box under argon and stored at -40°C.

[0066] Example 6. Deposition of a coating with a nitrogen- and oxygen-containing crosslinked layer onto an activated fluoropolymer surface This example describes the deposition of a coating having a nitrogen- and oxygen-containing crosslinked layer on an activated fluoropolymer surface as described herein. Specifically, the nitrogen- and oxygen-containing crosslinked layer is deposited on the activated fluoropolymer surface by PECVD using a mixture of ethylene (C2H4) at a flow rate of about 20 sccm, CO2 at a flow rate of about 20 sccm, and NH3 at a flow rate of about 5 sccm under a pressure of 80 Pa and an RF power of 20 W for 15 minutes. The coated activated fluoropolymer surface is sealed in a low-oxygen permeable bag in a glove box under argon and stored at -40°C.

[0067] Example 7. Deposition of a coating with a nitrogen-containing functional layer onto an activated fluoropolymer surface This example describes the deposition of a coating having a nitrogen-containing functional layer on an activated fluoropolymer surface as described herein. Specifically, the nitrogen-containing functional layer is deposited on the activated fluoropolymer surface by PECVD using a mixture of ethylene (C2H4) at a flow rate ranging from 20 to 10 sccm (linearly ramped over 90 seconds) and NH3 at a flow rate of approximately 15 sccm under a pressure of 80 Pa and an RF power of 20 W for 90 seconds. The coated activated fluoropolymer surface is sealed in a low-oxygen permeable bag in a glove box under argon and stored at -40°C.

[0068] Example 8. Deposition of a coating having a nitrogen- and oxygen-containing crosslinked layer and a nitrogen-containing functional layer onto an activated fluoropolymer surface This example describes the deposition of a coating having an oxygen-rich crosslinked layer and a nitrogen-rich functional layer on an activated fluoropolymer surface as described herein. First, an oxygen-containing crosslinked layer is deposited on the activated fluoropolymer surface by PECVD using a mixture of ethylene (C2H4) at a flow rate of about 20 sccm, CO2 at a flow rate of about 20 sccm, and NH3 at a flow rate of about 5 sccm, at a pressure of 80 Pa and an RF power of 20 W for 3 minutes. Next, a nitrogen-containing functional layer is deposited on the nitrogen- and oxygen-containing crosslinked layer by PECVD using a mixture of ethylene (C2H4) at a flow rate ranging from about 20 to about 10 sccm (linearly ramped over 1.5 minutes) and NH3 at a flow rate of about 15 sccm, at a pressure of 80 Pa and an RF power of 20 W for 1.5 minutes. The coated activated fluoropolymer surface is sealed in a low-oxygen-permeable bag in a glove box under argon and stored at -40°C.

[0069] Example 9. Elemental analysis of oxygen and nitrogen on the surfaces described in Examples 1-8 Elemental analysis was performed on the surfaces of Examples 1-8 by X-ray photoelectron spectroscopy (XPS). For XPS analysis, a Thermo Scientific K-Alpha instrument equipped with a single-wavelength Al Kα radiation X-ray source was used. The coated surfaces were mounted on a vacuum transfer module in a glove box and transferred to the XPS instrument to avoid any exposure to air. Survey spectra were obtained with the flood gun on, using a 400 μM spot size, a pass energy of 160 eV, and a dwell time of 200 ms. High-resolution spectra were also obtained using a pass energy of 20 eV and a dwell time of 200 ms. Surface composition was determined using Thermo Fisher Scientific Avantage Software (version: 5.9922).

[0070] Figure 4 shows the elemental percentages of oxygen and nitrogen on the surfaces described in Examples 1 to 8. Note that the data shows a small degree of secondary contamination between the examples.

[0071] The O / C and N / C atomic ratios of the coatings of Examples 3-8 are shown below in Table 1. There is some nitrogen present in the coating of Example 6 due to contamination.

[0072] [Table 1]

[0073] Example 10. Water contact angles of the surfaces described in Examples 1-8 The water contact angles of the surfaces of Examples 1 to 8 were measured.

[0074] Water contact angles were measured using a goniometer (Future Digital Scientific Corp.) connected to a video camera system and computer software (SCA 2.0). Contact angles were measured using the sessile drop method (3 μL Milli-Q water droplets) at room temperature. Average values ​​from five measurements were calculated and reported.

[0075] FIG. 5 shows the water contact angles of the surfaces described in Examples 1 to 8, respectively.

[0076] Example 11. Coating thickness of the coatings described in Examples 3-8 The thickness of the coatings in Examples 3-8 was measured using a Dektak XT (Veeco Sloan Technology) profilometer. Waviness in the FEP prevented measurements of plasma polymer thicknesses below 1 μm. Therefore, the coatings were deposited on silicon wafers for profilometry experiments. A mask covering the silicon wafer was used to create a step between the treated and untreated surfaces during plasma coating. Thickness was measured using a 12.5 μm radius stylus at 3 mg force. Each measurement was taken at room temperature with a resolution of 0.1 μm / point across the step area.

[0077] FIG. 6 shows exemplary thicknesses of coatings disposed on surfaces described in Examples 3-8, respectively.

[0078] Example 12. Effect of sterilization on surfaces described in Examples 5 and 8 This example describes the effect of sterilization by autoclave on the surfaces described in Examples 5 and 8. The effect was determined by the change in elemental percentage of oxygen and / or nitrogen on the surface upon sterilization, and by the change in thickness of the coating placed on the surface upon sterilization.

[0079] For the surface described in Example 5, the elemental percentage of oxygen on the unsterilized surface was determined to be about 29.59 atomic weight %, and the elemental percentage of oxygen on the sterilized surface was determined to decrease to about 25.24 atomic weight %. Therefore, a change of about 14.7 atomic weight % in the elemental percentage of oxygen on the surface can be considered a range for maintaining the stability of the coating on the surface.

[0080] For the surface described in Example 8, the elemental percentage of nitrogen on the unsterilized surface was determined to be about 18.79 atomic weight %, and the elemental percentage of nitrogen on the sterilized surface was determined to decrease to about 13.97 atomic weight %. Therefore, a change of about 25.7 atomic weight % in the elemental percentage of nitrogen on the surface can be considered a range for maintaining the stability of the coating on the surface.

[0081] The thickness of a coating disposed on a surface tends to increase after surface sterilization. Without being bound by theory, the inventors speculate that this may be due to thermal expansion and swelling of the fluoropolymer layer on the surface. For example, the thickness of the coating on a sterilized surface as described in Example 5 is determined to increase by about 59.7% compared to a non-sterilized surface. Furthermore, the thickness of the coating on a sterilized surface as described in Example 8 is determined to increase by about 1.1% compared to a non-sterilized surface.

[0082] Example 13. Cell attachment and proliferation of human mesenchymal stromal cells (hMSCs) on coated surfaces described in Examples 5 and 8, respectively This example describes cell attachment and proliferation of hMSCs on the coated surfaces described in Examples 5 and 8, respectively. For comparison, this example also describes cell attachment and proliferation of hMSCs on unactivated (e.g., untreated) fluoropolymer surfaces, activated (e.g., treated) fluoropolymer surfaces, and commercially available tissue culture polystyrene (TCPS) surfaces (e.g., Sarstedt, red and yellow) under similar experimental conditions.

[0083] Cell adhesion and proliferation were measured by measuring the cell seeding density (cells / cm) of cells attached on the surface. 2 ) can be determined. For cell culture testing, custom wells with various FEP surfaces were prepared by attaching FEP film to the bottom of CultureWell™ 8-well removable chamber slides (Gracebio) under sterile conditions. Human bone marrow-derived mesenchymal stromal cells (hMSCs, Poietics™, Lonza) were cultured using the StemMACS™ MSC Expansion Medium XF Kit (StemMACS™, Miltenyi Biotech). Frozen cells were thawed and plated in TCPS T-75 flasks (Sarstedt™, red cap) at 5,000–6,000 cells / cm. 2 Cells were seeded at a density of 1000 x 1000 cells / cm on FEP (untreated), FEP (treated), O-GPPC, N-hyb-GPPC, TCPS (Sarstedt®, red), and TCPS (Sarstedt®, yellow) surfaces, with the medium changed on day 3. When cells reached confluence, they were washed twice with Dulbecco's phosphate-buffered saline (DPBS) before being removed from the surface with TrypLE (Thermo Fisher Scientific). Cells were plated at 5000 cells / cm on FEP (untreated), FEP (treated), O-GPPC, N-hyb-GPPC, TCPS (Sarstedt®, red), and TCPS (Sarstedt®, yellow) surfaces. 2The plated cells were incubated at 37°C and 5% CO2. The proliferating cells on days 1 and 3 were fixed, permeabilized, and stained. DAPI was used to stain the nuclei of adherent cells. Fluorescent images were acquired from 21 positions within each well using an Olympus IX81 microscope (10x objective). The DAPI-stained adherent cells were analyzed and counted using ImageJ.

[0084] Figure 7 shows cell adhesion and proliferation of hMSCs on the coated surfaces described in Examples 5 and 8 compared to unactivated, activated, and commercially available TCPS surfaces, respectively. Overall, Figure 7 shows that hMSCs tend to adhere better to both coated surfaces compared to unactivated, activated, and commercially available TCPS surfaces under similar experimental conditions.

[0085] Specifically, Figure 7 shows that the cell seeding density of hMSCs on the coated surface is higher than the cell seeding density on the other tested surfaces (i.e., unactivated fluoropolymer surface, activated fluoropolymer surface, and TCPS surface) on day 1. Figure 8 also shows that the cell seeding density of hMSCs on the coated surface on day 3 is more than three-fold higher than the cell seeding density on the same surface on day 1, and that the cell seeding density of cells on the coated surface remains higher than the cell seeding density on the other tested surfaces on day 3. Thus, Figure 7 indicates that the surface chemistry of the cell culture surface, e.g., whether the surface is activated or unactivated and / or whether the surface is coated or uncoated, can significantly affect cell adhesion and proliferation of hMSCs on the surface, with coated surfaces tending to significantly promote cell adhesion and proliferation under similar experimental conditions.

[0086] Example 14. Cell adhesion and proliferation of monocyte-derived dendritic cells on coated surfaces described in Examples 5 and 8, respectively This example describes the cell attachment and proliferation of monocyte-derived dendritic cells on the coated surfaces described in Examples 5 and 8, respectively. For comparison, this example also describes the cell attachment and proliferation of monocyte-derived dendritic cells on unactivated (e.g., untreated) fluoropolymer surfaces, activated (e.g., treated) fluoropolymer surfaces, and commercially available TCPS surfaces under similar experimental conditions.

[0087] Isolation of primary human monocytes from fresh whole blood: Primary human monocytes were obtained through blood donations from healthy donors. Fresh blood was collected at McGill University Health Center and transported to the Stem Cell Bioprocessing Laboratory for further processing. The blood was first diluted 1:1 with DPBS supplemented with 2% human serum albumin (HSA, Sigma, catalog number A9080). The diluted blood was then gently layered on top of Histopaque®-1077 (Sigma, catalog number 10771) in a SepMate™-50 tube (STEMCELL™ Technologies, catalog number 85450) and subjected to density gradient centrifugation at 1200 × g. The centrifuged blood consisted of multiple layers. The buffy coat layer containing peripheral blood mononuclear cells (PBMCs) was collected into a new 50 mL conical tube (Fisher, catalog number 1443222). The tube was centrifuged at 300 × g for 8 minutes, after which the supernatant was aspirated and the cell pellet, consisting of the desired PBMCs, was resuspended in the residual fluid. Cells from a single donor were pooled into one tube and washed twice by filling to a volume of 50 mL with DPBS supplemented with 2% HSA and centrifuging at 300 × g for 8 minutes. The supernatant was aspirated, and the cell pellet was resuspended in a CryoStor® CS10 (STEMCELL™ Technologies, catalog number 07959) to a cell concentration of 0.5–50 × 106 cells / mL. The vials were kept in a -80°C freezer for 24 hours and then transferred to liquid nitrogen for long-term storage after 24 hours.

[0088] Monocyte enrichment from frozen PBMCs: On the day of the experiment, frozen PBMCs were thawed in a 37°C water bath and then diluted 1:1 in Plasma-lyte A (ThermoFisher, catalog no. NC1531549) containing 10% heat-inactivated umbilical cord plasma. The contents of the cryovial were transferred to a 15 mL conical tube and centrifuged at 300 × g for 5 minutes to remove the DMSO in CryoStor® medium, which is toxic to the cells. The cells were then resuspended at a concentration of 100 million cells / mL in DPBS supplemented with 2% HSA and 1 mM EDTA (ThermoFisher, catalog no. 15575020). Monocytes were then positively selected using the EasySep™ Human CD14 Positive Selection Kit II (STEMCELL™ Technologies). Monocytes were enriched by immunomagnetic cell sorting by labeling CD14+ cells with a cocktail of tetrameric antibodies targeting the CD14 antigen and dextran-coated magnetic particles according to the manufacturer's protocol. Isolated monocytes were suspended at a concentration of 1.0 x 106 cells / mL in ImmunoCult™-ACF dendritic cell medium (STEMCELL™ Technologies) containing differentiation factors (GM-CSF 50 ng / mL; IL-4 35 ng / mL).

[0089] Cell adhesion, differentiation, and maturation of monocyte-derived dendritic cells: For cell culture studies, custom wells with various FEP surfaces were prepared by attaching FEP film to the bottom of CultureWell™ 8-well removable chamber slides (Gracebio) under sterile conditions.

[0090] Day 0: Cells were seeded at a density of 1,500 cells / mm2 onto FEP (untreated), FEP (treated), O-GPPC, N-hyb-GPPC, and TCPS (Sarstedt®, red) surfaces in ImmunoCult™-ACF dendritic cell medium (STEMCELL™ Technologies) containing differentiation factors (GM-CSF 50 ng / mL; IL-4 35 ng / mL). Seeded cells were incubated at 37°C and 5% CO2 and allowed to adhere for 2 hours.

[0091] Quantification of Adherent Cells: After incubation, nonadherent cells were aspirated from each well, and adherent cells were rinsed off the surface by washing three times with DPBS. Adherent cells were stained by incubating them in ethidium homodimer and Hoechst mixture diluted 1:400 in DPBS for 15 minutes. Stained cells were counted by acquiring fluorescent images from 21 positions within each well using an Olympus IX81 inverted fluorescent microscope (10x objective). Stained adherent cells were analyzed and counted using ImageJ.

[0092] Day 3 - Differentiation Medium Change: Fresh Immunocult Differentiation Medium was prepared with 2x differentiation factors (GM-CSF = 50 ng / mL; IL-4 = 35 ng / mL). Samples were removed from the incubator and Differentiation Medium was added to each well. The chamber was gently shaken to mix the medium in the wells, and the cells were again incubated at 37°C and 5% CO2.

[0093] Day 5 - Quantification of Adherent Cells: After incubation, nonadherent cells were aspirated from each well, and adherent cells were rinsed off the surface by washing three times with DPBS. Adherent cells were stained by incubating them in ethidium homodimer and Hoechst mixture diluted 1:400 in DPBS for 15 minutes. Stained cells were counted by acquiring fluorescent images from 21 positions within each well using an Olympus IX81 inverted fluorescent microscope (10x objective). Stained adherent cells were analyzed and counted using ImageJ.

[0094] Day 5 - Maturation Medium Change: For dendritic cell maturation, fresh Immunocult maturation medium was prepared with differentiation factors (GM-CSF = 50 ng / mL; IL-4 = 35 ng / mL) and maturation factors (MPLA 2.5 μg / mL, IFN-γ 1000 U / mL). Samples were removed from the incubator, and the medium containing non-adherent cells was aspirated from the wells and transferred to labeled 15 mL centrifuge tubes. To avoid death of adherent cells, maturation medium was immediately added to empty wells. The aspirated medium was centrifuged at 300 × g for 5 minutes in the 15 mL tubes. The supernatant was aspirated, and the cell pellet was resuspended in the required maturation medium. The resuspended cell suspension was added to each well, and the cells were incubated at 37 °C and 5% CO2.

[0095] Day 7 - Quantification of Adherent Cells: After incubation, nonadherent cells were aspirated from each well, and adherent cells were rinsed off the surface by washing three times with DPBS. Adherent cells were stained by incubating them in ethidium homodimer and Hoechst mixture diluted 1:400 in DPBS for 15 minutes. Stained cells were counted by acquiring fluorescent images from 21 positions within each well using an Olympus IX81 inverted fluorescent microscope (10x objective). Stained adherent cells were analyzed and counted using ImageJ.

[0096] Figure 8 shows cell adhesion and proliferation of monocyte-derived dendritic cells on the coated surfaces described in Examples 5 and 8, compared to unactivated fluoropolymer surfaces, activated fluoropolymer surfaces, and commercial TCPS surfaces, respectively. Overall, Figure 8 shows that cells tend to adhere better to both coated surfaces compared to unactivated fluoropolymer surfaces, and that cell adhesion of cells on both coated surfaces is comparable to cell adhesion on activated fluoropolymer surfaces and commercial TCPS surfaces under similar experimental conditions.

[0097] Specifically, Figure 8 shows that at 2 hours, the cell seeding density of monocyte cells on the coated surface described in Example 5 is more than twice that on an unactivated fluoropolymer surface. Figure 8 also shows that at 2 hours, the cell seeding density of monocyte cells on the coated surface described in Example 8 is approximately 1.5-fold higher than that on an unactivated fluoropolymer surface and comparable to that on an activated fluoropolymer surface and a commercial TCPS surface. Furthermore, at either day 5 or day 7, the cell seeding density of dendritic cells on all of the tested surfaces is shown to be lower than that at 2 hours, but cell adhesion of cells on both coated surfaces is comparable to that on an activated fluoropolymer surface and a commercial TCPS surface under similar experimental conditions.

[0098] Various aspects of the present disclosure are further illustrated by the following list of numbered embodiments, which may be combined in any combination and in any number that is not logically or technically consistent. Embodiment 1. A surface suitable for cell culture, comprising: a substrate having a fluoropolymer surface; 1. A surface comprising: an amorphous hydrogenated carbon coating disposed on a fluoropolymer surface of a substrate, the amorphous hydrogenated carbon coating having a first thickness zone proximate to and extending from the fluoropolymer surface and a second thickness zone distal to the fluoropolymer surface and at a surface of the amorphous hydrogenated carbon coating, the amorphous hydrogenated carbon coating having a higher combined concentration of oxygen and nitrogen in the second thickness zone than in the first thickness zone. Embodiment 2. The surface of claim 1, wherein the fluoropolymer surface is a fluorinated ethylene propylene (FEP) surface. Embodiment 3. The surface of claim 1, wherein the fluoropolymer surface is a polytetrafluoroethylene (PTFE) surface, a perfluoroalkoxy (PFA) surface, an ethylene tetrafluoroethylene (ETFE) surface, a polyvinylidene fluoride (PVDF) surface, a polychlorotrifluoroethylene (PCTFE) surface, an ethylene chlorotrifluoroethylene (ECTFE) surface, an ethylene fluorinated ethylene propylene (EFEP) surface, a perfluoropolyether (PFPE) surface, a modified polytetrafluoroethylene (TFM) surface, a polyvinyl fluoride surface, or a combination of any two or more thereof. Embodiment 4. The surface of any one of claims 1 to 3, wherein the fluoropolymer surface is the surface of a fluoropolymer-silicone laminate film. Embodiment 5. The fluoropolymer surface has a viscosity of at least 1500 cc / m 2 -day-atm, e.g., at least 1800cc / m 2 -day-atm, or 1500-20000cc / m 2 -day-atm, or 1800-16000cc / m 2 5. The surface according to claim 1, which is the surface of a film having an oxygen permeability in the range of -day-atm. Embodiment 6. The fluoropolymer surface has a viscosity of at least 3500 cc / m 2 -day-atm, e.g., 4000cc / m 2 -day-atm, or 3500-25000cc / m 2 -Sun-ATM, 4000~23000cc / m 2 6. The surface according to claim 1, which is the surface of a film having a carbon dioxide permeability in the range of -day-atm. Embodiment 7. The surface of any one of claims 1 to 6, wherein the fluoropolymer surface is an activated fluoropolymer surface. Embodiment 8. The surface of claim 7, wherein the activated fluoropolymer surface has an oxygen concentration of less than 5 atomic % and a nitrogen concentration of less than 10 atomic %. Embodiment 9. The surface of claim 7 or 8, wherein the activated fluoropolymer surface has a water contact angle in the range of 60° to 120°. Embodiment 10. The surface of any one of claims 7 to 9, wherein the fluoropolymer surface is activated by plasma treatment. Embodiment 11. The surface of claim 10, wherein the plasma is an ammonia plasma, a CO2 plasma, or a plasma of a combination of ammonia and CO2. Embodiment 12. The surface of claim 10, wherein the plasma is an oxygen plasma, a nitrogen plasma, or a plasma of a combination of oxygen and nitrogen. Embodiment 13. The surface of any one of claims 7 to 9, wherein the fluoropolymer surface is activated by a corona treatment, such as a C treatment. Embodiment 14. The surface of any one of claims 1 to 13, wherein the amorphous hydrogenated carbon coating has a thickness in the range of 10 to 200 nm, e.g., 10 to 100 nm, or 10 to 75 nm, or 10 to 50 nm, or 15 to 200 nm, or 15 to 100 nm, or 15 to 75 nm, or 15 to 50 nm, or 20 to 200 nm, or 20 to 100 nm, or 20 to 75 nm, or 20 to 50 nm. Embodiment 15. The surface of any one of claims 1 to 14, wherein the amorphous hydrogenated carbon coating has a hydrogen to carbon ratio in the range of at least 1, e.g., at least 1.2, or at least 1.4. Embodiment 16. The surface of any one of claims 1 to 15, wherein the first thickness zone of the amorphous hydrogenated carbon coating has a thickness in the range of 8 to 190 nm, e.g., 8 to 100 nm, or 8 to 75 nm, or 8 to 50 nm, or 15 to 190 nm, or 15 to 100 nm, or 15 to 75 nm, or 15 to 50 nm, or 20 to 190 nm, or 20 to 100 nm, or 20 to 75 nm, or 20 to 50 nm. Embodiment 17. The surface of any one of claims 1 to 16, wherein in the first thickness zone, the amorphous hydrogenated carbon coating has a combined oxygen and nitrogen concentration that is at least 3 atomic % lower, e.g., at least 4 atomic % lower, or at least 5 atomic % lower, or at least 6 atomic % lower, or at least 7 atomic % lower, than the combined oxygen and nitrogen concentration in the second thickness zone. Embodiment 18. The surface of any one of claims 1 to 17, wherein the amorphous hydrogenated carbon coating has a combined oxygen and nitrogen concentration in the first thickness zone of at least 5 atomic %, e.g., at least 10 atomic %, or at least 15 atomic %. Embodiment 19. The surface of any one of claims 1 to 18, wherein the second thickness zone of the amorphous hydrogenated carbon coating has a thickness in the range of 2 to 100 nm, e.g., 2 to 50 nm, or 2 to 35 nm, or 2 to 20 nm, or 2 to 10 nm, or 5 to 100 nm, or 5 to 50 nm, or 5 to 35 nm, or 5 to 20 nm, or 5 to 10 nm, or 10 to 100 nm, or 10 to 50 nm, or 10 to 35 nm, or 10 to 20 nm, or 20 to 100 nm, or 20 to 50 nm. Embodiment 20. The surface of any one of claims 1 to 19, wherein in the second thickness zone, the amorphous hydrogenated carbon coating has a combined oxygen and nitrogen concentration of at least 10 atomic %, e.g., at least 15 atomic %, or at least 20 atomic %. Embodiment 21. The surface of any one of claims 1 to 20, wherein in the first thickness zone, the amorphous hydrogenated carbon coating comprises oxygen. Embodiment 22. The surface of claim 21, wherein the amorphous hydrogenated carbon coating in the first thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and oxygen. Embodiment 23. The surface of claim 21 or 22, wherein in the second thickness zone, the amorphous hydrogenated carbon coating comprises oxygen. Embodiment 24. The surface of any one of claims 21 to 23, wherein the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, such as at least 95 atomic %, carbon and oxygen. Embodiment 25. The surface of claim 21 or 22, wherein in the second thickness zone, the amorphous hydrogenated carbon coating comprises nitrogen. Embodiment 26. The surface of any one of claims 21, 22, and 25, wherein the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and nitrogen. Embodiment 27. The surface of claim 21 or 22, wherein in the second thickness zone, the amorphous hydrogenated carbon coating comprises oxygen and nitrogen. Embodiment 28. The surface of any one of claims 21, 22, and 27, wherein the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon, oxygen, and nitrogen. Embodiment 29. The surface of any one of claims 1 to 20, wherein in the first thickness zone, the amorphous hydrogenated carbon coating comprises oxygen and nitrogen. Embodiment 30. The surface of claim 29, wherein the amorphous hydrogenated carbon coating in the first thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon, oxygen, and nitrogen. Embodiment 31. The surface of claim 29 or 30, wherein in the second thickness zone, the amorphous hydrogenated carbon coating comprises oxygen. Embodiment 32. The surface of any one of claims 29 to 31, wherein at least 90 atomic %, e.g., at least 95 atomic %, of the amorphous hydrogenated carbon coating in the second thickness zone is composed of carbon and oxygen. Embodiment 33. The surface of claim 29 or 30, wherein in the second thickness zone, the amorphous hydrogenated carbon coating comprises nitrogen. Embodiment 34. The surface of any one of claims 29, 30, and 33, wherein the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon and nitrogen. Embodiment 35. The surface of claim 29 or 30, wherein in the second thickness zone, the amorphous hydrogenated carbon coating comprises oxygen and nitrogen. Embodiment 36. The surface of any one of claims 29, 30, and 35, wherein the amorphous hydrogenated carbon coating in the second thickness zone is composed of at least 90 atomic %, e.g., at least 95 atomic %, carbon, oxygen, and nitrogen. Embodiment 37. The surface of any one of claims 1 to 20, wherein in the first thickness zone, the amorphous hydrogenated carbon coating comprises nitrogen. Embodiment 38. The surface of claim 37, wherein at least 90 atomic %, e.g., at least 95 atomic %, of the amorphous hydrogenated carbon coating in the first thickness zone is composed of carbon and nitrogen. Embodiment 39. The surface of claim 37 or 38, wherein in the second thickness zone, the amorphous hydrogenated carbon coating comprises oxygen. Embodiment 40. The surface of any one of claims 37 to 39, wherein at least 90 atomic %, e.g., at least 95 atomic %, of the amorphous hydrogenated carbon coating in the second thickness zone is composed of carbon and oxygen. Embodiment 41. The surface of claim 37 or 38, wherein in the second thickness zone, the amorphous hydrogenated carbon coating comprises nitrogen. Embodiment 42. The surface of any one of claims 37, 38, and 41, wherein at least 90 atomic %, e.g., at least 95 atomic %, of the amorphous hydrogenated carbon coating in the second thickness zone is composed of carbon and nitrogen. Embodiment 43. The surface of claim 37 or 38, wherein in the second thickness zone, the amorphous hydrogenated carbon coating comprises oxygen and nitrogen. Embodiment 44. The surface of any one of claims 37, 38, and 43, wherein at least 90 atomic %, e.g., at least 95 atomic %, of the amorphous hydrogenated carbon coating in the second thickness zone is composed of carbon, nitrogen, and oxygen. Embodiment 45. The surface of any one of claims 1 to 44, wherein the first thickness zone of the amorphous hydrogenated carbon coating is contiguous with the second thickness zone of the amorphous hydrogenated carbon coating. Embodiment 46. The surface of any one of claims 1 to 44, wherein one or more additional thickness zones of amorphous hydrogenated carbon coating are disposed between the first thickness zone of amorphous hydrogenated carbon coating and the second thickness zone of amorphous hydrogenated carbon coating. Embodiment 47. The surface of claim 46, wherein the one or more additional thickness zones is a third thickness zone disposed between and contiguous with the first thickness zone of the amorphous hydrogenated carbon coating and the second thickness zone of the amorphous hydrogenated carbon coating. Embodiment 48. The surface of claim 47, wherein the second thickness zone comprises nitrogen and the third thickness zone comprises oxygen and / or nitrogen, and wherein the amorphous hydrogenated carbon coating has a higher combined concentration of oxygen and nitrogen in the third thickness zone than in the first thickness zone. Embodiment 49. The surface of any one of claims 1 to 48, which is the inner surface of a cell culture vessel, such as a cell culture bag, cell culture flask, cell culture vial, cell culture tube, or cell culture tubing. Embodiment 50. A method of making a surface suitable for cell culture, such as the surface of any one of claims 1 to 49, comprising the steps of: depositing an amorphous hydrogenated carbon coating on a fluoropolymer surface of a substrate, said deposition comprising: depositing a first thickness of the amorphous hydrogenated carbon coating by chemical vapor deposition using a combination of a hydrocarbon gas and one or more oxygen and / or nitrogen containing plasma reactive gases (e.g., CO and / or NH) in a first ratio of hydrocarbon gas to oxygen and / or nitrogen containing plasma reactive gas in a first zone of the amorphous hydrogenated carbon coating, the first zone being adjacent to and extending from the fluoropolymer surface; depositing a second thickness of the amorphous hydrogenated carbon coating in a second zone of the amorphous hydrogenated carbon coating by chemical vapor deposition (e.g., PECVD) using a combination of a hydrocarbon gas (e.g., ethylene or propylene) and one or more oxygen- and / or nitrogen-containing plasma reactive gases (e.g., CO and / or NH) in a second ratio of hydrocarbon gas to oxygen- and / or nitrogen-containing plasma reactive gas that is less than the first ratio of hydrocarbon gas to oxygen- and / or nitrogen-containing plasma reactive gas, wherein the second zone is distal from the fluoropolymer surface and at a surface of the amorphous hydrogenated carbon coating, and wherein the amorphous hydrogenated carbon coating has a higher combined concentration of oxygen and nitrogen in the second zone of the amorphous hydrogenated carbon coating than in the first zone of the amorphous hydrogenated carbon coating. Embodiment 51. The method of claim 50, further comprising, prior to depositing the second thickness of the amorphous hydrogenated carbon coating in the second zone of the amorphous hydrogenated carbon coating, depositing a third thickness of the amorphous hydrogenated carbon coating in a third zone of the amorphous hydrogenated carbon coating by chemical vapor deposition (e.g., PECVD) using a combination of a hydrocarbon gas (e.g., ethylene or propylene) and one or more oxygen- and / or nitrogen-containing plasma reactive gases (e.g., CO2 and / or NH3) in a third ratio of hydrocarbon gas to oxygen- and / or nitrogen-containing plasma reactive gas that is less than the first ratio of hydrocarbon gas to oxygen- and / or nitrogen-containing plasma reactive gas, wherein the third zone is between and contiguous with the first and second zones, and the amorphous hydrogenated carbon coating has a higher combined concentration of oxygen and nitrogen in the third zone than in the first zone of the amorphous hydrogenated carbon coating. Embodiment 52. The method of claim 50 or 51, wherein the chemical vapor deposition is plasma-enhanced chemical vapor deposition. Embodiment 53. The method of any one of claims 50 to 52, wherein the hydrocarbon gas is an alkene, such as propylene or ethylene. Embodiment 54. The method of any one of claims 50-53, wherein the fluoropolymer surface of the substrate onto which the amorphous hydrogenated carbon coating is deposited is an activated fluoropolymer surface. Embodiment 55. The method of claim 54, further comprising activating the fluoropolymer surface. Embodiment 56. The method of claim 55, wherein the fluoropolymer surface is activated by plasma treatment, such as treatment with NH3 plasma, CO2 plasma, a combination of NH3 and CO2 plasma, oxygen plasma, nitrogen plasma, or a combination of oxygen and nitrogen plasma. Embodiment 57. The method of claim 55, wherein the fluoropolymer surface is activated by corona treatment, such as C treatment. Embodiment 58. The method of any one of claims 54 to 57, wherein the activated fluoropolymer surface has an oxygen concentration of less than 5 atomic % and a nitrogen concentration of less than 10 atomic %. Embodiment 59. The method of any one of claims 54 to 58, wherein the activated fluoropolymer surface has a water contact angle in the range of 60° to 120°. Embodiment 60. A cell culture surface (e.g., as described in any one of claims 1 to 49) produced by the method of any one of claims 50 to 59. Embodiment 61. A method for culturing adherent cells, comprising incubating the surface of any one of claims 1 to 49 and 60 with adherent cells and growth medium. Embodiment 62. The method of claim 61, wherein the adherent cells are stem cells, e.g., mesenchymal stromal cells. Embodiment 63. The method of claim 61, wherein the adherent cells are dendritic cells, e.g., monocyte-derived dendritic cells. Embodiment 64. The method of any one of claims 61 to 63, wherein the growth medium is a basal medium. Embodiment 65. The method of any one of claims 61 to 64, wherein the growth medium comprises a buffer (e.g., sodium bicarbonate), glutamine, and growth factors. Embodiment 66. The method of any one of claims 61 to 65, wherein the growth medium has a pH in the range of 7.2 to 7.4. Embodiment 67. The method of any one of claims 61 to 66, wherein the incubation temperature for culturing adherent cells is in the range of 35 to 39°C (e.g., 37°C). Embodiment 68. The method of any one of claims 61 to 67, wherein the surface is sterilized before being incubated with the cells. Embodiment 68. The method of claim 68, wherein sterilization is performed by autoclaving.

Claims

1. 1. A surface suitable for cell culture, comprising: a substrate having a fluoropolymer surface; an amorphous hydrogenated carbon coating disposed on the fluoropolymer surface of the substrate, the amorphous hydrogenated carbon coating having a first thickness zone proximate to and extending from the fluoropolymer surface and a second thickness zone distal to the fluoropolymer surface and at a surface of the amorphous hydrogenated carbon coating, the amorphous hydrogenated carbon coating having a higher combined concentration of oxygen and nitrogen in the second thickness zone than in the first thickness zone.

2. The surface of claim 1 , wherein the fluoropolymer surface is a fluorinated ethylene propylene (FEP) surface.

3. The fluoropolymer surface has a density of at least 1500 cc / m ２ -day-atm oxygen permeability and at least 3500 cc / m ２ 2. The surface of claim 1, which is the surface of a film having a carbon dioxide permeability of -day-atm.

4. 10. The surface of claim 1, wherein the fluoropolymer surface is an activated fluoropolymer surface having a water contact angle in the range of 60° to 120°.

5. The surface of claim 4 , wherein the fluoropolymer surface is activated by plasma treatment.

6. The surface of claim 1 , wherein the amorphous hydrogenated carbon coating has a thickness in the range of 10 to 200 nm.

7. The surface of claim 1 , wherein the amorphous hydrogenated carbon coating has a hydrogen to carbon ratio in the range of at least 1.

8. The surface of claim 1 , wherein the first thickness zone of the amorphous hydrogenated carbon coating has a thickness in the range of 8 to 190 nm.

9. 10. The surface of claim 1, wherein the amorphous hydrogenated carbon coating has a combined oxygen and nitrogen concentration that is at least 3 atomic % less than the combined oxygen and nitrogen concentration in the second thickness zone.

10. 2. The surface coating of claim 1, wherein the amorphous hydrogenated carbon coating has a combined oxygen and nitrogen concentration in the first thickness zone of at least 5 atomic %, e.g., at least 10 atomic %, or at least 15 atomic %.

11. 2. The surface of claim 1, wherein the amorphous hydrogenated carbon coating has a thickness in the second thickness zone in the range of 2 to 100 nm, for example, 2 to 50 nm, or 2 to 35 nm, or 2 to 20 nm, or 2 to 10 nm, or 5 to 100 nm, or 5 to 50 nm, or 5 to 35 nm, or 5 to 20 nm, or 5 to 10 nm, or 10 to 100 nm, or 10 to 50 nm, or 10 to 35 nm, or 10 to 20 nm, or 20 to 100 nm, or 20 to 50 nm.

12. 10. The surface of claim 1, wherein in the second thickness zone, the amorphous hydrogenated carbon coating has a combined oxygen and nitrogen concentration of at least 10 atomic %, e.g., at least 15 atomic %, or at least 20 atomic %.

13. 10. The surface of claim 1, wherein a third thickness zone is disposed between and contiguous with the first thickness zone of the amorphous hydrogenated carbon coating and the second thickness zone of the amorphous hydrogenated carbon coating.

14. 14. The surface of claim 13, wherein the second thickness zone comprises nitrogen and the third thickness zone comprises oxygen and / or nitrogen, and wherein the amorphous hydrogenated carbon coating has a higher combined concentration of oxygen and nitrogen in the third thickness zone than in the first thickness zone.

15. 10. The surface of claim 1, which is the inner surface of a cell culture vessel.

16. 10. A method for preparing a surface suitable for cell culture according to claim 1, comprising: depositing an amorphous hydrogenated carbon coating on a fluoropolymer surface of a substrate, said depositing comprising: Chemical vapor deposition involves the deposition of a hydrocarbon gas and one or more oxygen- and / or nitrogen-containing plasma reactive gases (e.g., CO ２ and / or NH ３ ) in a first ratio of hydrocarbon gas to oxygen and / or nitrogen containing plasma reactive gas to deposit a first thickness of the amorphous hydrogenated carbon coating in a first zone of the amorphous hydrogenated carbon coating, the first zone being adjacent to and extending from the fluoropolymer surface; Chemical vapor deposition (e.g., PECVD) involves the deposition of a hydrocarbon gas (e.g., ethylene or propylene) and one or more oxygen- and / or nitrogen-containing plasma reactive gases (e.g., CO ２ and / or NH ３ ) in a second ratio of hydrocarbon gas to oxygen and / or nitrogen containing plasma reactive gas that is less than the first ratio of hydrocarbon gas to oxygen and / or nitrogen containing plasma reactive gas to deposit a second thickness of the amorphous hydrogenated carbon coating in a second zone of the amorphous hydrogenated carbon coating, the second zone being distal from the fluoropolymer surface and at a surface of the amorphous hydrogenated carbon coating, the amorphous hydrogenated carbon coating having a higher combined concentration of oxygen and nitrogen in the second zone of the amorphous hydrogenated carbon coating than in the first zone of the amorphous hydrogenated carbon coating.

17. 1. A method of preparing a surface suitable for cell culture, comprising: depositing an amorphous hydrogenated carbon coating on a fluoropolymer surface of a substrate, said depositing comprising: Chemical vapor deposition involves the deposition of a hydrocarbon gas and one or more oxygen- and / or nitrogen-containing plasma reactive gases (e.g., CO ２ and / or NH ３ ) in a first ratio of hydrocarbon gas to oxygen and / or nitrogen containing plasma reactive gas to deposit a first thickness of the amorphous hydrogenated carbon coating in a first zone of the amorphous hydrogenated carbon coating, the first zone being adjacent to and extending from the fluoropolymer surface; Chemical vapor deposition (e.g., PECVD) involves the deposition of a hydrocarbon gas (e.g., ethylene or propylene) and one or more oxygen- and / or nitrogen-containing plasma reactive gases (e.g., CO ２ and / or NH ３ ) in a second ratio of hydrocarbon gas to oxygen and / or nitrogen containing plasma reactive gas that is less than the first ratio of hydrocarbon gas to oxygen and / or nitrogen containing plasma reactive gas to deposit a second thickness of the amorphous hydrogenated carbon coating in a second zone of the amorphous hydrogenated carbon coating, the second zone being distal from the fluoropolymer surface and at a surface of the amorphous hydrogenated carbon coating, the amorphous hydrogenated carbon coating having a higher combined concentration of oxygen and nitrogen in the second zone of the amorphous hydrogenated carbon coating than in the first zone of the amorphous hydrogenated carbon coating.

18. 18. The method of claim 17, wherein the hydrocarbon gas is an alkene.

19. 20. A cell culture surface made by the method of claim 17.

20. 10. A method for culturing adherent cells, comprising incubating the surface of claim 1 with adherent cells and growth medium.

Images