WO2023245813A1

WO2023245813A1 - Composition and use thereof in preparation of cell membrane

Info

Publication number: WO2023245813A1
Application number: PCT/CN2022/108694
Authority: WO
Inventors: 高博韬; 关淑文
Original assignee: 广东省科学院生物与医学工程研究所
Priority date: 2022-06-22
Filing date: 2022-07-28
Publication date: 2023-12-28
Also published as: CN115369079A; CN115369079B

Abstract

Provided are a composition and the use thereof in preparation of a cell membrane. The composition comprises an ITS cell culture additive, dexamethasone and vitamin C. The composition can be used to prepare a cell membrane by means of a general cell culture dish; and compared with the conventional preparation of the cell membrane by means of a temperature-sensitive culture dish, by adopting the composition, the cost of preparing a cell membrane can be greatly reduced, the number of cells is increased, and the success rate of preparing the cell membrane is improved. The present invention can be better used in the fields of tissue engineering and regenerative medicine (repairing damaged tissues and/or organs, and constructing three-dimensional tissues and/or organs), and is expected to achieve commercial large-scale production.

Description

A composition and its application in preparing cell films

Technical field

The invention belongs to the technical field of cell culture, and specifically relates to a composition and its application in preparing cell films.

Background technique

Cell membrane is a dense tissue composed of cells and extracellular matrix. The cell membrane retains cell-cell and cell-matrix connections, can effectively maintain cell-cell interactions, and completely retains the precise and orderly network structure of the extracellular matrix. Growth factors, cytokines and other active factors loaded in the extracellular matrix can regulate cell behavior, influence cell fate, promote tissue repair and regeneration, and improve inflammation. Therefore, cell films are often used to repair and replace tissue or organ defects, construct three-dimensional tissues or organs, etc., and have broad application prospects in the fields of tissue engineering and regenerative medicine.

Currently, the main method for making cell films is to use temperature-sensitive culture dishes. The bottom of the temperature-sensitive culture dish is coated with poly(N-isopropylacrylamide) (PIPAAm), which is hydrophobic at 37°C and hydrophilic at 20°C. When the temperature drops from 37°C to 20°C, PIPAAm changes from hydrophobic to hydrophilic, causing the cells to detach from the bottom of the culture dish and obtain a cell film. However, using temperature-sensitive culture dishes to make cell films requires a low-temperature culture process. Low-temperature culture can lead to a decrease in cell viability and even functional changes in some fragile cells. Secondly, temperature-sensitive culture dishes are expensive. Therefore, it is particularly important to develop a composition and method for stably preparing cell films using ordinary cell culture dishes.

Contents of the invention

The object of the first aspect of the present invention is to provide a composition.

The second object of the present invention is to provide a culture medium.

The object of the third aspect of the present invention is to provide the use of the composition of the first aspect and/or the culture medium of the second aspect in preparing cell films or preparing products for cell film preparation.

The fourth object of the present invention is to provide a method for preparing cell films.

The fifth aspect of the present invention aims to provide a cell membrane.

The object of the sixth aspect of the present invention is to provide the composition of the first aspect, the kit of the second aspect of the present invention, the method of the fourth aspect of the present invention, and/or the application of the cell membrane of the fifth aspect of the present invention.

In order to achieve the above objects, the technical solutions adopted by the present invention are:

A first aspect of the present invention provides a composition comprising: ITS cell culture supplement, dexamethasone and vitamin C.

Preferably, the composition further comprises polysucrose.

Preferably, the Ficollose is at least one of Ficoll 400 (Ficoll 400) and Ficoll 70 (Ficoll 70); further, Ficoll 400 (Ficoll 400).

A second aspect of the present invention provides a culture medium comprising the composition of the first aspect of the present invention.

Preferably, the basal medium of the medium is at least one of DMEM high-sugar medium, DMEM-F12 medium, RPMI1640 medium, and DMEM low-sugar medium; further, it is DMEM high-glucose medium.

Preferably, the basal culture medium contains at least one of fetal bovine serum and antibiotics; further preferably, the basal culture medium contains fetal bovine serum and antibiotics.

Preferably, the concentration of fetal bovine serum in the basal culture medium is 8-12 v/v%.

Preferably, the antibiotic includes at least one of penicillin, gentamicin, amphotericin B, kanamycin, tetracycline, and streptomycin; further includes at least one of penicillin and streptomycin; further Contains penicillin and streptomycin.

Preferably, the concentration of the antibiotic in the basal culture medium is 0.5-1.5 v/v%.

Preferably, the concentration of the ITS cell culture additive in the culture medium is 0.1-2.0 v/v%; further, the concentration is 1-2.0 v/v%.

Preferably, the concentration of dexamethasone in the culture medium is 0.001-0.04 μg/mL.

Preferably, the concentration of vitamin C in the culture medium is 1-500 μg/mL; further, the concentration is 50-500 μg/mL.

Preferably, the concentration of polysucrose in the culture medium is 0 to 100 mg/mL, excluding 0; 25 mg/mL to 100 mg/mL.

A third aspect of the present invention provides the use of the composition of the first aspect and/or the culture medium of the second aspect in any one of (1) to (4);

(1) Preparation of cell membrane;

(2) Prepare products for cell film preparation;

(3) Promote cell proliferation;

(4) Prepare products that promote cell proliferation.

Preferably, the cells include: at least one of skin fibroblasts, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, periodontal ligament mesenchymal stem cells, and umbilical cord mesenchymal stem cells. ;further to skin fibroblasts.

A fourth aspect of the present invention provides a method for preparing a cell film, which includes the step of using the composition of the first aspect of the present invention and/or the culture medium of the second aspect of the present invention.

Preferably, the method includes the following steps: inoculating cells into a basal medium for first culture, and then replacing the basal medium containing the composition of the first aspect of the present invention and/or the second aspect of the present invention. The composition was cultured for a second time to obtain a cell film.

Preferably, the conditions for the first culture are 33-39°C and 3-7% CO ₂ for 18-30 hours.

Preferably, the conditions for the second culture are 33-39°C and 3-7% _CO2 for 4-15 days.

Preferably, the medium is changed every 1 to 3 days during the second culture process.

Preferably, the basal culture medium is at least one of DMEM high sugar culture medium, DMEM-F12 culture medium, RPMI1640 culture medium, and DMEM low sugar culture medium; further, it is DMEM high sugar culture medium.

Preferably, the seeding density of the cells is 3.5 to 750,000/mL.

Preferably, the cells are cultured using ordinary cell culture dishes.

Preferably, after the second culture is completed, the step of peeling off the cell membrane is also included.

Preferably, the step of peeling off the cell film is: gently blowing the bottom edge of the culture dish with a pipette.

A fifth aspect of the present invention provides a cell film obtained by the method of the fourth aspect of the present invention.

A sixth aspect of the present invention provides the composition of the first aspect of the present invention, the kit of the second aspect of the present invention, the method of the fourth aspect of the present invention and/or the cell membrane of the fifth aspect of the present invention. Application in any one of (1) to (3);

(1) Preparation of products for repairing damaged tissues and/or organs;

(2) Construct three-dimensional tissues and/or organs;

(3) Preparation of products for constructing three-dimensional tissues and/or organs.

The beneficial effects of the present invention are:

The present invention discloses a composition for the first time, including: ITS cell culture additive, dexamethasone and vitamin C; using this composition, cell films can be prepared through ordinary cell culture dishes, compared with the traditional preparation using temperature-sensitive culture dishes. Cell film, the use of this composition can greatly reduce the cost of preparing cell films, increase the number of cells, and improve the success rate of preparing cell films, which is beneficial to better application in the fields of tissue engineering and regenerative medicine (repairing damaged tissue and/or or organs, constructing three-dimensional tissues and/or organs), and is expected to achieve commercial mass production.

The present invention further defines that the composition further includes: polysucrose, thereby further improving the quality (integrity) of the cell film produced and increasing the number of cells.

Description of the drawings

Figure 1 shows Effect Example 1, Effect Example 2, Effect Comparative Example 1, Effect Comparative Example 2, Effect Comparative Example 3, Effect Comparative Example 4, Effect Comparative Example 5, Effect Comparative Example 6, Effect Comparative Example 7, and Effect Comparative Example. Visual diagram of the cell film prepared in Example 8 and Comparative Example 9: A is a visual diagram of the cell film prepared in Comparative Example 2; B is a visual diagram of the cell film prepared in Comparative Example 3; C is A visual diagram of the cell film prepared in Comparative Example 4; D is a visual diagram of the cell film prepared in Comparative Example 5; E is a visual diagram of the cell film prepared in Comparative Example 6; F is a visual diagram of the cell film prepared in Comparative Example 7. A visual diagram of the cell film obtained; G is a visual diagram of the cell film prepared in Comparative Example 8; H is a visual diagram of the cell film prepared in Comparative Example 9; I is a visual diagram of the cell film prepared in Comparative Example 1 Visual diagram; J is a visual diagram of the cell film prepared in Effect Example 2; K is a visual diagram of the cell film prepared in Effect Comparative Example 1.

Figure 2 shows Effect Example 1, Effect Example 2, Effect Comparative Example 2, Effect Comparative Example 3, Effect Comparative Example 4, Effect Comparative Example 5, Effect Comparative Example 6, Effect Comparative Example 7, Effect Comparative Example 8 and Effect Comparative Example. Cell viability test results of ratio 9: * indicates p<0.05.

Detailed ways

The content of the present invention will be further described in detail below through specific examples.

The materials, reagents, etc. used in this example are reagents and materials obtained from commercial sources unless otherwise specified.

The ITS cell culture supplement (100×) in this example was purchased from Cyagen, dexamethasone was purchased from McLean, vitamin C was purchased from Solarbio Life Science, Ficoll 400 was purchased from GE Healthcare, and NHDF cells were purchased from ATCC, with a diameter of 35 mm. Ordinary cell culture dishes were purchased from Corning; temperature-sensitive culture dishes with a diameter of 35 mm were purchased from CellSeed.

Example 1 A culture medium

A culture medium, which is a basic medium containing ITS cell culture additives, dexamethasone and vitamin C, wherein the basic medium is a base medium containing 10v/v% fetal bovine serum, 1v/v% penicillin-streptomycin DMEM high glucose medium, the concentration of ITS cell culture additives in the medium is 1v/v%, the concentration of dexamethasone in the medium is 0.04μg/mL, and the concentration of vitamin C in the medium is 50μg/mL.

The preparation method of the above culture medium is as follows: adding ITS cell culture supplement, dexamethasone and vitamin C to the basic culture medium, and filtering using a 0.22 μm sterile filter (purchased from Millipore) to obtain the culture medium.

Example 2 A culture medium

A culture medium, which is a basal culture medium containing ITS cell culture additives, dexamethasone, Ficoll 400 and vitamin C, wherein the basal culture medium is a basal culture medium containing 10v/v% fetal bovine serum, 1v/v% cyanide Streptomycin's DMEM high-glucose medium, the concentration of ITS cell culture additive in the medium is 1v/v%, the concentration of dexamethasone in the medium is 0.04μg/mL, and the concentration of vitamin C in the medium The concentration of Ficoll 400 in this medium is 50μg/mL, and the concentration of Ficoll 400 in this medium is 25mg/mL.

The preparation method of the above-mentioned culture medium is as follows: add ITS cell culture supplement, dexamethasone, Ficoll 400 and vitamin C to the basic culture medium, and filter using a 0.22 μm sterile filter (purchased from Millipore) to obtain the culture medium.

Example 3 A culture medium

A culture medium, which is a basic medium containing ITS cell culture additives, dexamethasone and vitamin C, wherein the basic medium is a base medium containing 10v/v% fetal bovine serum, 1v/v% penicillin-streptomycin DMEM high-glucose medium, the concentration of ITS cell culture additives in the medium is 0.1v/v%, the concentration of dexamethasone in the medium is 1ng/mL, and the concentration of vitamin C in the medium is 1μg/mL.

Example 4 A culture medium

A culture medium, which is a basic medium containing ITS cell culture additives, dexamethasone and vitamin C, wherein the basic medium is a base medium containing 10v/v% fetal bovine serum, 1v/v% penicillin-streptomycin DMEM high-glucose medium, the concentration of ITS cell culture additives in the medium is 2v/v%, the concentration of dexamethasone in the medium is 0.04μg/mL, and the concentration of vitamin C in the medium is 500μg/mL.

Example 5 A culture medium

A culture medium, which is a basal culture medium containing ITS cell culture additives, dexamethasone, Ficoll 400 and vitamin C, wherein the basal culture medium is a basal culture medium containing 10v/v% fetal bovine serum, 1v/v% cyanide Streptomycin's DMEM high-glucose medium, the concentration of ITS cell culture additive in this medium is 2v/v%, the concentration of dexamethasone in this medium is 0.04μg/mL, and the concentration of vitamin C in this medium The concentration of Ficoll 400 in this medium is 500μg/mL, and the concentration of Ficoll 400 in this medium is 100mg/mL.

Comparative Example 1 A culture medium

A culture medium, which is a DMEM high-glucose culture medium containing 10v/v% fetal bovine serum and 1v/v% penicillin-streptomycin.

Comparative Example 2 One culture medium

A culture medium, the culture medium is a basal culture medium containing Ficoll 400, wherein the basal culture medium is a DMEM high-glucose culture medium containing 10v/v% fetal bovine serum and 1v/v% penicillin-streptomycin, and Ficoll 400 is The concentration in this medium is 25 mg/mL.

The preparation method of the above culture medium is as follows: add Ficoll 400 to the basic culture medium, and filter it using a 0.22 μm sterile filter (purchased from Millipore) to obtain the culture medium.

Comparative Example 3 One culture medium

A culture medium, which is a basal culture medium containing vitamin C, wherein the basal culture medium is a DMEM high-glucose culture medium containing 10v/v% fetal bovine serum and 1v/v% penicillin-streptomycin, and vitamin C is present in The concentration in this medium is 50 μg/mL.

The preparation method of the above culture medium is as follows: adding vitamin C to the basic culture medium, filtering using a 0.22 μm sterile filter (purchased from Millipore) to obtain the culture medium.

Comparative Example 4 One culture medium

A culture medium, which is a basal culture medium containing Ficoll 400 and vitamin C, wherein the basal culture medium is a DMEM high-glucose culture medium containing 10v/v% fetal calf serum and 1v/v% penicillin-streptomycin, The concentration of vitamin C in this medium is 50 μg/mL, and the concentration of Ficoll 400 in this medium is 25 mg/mL.

The preparation method of the above culture medium is as follows: add Ficoll 400 and vitamin C to the basic culture medium, and filter it using a 0.22 μm sterile filter (purchased from Millipore) to obtain the culture medium.

Comparative Example 5 One culture medium

A culture medium, which is a basal culture medium containing ITS cell culture additives and vitamin C, wherein the basal culture medium is DMEM high sugar containing 10v/v% fetal bovine serum and 1v/v% penicillin-streptomycin. The concentration of vitamin C in the culture medium is 50 μg/mL, and the concentration of ITS cell culture additive in the culture medium is 1 v/v%.

The preparation method of the above culture medium is as follows: adding ITS cell culture additives and vitamin C to the basic culture medium, and filtering using a 0.22 μm sterile filter (purchased from Millipore) to obtain the culture medium.

Comparative Example 6 One culture medium

A culture medium, which is a basal culture medium containing dexamethasone and vitamin C, wherein the basal culture medium is a DMEM high-glucose culture medium containing 10v/v% fetal calf serum and 1v/v% penicillin-streptomycin. , the concentration of vitamin C in the culture medium is 50 μg/mL, and the concentration of dexamethasone in the culture medium is 0.04 μg/mL.

The preparation method of the above culture medium is as follows: adding dexamethasone and vitamin C to the basic culture medium, filtering using a 0.22 μm sterile filter (purchased from Millipore) to obtain the culture medium.

Comparative Example 7 One culture medium

A culture medium, which is a basal culture medium containing ITS cell culture additives, wherein the basal culture medium is a DMEM high-glucose culture medium containing 10v/v% fetal calf serum and 1v/v% penicillin-streptomycin, The concentration of ITS cell culture supplement in this medium is 1 v/v%.

The preparation method of the above-mentioned culture medium is as follows: adding ITS cell culture additives to the basic culture medium, and filtering using a 0.22 μm sterile filter (purchased from Millipore) to obtain the culture medium.

Comparative Example 8 One culture medium

A culture medium, the culture medium is a basal culture medium containing dexamethasone, wherein the basal culture medium is a DMEM high-glucose culture medium containing 10v/v% fetal calf serum, 1v/v% penicillin-streptomycin, and dexamethasone. The concentration of metasone in this medium is 0.04 μg/mL.

The preparation method of the above culture medium is as follows: adding dexamethasone to the basic culture medium, filtering using a 0.22 μm sterile filter (purchased from Millipore) to obtain the culture medium.

Effect Example Preparing cell films using different culture dishes and/or culture media

Effect Example 1

1. Pass NHDF cells (purchased from ATCC) to the 6th passage, and inoculate them onto a 35mm diameter ordinary cell culture dish. The number of cells inoculated is 1 million; add 2 mL of basic medium (the basic medium contains 10v/v% fetus DMEM high glucose medium containing bovine serum, 1v/v% penicillin and streptomycin); place the cells in a 37°C, 5% CO ₂ incubator for 24 hours;

2. Change the medium after 24 hours, add 2 mL of the culture medium of Example 1, and culture it in a 37°C, 5% CO2 incubator for 5 days. Change the medium once every 2 days. After 5 days, take out the cells and use a pipette to absorb an appropriate amount. Basic medium, gently pipe the bottom edge of the culture dish, and peel off the NHDF cell film, as shown in I in Figure 1. The resulting cell film is slightly damaged.

Effect Example 2

2. Change the medium after 24 hours, add 2 mL of the culture medium of Example 2, and culture it in a 37°C, 5% CO2 incubator for 5 days. Change the medium once every 2 days. After 5 days, take out the cells and use a pipette to absorb an appropriate amount. basal culture medium, gently pipe the bottom edge of the culture dish, and peel off the NHDF cell film, as shown in J in Figure 1. The obtained cell film is intact and intact.

Effect Example 3

2. Change the medium after 24 hours, add 2 mL of the culture medium of Example 3, and culture it in a 37°C, 5% CO2 incubator for 5 days. Change the medium once every 2 days. After 5 days, take out the cells and use a pipette to absorb an appropriate amount. Basic medium, gently pipe the bottom edge of the culture dish, and peel off the NHDF cell film. The resulting cell film is slightly damaged.

Effect Example 4

2. Change the medium after 24 hours, add 2 mL of the culture medium of Example 4, and culture it in a 37°C, 5% CO2 incubator for 5 days. Change the medium once every 2 days. After 5 days, take out the cells and use a pipette to absorb an appropriate amount. Basic medium, gently pipe the bottom edge of the culture dish, and peel off the NHDF cell film. The resulting cell film is slightly damaged.

Effect Example 5

2. Change the medium after 24 hours, add 2 mL of the culture medium of Example 5, and culture it in a 37°C, 5% CO2 incubator for 5 days. Change the medium once every 2 days. After 5 days, take out the cells and use a pipette to absorb an appropriate amount. Basic medium, gently pipe the bottom edge of the culture dish, and peel off the NHDF cell film. The resulting cell film is slightly damaged.

Effect comparison example 1

1. Pass NHDF cells (purchased from ATCC) to the 6th passage, and inoculate them onto a 35mm diameter temperature-sensitive culture dish. The number of cells inoculated is 1 million; add 2 mL of basic medium (the basic medium contains 10v/v% Fetal bovine serum, 1v/v% penicillin-streptomycin in DMEM high-glucose medium (i.e., the medium in Comparative Example 1); the cells were cultured in a 37°C, 5% _CO2 incubator for 6 days, every 2 days The medium was changed once. After 6 days, the cells were taken out. Use a pipette to absorb an appropriate amount of basal culture medium. Gently pipe the bottom edge of the culture dish to peel off the NHDF cell film. As shown in K in Figure 1, the resulting cell film was seriously damaged.

Effect comparison example 2

1. Pass NHDF cells (purchased from ATCC) to the 6th passage, and inoculate them onto a 35mm diameter ordinary cell culture dish. The number of cells inoculated is 1 million; add 2 mL of basic medium (the basic medium contains 10v/v% fetus DMEM high-glucose medium with bovine serum and 1v/v% penicillin and streptomycin (i.e., the medium in Comparative Example 1); the cells were cultured in a 37°C, 5% _CO2 incubator for 6 days, and the cells were replaced every 2 days. Solution 1 time, take out the cells after 6 days, use a pipette to absorb an appropriate amount of basal culture medium, gently pipe the bottom edge of the culture dish, and peel off to obtain the NHDF cell film. As shown in A in Figure 1, the cell film cannot be obtained.

Effect comparison example 3

2. Change the medium after 24 hours, add 2 mL of the culture medium of Comparative Example 2, place it in a 37°C, 5% CO2 incubator for 5 days, change the medium once every 2 days, take out the cells after 5 days, and use a pipette to absorb an appropriate amount. Basic medium, gently pipe the bottom edge of the culture dish, and peel off to obtain the NHDF cell film. As shown in B in Figure 1, the cell film cannot be obtained.

Effect comparison example 4

2. Change the medium after 24 hours, add 2 mL of the culture medium of Comparative Example 3, place it in a 37°C, 5% CO2 incubator for 5 days, change the medium once every 2 days, take out the cells after 5 days, and use a pipette to absorb an appropriate amount. Basic medium, gently pipe the bottom edge of the culture dish, and peel off to obtain the NHDF cell film. As shown in C in Figure 1, the cell film cannot be obtained.

Effect comparison example 5

2. Change the medium after 24 hours, add 2 mL of the culture medium of Comparative Example 4, place it in a 37°C, 5% CO2 incubator for 5 days, change the medium once every 2 days, take out the cells after 5 days, and use a pipette to absorb an appropriate amount. Basic medium, gently pipe the bottom edge of the culture dish, and peel off the NHDF cell film. As shown in D in Figure 1, the cell film is damaged.

Effect comparison example 6

2. Change the medium after 24 hours, add 2 mL of the culture medium of Comparative Example 5, place it in a 37°C, 5% CO2 incubator for 5 days, change the medium once every 2 days, take out the cells after 5 days, and use a pipette to absorb an appropriate amount. Basic medium, gently pipe the bottom edge of the culture dish, and peel off the NHDF cell film, as shown in E in Figure 1. The resulting cell film is seriously damaged.

Effect comparison example 7

2. Change the medium after 24 hours, add 2 mL of the culture medium of Comparative Example 6, and culture it in a 37°C, 5% CO ₂ incubator for 5 days. Change the medium once every 2 days. After 5 days, take out the cells and pipette them. Use an appropriate amount of basal culture medium, gently pipe the bottom edge of the culture dish, and peel off the NHDF cell film. As shown in F in Figure 1, the resulting cell film is seriously damaged.

Effect comparison example 8

2. Change the medium after 24 hours, add 2 mL of the culture medium of Comparative Example 7, and culture it in a 37°C, 5% CO ₂ incubator for 5 days. Change the medium once every 2 days. After 5 days, take out the cells and pipette them. Use an appropriate amount of basic culture medium, gently pipe the bottom edge of the culture dish, and peel off to obtain the NHDF cell film. As shown in G in Figure 1, the cell film cannot be obtained.

Effect comparison example 9

2. Change the medium after 24 hours, add 2 mL of the culture medium of Comparative Example 8, and culture it in a 37°C, 5% CO ₂ incubator for 5 days. Change the medium once every 2 days. After 5 days, take out the cells and pipette them. Use an appropriate amount of basic culture medium, gently pipe the bottom edge of the culture dish, and peel off to obtain the NHDF cell film. As shown in H in Figure 1, the cell film cannot be obtained.

According to effect example 1, effect example 2, effect comparison example 1, effect comparison example 2, effect comparison example 3, effect comparison example 4, effect comparison example 5, effect comparison example 6, effect comparison example 7, effect comparison example 8 Compared with the cell film preparation method of Comparative Example 9, excluding the same consumables used by the 11 methods, the costs of preparing NHDF cell films by the 11 methods were calculated. The prices of each consumable are shown in Table 1. The costs of preparing NHDF cell films by 11 methods are shown in Table 2. It can be seen that the cost of obtaining NHDF cell films in Comparative Example 1 is 114.4 times that of Effective Example 1 and 57.3 times that of Effective Example 2. In terms of the success rate of obtaining cell films (Effective Examples 1 to 5 and Effective Comparative Examples 1 to 9 were repeated 10 times each), the definition of successfully obtaining a cell film is that no part of the cell film is lost during the process of peeling off the cell film. Effect Example 1 The cell membrane is indeed damaged at the edge, but only the edge is broken or cracked, and no part of the fragments is lost. When the cell membrane loses part of the fragments during the peeling process, it is defined as unable to be successfully obtained), Effect Example 1 and Effect Implementation The success rate of Example 2 reached 100%, but the integrity of the cell membrane obtained in Example 2 was better (Figure 1). The success rate of Effect Comparative Example 5 and Effect Comparative Example 7 is 67%, the success rate of Effect Comparative Example 6 is 50%, the success rate of Effect Comparative Example 5 to 7 is relatively low, and the success rate of Effect Comparative Example 6 and Effect Comparative Example 7 The resulting cell film was severely damaged and its integrity was far inferior to that of Example 2; the success rate of Comparative Example 1 using a temperature-sensitive culture dish was only 33%; while the success rate of Comparative Examples 2 to 3 and 8 to 9 was 0 . The costs and success rates of obtaining cell films in Effect Examples 3, 4, and 5 are similar to Effect Examples 1 and 2.

Effect Example 1, Effect Example 2, Effect Comparative Example 2, Effect Comparative Example 3, Effect Comparative Example 4, Effect Comparative Example 5, Effect Comparative Example 6, Effect Comparative Example 7, Effect Comparative Example 8 and Effect Comparative Example 9. Carry out cell viability detection (each treatment is repeated 3 times, and each time point detection is repeated 3 times). For specific methods, refer to the CCK-8 (purchased from Beyotime) reagent test instructions. After adding the specific culture medium in step 2) The cell viability was detected at 24h, 72h and 120h (the effect comparison example 2 was at 48h, 96h and 144h after adding the basal medium). As shown in Figure 2, as time goes by, the difference between the cell activity of Effect Example 2 and the comparative example gradually increases, that is, when cultured for 120 hours in a basic medium containing ITS, dexamethasone, Ficoll 400 and vitamin C , the cell activity reached the peak, and except for Effect Comparative Example 6, the cell activity of Effect Example 2 was significantly higher than that of all Effect Comparative Examples and Effect Example 1. Although there is no statistical difference between the cell activity of Effective Example 2 and Effective Comparative Example 6, judging from the effect of the peeled cell film (as shown in Figure 1), Effective Example 2 obviously has better integrity and a higher success rate. The cell activities of Effect Examples 3, 4, and 5 were similar to Effect Examples 1 and 2.

Table 1 Details of consumables used in Examples and Comparative Examples

	规格Specification	品牌brand	价格(元)Price (yuan)
温度敏感型培养皿Temperature sensitive petri dish	35mm35mm	CellSeedCellSeed	346346
维生素CVitamin C	25g25g	Solarbio Life ScienceSolarbio Life Science	6868
Ficoll 400Ficoll 400	100g100g	GE HealthcareGE Healthcare	20082008
ITS(100×)ITS(100×)	10mL10mL	CyagenCyagen	269269
地塞米松Dexamethasone	5g5g	麦克林McLean	212212
普通细胞培养皿Ordinary cell culture dish	35mm35mm	康宁Corning	1.411.41

Table 2 Cost and success rate of making cell films

	成功率(％)Success rate(%)	成本(元)Cost (yuan)
效果实施例1Effect Example 1	100100	3.0248261763.024826176
效果实施例2Effect Example 2	100100	6.0368261766.036826176
效果对比例1Effect comparison example 1	3333	346346
效果对比例2Effect comparison example 2	00	1.411.41
效果对比例3Effect comparison example 3	00	4.4224.422
效果对比例4Effect comparison example 4	1717	1.4108161.410816
效果对比例5Effect comparison example 5	6767	4.4228164.422816
效果对比例6Effect comparison example 6	5050	3.0248163.024816
效果对比例7Effect comparison example 7	6767	1.4108261761.410826176
效果对比例8Effect comparison example 8	00	3.0243.024
效果对比例9Effect comparison example 9	00	1.4100101761.410010176

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims

A composition includes: ITS cell culture supplement, dexamethasone and vitamin C.
The composition according to claim 1, characterized in that: the composition further comprises: polysucrose;

Preferably, the polysucrose is at least one of polysucrose 400 and polysucrose 70.
A culture medium comprising the composition of claim 1 or 2.
The culture medium according to claim 3, characterized in that:

The concentration of the ITS cell culture additive in the culture medium is 0.1-2.0v/v%;

Preferably, the concentration of dexamethasone in the culture medium is 0.001 to 0.04 μg/mL;

Preferably, the concentration of vitamin C in the culture medium is 1 to 500 μg/mL.
The culture medium according to claim 4, characterized in that:

The concentration of polysucrose in the culture medium is 0 to 100 mg/mL, excluding 0;

Preferably, the basal medium of the medium is at least one of DMEM high-glucose medium, DMEM-F12 medium, RPMI1640 medium, and DMEM low-glucose medium.
The application of the composition according to any one of claims 1 to 2 and/or the culture medium according to any one of claims 3 to 5 in any one of (1) to (4);

(1) Preparation of cell membrane;

(2) Prepare products for cell film preparation;

(3) Promote cell proliferation;

(4) Prepare products that promote cell proliferation.
A method for preparing a cell film, comprising the step of using the composition according to any one of claims 1 to 2 and/or the culture medium according to any one of claims 3 to 5.
The method according to claim 7, characterized in that:

The method includes the following steps: inoculating cells into a basal medium for first culture, and then replacing the basal medium containing the composition of any one of claims 1 to 2 and/or claims 3 to The composition described in any one of 5 is cultured for a second time to obtain a cell film;

Preferably, the cells include: at least one of skin fibroblasts, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, periodontal ligament mesenchymal stem cells, and umbilical cord mesenchymal stem cells. .
A cell film prepared by the method described in claim 7 or 8.
The application of at least one of (a1) to (a4) in any one of (1) to (3);

(a1) The composition according to any one of claims 1 to 2;

(a2) The kit according to any one of claims 3 to 5;

(a3) The method of claim 7 or 8;

(a4) The cell membrane according to claim 9;

(1) Preparation of products for repairing damaged tissues and/or organs;

(2) Construct three-dimensional tissues and/or organs;

(3) Preparation of products for constructing three-dimensional tissues and/or organs.