Type I collagen coating method for promoting cell adherent proliferation

Abstract

The invention discloses an I-type collagen coating method for promoting cell adherent proliferation, and belongs to the technical field of cell culture. According to the method, collagen is not prone to falling off, no obvious adverse effect is generated on the biological activity of the collagen, and coating is easy and fast, 60%-80% (v / v) ethyl alcohol is used as a solvent to dissolve or dilute the collagen I, and a collagen I solution is obtained; the culture vessel is coated with the I-type collagen solution as a coating solution, the culture vessel coated with the I-type collagen is obtained, the whole process is carried out at room temperature, a specific environment does not need to be provided, the defect that an existing traditional coating mode needs to be carried out under ice is overcome, and the culture vessel can be used for promoting cell proliferation and adhesion, is suitable for culture of various cells and has good application prospects. And the cost is reduced.

Description

Technical Field

[0001] The invention relates to a type I collagen coating method for promoting cell adhesion and proliferation, and belongs to the technical field of cell culture. Background Art

[0002] In the early days of cell culture technology, scientists began to try various methods to simulate the cell growth environment in the body. Type I collagen has gradually become a hot topic of research due to its similarity to the extracellular matrix. Early coating technology was relatively simple, mainly fixing collagen on culture dishes or culture plates by physical adsorption or chemical cross-linking. With the development of biotechnology and materials science, type I collagen coating technology has been continuously improved, and the uniformity and stability of collagen on the surface of consumables have been improved by optimizing the coating process. At the same time, a variety of different specifications and types of culture consumables have been developed, such as 96-well plates, culture dishes, etc., to meet different experimental needs.

[0003] The physical adsorption method uses the physical interaction between collagen molecules and the surface of consumables (such as van der Waals forces, hydrogen bonds, etc.) to adsorb collagen on the surface of consumables. This method is simple and easy, but there may be problems with collagen shedding or uneven distribution. The chemical cross-linking method connects collagen to the surface of consumables through chemical bonds to form a stable coating layer. This method can significantly improve the stability and durability of collagen on the surface of consumables, but it may have a certain effect on the biological activity of collagen. The hydrogel method uses hydrogel as a carrier, embeds collagen in it, and then coats it on the surface of consumables. This method can form a more uniform coating layer while maintaining the biological activity of collagen. However, the preparation and coating process of hydrogels may be relatively complicated.

[0004] Therefore, it is crucial to be able to simply, quickly and stably coat type I collagen consumables on cell culture plates or culture dishes without affecting the biological activity of collagen. Summary of the invention

[0005] To solve the above problems, the present invention provides a type I collagen coating method for promoting cell adhesion and proliferation. A specific type I collagen is selected in combination with a room temperature coating process, and a suitable solvent, ethanol, is screened out to achieve traceless and stable coating of type I collagen. The coating method is simple and suitable for large-scale use.

[0006] The first object of the present invention is to provide a method for coating type I collagen, comprising the following steps: diluting type I collagen with 60% to 80% (v / v) ethanol as a solvent to obtain a type I collagen solution, coating a culture vessel with the type I collagen solution as a coating liquid to obtain a culture vessel coated with type I collagen.

[0007] Furthermore, the solvent is 70% (v / v) ethanol.

[0008] Furthermore, the concentration of the type I collagen solution is 0.01-0.1 mg / mL.

[0009] Further, the ratio of type I collagen solution to the surface area of ​​the culture vessel is 0.4-10 μg / cm 2 Add type I collagen solution.

[0010] Furthermore, the coating time is 6-24 hours, preferably 6 hours.

[0011] Furthermore, the coating temperature is 20-25°C.

[0012] Furthermore, before the coating step, the method further includes rinsing the cell culture vessel with PBS.

[0013] Furthermore, after the coating step, the method further includes removing excess collagen solution and drying the solution.

[0014] Furthermore, the culture vessel coated with type I collagen is used for cell culture; the cells include but are not limited to human fibrosarcoma cells.

[0015] The second object of the present invention is to provide the use of the culture vessel coated with type I collagen prepared by the coating method in cell culture.

[0016] Beneficial effects of the present invention:

[0017] The present invention provides a new type I collagen coating method, innovatively dilutes type I collagen with a specific concentration of ethanol as a solvent, and uses a room temperature coating method to achieve a simple and fast traceless coating, which overcomes the problem of collagen shedding in the current physical adsorption method, and overcomes the defects of the chemical cross-linking method that may have a certain impact on the biological activity of collagen and the complex preparation and coating process faced by the hydrogel method. The coating of the present invention is carried out at room temperature throughout the process, without providing a specific environment, overcoming the defect that the current traditional coating method needs to be carried out under ice, and the obtained culture vessel coated with type I collagen can be used to promote cell proliferation and adhesion, is suitable for the culture of a variety of cells, reduces costs, and has a wide range of application prospects in cell culture and related research. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a comparison chart of the effects of different solvent-coated type I collagen methods on cell culture.

[0019] Figure 2This is a rough range test comparing the effects of different concentrations of type I collagen and coating time on cell culture.

[0020] Figure 3 A comparison chart to accurately test the effects of different concentrations of type I collagen and coating time on cell culture. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0022] The scheme involved in the present invention is as follows:

[0023] In order to solve the shortcoming that type I collagen is easily detached due to physical adsorption and ensure the uniformity between parallel holes, the present invention proposes a new method for coating type I collagen:

[0024] 60% to 80% (v / v) ethanol is used as a solvent to dissolve or dilute type I collagen to obtain a type I collagen solution, and the type I collagen solution is used as a coating liquid to coat a culture vessel to obtain a culture vessel coated with type I collagen.

[0025] At present, the commonly used solvent for coating type I collagen is acetic acid, which helps maintain the structure and function of collagen. However, since the dynamic response of collagen molecules to acetic acid is related to the change in the size of collagen fibers, this process is not stable, resulting in the inability to strictly control the coating process, and some studies have shown that the presence of acetic acid residues may reduce cell proliferation. In addition, the acetic acid coating process needs to be kept at a low temperature (such as on ice) to avoid rapid gelation of collagen at room temperature, which increases the complexity of the operation.

[0026] In the present invention, acetic acid is replaced with an ethanol solution in a specific concentration range as a solvent for coating type I collagen: ethanol and acetic acid have different solubility for type I collagen, and the use of ethanol will change the solubility and stability of collagen. At the same time, it is well known that the coating effect and stability of collagen depend on a specific pH value. Acetic acid is acidic, while ethanol provides a neutral environment. Replacing it with ethanol may affect the coating. The present invention has been verified to find that ethanol as a coating solvent can make type I collagen more uniform and adsorb more tightly, ensuring the parallelism between holes. More importantly, even if ethanol is successfully used for coating, it is necessary to verify whether it will have a negative impact on the adhesion and growth of cells: existing studies have shown that ethanol can inhibit cell proliferation and may cause cell apoptosis by inducing the production of intracellular reactive oxygen species, so it is generally not thought of using ethanol as a coating solvent. However, it was unexpectedly found in the present invention that the cell culture plate coated with type I collagen dissolved in ethanol had no obvious adverse effect on the growth of human cells, and the proliferation and growth of cells were better than those using acetic acid.

[0027] Preferably, 60% to 80% (v / v) ethanol is used as the solvent, for example 65% (v / v), 70% (v / v), 75% (v / v), 80% (v / v), 85% (v / v), 90% (v / v), 95% (v / v), 100% (v / v) anhydrous ethanol, etc., including but not limited to the concentration values ​​listed above. The most preferred solvent is 70% (v / v) ethanol.

[0028] Preferably, the concentration of the type I collagen solution is 0.01-0.1 mg / mL.

[0029] Preferably, the ratio of type I collagen solution to the surface area of ​​the culture vessel is 0.4-10 μg / cm 2 Add type I collagen solution. For example, 1.5 μg / cm 2 , 2μg / cm 2 , 2.5μg / cm 2 , 3μg / cm 2 , 3.5μg / cm 2 , 4μg / cm 2 , 4.5μg / cm 2 , 5μg / cm 2 , 5.5μg / cm 2 , 6μg / cm 2 、6.5μg / cm 2 , 7μg / cm 2 , 7.5μg / cm 2 , 8μg / cm 2 , 8.5μg / cm 2 , 9μg / cm 2, 9.5μg / cm 2 Etc., including but not limited to the ratios listed above.

[0030] In the present invention, the concentration of type I collagen solution directly affects the coating thickness. A higher concentration (higher than 10 μg / cm 2 ) will lead to the formation of a thicker collagen layer, which will lead to excessive rigidity and a decrease in cell proliferation rate. The ratio of type I collagen to surface area will also affect the thickness and uniformity of the coating. Different culture vessels require different volumes of collagen solution to ensure uniform coating.

[0031] In terms of the effect on cell growth, collagen has a significant effect on cell growth, and suitably coated type I collagen can better maintain cell proliferation and promote morphogenesis. More importantly, the collagen selected in the present invention is type I collagen. As is well known, the type of collagen will also affect cell growth. For example, although Cellmatrix Type IA and Type IP are both type I collagen, the coating layers prepared at the same concentration have different strengths, thereby affecting cell proliferation and morphology. The type I collagen selected in the present invention is combined with ethanol as a solvent coating process, which retains the original advantages of type I collagen and realizes the simple coating of this type of type I collagen.

[0032] Preferably, the coating time is 6-24 hours, for example 6.5 hours, 7 hours, 7.5 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, etc., including but not limited to the time lengths listed above.

[0033] Preferably, the coating temperature is 20-25°C. For example, 21°C, 22°C, 23°C, 24°C, 25°C, etc., including but not limited to the temperatures listed above. Collagen forms fibers and gels under physiological conditions. Temperature has a significant effect on the gelation rate and gel strength of collagen. At different temperatures, the behavior of collagen is completely different, which in turn affects the coating effect. At the same time, the structure of collagen is closely related to temperature, which not only affects the stability of collagen, but also affects the supportive structural network provided by collagen for cell proliferation and adhesion. Therefore, in the present invention, not only room temperature is selected for the convenience of operation, but also it is found that the appropriate solvent and coating method at this temperature can minimize the impact on cell behavior.

[0034] Preferably, before the coating step, the method further comprises rinsing the cell culture vessel with PBS.

[0035] Preferably, after the coating step, the method further comprises the steps of removing excess collagen solution and drying.

[0036] Preferably, the culture vessel coated with type I collagen is used for cell culture; the cells include but are not limited to human fibrosarcoma cells. Different types of cells have different adhesion and proliferation activities to collagen. Therefore, when selecting type I collagen coated consumables, it is necessary to carefully select according to specific experimental requirements and cell types.

[0037] Example 1

[0038] (1) Dilute type I collagen to 0.063 mg / mL with ultrapure water, PBS, 70% (v / v) ethanol, and 0.02 M acetic acid, respectively;

[0039] (2) Take a sterile cell culture plate and rinse it with PBS. 2 Add the diluted type I collagen solution and shake gently to ensure that the bottom is completely covered with liquid;

[0040] (3) Allow to stand at room temperature (20-25°C) for 6 hours for coating;

[0041] (4) After the coating is completed, the excess collagen solution in the cell culture plate is aspirated and dried to obtain a coated cell plate;

[0042] (5) Add cell suspension: 100 μL containing 1×10 5 Add a suspension of HT1080 cells (human fibrosarcoma cells) to the coated cell plate;

[0043] (6) Adding culture medium: Add 1 mL of SFM serum-free culture medium to the cell plate in step (5) and observe cell growth after 24 hours.

[0044] Microscopic observation of the growth of HT1080 cells in different groups Figure 1 As can be seen from the figure, when the remaining coating solution was directly sucked away after coating, the ddH2O, 70% ethanol and 0.02M acetic acid groups dried without traces, and the cell culture effect of the ddH2O and 70% ethanol groups was equivalent to the normal level; only the 70% ethanol group ensured the uniformity of coating and could be dried quickly. Therefore, 70% ethanol was finally selected to dilute type I collagen for coating the cell plate.

[0045] Example 2

[0046] (1) Dilute type I collagen to 0.063 mg / mL with 70% (v / v) ethanol;

[0047] (2) Take a sterile cell culture plate and rinse it with PBS. 2 , 2μg / cm 2, 10μg / cm 2 Add the diluted type I collagen solution and shake gently to ensure that the bottom is completely covered with liquid;

[0048] (3) Allow to stand at room temperature (20-25°C) for 6 h and 24 h respectively for coating;

[0049] (4) After the coating is completed, the excess collagen solution in the cell culture plate is aspirated and dried to obtain a coated cell plate;

[0050] (5) Add cell suspension: 100 μL containing 1×10 5 Add a suspension of HT1080 cells (human fibrosarcoma cells) to the coated cell plate;

[0051] (6) Adding culture medium: Add 1 mL of SFM serum-free culture medium to the cell plate in step (5) and observe cell growth after 24 hours.

[0052] Microscopic observation of the growth of HT1080 cells in different groups Figure 2 As can be seen from the figure, at 0 and 0.4 μg / cm 2 , 2μg / cm 2 , 10μg / cm 2 Under coating, only 2μg / cm 2 , 10μg / cm 2 The number of cells attached to the wall is close to the same, while 0.4 μg / cm 2 The number of cells attached to the wall was significantly reduced under the concentration coating, so it can be inferred that 2-10μg / cm 2 The concentration is already saturated at 0.4 μg / cm 2 The following concentrations could no longer meet the cell adhesion requirements. Therefore, 0.4 μg / cm 2 ~2 μg / cm 2 , 6h to 24h for further optimization of coating concentration and coating time.

[0053] Example 3

[0054] (1) Dilute type I collagen to 0.063 mg / mL with 70% (v / v) ethanol;

[0055] (2) Take a sterile cell culture plate and rinse it with PBS. 2 , 1μg / cm 2 , 2μg / cm 2 Add the diluted type I collagen solution and shake gently to ensure that the bottom is completely covered with liquid;

[0056] (3) Allow to stand at room temperature (20-25°C) for 6 h, 18 h, and 24 h for coating;

[0057] (4) After the coating is completed, the excess collagen solution in the cell culture plate is aspirated and dried to obtain a coated cell plate;

[0058] (5) Add cell suspension: 100 μL containing 1×10 5 Add a suspension of HT1080 cells (human fibrosarcoma cells) to the coated cell plate;

[0059] (6) Adding culture medium: Add 1 mL of SFM serum-free culture medium to the cell plate in step (5) and observe cell growth after 24 hours.

[0060] Microscopic observation of the growth of HT1080 cells in different groups Figure 3 As can be seen from the figure, at 0 and 0.4 μg / cm 2 , 1μg / cm 2 , 2μg / cm 2 Under coating, 1μg / cm 2 and 2 μg / cm 2 Still maintain consistent cell attachment number, and 0.4μg / cm 2 The number of cells attached to the wall was significantly reduced, so 1 μg / cm 2 The conditions were suitable; no significant difference was found in the number of cells attached to the wall at 6, 18 and 24 h of coating. Considering the performance of the coated cells and the convenience of calculation, the 1 μg / cm 2 , 6h are the optimal coating conditions in experimental tests.

[0061] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for coating with type I collagen to promote cell adhesion and proliferation, characterized in that: The method comprises the following steps: using 60% to 80% (v / v) ethanol as a solvent to dilute type I collagen to obtain a type I collagen solution, using the type I collagen solution as a coating liquid to coat a culture vessel to obtain a culture vessel coated with type I collagen.

2. The coating method according to claim 1, characterized in that The concentration of the type I collagen solution is 0.01-0.1 mg / mL; the ratio of the type I collagen solution to the surface area of ​​the culture vessel is 0.4-10 μg / cm 2 Add type I collagen solution to the culture dish.

3. The coating method according to claim 1, characterized in that The coating time is 6-24h.

4. The coating method according to claim 1, characterized in that The coating temperature is 20-25°C.

5. The coating method according to claim 1, characterized in that The solvent is 70% (v / v) ethanol.

6. The coating method according to claim 1, characterized in that Before the coating step, the step of rinsing the cell culture vessel with PBS is also included.

7. The coating method according to claim 1, characterized in that After the coating step, the steps of removing excess collagen solution and drying are also included.

8. A culture vessel coated with type I collagen prepared by the coating method according to any one of claims 1 to 7.

9. Use of the culture vessel according to claim 8 in cell culture.

10. The use according to claim 9, characterized in that: When the culture vessel coated with type I collagen is used for cell culture, culture medium and cells to be cultured are added to the culture vessel for cell culture; Preferably, the cells to be cultured include human fibrosarcoma cells.

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