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

Abstract

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

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 384,466, filed on November 21, 2022, which is hereby incorporated by reference in its entirety.

[0003] Background of the Present Disclosure Technical Field

[0004] The present disclosure generally relates to coatings for cell culture devices. More specifically, the present disclosure relates to coated surfaces suitable for cell culture, methods for preparing such surfaces, and methods for culturing adherent cells on such surfaces. Background Art

[0005] In vitro cell culture is a complex process in which cells are grown under controlled conditions, which are typically outside of the natural environment but under conditions similar enough to in - vivo conditions to allow growth and, in the case of adherent cells, to allow adhesion to a growth surface. It is typically cells derived from multicellular eukaryotes, especially cultured animal cells. However, cells from plants, fungi, and insects, as well as microorganisms including viruses, bacteria, and protists, can also be cultured.

[0006] In vitro cell culture can not only provide materials for research but also for various applications in pharmacology, physiology, and toxicology. Cell culture can also be desirably used in bioprocessing and cell therapy.

[0007] In a typical cell - culture process, cells can be grown in an incubator and maintained at an appropriate temperature and gas mixture. Generally, mammalian cells are incubated at 37 °C, where the pH is maintained between 7.2 and 7.4. The pH can be controlled using a bicarbonate - buffer system in the medium in combination with an incubator atmosphere of about 5 vol% to 7 vol% carbon dioxide. Carbon dioxide reacts with water to form carbonic acid, which in turn interacts with bicarbonate ions in the medium to form a buffer system that can maintain the pH near physiological levels. For many desired cell types, oxygen is necessary for cell metabolism and growth. For each cell type, the culture conditions may vary, and changing the conditions for a specific cell type may result in the expression of different phenotypes. For example, the bicarbonate - based buffer can be replaced with monosodium and disodium or trisodium phosphate buffers, chloride and ammonia buffers, lactate, or organic buffers such as HEPES, etc.

[0008] Commercially available cell culture containers in the form of cell culture bags are a conventional form for cell culture. Cell culture bags have the advantage of being disposable, reducing preparation and cleaning times. Additionally, cell culture bags are pre-sterilizable, inexpensive, easy to use, and require minimal storage and usage space. Disposables also help reduce the risk of cell culture contamination and environmental pollution.

[0009] Cell culture bags typically have an inner surface of fluoropolymer. Fluoropolymer surfaces are highly advantageous because they can have very low levels of leachable organic materials, which can be important in many cell culture applications. Fluoropolymer surfaces also resist protein adsorption. From the perspective of manufacturability and sterilizability, fluoropolymer surfaces are further advantageous.

[0010] However, fluoropolymer surfaces generally have a low surface energy, and thus, adherent cells may have difficulty adhering to such surfaces. This can render cell culture systems (e.g., cell culture devices) based on fluoropolymer surfaces much less effective for culturing adherent cells.

[0011] Accordingly, there remains a need for a surface suitable for cell culture, particularly for adherent cell culture. SUMMARY OF THE INVENTION

[0012] Accordingly, one aspect of the present disclosure is a surface suitable for cell culture. The surface includes a substrate having a fluoropolymer surface; and

[0013] an amorphous hydrogenated carbon coating disposed on the fluoropolymer surface of the substrate, the amorphous hydrogenated carbon coating having a first thickness region and a second thickness region, the first thickness region being adjacent to and extending from the fluoropolymer surface, the second thickness region being remote from the fluoropolymer surface and at the surface of the amorphous hydrogenated carbon coating, the amorphous hydrogenated carbon coating having a higher total concentration of oxygen and nitrogen in the second thickness region than in the first thickness region.

[0014] Another aspect of the present disclosure is a method for preparing a surface suitable for cell culture, e.g., as further described herein. The method includes

[0015] depositing an amorphous hydrogenated carbon coating on the fluoropolymer surface of the substrate of the surface, the deposition including:

[0016] depositing a first thickness of amorphous hydrogenated carbon coating in a first region thereof using a combination of a hydrocarbon gas (e.g., ethylene or propylene) and CO2 at a first ratio of hydrocarbon gas to CO2 via chemical vapor deposition (e.g., plasma-enhanced chemical vapor deposition (PECVD)), wherein the first region is adjacent to and extends from the fluoropolymer surface; and

[0017] A second thickness of an amorphous hydrogenated carbon coating is deposited in its second region via 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, wherein the second region is remote from the fluoropolymer surface and is at the surface of the amorphous hydrogenated carbon coating, and wherein the amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in its second region than in its first region.

[0018] Another aspect of the present disclosure is a method for culturing adherent cells, the method comprising incubating a surface as described herein with adherent cells and a growth medium.

[0019] 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).

[0020] Based on the description provided herein, other aspects of the present disclosure will be apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic cross-sectional view of a surface suitable for cell culture applications according to one embodiment of the present disclosure.

[0022] Figure 2 is a schematic cross-sectional view of a surface suitable for cell culture applications according to another embodiment of the present disclosure.

[0023] Figure 3 Schematic plan views and schematic cross-sectional views of a cell culture vessel of the present disclosure are provided.

[0024] Figure 4 is a graph showing the elemental percentages of oxygen and nitrogen on the surfaces described in Examples 1-8.

[0025] Figure 5 is a graph showing the water contact angles of the surfaces described in Examples 1-8.

[0026] Figure 6 is a graph showing the exemplary thicknesses of the coatings disposed on the surfaces described in Examples 3-8.

[0027] Figure 7 is a graph showing cell adhesion and proliferation of hMSCs on the coated surfaces described in Examples 5 and 8 compared to on unactivated fluoropolymer surfaces, activated fluoropolymer surfaces, and commercially available TCPS surfaces.

[0028] Figure 8 It is a graph showing cell adhesion and proliferation of monocyte-derived dendritic cells on the coated surfaces described in Examples 5 and 8, respectively, compared to on an unactivated fluoropolymer surface, an activated fluoropolymer surface, and a commercially available TCPS surface. Detailed Description

[0029] The inventors have noted various ways that can be used to treat fluoropolymer surfaces to provide improved adhesion for adherent cells. Treating a fluoropolymer surface with plasma can increase hydrophilicity (e.g., as measured by 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 desired surface for culturing adherent cells.

[0030] A cell culture device can be coated with a pro-adherent protein (such as laminin, fibronectin, or collagen). These proteins are typically obtained from animals or humans and must be purified and characterized before being used in clinical applications. However, the purification and characterization process can be expensive and time-consuming, which ultimately increases the cost of the cell culture device.

[0031] The inventors have noted that a cell culture surface having a relatively high concentration of nitrogen- and / or oxygen-containing functional groups (e.g., amine and / or carboxylic acid functional groups) on its surface can help enhance cell adhesion and proliferation. For example, a cell culture device having amine and / or carboxylic acid functional groups can generally exhibit better cell culture kinetics in serum-free cell culture than a cell culture device without such functional groups. However, since amines and carboxylic acids can react with oxygen and also cause surface reorganization, the amount of these functional groups on the surface may decrease over time and thus reduce the surface function of the cell culture device.

[0032] The inventors have determined that a desired cell culture system for adherent cell culture can be provided by coating the surface of a fluoropolymer layer with a coating having nitrogen- and / or oxygen-containing functional groups. Plasma polymerization can be used to provide such coatings in the form of amorphous coatings. However, the inventors have determined that such coatings may be unstable during autoclaving processes, which are commonly used to sterilize cell culture surfaces, if a uniform coating with an ideally high amount of nitrogen- and / or oxygen-containing functional groups is provided on the surface. The inventors have determined that a coating having a lower amount of nitrogen- and / or oxygen-containing functional groups near the fluoropolymer surface and a higher amount of nitrogen- and / or oxygen-containing functional groups away from 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 desired properties for cell culture at the coating surface but also provides a coating that is tough enough to withstand autoclaving. The inventors have noted that the coatings described herein can be functionalized to provide surfaces that are particularly suitable for adherent cell culture while being sterilizable (e.g., by autoclaving), protein-free, and relatively simple and inexpensive to manufacture.

[0033] Accordingly, one aspect of the present disclosure is a surface suitable for cell culture applications. The surface can 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 can be the inner surface of a cell culture device. The surface includes a substrate having a fluoropolymer surface. The surface further includes an amorphous hydrogenated carbon coating disposed on the fluoropolymer surface of the substrate. The amorphous hydrogenated carbon coating has a first thickness region and a second thickness region, the first thickness region being adjacent to and extending from the fluoropolymer surface, and the second thickness region being away from the fluoropolymer surface and at the surface of the amorphous hydrogenated carbon coating. Notably, the amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in the second thickness region than in the first thickness region. As used herein, the term "concentration" means the atomic weight-based elemental percentage.

[0034] One embodiment of the surface according to the present disclosure is in Figure 1is shown in a schematic cross-sectional view. In this embodiment, the surface 100 for cell culture applications includes a substrate 110 having a fluoropolymer surface 112. The surface 100 also 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 region 122 and a second thickness region 124. The first thickness region is adjacent to and extends from the fluoropolymer surface 112, and the second thickness region is remote from the fluoropolymer surface 112 and is at the surface 126 of the amorphous hydrogenated carbon coating 120. The amorphous hydrogenated carbon coating 120 has a higher total concentration of oxygen and nitrogen in the second thickness region 124 than in the first thickness region 122. It is noted that Figure 1 the embodiment of shows the first thickness region 122 adjacent to the second thickness region 124.

[0035] A variety of fluoropolymers can be used to provide the fluoropolymer surface. In various desired embodiments as further described herein, the fluoropolymer surface is a fluorinated ethylene propylene (FEP) surface. Optionally, other fluoropolymers can be used. For example, in various embodiments as further described 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.

[0036] In various desired embodiments as further described herein, the fluoropolymer surface is the surface of a fluoropolymer-silicone laminate film. The inventors have noted 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 that provides an FEP fluoropolymer surface. The silicone-fluoropolymer laminate is described in U.S. Patent No. 9,926,524, which is hereby incorporated by reference in its entirety. Cell culture bags based on the silicone-fluoropolymer laminate are available commercially under the trade name from Saint-Gobain Performance Polymer Products Corporation.

[0037] As will be understood by one of ordinary skill in the art, to allow the cell culture medium to absorb oxygen from the atmosphere and desorb CO2 to the atmosphere through the surface of the cell culture, it may be desirable to make the cell culture system have a certain degree of oxygen and CO2 permeability.

[0038] Thus, in various desired embodiments as further described herein, the fluoropolymer surface as described herein is the surface of a membrane having an oxygen permeability of at least 1500 cc / m 2 -day-atm, for example, at least 1800 cc / m 2 -day-atm, or in the range of 1500 cc / m 2 -day-atm to 20000 cc / m 2 -day-atm or 1800 cc / m 2 -day-atm to 16000 cc / m 2 -day-atm. The oxygen permeability is measured using a MOCON OxTran 220 O2TR analyzer under the following test conditions in accordance with ASTM D3985: Temperature: 23 °C; Test gas: 10% O2 in N2; Humidity: 0% relative humidity on both sides of the membrane; Carrier gas flow rate: 20 sccm N2; Test area: 5 cm 2 ; Inspection period: 15 minutes.

[0039] In various desired embodiments as further described herein, the fluoropolymer surface as described herein is the surface of a membrane having a CO2 permeability of at least 3500 cc / m 2 -day-atm, for example, 4000 cc / m 2 -day-atm, or in the range of 3500 cc / m 2 -day-atm to 25000 cc / m 2 -day-atm, 4000 cc / m 2 -day-atm to 23000 cc / m 2 -day-atm. The CO2 permeability is measured using a MOCON Permatran-C 441 CO2TR analyzer under the following test conditions in accordance with ASTM F2476: Temperature: 23 °C; Test gas: 100% CO2; Humidity: 0% relative humidity on both sides of the membrane; Carrier gas flow rate: 50 sccm N2; Test area: 5 cm 2 ; Inspection period: 30 minutes.

[0040] In various desired embodiments as further described herein, the fluoropolymer surface as described herein is the surface of a membrane having a water vapor transmission rate (WVTR) in the range of 0.65 g / m 2 -day-atm to 1 g / m2 - per day - atm, for example, at 0.72 g / m 2 - per day - atm to 0.94 g / m 2 - per day - atm. WVTR was measured using a MOCON Permatran W700 water vapor analyzer under the following test conditions in accordance with ASTM F1249: Temperature: 23 °C; Humidity: 100% RH on the test gas side; 0% RH on the detector side of the membrane; Carrier gas flow rate: 10 sccm N2; Test area: 50 cm 2 ; Inspection period: 60 minutes.

[0041] The inventors have noted that the adhesion of an amorphous hydrogenated carbon coating to a fluoropolymer surface can be improved by using an activated fluoropolymer surface (e.g., a treated fluoropolymer surface). Thus, in various desired embodiments as further described herein, the fluoropolymer surface is an activated fluoropolymer surface. The activated fluoropolymer surface provides oxygen - and / or nitrogen - containing functional groups. Without wishing to be bound by theory, the inventors speculate that such functional groups provide a reactive surface from which, for example, an amorphous film can be grown by plasma polymerization. The amorphous film can be covalently bonded to the activated fluoropolymer surface.

[0042] A variety of processing techniques can be used to provide an activated fluoropolymer surface. For example, plasma treatment can be used, such as treatment with ammonia (NH3) plasma, CO2 plasma, or a plasma of a combination of NH3 and CO2. The inventors have determined that treatment with NH3 plasma can better increase the hydrophilicity and biocompatibility of the fluoropolymer surface compared to treatment of the fluoropolymer surface with CO2 plasma, and thus can better promote cell adhesion and proliferation on the cell culture surface. In other embodiments, the plasma can be an oxygen plasma, a nitrogen plasma, or a plasma of a combination of oxygen and nitrogen. Without wishing to be bound by theory, the inventors speculate that plasma treatment can not only add nitrogen - and / or oxygen - containing functional groups at the fluoropolymer surface, but also remove some fluorine from the fluoropolymer surface.

[0043] In other embodiments, the fluoropolymer surface can be activated (e.g., treated) by corona treatment such as C treatment. As used herein, corona treatment (also known as corona discharge) is a plasma treatment of a surface in an atmosphere containing an organic gas. The organic gas can be based on, for example, a ketone or an alcohol. 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 increases the adhesion of the fluoropolymer layer to the layer with which it is in direct contact. In one embodiment, the treatment increases the adhesion of the fluoropolymer layer to a polymer layer. In an exemplary embodiment, the treatment includes C treatment of a fluoropolymer capable of undergoing C treatment. C treatment is further disclosed in U.S. Patent Nos. 6,726,979 and 8,559,100, each of which is hereby incorporated by reference in its entirety.

[0044] In various embodiments as further described 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 and is thus based on the atomic percentage of all atoms other than hydrogen, which is understood by one of ordinary skill in the art to generally not be detectable by XPS.

[0045] In various embodiments as further described herein, the activated fluoropolymer surface can desirably have a reduced water contact angle. As will be understood by one of ordinary skill in the art, surfaces with a higher amount of nitrogen- and / or oxygen-containing functional groups generally have a lower water contact angle. Thus, the water contact angle can be used to measure the degree of activation of the fluoropolymer surface. In various embodiments as further described herein, the activated fluoropolymer surface has a water contact angle in the range of 60° to 120°.

[0046] As described above, an amorphous hydrogenated carbon coating is disposed on the fluoropolymer surface (e.g., the activated fluoropolymer surface) of the substrate. As used herein, an amorphous hydrogenated carbon coating is a coating having a significant hydrogen and carbon content and is essentially substantially non-crystalline. As described in detail below, such coatings can be prepared by the polymerization of polymerizable hydrocarbons (e.g., ethylene or propylene) under chemical vapor deposition conditions, e.g., by plasma enhanced chemical vapor deposition (PECVD).

[0047] One of ordinary skill in the art can determine the desired thickness of the amorphous hydrogenated carbon coating based on the disclosure herein. In various desired embodiments as further described herein, the thickness of the amorphous hydrogenated carbon coating is in the range of 10 nm to 200 nm, for example, 10 nm to 100 nm, or 10 nm to 75 nm, or 10 nm to 50 nm, or 15 nm to 200 nm, or 15 nm to 100 nm, or 15 nm to 75 nm, or 15 nm to 50 nm, or 20 nm to 200 nm, or 20 nm to 100 nm, or 20 nm to 75 nm, or 20 nm to 50 nm. However, other thicknesses can also be used.

[0048] In various embodiments, the amorphous hydrogenated carbon coatings described herein have few (if any) graphitic characteristics. In various desired embodiments as further described herein, the ratio of hydrogen to carbon in the amorphous hydrogenated carbon coating is at least 1, for example, at least 1.2 or at least 1.4. The ratio of hydrogen to carbon can be measured as described in the following article: “Chemical Characterisation of Nitrogen-Rich Plasma-Polymer Films Deposited in Dielectric Barrier Discharges at Atmospheric Pressure” by P.-L. Girard-Lauriault et al., Plasma Process. Polym., 5, 631-44 (2008), which is hereby incorporated by reference in its entirety.

[0049] As described above, it may be advantageous to have a lower amount of nitrogen- and / or oxygen-containing functional groups near the fluoropolymer surface and a higher amount of nitrogen- and / or oxygen-containing functional groups away from 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 and thus can provide a stable polymer backbone and thereby enhance the stability of the coating. Thus, in Figure 1 an embodiment, the amorphous hydrogenated carbon coating 120 has a higher hydrocarbon concentration in the first thickness region 122 than in the second thickness region 124 and forms more crosslinked layers in the first thickness region 122. In various desired embodiments as further described herein, the total concentration of oxygen and nitrogen in the first thickness region of the amorphous hydrogenated carbon coating is at least 3 atomic % lower than the total concentration of oxygen and nitrogen in the second thickness region, for example, at least 4 atomic %, or at least 5 atomic %, or at least 6 atomic %, or at least 7 atomic %. However, in many embodiments, the total concentration of oxygen and nitrogen in the first thickness region of the amorphous hydrogenated carbon coating is at least 5 atomic %, for example, at least 10 atomic % or at least 15 atomic %.

[0050] A person of ordinary skill in the art can determine the desired thickness of the first thickness region of the amorphous hydrogenated carbon coating based on the disclosure herein. In various desired embodiments as further described herein, the thickness of the first thickness region of the amorphous hydrogenated carbon coating is in the range of 8 nm to 190 nm, for example, 8 nm to 100 nm, or 8 nm to 75 nm, or 8 nm to 50 nm, or 15 nm to 190 nm, or 15 nm to 100 nm, or 15 nm to 75 nm, or 15 nm to 50 nm, or 20 nm to 190 nm, or 20 nm to 100 nm, or 20 nm to 75 nm, or 20 nm to 50 nm. However, other thicknesses may also be used.

[0051] In addition, as described above, it is advantageous to keep the coating away from the fluoropolymer surface and have a higher amount of nitrogen- and / or oxygen-containing functional groups at the surface of the coating. Thus, the coating can provide a more highly functionalized layer at the surface of the coating. The inventors have determined that, for example, in the presence of carboxylic acid and / or amine functional groups, the more functionalized layer of the coating can enhance the functional properties of the coating, especially with respect to the adhesion of adherent cells. Thus, in Figure 1 an embodiment, the amorphous hydrogenated carbon coating 120 includes a higher oxygen and / or nitrogen concentration in the second thickness region 124 than in the first thickness region 122 and can thus provide a more functionalized layer in the second thickness region 124. In various desired embodiments as further described herein, the total concentration of oxygen and nitrogen in the second thickness region of the amorphous hydrogenated carbon coating is at least 10 atomic %, for example, at least 15 atomic % or at least 20 atomic %.

[0052] A person of ordinary skill in the art can determine the desired thickness of the second thickness region of the amorphous hydrogenated carbon coating based on the disclosure herein. In various desired embodiments as further described herein, the thickness of the second thickness region of the amorphous hydrogenated carbon coating is in the range of 2 nm to 100 nm, for example, 2 nm to 50 nm, or 2 nm to 35 nm, or 2 nm to 20 nm, or 5 nm to 100 nm, or 5 nm to 50 nm, or 5 nm to 35 nm, or 5 nm to 20 nm, or 5 nm to 10 nm, or 10 nm to 100 nm, or 10 nm to 50 nm, or 10 nm to 35 nm, or 10 nm to 20 nm, or 20 nm to 100 nm, or 20 nm to 50 nm. However, other thicknesses may also be used.

[0053] In various desired embodiments as further described herein, the amorphous hydrogenated carbon coating contains oxygen near the fluoropolymer surface (e.g., in a first thickness region). Thus, in various embodiments, the amorphous hydrogenated carbon coating in its first thickness region is composed of at least 90 atomic percent, such as at least 95 atomic percent, of carbon and oxygen (i.e., hydrogen is deducted from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating may contain oxygen away from the fluoropolymer surface and at the surface of the coating (e.g., in a second thickness region). Thus, in various such embodiments, the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic percent, such as at least 95 atomic percent, of carbon and oxygen (i.e., hydrogen is deducted from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating may contain nitrogen away from the fluoropolymer surface and at the surface of the coating (e.g., in a second thickness region). Thus, in various such embodiments, the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic percent, such as at least 95 atomic percent, of carbon and nitrogen (i.e., hydrogen is deducted from the analysis as described above). And in various such embodiments, the amorphous hydrogenated carbon coating may contain nitrogen and oxygen away from the fluoropolymer surface and at the surface of the coating (e.g., in a second thickness region). Thus, in various such embodiments, the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic percent, such as at least 95 atomic percent, of carbon, nitrogen, and oxygen (i.e., hydrogen is deducted from the analysis as described above).

[0054] In various desired embodiments as further described herein, the amorphous hydrogenated carbon coating contains oxygen and nitrogen near the fluoropolymer surface (e.g., in a first thickness region). Thus, in various embodiments, the amorphous hydrogenated carbon coating in its first thickness region is composed of at least 90 atomic percent, such as at least 95 atomic percent, of carbon, nitrogen, and oxygen (i.e., hydrogen is deducted from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating may contain oxygen away from the fluoropolymer surface and at the surface of the coating (e.g., in a second thickness region). Thus, in various such embodiments, the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic percent, such as at least 95 atomic percent, of carbon and oxygen (i.e., hydrogen is deducted from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating may contain nitrogen away from the fluoropolymer surface and at the surface of the coating (e.g., in a second thickness region). Thus, in various such embodiments, the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic percent, such as at least 95 atomic percent, of carbon and nitrogen (i.e., hydrogen is deducted from the analysis as described above). And in various such embodiments, the amorphous hydrogenated carbon coating may contain nitrogen and oxygen away from the fluoropolymer surface and at the surface of the coating (e.g., in a second thickness region). Thus, in various such embodiments, the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic percent, such as at least 95 atomic percent, of carbon, nitrogen, and oxygen (i.e., hydrogen is deducted from the analysis as described above).

[0055] In various desired embodiments as further described herein, the amorphous hydrogenated carbon coating contains nitrogen adjacent to the fluoropolymer surface (e.g., in a first thickness region). Thus, in various embodiments, the amorphous hydrogenated carbon coating in its first thickness region is composed of at least 90 atomic %, such as at least 95 atomic %, of carbon and nitrogen (i.e., hydrogen is deducted from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating may contain oxygen away from the fluoropolymer surface and at the surface of the coating (e.g., in a second thickness region). Thus, in various such embodiments, the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic %, such as at least 95 atomic %, of carbon and oxygen (i.e., hydrogen is deducted from the analysis as described above). In various such embodiments, the amorphous hydrogenated carbon coating may contain nitrogen away from the fluoropolymer surface and at the surface of the coating (e.g., in a second thickness region). Thus, in various such embodiments, the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic %, such as at least 95 atomic %, of carbon and nitrogen (i.e., hydrogen is deducted from the analysis as described above). And in various such embodiments, the amorphous hydrogenated carbon coating may contain nitrogen and oxygen away from the fluoropolymer surface and at the surface of the coating (e.g., in a second thickness region). Thus, in various such embodiments, the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic %, such as at least 95 atomic %, of carbon, nitrogen and oxygen (i.e., hydrogen is deducted from the analysis as described above).

[0056] In various embodiments, the first thickness region of the amorphous hydrogenated carbon coating is adjacent to the second thickness region of the amorphous hydrogenated carbon coating. For example, in Figure 1 the embodiment of, the first thickness region 122 is adjacent to the second thickness region 124.

[0057] However, in other embodiments, one or more additional thickness regions of the amorphous hydrogenated carbon coating are provided between the first thickness region and the second thickness region of the amorphous hydrogenated carbon coating. For example, in various embodiments, one or more additional thickness regions are a third thickness region provided between and adjacent to the first thickness region and the second thickness region. One such embodiment of the surface according to the present disclosure is in Figure 2is shown in a schematic cross-sectional view. In this embodiment, the surface 200 for cell culture applications includes a substrate 210 having a fluoropolymer surface 212. The surface 200 also includes an amorphous hydrogenated carbon coating 220 disposed on the fluoropolymer surface 212 of the substrate 210. The amorphous hydrogenated carbon coating 220 has a first thickness region 222 and a second thickness region 224. The first thickness region is adjacent to and extends from the fluoropolymer surface 212, and the second thickness region is away from the fluoropolymer surface 212 and is at the surface 226 of the amorphous hydrogenated carbon coating 220. The amorphous hydrogenated carbon coating 220 has a higher total concentration of oxygen and nitrogen in the second thickness region 224 than in the first thickness region 222. Notably, in this embodiment, a third thickness region 228 is disposed between and adjacent to the first thickness region 222 and the second thickness region 224.

[0058] One or more thickness regions disposed between the first region and the second region can have a variety of compositions. Notably, in some embodiments, the third thickness region can contain oxygen and / or nitrogen, and the amorphous hydrogenated carbon coating can have a higher total concentration of oxygen and nitrogen in the third thickness region than in the first thickness region.

[0059] The surfaces described herein can be used in a variety of cell culture systems. For example, in various embodiments as further described herein, the surface is the inner surface of a cell culture container. A variety of cell culture containers (especially those made of fluoropolymer materials) can be adapted to the surfaces of the present invention, for example, by depositing an amorphous hydrogenated carbon coating thereon. For example, in various desired embodiments, the cell culture container is a cell culture bag. In Figure 3 One such embodiment is shown in a schematic top view (the top of the figure) and a schematic cross-sectional view (the bottom of the figure). Here, the cell culture bag 350 is formed by two polymer films 355 having fluoropolymer surfaces, which are laminated together at their edges 358. The surface 300 as described herein is disposed at the inner surface of the cell culture bag. In some embodiments, the coating may not be present at the laminated edge so as not to interfere with lamination. Many of the above-mentioned fluoropolymers can be suitable for such bags. The various fluoropolymer materials described above (including fluoropolymer-silicone laminates) can be suitable for such cell culture bags. Of course, other cell culture containers can be adapted to be used with the surfaces described herein (e.g., in the form of flasks, vials, tubes, and tubing).

[0060] Another aspect of the present disclosure is a method for preparing a surface suitable for cell culture applications (e.g., a surface as described herein). The method includes depositing an amorphous hydrogenated carbon coating on a fluoropolymer surface of a substrate. The deposition includes 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) to deposit a first thickness of the amorphous hydrogenated carbon coating in a first region thereof via chemical vapor deposition (e.g., PECVD) at a first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas. The first region is adjacent to and extends from the fluoropolymer surface. The deposition further includes 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) to deposit a second thickness of the amorphous hydrogenated carbon coating in a second region thereof via chemical vapor deposition (e.g., PECVD) at a second ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas, the second ratio being lower than the first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas (e.g., CO2 and / or NH3). The second region is remote from the fluoropolymer surface and is at the surface of the amorphous hydrogenated carbon coating. Notably, the deposited amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in its second region than in its first region.

[0061] The amorphous hydrogenated carbon coatings of the first thickness and the second thickness can be deposited without an intervening layer therebetween, e.g., to provide a surface in which the first region is adjacent to the second region, as described with respect to Figure 1 that described. In other embodiments, one or more thicknesses of the amorphous hydrogenated carbon coating can be deposited between the first thickness and the second thickness, as described above with respect to Figure 2 that described. For example, in various embodiments as further described herein, the method can further include depositing a third thickness of the amorphous hydrogenated carbon coating in a third region thereof via chemical vapor deposition (e.g., PECVD) at a third ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas, the third ratio being lower than the first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas, 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), before depositing the second thickness of the amorphous hydrogenated carbon coating in its second region as described herein. In this embodiment, after depositing the second thickness of the amorphous hydrogenated carbon coating in its second region as described herein, the third region is between and adjacent to the first region and the second region of the amorphous hydrogenated carbon coating. Notably, the amorphous hydrogenated carbon coating can have a higher total concentration of oxygen and nitrogen in its third region than in its first region.

[0062] One of ordinary skill in the art can adjust 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, a hydrocarbon gas or plasma reactive gas need not be a gas under ambient conditions; rather, it must be a gas under the chemical vapor deposition conditions used.

[0063] A variety of hydrocarbon gases can be used. For example, chemical vapor deposition of olefins (such as propylene or ethylene or 1,3 - butadiene) can be used to provide an amorphous hydrogenated carbon coating. However, other hydrocarbon gases can be used, such as alkynes (e.g., acetylene, methylacetylene) and alkanes (e.g., methane, ethane, propane).

[0064] Similarly, a variety of oxygen - containing and / or nitrogen - containing gases can be used, provided that they are reactive under plasma conditions. A variety of 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 an oxygen content to the deposited material. Carbon dioxide is a particularly desirable oxygen - containing gas. Oxygen can be used, but at higher concentrations it can act as an etchant. Similarly, a variety of nitrogen - containing gases can be used to provide a nitrogen content to the deposited material, such as amines (e.g., methylamine, ethylamine, allylamine), imines (e.g., methylimine) and nitriles (e.g., acetonitrile). Ammonia is a particularly desirable nitrogen - containing gas. Some plasma - reactive compounds can include both oxygen and nitrogen, for example, nitrogen oxides and amides (e.g., formamide, acetamide).

[0065] By varying the relative amounts of the hydrocarbon gas, oxygen - containing gas, and nitrogen - containing gas, one of ordinary skill in the art can provide varying concentrations of oxygen and / or nitrogen between the various regions of the coating to provide the various regions described herein. For example, in some embodiments, the mass ratio of the hydrocarbon gas to the oxygen - containing gas (e.g., ethylene:CO2) can be in the range of 0.5 - 20, and in some embodiments, the mass ratio of the hydrocarbon gas to the nitrogen - containing gas (e.g., ethylene:NH3) can be in the range of 0.1 - 3. 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 the first region than in the deposition of the second region to provide a more highly cross - linked layer in the first region and a more highly functionalized layer in the second region.

[0066] Note that the variation in the content of oxygen and nitrogen can be continuous; the concentration need not be uniform within any region as described herein.

[0067] As noted above, it may be desirable to coat an activated fluoropolymer surface. As noted above, an activated fluoropolymer surface can improve the adhesion of a coating to the fluoropolymer surface. Thus, in various embodiments of the methods described herein, the fluoropolymer surface of a substrate on which an amorphous hydrogenated carbon coating is deposited is an activated fluoropolymer surface, e.g., as described above. In various embodiments, the method further includes activating the fluoropolymer surface. The fluoropolymer surface can be activated by plasma treatment, such as by treatment with an NH3 plasma, a CO2 plasma, or a plasma of a combination of NH3 and CO2. In other embodiments, the plasma is an oxygen plasma, a nitrogen plasma, or a plasma of a combination of oxygen and nitrogen. In other embodiments, the fluoropolymer surface can be activated by corona treatment (such as C treatment). In various embodiments as further described 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 further described herein, the activated fluoropolymer surface can have a water contact angle in the range of 60° to 120°.

[0068] Another aspect of the present disclosure is a method for culturing adherent cells, the method including incubating a surface as described herein with adherent cells and a growth medium. For example, the surface can be the surface of a cell culture device (e.g., a cell culture bag, a cell culture tube, a cell culture vial, or cell culture tubing), for example.

[0069] A variety of adherent cells can be cultured using the surface 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 of ordinary skill in the art will identify other cell types suitable for growth.

[0070] Those of ordinary skill in the art will identify a suitable growth medium for culturing adherent cells. In various desired embodiments as further described herein, the growth medium is a basal medium. In various embodiments, the growth medium includes a buffer (e.g., sodium bicarbonate), glutamine, and growth factors. In various desired embodiments as further described herein, the pH of the growth medium is in the range of 7.2 - 7.4.

[0071] Those of ordinary skill in the art will identify appropriate incubation conditions for the growth methods described herein. For example, in various desired embodiments as further described herein, the incubation temperature for culturing adherent cells is in the range of 35°C to 39°C (e.g., 37°C).

[0072] It should be noted that the surfaces and containers described herein can be sterilized before being used for cell culture. For example, sterilization can be carried out by autoclaving. A significant advantage is that the various surfaces described herein can be sterilized by autoclaving before use while still maintaining significant functionality.

[0073] Example

[0074] The following examples are used to illustrate various preferred embodiments and aspects of the present disclosure and should not be construed as limiting its scope.

[0075] Example 1. Activating the surface of a fluoropolymer using CO2 plasma

[0076] This example describes the use of CO2 plasma to activate a fluoropolymer surface. First, the fluoropolymer surface (here, a fluorinated ethylene propylene (FEP) polymer surface) was cleaned for 30 minutes using 70% ethanol in an ultrasonic bath sonicator, then the fluoropolymer surface was rinsed with reverse osmosis water, and the fluoropolymer surface was dried overnight under vacuum. The cleaned fluoropolymer surface was placed on an electrode and loaded into a PECVD reactor. The PECVD reactor is generally described in M. Buddhadasa and P.-L. Girard-Lauriault's "Plasma co-polymerisation of ethylene, 1,3-butadiene and ammonia mixtures: Amine content and water stability", Thin Solid Films, 591, 76 - 85 (2015), which is hereby incorporated by reference in its entirety. The surface was 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 was sealed in a low-oxygen permeation bag in a glove box under argon and stored at -40 °C. Although in this example, the activated fluoropolymer surface was removed from the PECVD reactor and stored, those of ordinary skill in the art will understand that activation and subsequent coating deposition can be carried out sequentially in the PECVD reactor.

[0077] Example 2. Activating the surface of a fluoropolymer using ammonia (NH3) plasma

[0078] This example describes the use of NH3 plasma to activate (e.g., treat) a fluoropolymer surface. First, the fluoropolymer surface (here, a fluorinated ethylene propylene (FEP) polymer surface) was cleaned in an ultrasonic bath sonicator with 70% ethanol for 30 minutes, then rinsed with reverse osmosis water, and dried under vacuum overnight. The cleaned fluoropolymer surface was placed on an electrode and loaded into a PECVD reactor. The surface was 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 was sealed in a low oxygen permeation bag inside a glove box under argon and stored at -40 °C. Although in this example, the activated fluoropolymer surface was removed from the PECVD reactor and stored, those of ordinary skill in the art will understand that activation and subsequent coating deposition can be carried out sequentially in the PECVD reactor.

[0079] Example 3. Depositing a coating with an oxygen-containing crosslinked layer onto the activated fluoropolymer surface

[0080] This example describes the deposition of an amorphous hydrogenated carbon coating having an oxygen-containing crosslinked layer onto the activated fluoropolymer surface as described herein. In particular, a mixture of ethylene (C2H4) at a flow rate of approximately 5 sccm and CO2 at a flow rate of approximately 40 sccm was used to deposit an oxygen-rich crosslinked layer onto the activated fluoropolymer surface via PECVD at a pressure of 80 Pa and an RF power of 20 W for 10 minutes. The coated fluoropolymer surface was sealed in a low oxygen permeation bag inside a glove box under argon and stored at -40 °C.

[0081] Example 4. Depositing a coating with an oxygen-containing functional layer onto the activated fluoropolymer surface

[0082] This example describes the deposition of a coating having an oxygen-containing functional layer onto the activated fluoropolymer surface as described herein. In particular, a mixture of ethylene (C2H4) at a flow rate of approximately 5 sccm - 2.5 sccm (linearly ramping over a 10-minute period) and CO2 at a flow rate of approximately 40 sccm was used to deposit the oxygen-containing functional layer onto the activated fluoropolymer surface via PECVD at a pressure of 80 Pa and an RF power of 20 W for up to 10 minutes. The coated fluoropolymer surface was sealed in a low oxygen permeation bag inside a glove box under argon and stored at -40 °C.

[0083] Example 5. Depositing a coating with an oxygen-containing crosslinked layer and an oxygen-containing functional layer onto the activated fluoropolymer surface on

[0084] This example describes depositing a coating having an oxygen-containing crosslinked layer and an oxygen-containing functional layer onto an activated fluoropolymer surface as described herein. First, a mixture of ethylene (C2H4) with a flow rate of about 5 sccm and CO2 with a flow rate of about 40 sccm is used to deposit the oxygen-containing crosslinked layer onto the activated fluoropolymer surface via PECVD at a pressure of 80 Pa and an RF power of 20 W for 5 minutes. Then, a mixture of ethylene (C2H4) with a flow rate ranging from about 5 sccm to 2.5 sccm (linearly ramping down within 1.5 minutes) and CO2 with a flow rate of about 40 sccm is used to deposit the oxygen-containing functional layer onto the activated oxygen-rich crosslinked layer via PECVD at a pressure of 80 Pa and an RF power of 20 W for 1.5 minutes. The coated fluoropolymer surface is sealed in a low-oxygen permeation bag in a glove box under argon and stored at -40 °C.

[0085] Example 6. Depositing a coating with a nitrogen- and oxygen-containing crosslinked layer onto the activated fluoropolymer surface

[0086] This example describes depositing a coating having a nitrogen- and oxygen-containing crosslinked layer onto an activated fluoropolymer surface as described herein. In particular, a mixture of ethylene (C2H4) with a flow rate of 20 sccm, CO2 with a flow rate of about 20 sccm, and NH3 with a flow rate of about 5 sccm is used to deposit the nitrogen- and oxygen-containing crosslinked layer onto the activated fluoropolymer surface via PECVD at a pressure of 80 Pa and an RF power of 20 W for 15 minutes. The coated fluoropolymer surface is sealed in a low-oxygen permeation bag in a glove box under argon and stored at -40 °C.

[0087] Example 7. Depositing a coating with a nitrogen-containing functional layer onto the activated fluoropolymer surface

[0088] This example describes depositing a coating having a nitrogen-containing functional layer onto an activated fluoropolymer surface as described herein. In particular, a mixture of ethylene (C2H4) with a flow rate ranging from 20 sccm to 10 sccm (linearly ramping down within 90 seconds) and NH3 with a flow rate of about 15 sccm is used to deposit the nitrogen-containing functional layer onto the activated fluoropolymer surface via PECVD at a pressure of 80 Pa and an RF power of 20 W for 90 seconds. The coated fluoropolymer surface is sealed in a low-oxygen permeation bag in a glove box under argon and stored at -40 °C.

[0089] Example 8. Depositing a coating with a nitrogen- and oxygen-containing crosslinked layer and a nitrogen-containing functional layer onto the activated fluoropolymer surface

[0090] This example describes depositing a coating having an oxygen-rich crosslinked layer and a nitrogen-rich functional layer onto an activated fluoropolymer surface as described herein. First, an oxygen-containing crosslinked layer is deposited onto the activated fluoropolymer surface via PECVD for 3 minutes at a pressure of 80 Pa and an RF power of 20 W 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. Then, a nitrogen-containing functional layer is deposited onto the nitrogen- and oxygen-containing crosslinked layer via PECVD for 1.5 minutes at a pressure of 80 Pa and an RF power of 20 W using a mixture of ethylene (C2H4) in the flow rate range of about 20 sccm to about 10 sccm (linearly ramping within 1.5 minutes) and NH3 at a flow rate of about 15 sccm. The coated fluoropolymer surface is sealed in a low-oxygen permeation bag in a glove box under argon and stored at -40 °C.

[0091] Example 9. Elemental analysis of oxygen and nitrogen on the surfaces described in Examples 1 to 8

[0092] Elemental analysis of the surfaces of Examples 1 to 8 was performed by X-ray photoelectron spectroscopy (XPS). For XPS analysis, a Thermo Scientific K-Alpha instrument equipped with a monochromatic Al Kα radiation X-ray source was used. The coated surface was mounted on a vacuum transfer module in a glove box and transported to the XPS instrument to avoid exposure to air under any circumstances. A survey spectrum was obtained at a 400 μM spot size with a pass energy of 160 eV and a dwell time of 200 ms with the Flood Gun (retarding electron gun) on. In addition, high-resolution spectra were obtained with a pass energy of 20 eV and a dwell time of 200 ms. The surface composition was determined using Thermo Fisher Scientific Avantage software (version: 5.9922).

[0093] Figure 4 is a graph showing the elemental percentages of oxygen and nitrogen on the surfaces described in Examples 1 to 8. Note that the data indicate a minor degree of cross-contamination between the examples.

[0094] The O / C and N / C atomic ratios of the coatings of Examples 3 to 8 are shown in Table 1 below. Some nitrogen is present in the coating of Example 6 due to contamination.

[0095] Table 1

[0096] Sample O / C N / C Example 3 0.35 0.01 Example 4 0.46 0.01 Example 5 0.43 0.02 Example 6 0.08 0.07 Example 7 0.03 0.26 Example 8 0.05 0.24

[0097] Example 10. Water contact angle of the surfaces described in Examples 1 to 8

[0098] The water contact angles of the surfaces of Examples 1 to 8 were measured.

[0099] The water contact angle was measured using a goniometer (FutureDigitalScientific Corp.) connected to a camera system and computer software (SCA 2.0). The contact angle was measured at room temperature using the sessile drop method (3 μL Milli-Q water (ultrapure water) drops). The average value from 5-point measurements was calculated and reported.

[0100] Figure 5 Figures respectively show the water contact angles of the surfaces described in Examples 1 to 8.

[0101] Example 11. Coating thickness of the coatings described in Examples 3 to 8

[0102] The thickness of the coatings of Examples 3 to 8 was measured using a Dektak XT (Veeco Sloan Technology) profilometer. The corrugation of FEP prevented the measurement of plasma polymer thickness below 1 μm. Therefore, the coatings were deposited on silicon wafers for profilometry experiments. During plasma coating, a mask covering the silicon wafers was used to create a step between the treated and untreated surfaces. The thickness was measured using a stylus with a radius of 12.5 μm at a force of 3 mg. At room temperature, each measurement was obtained at a resolution of 0.1 μm / point in the stepped area.

[0103] Figure 6 Figures respectively show exemplary thicknesses of the coatings provided on the surfaces described in Examples 3 to 8.

[0104] Example 12. Sterilization effect on the surfaces described in Examples 5 and 8

[0105] This example describes the effect of autoclaving the surfaces described in Examples 5 and 8. The effect was determined by the change in the elemental percentage of oxygen and / or nitrogen on the surface after sterilization, and by the change in the thickness of the coatings provided on the surface after sterilization.

[0106] Regarding the surface described in Example 5, the elemental percentage of oxygen on the non-sterilized surface was determined to be approximately 29.59 atomic weight %, and the elemental percentage of oxygen on the sterilized surface was determined to have decreased to approximately 25.24 atomic weight %. Therefore, the change in the elemental percentage of oxygen on the surface of approximately 14.7 atomic weight % can be considered to be the range for maintaining the stability of the coating on the surface.

[0107] Regarding the surface described in Example 8, the elemental percentage of nitrogen on the non-sterilized surface was determined to be approximately 18.79 atomic weight %, and the elemental percentage of nitrogen on the sterilized surface was determined to have decreased to approximately 13.97 atomic weight %. Therefore, the change in the elemental percentage of nitrogen on the surface of approximately 25.7 atomic weight % can be considered to be the range for maintaining the stability of the coating on the surface.

[0108] After surface sterilization, the thickness of the coating provided on the surface tends to increase. Without wishing to be bound by theory, the inventors speculate that this may be due to the thermal expansion and swelling of the fluoropolymer layer at the surface. For example, the thickness of the coating on the sterilized surface as described in Example 5 was determined to increase by about 59.7% compared to the non-sterilized surface. In addition, the thickness of the coating on the sterilized surface as described in Example 8 was determined to increase by about 1.1% compared to the non-sterilized surface.

[0109] Example 13. Cell adhesion and proliferation of human mesenchymal stromal cells (hMSCs) on the coated surfaces described in Examples 5 and 8 respectively

[0110] This example describes the cell adhesion and proliferation of hMSCs on the coated surfaces described in Examples 5 and 8, respectively. For comparison, this example also describes the cell adhesion 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.

[0111] Cell adhesion and proliferation can be determined by the cell seeding density (cells / cm 2 ) of the cells adhered to the surface. For cell culture studies, custom wells with various FEP surfaces were prepared by mounting FEP membranes onto the bottom of a CultureWell TM 8-well removable chamber slide (Gracebio). Human bone marrow-derived mesenchymal stromal cells (hMSCs, Poietics TM MSC expansion medium XF kit (StemMACS TM , Miltenyi Biotech) were used to culture the cells. The cryopreserved cells were thawed and seeded at a density of 5,000 to 6,000 cells / cm TM into a TCPS T-75 flask ( 2 , red cap), and the medium was changed on day 3. When the cells reached confluence, they were washed twice in Dulbecco's phosphate-buffered saline (DPBS) and then removed from the surface using TrypLE (Thermo Fisher Scientific). The cells were seeded at 5000 cells / cm 2 onto the following surfaces: FEP (untreated), FEP (treated), O-GPPC, N-hyb-GPPC, TCPS ( TM , red) and TCPS ( , red) and TCPS ( , yellow). The inoculated cells were incubated at 37 °C and 5% CO2. The cells amplified on the 1st and 3rd days 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). ImageJ was used to analyze and count the DAPI-stained adherent cells.

[0112] Figure 7 is a graph showing cell adhesion and proliferation of hMSCs on the coated surfaces described in Examples 5 and 8, respectively, compared to those on an unactivated fluoropolymer surface, an activated fluoropolymer surface, and a commercially available TCPS surface. Generally, Figure 7 shows that under similar experimental conditions, hMSCs tend to adhere better on the two coated surfaces compared to the unactivated fluoropolymer surface, the activated fluoropolymer surface, and the commercially available TCPS surface.

[0113] Specifically, Figure 7 shows that on the 1st day, the cell seeding density of hMSCs on the coated surfaces is higher than that of hMSCs on other test surfaces (i.e., the unactivated fluoropolymer surface, the activated fluoropolymer surface, and the TCPS surface). Figure 8 Also shows that on the 3rd day, the cell seeding density of hMSCs on the coated surfaces is more than three times that on the 1st day on the same surfaces, and on the 3rd day, the cell seeding density of the cells on the coated surfaces is still higher than that of the cells on other test surfaces. Therefore, 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, where the coated surfaces tend to significantly promote cell adhesion and proliferation under similar experimental conditions.

[0114] Example 14. Cell adhesion and proliferation of monocyte-derived dendritic cells on the coated surfaces described in Examples 5 and 8 respectively

[0115] This example describes cell adhesion and proliferation of monocyte-derived dendritic cells on the coated surfaces described in Examples 5 and 8, respectively. For comparison, this example also describes cell adhesion and proliferation of monocyte-derived dendritic cells on an unactivated (e.g., untreated) fluoropolymer surface, an activated (e.g., treated) fluoropolymer surface, and a commercially available TCPS surface under similar experimental conditions.

[0116] Isolating primary human monocytes from fresh whole blood: Primary human monocytes were obtained from blood donations of healthy donors. Fresh blood was collected at the McGill University Health Center and transported to the Stem Cell Bioprocessing Laboratory for further processing. First, the blood was diluted at a 1:1 ratio in DPBS supplemented with 2% human serum albumin (HSA, Sigma, catalog number A9080), and then the diluted blood was gently layered on top of TM -50 tubes (STEMCELL TM Technologies, catalog number 85450) containing -1077 (Sigma, catalog number 10771) and exposed to density gradient centrifugation at 1200×g. The centrifuged blood consisted of multiple layers. The buffy coat 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 remaining volume. Cells from a single donor were pooled into one tube and washed twice by filling the volume to 50 mL with DPBS supplemented with 2% HSA and centrifuged at 300×g for 8 minutes. The supernatant was aspirated and the cell pellet was resuspended in CS10 (STEMCELL TM Technologies, catalog number 07959) to a cell concentration of 0.5 - 50×10^6 cells / ml. The vials were kept in an -80°C freezer for 24 hours and then transferred to liquid nitrogen for long-term storage.

[0117] Enriching monocytes from cryopreserved PBMC: On the day of the experiment, the frozen PBMCs were thawed in a 37°C water bath and then diluted at a 1:1 ratio in Plasma-lyte A (Thermo Fisher, catalog number NC1531549) with 10% heat-inactivated umbilical cord plasma. The contents of the frozen vial were transferred to a 15 mL conical tube and then centrifuged at 300×g for 5 minutes to remove DMSO, which is toxic to the cells in the medium. The cells were then resuspended at a concentration of 100 million cells / mL in DPBS supplemented with 2% HSA and 1 mM EDTA (Thermo Fisher, catalog number 15575020), and then EasySep TM Human CD14 Positive Selection Kit II (STEMCELL TMTechnologies) perform positive selection on monocytes. Based on the manufacturer's protocol, CD14+ cells are labeled with a mixture of a tetrameric antibody targeting the CD14 antigen and dextran-coated magnetic particles by immunomagnetic cell sorting to enrich monocytes. The isolated monocytes are suspended in ImmunoCult TM -ACF dendritic cell medium (STEMCELL TM Technologies) at a concentration of 1.0 × 106 cells / mL.

[0118] Cell adhesion, differentiation and maturation of monocyte-derived dendritic cells : For cell culture studies, custom wells with various FEP surfaces are prepared by mounting an FEP membrane onto the bottom of a CultureWell TM 8-well removable chamber slide (Gracebio) under sterile conditions.

[0119] Day 0 : In ImmunoCult TM -ACF dendritic cell medium (STEMCELL TM Technologies) containing differentiation factors (GM-CSF 50 ng / mL; IL-4 35 ng / mL), cells are seeded onto the following surfaces at a density of 1,500 cells / mm 2 : FEP (untreated), FEP (treated), O-GPPC, N-hyb-GPPC, and TCPS ( , red). The seeded cells are incubated at 37 °C and 5% CO2 and allowed to adhere for 2 hours.

[0120] Quantification of adherent cells : After incubation, non-adherent cells are aspirated from each well and the adherent cells are rinsed off the surface by washing three times with DPBS. The adherent cells are stained by incubation in ethidium homodimer, and the Hoechst mixture is diluted 1:400 in DPBS for 15 minutes. The stained cells are enumerated by acquiring fluorescence images from 21 positions within each well using an Olympus IX81 inverted fluorescence microscope (10X objective). ImageJ is used to analyze and count the stained adherent cells.

[0121] Day 3 - Replacement of differentiation medium: Prepare fresh Immunocult differentiation medium with 2X differentiation factors (GM-CSF = 50 ng / mL; IL-4 = 35 ng / mL). Remove the samples from the incubator and add the differentiation medium to the corresponding wells. Gently shake the chamber to mix the medium within the wells and incubate the cells again at 37 °C and 5% CO2.

[0122] Day 5 - Quantification of adherent cells : After incubation, aspirate the non-adherent cells from each well and rinse them off the surface by washing the adherent cells three times with DPBS. Stain the adherent cells by incubating them in ethidium homodimer and dilute the Hoechst mixture 1:400 in DPBS for 15 minutes. Enumerate the stained cells by acquiring fluorescence images from 21 positions within each well using an Olympus IX81 inverted fluorescence microscope (10X objective). Use ImageJ to analyze and count the stained adherent cells.

[0123] Day 5 - Replacement of maturation medium : To mature dendritic cells, prepare fresh Immunocult maturation medium 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). Remove the samples from the incubator and aspirate the medium containing non-adherent cells from the wells and transfer it to a labeled 15 mL centrifuge tube. To avoid death of the adherent cells, immediately add the maturation medium to the empty wells. Centrifuge the aspirated medium in the 15 mL tube at 300 × g for 5 minutes. Aspirate the supernatant and resuspend the cell pellet in the required maturation medium. Add the resuspended cell suspension to the corresponding wells and incubate the cells at 37 °C and 5% CO2.

[0124] Day 7 - Quantification of adherent cells : After incubation, aspirate the non-adherent cells from each well and rinse them off the surface by washing the adherent cells three times with DPBS. Stain the adherent cells by incubating them in ethidium homodimer and dilute the Hoechst mixture 1:400 in DPBS for 15 minutes. Enumerate the stained cells by acquiring fluorescence images from 21 positions within each well using an Olympus IX81 inverted fluorescence microscope (10X objective). Use ImageJ to analyze and count the stained adherent cells.

[0125] Figure 8 is a graph showing cell adhesion and proliferation of monocyte-derived dendritic cells on the coated surfaces described in Examples 5 and 8, respectively, compared to on an unactivated fluoropolymer surface, an activated fluoropolymer surface, and a commercially available TCPS surface. Generally,Figure 8 It is shown that, under similar experimental conditions, compared with the unactivated fluoropolymer surface, cells tend to adhere better on the two coated surfaces, and cell adhesion on the two coated surfaces is comparable to cell adhesion on the activated fluoropolymer surface and the commercially available TCPS surface.

[0126] Specifically, Figure 8 It is shown that at 2 hours, the cell seeding density of monocytes on the coated surface described in Example 5 is more than twice the cell seeding density of monocytes on the unactivated fluoropolymer surface. Figure 8 It is also shown that at 2 hours, the cell seeding density of monocytes on the coated surface described in Example 8 is about 1.5 times the cell seeding density of monocytes on the unactivated fluoropolymer surface and is comparable to the cell seeding density of monocytes on the activated fluoropolymer surface and the commercially available TCPS surface. In addition, on the 5th or 7th day, the cell seeding density of dendritic cells on all tested surfaces is shown to be lower than the cell seeding density at 2 hours, but under similar experimental conditions, cell adhesion on the two coated surfaces is shown to be comparable to cell adhesion on the activated fluoropolymer surface and the commercially available TCPS surface.

[0127] The various aspects of the present disclosure are further described by the following list of enumerated embodiments, which can be combined in any combination and in any number that is logically or technically inconsistent.

[0128] Embodiment 1. A surface suitable for cell culture, the surface comprising:

[0129] a substrate having a fluoropolymer surface; and

[0130] an amorphous hydrogenated carbon coating disposed on the fluoropolymer surface of the substrate, the amorphous hydrogenated carbon coating having a first thickness region and a second thickness region, the first thickness region being adjacent to and extending from the fluoropolymer surface, the second thickness region being remote from the fluoropolymer surface and at the surface of the amorphous hydrogenated carbon coating, the amorphous hydrogenated carbon coating having a higher total concentration of oxygen and nitrogen in the second thickness region than in the first thickness region.

[0131] Embodiment 2. The surface according to claim 1, wherein the fluoropolymer surface is a fluorinated ethylene propylene (FEP) surface.

[0132] Embodiment 3. The surface according to 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.

[0133] Embodiment 4. The surface according to any one of claims 1 to 3, wherein the fluoropolymer surface is the surface of a fluoropolymer-silicone laminate film.

[0134] Embodiment 5. The surface according to any one of claims 1 to 4, wherein the fluoropolymer surface is the surface of a film having an oxygen permeability of at least 1500 cc / m 2 -day-atm, for example, at least 1800 cc / m 2 -day-atm, or in the range of 1500 cc / m 2 -day-atm to 20000 cc / m 2 -day-atm or 1800 cc / m 2 -day-atm to 16000 cc / m 2 -day-atm.

[0135] Embodiment 6. The surface according to any one of claims 1 to 5, wherein the fluoropolymer surface is the surface of a film having a carbon dioxide permeability of at least 3500 cc / m 2 -day-atm, for example, 4000 cc / m 2 -day-atm, or in the range of 3500 cc / m 2 -day-atm to 25000 cc / m 2 -day-atm, 4000 cc / m 2 -day-atm to 23000 cc / m 2 -day-atm.

[0136] Embodiment 7. The surface according to any one of claims 1 to 6, wherein the fluoropolymer surface is an activated fluoropolymer surface.

[0137] Embodiment 8. The surface according to 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 %.

[0138] Embodiment 9. The surface according to claim 7 or claim 8, wherein the activated fluoropolymer surface has a water contact angle in the range of 60° to 120°.

[0139] Embodiment 10. The surface according to any one of claims 7 to 9, wherein the fluoropolymer surface is activated by plasma treatment.

[0140] Embodiment 11. The surface according to claim 10, wherein the plasma is an ammonia plasma, a CO2 plasma, or a plasma of a combination of ammonia and CO2.

[0141] Embodiment 12. The surface according to claim 10, wherein the plasma is an oxygen plasma, a nitrogen plasma, or a plasma of a combination of oxygen and nitrogen.

[0142] Embodiment 13. The surface according to any one of claims 7 to 9, wherein the fluoropolymer surface is activated by corona treatment such as C treatment.

[0143] Embodiment 14. The surface according to any one of claims 1 to 13, wherein the thickness of the amorphous hydrogenated carbon coating is in the range of 10 nm to 200 nm, for example, 10 nm to 100 nm, or 10 nm to 75 nm, or 10 nm to 50 nm, or 15 nm to 200 nm, or 15 nm to 100 nm, or 15 nm to 75 nm, or 15 nm to 50 nm, or 20 nm to 200 nm, or 20 nm to 100 nm, or 20 nm to 75 nm, or 20 nm to 50 nm.

[0144] Embodiment 15. The surface according to any one of claims 1 to 14, wherein the ratio of hydrogen to carbon in the amorphous hydrogenated carbon coating is in the range of at least 1, for example, at least 1.2 or at least 1.4.

[0145] Embodiment 16. The surface according to any one of claims 1 to 15, wherein the thickness of the first thickness region of the amorphous hydrogenated carbon coating is in the range of 8 nm to 190 nm, for example, 8 nm to 100 nm, or 8 nm to 75 nm, or 8 nm to 50 nm, or 15 nm to 190 nm, or 15 nm to 100 nm, or 15 nm to 75 nm, or 15 nm to 50 nm, or 20 nm to 190 nm, or 20 nm to 100 nm, or 20 nm to 75 nm, or 20 nm to 50 nm.

[0146] Embodiment 17. A surface according to any one of claims 1 to 16, wherein in the first thickness region, the total concentration of oxygen and nitrogen of the amorphous hydrogenated carbon coating is at least 3 atomic% lower than the total concentration of oxygen and nitrogen in the second thickness region, for example, at least 4 atomic% lower, or at least 5 atomic% lower, or at least 6 atomic% lower, or at least 7 atomic% lower.

[0147] Embodiment 18. The surface coating of any one of claims 1 to 17, wherein the amorphous hydrogenated carbon coating has a total concentration of oxygen and nitrogen in the first thickness region of at least 5 atomic %, e.g., at least 10 atomic % or at least 15 atomic %.

[0148] Embodiment 19. A surface according to any one of claims 1 to 18, wherein the thickness of the second thickness region of the amorphous hydrogenated carbon coating is in the range of 2nm to 100nm, for example, 2nm to 50nm, or 2nm to 35nm, or 2nm to 20nm, or 2nm to 10nm, or 5nm to 100nm, or 5nm to 50nm, or 5nm to 35nm, or 5nm to 20nm, or 5nm to 10nm, or 10nm to 100nm, or 10nm to 50nm, or 10nm to 35nm, or 10nm to 20nm, or 20nm to 100nm, or 20nm to 50nm.

[0149] Embodiment 20. A surface according to any one of claims 1 to 19, wherein in the second thickness region, the total concentration of oxygen and nitrogen in the amorphous hydrogenated carbon coating is at least 10 atomic %, for example, at least 15 atomic % or at least 20 atomic %.

[0150] Embodiment 21. The surface of any one of claims 1 to 20, wherein, in the first thickness region, the amorphous hydrogenated carbon coating comprises oxygen.

[0151] Embodiment 22. The surface of claim 21 , wherein the amorphous hydrogenated carbon coating in its first thickness region consists of at least 90 atomic %, such as at least 95 atomic %, carbon and oxygen.

[0152] Embodiment 23. The surface of claim 21 or 22, wherein, in the second thickness region, the amorphous hydrogenated carbon coating comprises oxygen.

[0153] Embodiment 24. The surface of any one of claims 21 to 23, wherein the amorphous hydrogenated carbon coating in its second thickness region consists of at least 90 atomic %, such as at least 95 atomic %, carbon and oxygen.

[0154] Embodiment 25. The surface according to claim 21 or claim 22, wherein in the second thickness region, the amorphous hydrogenated carbon coating contains nitrogen.

[0155] Embodiment 26. The surface according to any one of claims 21, 22, and 25, wherein the amorphous hydrogenated carbon coating in its second thickness region consists of at least 90 atomic%, such as at least 95 atomic%, of carbon and nitrogen.

[0156] Embodiment 27. The surface according to claim 21 or claim 22, wherein in the second thickness region, the amorphous hydrogenated carbon coating contains oxygen and nitrogen.

[0157] Embodiment 28. The surface according to any one of claims 21, 22, and 27, wherein the amorphous hydrogenated carbon coating in its second thickness region consists of at least 90 atomic%, such as at least 95 atomic%, of carbon, oxygen, and nitrogen.

[0158] Embodiment 29. The surface according to any one of claims 1 to 20, wherein in the first thickness region, the amorphous hydrogenated carbon coating contains oxygen and nitrogen.

[0159] Embodiment 30. The surface according to claim 29, wherein the amorphous hydrogenated carbon coating in its first thickness region consists of at least 90 atomic%, such as at least 95 atomic%, of carbon, oxygen, and nitrogen.

[0160] Embodiment 31. The surface according to claim 29 or 30, wherein in the second thickness region, the amorphous hydrogenated carbon coating contains oxygen.

[0161] Embodiment 32. The surface according to any one of claims 29 to 31, wherein the amorphous hydrogenated carbon coating in its second thickness region consists of at least 90 atomic%, such as at least 95 atomic%, of carbon and oxygen.

[0162] Embodiment 33. The surface according to claim 29 or claim 30, wherein in the second thickness region, the amorphous hydrogenated carbon coating contains nitrogen.

[0163] Embodiment 34. The surface according to any one of claims 29, 30, and 33, wherein the amorphous hydrogenated carbon coating in its second thickness region consists of at least 90 atomic%, such as at least 95 atomic%, of carbon and nitrogen.

[0164] Embodiment 35. The surface according to claim 29 or claim 30, wherein in the second thickness region, the amorphous hydrogenated carbon coating contains oxygen and nitrogen.

[0165] Embodiment 36. The surface according to any one of claims 29, 30, and 35, wherein the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic %, for example at least 95 atomic %, of carbon, oxygen, and nitrogen.

[0166] Embodiment 37. The surface according to any one of claims 1 to 20, wherein in the first thickness region, the amorphous hydrogenated carbon coating contains nitrogen.

[0167] Embodiment 38. The surface according to claim 37, wherein the amorphous hydrogenated carbon coating in its first thickness region is composed of at least 90 atomic %, for example at least 95 atomic %, of carbon and nitrogen.

[0168] Embodiment 39. The surface according to claim 37 or claim 38, wherein in the second thickness region, the amorphous hydrogenated carbon coating contains oxygen.

[0169] Embodiment 40. The surface according to claims 37 to 39, wherein the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic %, for example at least 95 atomic %, of carbon and oxygen.

[0170] Embodiment 41. The surface according to claim 37 or claim 38, wherein in the second thickness region, the amorphous hydrogenated carbon coating contains nitrogen.

[0171] Embodiment 42. The surface according to claims 37, 38, and 41, wherein the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic %, for example at least 95 atomic %, of carbon and nitrogen.

[0172] Embodiment 43. The surface according to claim 37 or claim 38, wherein in the second thickness region, the amorphous hydrogenated carbon coating contains oxygen and nitrogen.

[0173] Embodiment 44. The surface according to claims 37, 38, and 43, wherein the amorphous hydrogenated carbon coating in its second thickness region is composed of at least 90 atomic %, for example at least 95 atomic %, of carbon, nitrogen, and oxygen.

[0174] Embodiment 45. The surface according to any one of claims 1 to 44, wherein the first thickness region of the amorphous hydrogenated carbon coating is adjacent to the second thickness region of the amorphous hydrogenated carbon coating.

[0175] Embodiment 46. The surface according to any one of claims 1 to 44, wherein one or more additional thickness regions of the amorphous hydrogenated carbon coating are provided between the first thickness region of the amorphous hydrogenated carbon coating and the second thickness region of the amorphous hydrogenated carbon coating.

[0176] Embodiment 47. The surface according to claim 46, wherein the one or more additional thickness regions are a third thickness region, and the third thickness region is provided between the first thickness region of the amorphous hydrogenated carbon coating and the second thickness region of the amorphous hydrogenated carbon coating and is adjacent to the first thickness region and the second thickness region.

[0177] Embodiment 48. The surface according to claim 47, wherein the second thickness region contains nitrogen, and wherein the third thickness region contains oxygen and / or nitrogen, and wherein the amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in the third thickness region than in the first thickness region.

[0178] Embodiment 49. The surface according to any one of claims 1 to 48, wherein the surface is the inner surface of a cell culture container, and the cell culture container is, for example, a cell culture bag, a cell culture flask, a cell culture vial, a cell culture tube, or a cell culture tubing.

[0179] Embodiment 50. A method for preparing a surface suitable for cell culture, the surface being, for example, the surface according to any one of claims 1 to 49, the method comprising:

[0180] Depositing an amorphous hydrogenated carbon coating on a fluoropolymer surface of a substrate, the deposition comprising:

[0181] Using a combination of a hydrocarbon gas and one or more oxygen- and / or nitrogen-containing plasma-reactive gases (e.g., CO2 and / or NH3) to deposit a first thickness of the amorphous hydrogenated carbon coating in a first region thereof via chemical vapor deposition at a first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas, wherein the first region is adjacent to and extends from the fluoropolymer surface; and

[0182] 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), deposit a second thickness of the amorphous hydrogenated carbon coating in its second region via chemical vapor deposition (e.g., PECVD) at a second ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas, the second ratio being lower than the first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas, wherein the second region is remote from the fluoropolymer surface and is at the surface of the amorphous hydrogenated carbon coating, and wherein the amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in its second region than in its first region.

[0183] Embodiment 51. The method according to claim 50, the method further comprising: before depositing the second thickness of the amorphous hydrogenated carbon coating in its second region, 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) to deposit a third thickness of the amorphous hydrogenated carbon coating in its third region via chemical vapor deposition (e.g., PECVD) at a third ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas, the third ratio being lower than the first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gas, wherein the third region is between the first region and the second region and is adjacent to the first region and the second region, and wherein the amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in its third region than in its first region.

[0184] Embodiment 52. The method according to claim 50 or claim 51, wherein the chemical vapor deposition is plasma-enhanced chemical vapor deposition.

[0185] Embodiment 53. The method according to any one of claims 50 to 52, wherein the hydrocarbon gas is an olefin, e.g., propylene or ethylene.

[0186] Embodiment 54. The method according to any one of claims 50 to 53, wherein the fluoropolymer surface of the substrate on which the amorphous hydrogenated carbon coating is deposited is an activated fluoropolymer surface.

[0187] Embodiment 55. The method according to claim 54, the method further comprising activating the fluoropolymer surface.

[0188] Embodiment 56. The method according to claim 55, wherein the fluoropolymer surface is activated by plasma treatment, for example, treated with NH3 plasma, CO2 plasma, plasma of a combination of NH3 and CO2, oxygen plasma, nitrogen plasma, or plasma of a combination of oxygen and nitrogen.

[0189] Embodiment 57. The method according to claim 55, wherein the fluoropolymer surface is activated by corona treatment, for example, C treatment.

[0190] Embodiment 58. The method according to 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 %.

[0191] Embodiment 59. The method according to any one of claims 54 to 58, wherein the activated fluoropolymer surface has a water contact angle in the range of 60° to 120°.

[0192] Embodiment 60. A surface for cell culture (for example, according to any one of claims 1 to 49), the surface being prepared by the method according to any one of claims 50 to 59.

[0193] Embodiment 61. A method for culturing adherent cells, the method comprising incubating the surface according to any one of claims 1 to 49 and 60 together with adherent cells and a growth medium.

[0194] Embodiment 62. The method according to claim 61, wherein the adherent cells are stem cells, for example, mesenchymal stromal cells.

[0195] Embodiment 63. The method according to claim 61, wherein the adherent cells are dendritic cells, for example, monocyte-derived dendritic cells.

[0196] Embodiment 64. The method according to any one of claims 61 to 63, wherein the growth medium is a basal medium.

[0197] Embodiment 65. The method according to any one of claims 61 to 64, wherein the growth medium contains a buffer (for example, sodium bicarbonate), glutamine, and growth factors.

[0198] Embodiment 66. The method according to any one of claims 61 to 65, wherein the pH of the growth medium is in the range of 7.2 - 7.4.

[0199] Embodiment 67. The method according to any one of claims 61 to 66, wherein the incubation temperature for culturing the adherent cells is in the range of 35 °C to 39 °C (for example, 37 °C).

[0200] Embodiment 68. The method according to any one of claims 61 to 67, wherein the surface is sterilized before incubation with the cells.

[0201] Embodiment 68. The method according to claim 68, wherein the sterilization is carried out by autoclaving.

Claims

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

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

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

4. The surface according to 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 according to claim 4, wherein The fluoropolymer surface is activated by plasma treatment.

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

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

8. The surface according to claim 1, wherein, The thickness of the first thickness region of the amorphous hydrogenated carbon coating is in the range of 8 nm to 190 nm.

9. The surface according to claim 1, wherein In the first thickness region, the total concentration of oxygen and nitrogen in the amorphous hydrogenated carbon coating is at least 3 atomic % lower than the total concentration of oxygen and nitrogen in the second thickness region.

10. The surface coating according to claim 1, wherein, The total concentration of oxygen and nitrogen in the amorphous hydrogenated carbon coating in the first thickness region is at least 5 atomic %, for example, at least 10 atomic % or at least 15 atomic %.

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

12. The surface according to claim 1, wherein, In the second thickness region, the total concentration of oxygen and nitrogen in the amorphous hydrogenated carbon coating is at least 10 atomic %, for example, at least 15 atomic % or at least 20 atomic %.

13. The surface according to claim 1, wherein, A third thickness region is disposed between the first thickness region and the second thickness region of the amorphous hydrogenated carbon coating and is adjacent to the first thickness region and the second thickness region.

14. The surface according to claim 13, wherein The second thickness region contains nitrogen, and wherein the third thickness region contains oxygen and / or nitrogen, and wherein the amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in the third thickness region than in the first thickness region.

15. The surface according to claim 1, wherein The surface is the inner surface of a cell culture vessel.

16. A method for preparing a surface suitable for cell culture according to claim 1, the method comprising: depositing an amorphous hydrogenated carbon coating on a fluoropolymer surface of a substrate, the deposition comprising: Deposit the first thickness of the amorphous hydrogenated carbon coating in its first region 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., CO2 and / or NH3) at a first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gases, wherein the first region is adjacent to and extends from the fluoropolymer surface; and Deposit the second thickness of the amorphous hydrogenated carbon coating in its second region 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) at a second ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gases, the second ratio being lower than the first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gases, wherein the second region is remote from the fluoropolymer surface and is at the surface of the amorphous hydrogenated carbon coating, and wherein the amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in its second region than in its first region.

17. A method for preparing a surface suitable for cell culture, the method comprising: Deposit an amorphous hydrogenated carbon coating on a fluoropolymer surface of a substrate, the deposition comprising: Deposit the first thickness of the amorphous hydrogenated carbon coating in its first region 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., CO2 and / or NH3) at a first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gases, wherein the first region is adjacent to and extends from the fluoropolymer surface; and Deposit the second thickness of the amorphous hydrogenated carbon coating in its second region 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) at a second ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gases, the second ratio being lower than the first ratio of the hydrocarbon gas to the oxygen- and / or nitrogen-containing plasma-reactive gases, wherein the second region is remote from the fluoropolymer surface and is at the surface of the amorphous hydrogenated carbon coating, and wherein the amorphous hydrogenated carbon coating has a higher total concentration of oxygen and nitrogen in its second region than in its first region.

18. The method according to claim 17, wherein The hydrocarbon gas is an olefin.

19. A surface for cell culture, the surface being prepared by the method according to claim 17.

20. A method for culturing adherent cells, the method comprising incubating the surface according to claim 1 with adherent cells and a growth medium.

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