A method for non-destructively peeling a cell sheet in culture from a cell culture surface of a culture container

Abstract

The present invention relates to a method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container, belongs to the field of biotechnology, and comprises the following steps: S1, preparing a digestion solution: mixing DMEM / F12 culture medium, dispase, collagenase, and ROCK inhibitor to obtain a digestion solution; S2, cell sheet digestion: adding a cell sheet to be peeled off to the digestion solution obtained in step S1, and incubating; S3, peeling off a cell sheet: affixing the cell sheet to be peeled off to a silicone gauze, then simultaneously clamping the cell sheet-silicone gauze and pulling it away from one end of a cell culture dish until it is separated from the cell culture surface of the cell culture dish, obtaining a cell sheet-silicone gauze, that is, completing the rapid and non-destructive separation of the cell sheet in culture from the cell culture surface of the culture dish. Compared with the prior art, the method of the present invention can make the cell sheet completely detached, and the bottom of the culture dish has less adhesion; the overall operation is convenient, the time is short, and it is suitable for the preparation and peeling off of a large number of cell sheets.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container. Background Art

[0002] Cell sheets are a scaffold-free tissue engineering technology that can be used to create transplantable two-dimensional (2D) and three-dimensional (3D) tissues and organs. Cell sheets composed of pure cells can not only avoid the adverse effects of scaffold materials, but can also be further assembled to form more complex three-dimensional functional tissues, and therefore have attracted much attention in the biomedical field.

[0003] The acquisition method after the construction of the cell sheet is mainly based on the culture substrate constructed with sensitive materials. By changing the temperature, enzymes, ions and other stimulation factors, the adhesion behavior of the substrate to the cells is adjusted to make the cells naturally detach, thereby obtaining the cell sheet. Among them, the UpCell temperature-responsive cell culture dish is more commonly used. The surface of the tissue culture polystyrene (TCPS) dish is modified with a thin layer of temperature-responsive polymer poly (N-isopropylacrylamide) (PNIPAAm). At room temperature, PNIPAAm combines with water and swells, releasing adherent cells and extracellular matrix. Separate the cells from the culture substrate that does not contain trypsin. This culture dish is expensive and not convenient in actual use. It brings additional operating steps and equipment requirements to the culture process, because under this condition, operations such as cell observation and fluid replacement need to be maintained at 37°C. What is important is that the detachment effect of the cell sheet is not consistent, which limits the large-scale preparation and harvest of cell sheets for industrialization. Summary of the invention

[0004] The purpose of the present invention is to overcome the defects of the above-mentioned prior art in that the detachment effect of the cell sheet is unstable and to provide a method for non-destructively detaching the cell sheet in culture from the cell culture surface of the culture container.

[0005] The purpose of the present invention can be achieved by the following technical solutions: The technical solution of the present invention is to provide a method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container, comprising the following steps: S1. Preparation of digestion solution: DMEM / F12 medium, dispase, collagenase, and ROCK inhibitor were mixed to obtain a digestion solution; S2. Cell sheet digestion: Discard the original culture medium of the cell sheet to be removed from the cell culture dish, add it to the digestion solution obtained in step S1, and incubate; S3. Cell sheet removal: The cell sheet to be peeled off is attached to the silicone gauze, and then the cell sheet-silicone gauze is clamped and separated from the cell culture surface of the cell culture dish to obtain the cell sheet-silicone gauze, thereby completing the rapid and non-destructive dissociation of the cultured cell sheet from the cell culture surface of the culture dish.

[0006] In some specific embodiments, in step S1, the DMEM / F12 culture medium, dispase, collagenase, and ROCK inhibitor are mixed according to 50 mL: 0.5-2.4 U / mL: 0.1-1 mg / mL: 1-20 uM to obtain a digestion solution.

[0007] In some specific embodiments, in step S2, the cell sheet to be peeled off is selected from any one of a cell sheet consisting of keratinocytes, a cell sheet consisting of fibroblasts, and a cell sheet consisting of mesenchymal stem cells.

[0008] In some specific embodiments, when the cell sheet to be peeled off is a cell sheet composed of keratinocytes, in the digestion solution, the final concentration of dispase is 2-2.4 U / mL, and the final concentration of collagenase is 0.1-0.3 mg / mL; When the cell sheet to be peeled off is composed of fibroblasts, the final concentration of dispase in the digestion solution is 0.5-1.4 U / mL, and the final concentration of collagenase is 0.8-1 mg / mL; When the cell sheet to be peeled off is a cell sheet composed of mesenchymal stem cells, in the digestion solution, the final concentration of dispase is 0.5-1 U / mL, and the final concentration of collagenase is 0.1-0.5 mg / mL.

[0009] In some specific embodiments, in step S2, the incubation conditions are: 5% CO2, temperature of 37°C, and time of 10 to 50 min.

[0010] In some specific embodiments, in step S3, the method for peeling off the cell sheet is specifically as follows: first, the edge of the cell sheet to be peeled off is lifted up from the cell culture surface of the cell culture dish, and the silicone gauze is placed in the cell culture dish containing the digestive fluid, so that the lifted side of the cell sheet is closely attached to the silicone gauze, and then the edge of the cell sheet-silicone gauze is clamped and separated from the cell culture surface of the cell culture dish to obtain the cell sheet-silicone gauze, thereby completing the rapid and non-destructive dissociation of the cultured cell sheet from the cell culture surface of the culture dish.

[0011] In some specific embodiments, after the cell sheet is dissociated from the cell culture surface of the culture dish, the method further includes washing and packaging the cell sheet.

[0012] In some specific embodiments, the cleaning method is as follows: placing the peeled cell sheet-silica gel in PBS buffer to rinse the digestive solution on both sides of the cell sheet-silica gel and the digestive solution between the cell sheet and the silica gel.

[0013] In some specific embodiments, the cell sheet and the silicone gauze are clamped and placed vertically in a PBS buffer solution, so that the cell sheet and the silicone gauze are separated and unfolded in the PBS buffer solution. After the separation and unfolding, the side of the cell sheet that is attached to the silicone gauze is in contact with the PBS buffer surface, thereby rinsing the digestive fluid between the cell sheet and the silicone gauze.

[0014] In some specific embodiments, the packaging method is as follows: the cell sheet-silicone gauze is placed in a new culture dish with the cell sheet on top and the silicone gauze on the bottom and sealed for storage.

[0015] After cleaning, the silicone gauze and the cell sheet are physically attached together by PBS buffer. In subsequent use, use tweezers to stick the cell sheet side of the cell sheet-silicone gauze to the skin wound, and then use tweezers to clamp the silicone gauze and gently peel it out to easily separate it from the cell sheet without any impact on the cell sheet.

[0016] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention is applicable to cell sheets formed by various cell types. By adjusting the concentrations of the enzyme, different cell sheets can be efficiently peeled off without damaging the cell sheets.

[0017] (2) Compared with the conventional peeling method, the present invention can completely detach the cell sheet with less adhesion to the bottom of the culture dish.

[0018] (3) The overall operation of the method of the present invention is convenient and time-saving, and is suitable for the preparation and separation of a large number of cell sheets. In addition, the harvested cell sheets have a high viability and are free of damage, which lays a foundation for the industrial preparation and harvesting of cell sheets. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The cell sheet-silicone gauze is peeled off from the cell culture surface of the culture dish in Example 1.

[0020] Figure 2 This is a schematic diagram of the process of rinsing the cell sheet-silicone gauze in Example 1.

[0021] Figure 3 The cell sheet residues on the bottom of the culture dish after the cell sheets were peeled off in Example 1 (A) and Comparative Example 1 (B) were observed using an optical microscope.

[0022] Figure 4The digestion of the cell sheets in the cell sheet-silicone gauze of Example 1 (B) and Comparative Example 1 (A) was observed with naked eyes. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0024] In the following examples and comparative examples, the sources of the raw materials used are described as follows: (1) Dispase: Thermofisher, 17105-041; (2) Collagenase I: Thermofisher, 17100-017.

[0025] The rest of the raw materials or processing techniques, unless otherwise specified, are conventional commercially available raw materials or conventional processing techniques in the art.

[0026] Example 1

[0027] This embodiment provides a method for peeling off a cell sheet composed of keratinocytes. Specifically, the keratinocytes are derived from fresh human hair follicle tissues that are digested and cultured to expand the cells (P1-P3). The cell sheet is obtained by culturing the expanded cells in sheets, and the method comprises the following steps: (1) Preparation of digestive fluid Prepare digestion solution: its components include 50 mL of commercial DMEM / F12 medium, 2.4 U / mL of dispase, 0.1 mg / mL of collagenase, and 5 uM of ROCK inhibitor based on a volume of 50 mL.

[0028] (2) Cell sheet digestion Discard the original culture medium of the cell sheet culture dish that is about to be peeled off, add the freshly prepared digestion solution mentioned above, put it in a 5% carbon dioxide incubator, incubate at 37°C for 50 minutes, and remove it from the carbon dioxide incubator after the end.

[0029] (3) Cell sheet-silicone gauze peeling Use tweezers to lift the edge of the cell sheet from the cell culture surface of the culture dish, and place the silicone gauze into the cell culture dish containing the digestive fluid so that the lifted side of the cell sheet is in close contact with the silicone gauze. Use tweezers to clamp the edge of the cell sheet-silicone gauze and gently pull it outward to quickly and damagelessly peel off the cell sheet-silicone gauze.

[0030] (4) Cleaning and packaging The peeled cell sheet-silicone gauze was placed in PBS and rinsed three times, and placed in a new culture dish with the cell sheet on top and the silicone gauze on the bottom. After sealing, it can be used immediately afterwards.

[0031] like Figure 1 Shown is a cell sheet-silicone gauze dissociated from the cell culture surface of a culture dish.

[0032] like Figure 2 The figure shows the process of washing the cell sheet. After the cell sheet and the silicone gauze are peeled off together, they must be washed with PBS before subsequent use to wash off the digestive solution. During this washing process, the cell sheet and the silicone gauze first wash off the digestive solution on the surface of the cell sheet, and then wash vertically in the washing solution, and the cell sheet and the silicone gauze will be briefly separated on the liquid surface ( Figure 2 As shown in the figure), vertical washing can wash away the digestion solution between the cell sheet and the silicone gauze, and also facilitate the flattening of the cell sheet on the silicone gauze.

[0033] Example 2

[0034] This embodiment provides a method for peeling off a cell sheet constructed by fibroblasts. Specifically, the fibroblasts are derived from human skin dermis tissue digested and cultured and expanded cells (P2-P5). The cell sheet is obtained by culturing the expanded cells in sheets, comprising the following steps: (1) Preparation of digestive fluid Prepare digestion solution: Its components include 50 mL of commercial DMEM / F12 medium, 1 U / mL dispase, 1 mg / mL collagenase, and 20 uM ROCK inhibitor based on a volume of 50 mL.

[0035] (2) Cell sheet digestion Discard the original culture medium of the cell sheet culture dish that is about to be peeled off, add the freshly prepared digestion solution mentioned above, put it in a 5% carbon dioxide incubator, incubate at 37°C for 20 minutes, and remove it from the 5% carbon dioxide incubator after the end.

[0036] (3) Cell sheet-silicone gauze peeling Use tweezers to lift the edge of the cell sheet from the cell culture surface of the culture dish, and place the silicone gauze into the cell culture dish containing the digestive fluid so that the lifted side of the cell sheet is in close contact with the silicone gauze. Use tweezers to clamp the edge of the cell sheet-silicone gauze and gently pull it outward to quickly and damagelessly peel off the cell sheet-silicone gauze.

[0037] (4) Cleaning and packaging The peeled cell sheet-silicone gauze was placed in PBS and rinsed 5 times to wash off the digestion solution, and then placed in a new culture dish with the cell sheet on top and the silicone gauze on the bottom. After sealing, it can be used immediately afterwards.

[0038] Example 3

[0039] This embodiment provides a method for peeling off a cell sheet constructed by umbilical cord mesenchymal stem cells. Specifically, the umbilical cord mesenchymal stem cells are derived from human umbilical cord tissue and cultured and expanded cells (P2-P5) after Wharton's jelly digestion. The cell sheet is obtained by culturing the expanded cells in sheets, comprising the following steps: (1) Preparation of digestive fluid Prepare digestion solution: Its components include 50 mL of commercial DMEM / F12 medium, 1 U / mL dispase, 0.5 mg / mL collagenase, and 10 uM ROCK inhibitor based on a volume of 50 mL.

[0040] (2) Cell sheet digestion After culture, discard the original culture medium from the cell sheet culture dish that is ready to be peeled off, add freshly prepared digestion solution, put it in a 5% carbon dioxide incubator, incubate at 37°C for 10 minutes, and remove it from the 5% carbon dioxide incubator after the end.

[0041] (3) Cell sheet-silicone gauze peeling Use tweezers to lift the edge of the cell sheet from the cell culture surface of the culture dish, and place the silicone gauze into the cell culture dish containing the digestive fluid so that the lifted side of the cell sheet is in close contact with the silicone gauze. Use tweezers to clamp the edge of the cell sheet-silicone gauze and gently pull it outward to quickly and damagelessly peel off the cell sheet-silicone gauze.

[0042] (4) Cleaning and packaging The peeled cell sheet-silicone gauze was placed in PBS and rinsed twice to wash off the digestion solution, and then placed in a new culture dish with the cell sheet on top and the silicone gauze on the bottom. After sealing, it can be used immediately afterwards.

[0043] Comparative Example 1 Compared with Example 1, most of the steps are the same, except that in step (3), the cell sheet is directly clamped away from the cell culture surface of the culture dish with tweezers, so that the cell sheet is peeled off from the cell culture surface of the culture dish.

[0044] like Figure 3 As shown in FIG. 1 , by observing the bottom of the culture dish with and without the silicone gauze removed through an optical microscope, it can be found that: for the cell sheet under the same culture conditions, only a small amount of cells remain on the culture dish after the silicone gauze is removed, as shown in FIG. Figure 3 As shown in A in the figure; without using silicone gauze, after scraping and peeling with tweezers, the number of residual cells on the culture dish is much greater than that of the silicone gauze peeling group, as shown in Figure 3 As shown in B.

[0045] The difference between the cell residues in Comparative Example 1 and Example 1 is that after enzymatic digestion, the cell sheet is still attached to the cell culture dish. Under the condition of not using silicone gauze (i.e., Comparative Example 1), the cell sheet is scraped off by the tip of tweezers to make it detached. In this process, the cell sheet is unevenly stressed, and the connection between cells is easily disconnected at the place where the stress is greater, forming residues at the bottom of the cell culture dish. When silicone gauze is used (i.e., Example 1), the cell sheet is attached to the silicone gauze when it is peeled off. During the peeling process, the force is uniform on the same plane, the overall interference is less, and the residue is relatively small. In addition, peeling off by combining silicone gauze will be faster than scraping, which is conducive to the peeling off of a large number of preparations.

[0046] Comparative Example 2 Compared with Example 1, most of the steps are the same, except that in step (1), the final concentration of dispase in the digestion solution is 2.4 U / mL, and the final concentration of collagenase is 1 mg / mL. The high-concentration enzyme ratio is used to digest the cell sheet, so that the cell sheet is loosened from the cell culture surface.

[0047] like Figure 4 As shown, by visually observing the cell sheets after digestion and peeling of Example 1 and Comparative Example 2, it can be found that: under the digestion conditions of Comparative Example 2 (the digestion solution contains a final concentration of 2.4 U / mL dispase and 1 mg / mL collagenase), the cell sheets derived from hair follicles are over-digested, and small cavities (such as Figure 4 ); under the digestion conditions of Example 1 (the digestion solution contained a final concentration of 2.4 U / mL dispase and 0.1 mg / mL collagenase), the hair follicle-derived cell sheet could be detached intact (as shown in FIG. Figure 4 (as shown in B in the figure).

[0048] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container, characterized in that: The steps include: S1. Preparation of digestion solution: DMEM / F12 medium, dispase, collagenase, and ROCK inhibitor were mixed to obtain a digestion solution; S2. Cell sheet digestion: Discard the original culture medium of the cell sheet to be removed from the cell culture dish, add the digestion solution obtained in step S1, and incubate; S3. Cell sheet removal: The cell sheet to be peeled off is attached to the silicone gauze, and then the cell sheet and the silicone gauze are simultaneously clamped and separated from the cell culture surface of the cell culture dish to obtain a cell sheet-silicone gauze, thereby completing the rapid and non-destructive peeling of the cultured cell sheet from the cell culture surface of the cell culture dish.

2. The method for non-destructively removing a cell sheet in culture from a cell culture surface of a culture container according to claim 1, characterized in that: In step S1, the DMEM / F12 culture medium, dispase, collagenase, and ROCK inhibitor are mixed according to 50 mL: 0.5-2.4 U / mL: 0.1-1 mg / mL: 1-20 uM to obtain a digestion solution.

3. The method for non-destructively removing a cell sheet in culture from a cell culture surface of a culture container according to claim 2, characterized in that: In step S2, the cell sheet to be peeled off is selected from any one of a cell sheet composed of keratinocytes, a cell sheet composed of fibroblasts, and a cell sheet composed of mesenchymal stem cells.

4. The method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container according to claim 3, characterized in that: In step S2, when the cell sheet to be peeled off is a cell sheet composed of keratinocytes, in the digestion solution, the final concentration of dispase is 2-2.4 U / mL, and the final concentration of collagenase is 0.1-0.3 mg / mL; When the cell sheet to be peeled off is composed of fibroblasts, in the digestion solution, the final concentration of dispase is 0.5-1.4 U / mL, and the final concentration of collagenase is 0.8-1 mg / mL; When the cell sheet to be peeled off is a cell sheet composed of mesenchymal stem cells, in the digestion solution, the final concentration of dispase is 0.5-1 U / mL, and the final concentration of collagenase is 0.1-0.5 mg / mL.

5. The method for non-destructively removing a cell sheet in culture from a cell culture surface of a culture container according to claim 1, characterized in that: In step S2, the incubation conditions are: 5% CO2, temperature of 37°C, and time of 10 to 50 min.

6. The method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container according to claim 1, characterized in that: In step S3, the method for peeling off the cell sheet is specifically as follows: first, the edge of the cell sheet to be peeled off is lifted up from the cell culture surface of the cell culture dish, and the silicone gauze is placed in the cell culture dish containing the digestive fluid, so that the lifted side of the cell sheet is closely attached to the silicone gauze, and then the cell sheet and the edge of the silicone gauze are clamped and separated from the cell culture surface of the cell culture dish to obtain the cell sheet-silicone gauze, thereby completing the rapid and non-destructive dissociation of the cultured cell sheet from the cell culture surface of the culture dish.

7. The method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container according to claim 1, characterized in that: After the cell sheets are dissociated from the cell culture surface of the culture dish, the process also includes washing and packaging the cell sheets.

8. The method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container according to claim 7, characterized in that: The cleaning method is as follows: the peeled cell sheet-silica gel gauze is placed in PBS buffer for rinsing, which is used to rinse the digestive fluid on both sides of the cell sheet-silica gel gauze and the digestive fluid between the cell sheet and the silica gel gauze.

9. The method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container according to claim 8, characterized in that: The cell sheet and silicone gauze were clamped and placed vertically in PBS buffer, so that the cell sheet and silicone gauze were separated and unfolded in the PBS buffer. After the cell sheet and the silicone gauze were separated and unfolded, the sides were in contact with the PBS buffer surface, thereby rinsing the digestive fluid between the cell sheet and the silicone gauze.

10. The method for non-destructively peeling off a cell sheet in culture from a cell culture surface of a culture container according to claim 7, characterized in that: The packaging method is as follows: the cell sheet-silicone gauze is placed in a new culture dish with the cell sheet on top and the silicone gauze on the bottom, and then sealed and stored.

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