Method for rapidly obtaining cell sheet through self-aggregation of high-concentration cells

Abstract

The invention belongs to the technical field of cell patches, and relates to a method for rapidly obtaining a cell patch through self-aggregation of high-concentration cells. According to the method, on the basis of a high-concentration cell suspension, high-concentration cells are subjected to standing culture on an ultra-low adsorption culture dish, so that the high-concentration cells are subjected to self-aggregation, and finally, the cell sheet with high integrity and uniform thickness is successfully harvested. The method has the following advantages: firstly, only a common complete culture medium is needed in the process of preparing the cell sheet, and a film-forming culture medium added with various components is not needed, so that the culture cost is saved; moreover, the film forming condition of the method is simple and convenient, special equipment is not needed, and manpower loss is also saved. In addition, according to the method, the film forming time is greatly shortened, and the film can be formed only by standing and culturing for several hours. The diaphragm prepared by the method is uniform in thickness, flat and continuous, and has certain mechanical strength. The method for obtaining the cell sheet is very simple and rapid, and has a wide application prospect in the field of cell sheets.

Description

Technical Field

[0001] The invention belongs to the technical field of cell membrane sheets and relates to a method for quickly obtaining a cell membrane sheet by self-aggregation of high-concentration cells. Background Art

[0002] Cell sheet technology is a method for producing dense cell tissues. This technology was originally proposed by Okano and his colleagues and has been widely used in cell-based therapy, drug discovery, regenerative medicine and tissue engineering. In typical cell sheet engineering technology, cells are cultured on a thermoresponsive surface. When the temperature drops from 37°C to room temperature (RT), the cells change from a hydrophobic cell adhesion state to a hydrophilic cell repulsion state. That is, when the cell monolayer grown on this functional thermoresponsive matrix is ​​transferred outside the cell culture incubator, the entire cell sheet is slightly separated and can be harvested as a monolayer with its intact extracellular matrix. In addition to thermal response, cell membrane sheets can also be obtained using different types of response systems, such as electrical stimulation response, light response, magnetic response, etc. In addition, mechanical blowing can also be used to peel off fused cells from the culture dish. Although these methods can separate fused cells from the culture dish surface without enzyme treatment to obtain complete continuous cell sheets. However, they have high requirements for equipment, long culture time of cell membrane sheets, large amount of culture medium used in the culture process, and cumbersome experimental process resulting in large manpower loss. Summary of the invention

[0003] In view of this, the present invention provides a method for rapidly obtaining a cell membrane sheet by self-aggregation of high-concentration cells. The method has simple membrane forming conditions, fast membrane forming speed, and the prepared cell membrane sheet has high integrity and uniform thickness.

[0004] The present invention provides a method for statically culturing high-concentration cells on an ultra-low adsorption culture dish to make them self-aggregate to obtain a cell membrane sheet with high integrity and uniform thickness. After obtaining a certain number of cells in a normal state by a conventional cell culture method, the cells are counted. After transferring a certain concentration of cells to an ultra-low adsorption well plate and mixing them, they are cultured in a carbon dioxide constant temperature incubator for several hours to obtain a spontaneously formed cell membrane sheet.

[0005] The specific technical solutions of the present invention are as follows:

[0006] S1) After the cells in normal state are cultured to a cell density of more than 80%, the cells are digested and set aside;

[0007] S2) Count the cells and adjust the cell suspension concentration to 200w cells / mL;

[0008] S3) Transfer 2 mL of cell suspension to an ultra-low attachment plate;

[0009] S4) After the cell suspension in the ultra-low adsorption well plate is mixed evenly, the well plate is gently transferred to a carbon dioxide constant temperature incubator and cultured for two hours;

[0010] The advantages of this method are as follows: First, in the process of preparing cell membranes, only ordinary complete culture medium is needed, and there is no need to use membrane-forming culture medium with multiple ingredients added, which greatly saves the culture cost. Furthermore, the membrane-forming conditions of this method are simple and do not require the use of special equipment, which also greatly saves manpower loss. In addition, the membrane-forming time of this method is short, and the membrane can be formed by static culture for several hours, which greatly shortens the membrane-forming time. In addition, the membrane prepared by this method has uniform thickness, flat and complete membrane, and has a certain mechanical strength. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 A diagram for the abstract;

[0012] Figure 2 Example 1 of manufacturing a human umbilical cord mesenchymal stem cell membrane sheet in an ultra-low adsorption 12-well plate and its microscope image;

[0013] Figure 3 This is a state diagram of normal human skin fibroblasts with different cell concentrations in an ultra-low adsorption 12-well plate after shaking for 5 minutes;

[0014] Figure 4 This is a microscope image of normal human skin fibroblasts with different cell concentrations in an ultra-low adsorption 12-well plate after shaking for 5 minutes;

[0015] Figure 5 This is a microscope image of Example 2 of manufacturing a normal human skin fibroblast cell sheet with different cell concentrations in an ultra-low adsorption 12-well plate;

[0016] Figure 6 This is a microscope picture of the preparation example 3 of the normal human skin fibroblast cell sheet statically cultured at different cell concentrations in an ultra-low adsorption 12-well plate;

[0017] Figure 7 Example 3 for preparing a cell sheet of normal human skin fibroblasts statically cultured at different cell concentrations in an ultra-low adsorption 12-well plate;

[0018] Figure 8 This is a diagram of the state of normal human skin fibroblast cell sheet preparation example 3 after fixation, which is statically cultured at different cell concentrations in an ultra-low adsorption 12-well plate. DETAILED DESCRIPTION

[0019] Example 1

[0020] (1) Cell culture: After the cryopreserved human umbilical cord mesenchymal stem cells are revived, they are cultured at 37° C. and 5% CO 2 . After several passages, the human umbilical cord mesenchymal stem cells are cultured until they are revitalized and can be used to prepare human umbilical cord mesenchymal stem cell membranes.

[0021] (2) Cell digestion: Normal human umbilical cord mesenchymal stem cells are cultured for three days until the cell density reaches more than 80%, and then the cells are digested and set aside.

[0022] (3) Cell counting: Perform cell counting and adjust the cell suspension concentration to 225w cell / mL.

[0023] (4) Transfer 4 mL of cell suspension to an ultra-low adsorption 12-well plate.

[0024] (5) Gently transfer the 12-well plate to a carbon dioxide constant temperature incubator and culture for five hours.

[0025] Example 2

[0026] (1) Cell culture: The frozen normal human skin fibroblasts are revived and cultured at 37°C, 5% CO2. After several passages, the normal human skin fibroblasts are cultured until they are revitalized and can be used to prepare normal human skin fibroblast cell sheets.

[0027] (2) Cell digestion: Normal human skin fibroblasts in normal condition were cultured for three days until the cell density reached 80% or more, and then the cells were digested and set aside.

[0028] (3) Cell counting: Cell counting was performed and the concentrations of the cell suspension were prepared to 100w cell / mL, 200w cell / mL, and 400w cell / mL, respectively.

[0029] (4) Take 2 mL of each cell concentration suspension and transfer it to an ultra-low adsorption 12-well plate and shake it on a shaker for 5 min.

[0030] (5) Gently transfer the 12-well plate to a carbon dioxide constant temperature incubator and incubate for two hours.

[0031] Example 3

[0032] (1) Cell culture: The frozen normal human skin fibroblasts are revived and cultured at 37°C, 5% CO2. After several passages, the normal human skin fibroblasts are cultured until they are revitalized and can be used to prepare normal human skin fibroblast cell sheets.

[0033] (2) Cell digestion: Normal human skin fibroblasts in normal condition were cultured for three days until the cell density reached 80% or more, and then the cells were digested and set aside.

[0034] (3) Cell counting: Perform cell counting and prepare the cell suspension concentrations to 100w cell / mL and 200w cell / mL respectively.

[0035] (4) Take 2 mL of each cell concentration suspension and transfer them to ultra-low adsorption 12-well plates.

[0036] (5) Gently transfer the 12-well plate to a carbon dioxide constant temperature incubator and incubate for two hours.

[0037] Example 4

[0038] Figure 2 The optical microscope is used to observe the morphology of the cell membrane sheet formed in Manufacturing Example 1. It can be seen from the microscope image that when the cell amount is 900w, it may be that the density of the cell suspension is uneven, and a cell membrane sheet of a certain thickness can be formed at this time, but the integrity of the cell membrane sheet is not high.

[0039] Example 5

[0040] Figure 3 After shaking for five minutes, cell aggregation was observed in all three concentrations of suspensions. Figure 4 The results of optical microscope observation show that the cell suspension has an obvious density gradient. After two hours of culture, the cell membrane morphology of Example 2 was observed using an optical microscope. The results are as follows Figure 5 The results showed that when the cell weight was 800w, the cell membrane was dense and complete, and the edge of the membrane was smooth. At this time, the mechanical properties of the cell membrane were better and could be blown; when the cell weight was 400w, the cell membrane was dense and complete, but partial folding occurred in this manufacturing example. At this time, the mechanical properties of the cell membrane were better and could be blown; when the cell weight was 200w, the local area of ​​the cell membrane was dense and complete, the edge of the membrane was tortuous, the mechanical properties were poor, and the cell membrane broke after blowing.

[0041] Example 6

[0042] The morphology of the cell membrane sheet of manufacturing example 3 was observed using an optical microscope. Figure 6 The results showed that when the cell weight was 400w, the cell membrane was dense and complete, and the membrane edge was smooth; when the cell weight was 200w, the cell membrane edge was tortuous. Figure 7 As shown in its appearance picture, through visual observation, the membrane formed by the oscillation group is thick in the middle and thin on both sides, while the thickness of the cell membrane obtained by static culture is more uniform. Figure 8 This is the morphology of the cell membrane after fixation. The results show that when the cell weight is 400w, the mechanical properties of the cell membrane are better, and the blowing operation can be performed, and the membrane is intact after fixation; when the cell weight is 200w, the mechanical properties of the cell membrane are poor, and the cell membrane is broken after blowing.

Claims

1. A method for rapidly obtaining cell membrane sheets by self-aggregation of high-concentration cells. The preparation method comprises the following steps: (1) Obtaining a cell suspension; (2) Counting cells and adjusting the concentration of the cell suspension to the target concentration; (3) Transfer 2 mL of cell suspension of different concentrations to ultra-low adsorption well plates; (4) After the cell suspension in the ultra-low adsorption well plate is mixed evenly, the well plate is gently transferred to a carbon dioxide constant temperature incubator and cultured for two hours.

2. The method for rapidly obtaining a cell membrane sheet as described in claim 1, wherein the preparation steps are as follows: (1) After culturing cells in a normal state until the cell density reaches 80% or more, the cells are digested and set aside for later use. When resuspending, only a small amount of culture medium is needed to resuspend the cells, and a cell suspension with a higher concentration is obtained.

3. The method for rapidly obtaining a cell membrane sheet as claimed in claim 1, wherein the preparation step: (2) specifically comprises: counting the cell suspension obtained in (1), and configuring the cell suspension concentrations to 200w cell / mL, 400w cell / mL, and 800w cell / mL, respectively.

4. The method for rapidly obtaining a cell membrane sheet as claimed in claim 1, wherein the preparation step: (3) is specifically: purchasing an ultra-low adsorption well plate, and transferring 2 mL of each of 200w cell / mL, 400w cell / mL, and 800w cell / mL cell suspensions to the ultra-low adsorption well plate.

5. The method for rapidly obtaining a cell membrane sheet as claimed in claim 1, wherein the preparation step (4) is specifically: fully shaking the ultra-low adsorption plate to make the cells evenly distributed. An optical microscope can be used to determine whether the cell density in the ultra-low adsorption plate is uniform. After ensuring that the cell suspension in the ultra-low adsorption plate is evenly mixed, the plate is gently transferred to a carbon dioxide constant temperature incubator and incubated for two hours.

6. The method for quickly obtaining a cell sheet as claimed in claim 1, which is applied in the field of cell sheets.

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