WO2023245813A1 - Composition and use thereof in preparation of cell membrane - Google Patents

Composition and use thereof in preparation of cell membrane Download PDF

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WO2023245813A1
WO2023245813A1 PCT/CN2022/108694 CN2022108694W WO2023245813A1 WO 2023245813 A1 WO2023245813 A1 WO 2023245813A1 CN 2022108694 W CN2022108694 W CN 2022108694W WO 2023245813 A1 WO2023245813 A1 WO 2023245813A1
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medium
cell
culture
culture medium
cells
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PCT/CN2022/108694
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Chinese (zh)
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高博韬
关淑文
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广东省科学院生物与医学工程研究所
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    • C12N5/0602Vertebrate cells
    • C12N5/0652Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656Adult fibroblasts
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    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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    • A61L27/3804Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
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Definitions

  • the invention belongs to the technical field of cell culture, and specifically relates to a composition and its application in preparing cell films.
  • 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.
  • 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.
  • PIPAAm poly(N-isopropylacrylamide)
  • PIPAAm poly(N-isopropylacrylamide)
  • the temperature-sensitive culture dishes require a low-temperature culture process. Low-temperature culture can lead to a decrease in cell viability and even functional changes in some fragile cells.
  • 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.
  • 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.
  • a first aspect of the present invention provides a composition comprising: ITS cell culture supplement, dexamethasone and vitamin C.
  • the composition further comprises polysucrose.
  • 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.
  • 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.
  • 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.
  • the concentration of fetal bovine serum in the basal culture medium is 8-12 v/v%.
  • 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.
  • the concentration of the antibiotic in the basal culture medium is 0.5-1.5 v/v%.
  • 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%.
  • the concentration of dexamethasone in the culture medium is 0.001-0.04 ⁇ g/mL.
  • the concentration of vitamin C in the culture medium is 1-500 ⁇ g/mL; further, the concentration is 50-500 ⁇ g/mL.
  • 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);
  • 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.
  • 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.
  • the conditions for the first culture are 33-39°C and 3-7% CO 2 for 18-30 hours.
  • the conditions for the second culture are 33-39°C and 3-7% CO2 for 4-15 days.
  • the medium is changed every 1 to 3 days during the second culture process.
  • 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.
  • 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.
  • the seeding density of the cells is 3.5 to 750,000/mL.
  • the cells are cultured using ordinary cell culture dishes.
  • the step of peeling off the cell membrane is also included.
  • 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);
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 0.04 ⁇ g/mL, and the concentration of vitamin C in the medium is 50 ⁇ g/mL.
  • 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 0.04 ⁇ g/mL
  • the concentration of vitamin C in the medium is 50 ⁇ g/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 ⁇ m sterile filter (purchased from Millipore) to obtain the culture medium.
  • a culture medium which is a basal culture medium containing ITS cell culture additives, dexamethasone, Ficoll 400 and vitamin C
  • 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 0.04 ⁇ g/mL
  • the concentration of vitamin C in the medium The concentration of Ficoll 400 in this medium is 50 ⁇ g/mL
  • 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 ⁇ m sterile filter (purchased from Millipore) to obtain the 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 1 ⁇ g/mL.
  • 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
  • the concentration of vitamin C in the medium is 1 ⁇ g/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 ⁇ m sterile filter (purchased from Millipore) to obtain the 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 0.04 ⁇ g/mL, and the concentration of vitamin C in the medium is 500 ⁇ g/mL.
  • 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 0.04 ⁇ g/mL
  • the concentration of vitamin C in the medium is 500 ⁇ g/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 ⁇ m sterile filter (purchased from Millipore) to obtain the culture medium.
  • a culture medium which is a basal culture medium containing ITS cell culture additives, dexamethasone, Ficoll 400 and vitamin C
  • 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 0.04 ⁇ g/mL
  • the concentration of vitamin C in this medium The concentration of Ficoll 400 in this medium is 500 ⁇ g/mL
  • the concentration of Ficoll 400 in this medium is 100mg/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 ⁇ m sterile filter (purchased from Millipore) to obtain the culture medium.
  • a culture medium which is a DMEM high-glucose culture medium containing 10v/v% fetal bovine serum and 1v/v% penicillin-streptomycin.
  • 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 ⁇ m sterile filter (purchased from Millipore) to obtain the 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 ⁇ g/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 ⁇ m sterile filter (purchased from Millipore) to obtain the 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 ⁇ g/mL
  • 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 ⁇ m sterile filter (purchased from Millipore) to obtain the 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 ⁇ g/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 ⁇ m sterile filter (purchased from Millipore) to obtain the 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 ⁇ g/mL
  • the concentration of dexamethasone in the culture medium is 0.04 ⁇ g/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 ⁇ m sterile filter (purchased from Millipore) to obtain the 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 ⁇ m sterile filter (purchased from Millipore) to obtain the 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.
  • concentration of metasone in this medium is 0.04 ⁇ g/mL.
  • the preparation method of the above culture medium is as follows: adding dexamethasone to the basic culture medium, filtering using a 0.22 ⁇ m sterile filter (purchased from Millipore) to obtain the culture medium.
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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.
  • NHDF cells purchased from ATCC
  • 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.
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • NHDF cells purchased from ATCC
  • 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;
  • 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
  • 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.
  • 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.

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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

一种组合物及其在制备细胞薄膜中的应用A composition and its application in preparing cell films 技术领域Technical field
本发明属于细胞培养技术领域,具体涉及一种组合物及其在制备细胞薄膜中的应用。The invention belongs to the technical field of cell culture, and specifically relates to a composition and its application in preparing cell films.
背景技术Background technique
细胞薄膜是一种由细胞和细胞外基质组成的致密组织。细胞薄膜保留了细胞-细胞、细胞-基质间的连接,可有效地维持细胞间相互作用,完整地保留了细胞外基质精密有序的网络结构。细胞外基质负载的生长因子、细胞因子等活性因子可调控细胞行为、影响细胞命运、促进组织修复再生和改善炎症。因此,细胞薄膜常用于组织或器官缺损位置的修复、替代,三维组织或器官的构建等,在组织工程和再生医学领域具有广泛的应用前景。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.
目前,制作细胞薄膜的主要方法是采用温度敏感型培养皿。温度敏感型培养皿底部涂有聚(N-异丙基丙烯酰胺)(PIPAAm),PIPAAm在37℃时具有疏水性,而在20℃时具有亲水性。当温度从37℃降为20℃时,PIPAAm由疏水性变成亲水性,导致细胞脱离培养皿底部,从而获得细胞薄膜。但是,使用温度敏感型培养皿制作细胞薄膜需要有一个低温培养过程,低温培养可导致细胞活力下降,一些脆弱的细胞甚至发生功能上的变化,其次,温度敏感型培养皿价格昂贵。因此,开发一种利用普通细胞培养皿稳定制备细胞薄膜的组合物及方法尤为重要。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.
发明内容Contents of the invention
本发明的第一方面的目的,在于提供一种组合物。The object of the first aspect of the present invention is to provide a composition.
本发明的第二方面的目的,在于提供一种培养基。The second object of the present invention is to provide a culture medium.
本发明的第三方面的目的,在于提供第一方面的组合物和/或第二方面的培养基在制备细胞薄膜或制备用于细胞薄膜制备的产品中的应用。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.
本发明的第四方面的目的,在于提供一种制备细胞薄膜的方法。The fourth object of the present invention is to provide a method for preparing cell films.
本发明的第五方面的目的,在于提供一种细胞薄膜。The fifth aspect of the present invention aims to provide a cell membrane.
本发明的第六方面的目的,在于提供第一方面的组合物、本发明第二方面的试剂盒、本发明第四个方面的方法和/或本发明第五个方面的细胞薄膜的应用。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.
为了实现上述目的,本发明所采取的技术方案是:In order to achieve the above objects, the technical solutions adopted by the present invention are:
本发明的第一个方面,提供一种组合物,包含:ITS细胞培养添加物、地塞米松和维生素C。A first aspect of the present invention provides a composition comprising: ITS cell culture supplement, dexamethasone and vitamin C.
优选地,所述组合物还包含:聚蔗糖。Preferably, the composition further comprises polysucrose.
优选地,所述聚蔗糖为聚蔗糖400(Ficoll400)、聚蔗糖70(Ficoll70)中的至少一种;进一步为聚蔗糖400(Ficoll400)。Preferably, the Ficollose is at least one of Ficoll 400 (Ficoll 400) and Ficoll 70 (Ficoll 70); further, Ficoll 400 (Ficoll 400).
本发明的第二个方面,提供一种培养基,包含本发明第一个方面的组合物。A second aspect of the present invention provides a culture medium comprising the composition of the first aspect of the present invention.
优选地,所述培养基的基础培养基为DMEM高糖培养基、DMEM-F12培养基、RPMI1640培养基、DMEM低糖培养基中的至少一种;进一步为DMEM高糖培养基。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.
优选地,所述基础培养基包含胎牛血清和抗生素中的至少一种;进一步优选地,所述基础培养基包含胎牛血清和抗生素。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.
优选地,所述胎牛血清在所述基础培养基中的浓度为8~12v/v%。Preferably, the concentration of fetal bovine serum in the basal culture medium is 8-12 v/v%.
优选地,所述抗生素包含青霉素、庆大霉素、两性霉素B、卡那霉素、四环素、链霉素中的至少一种;进一步包含青霉素、链霉素中的至少一种;更进一步包含青霉素和链霉素。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.
优选地,所述抗生素在所述基础培养基中的浓度为0.5~1.5v/v%。Preferably, the concentration of the antibiotic in the basal culture medium is 0.5-1.5 v/v%.
优选地,所述ITS细胞培养添加物在所述培养基中的浓度为0.1~2.0v/v%;进一步为1~2.0v/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%.
优选地,所述地塞米松在所述培养基中的浓度为0.001~0.04μg/mL。Preferably, the concentration of dexamethasone in the culture medium is 0.001-0.04 μg/mL.
优选地,所述维生素C在所述培养基中的浓度为1~500μg/mL;进一步为50~500μg/mL。Preferably, the concentration of vitamin C in the culture medium is 1-500 μg/mL; further, the concentration is 50-500 μg/mL.
优选地,所述聚蔗糖在所述培养基中的浓度为0~100mg/mL,不包含0;25mg/mL~100mg/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.
本发明的第三个方面,提供第一方面的组合物和/或第二方面的培养基在(1)~(4)中任一项的应用;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)制备细胞薄膜;(1) Preparation of cell membrane;
(2)制备用于细胞薄膜制备的产品;(2) Prepare products for cell film preparation;
(3)促进细胞增殖;(3) Promote cell proliferation;
(4)制备促进细胞增殖的产品。(4) Prepare products that promote cell proliferation.
优选地,所述细胞包含:皮肤成纤维细胞、骨髓间充质干细胞、脂肪间充质干细胞、牙髓间充质干细胞、牙周膜间充质干细胞、脐带间充质干细胞中的至少一种;进一步为皮肤成纤维细胞。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.
本发明的第四个方面,提供一种制备细胞薄膜的方法,包含:采用本发明第一个方面的组合物和/或本发明第二个方面的培养基的步骤。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.
优选地,所述方法包括如下步骤:将细胞接种至基础培养基中进行第一次培养,然后, 更换包含本发明第一个方面的组合物的基础培养基和/或本发明第二个方面的组合物进行第二次培养,得到细胞薄膜。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.
优选地,所述第一次培养的条件为33~39℃,3~7%CO 2下培养18~30h。 Preferably, the conditions for the first culture are 33-39°C and 3-7% CO 2 for 18-30 hours.
优选地,所述第二次培养的条件为33~39℃,3~7%CO 2下培养4~15天。 Preferably, the conditions for the second culture are 33-39°C and 3-7% CO2 for 4-15 days.
优选地,所述第二次培养过程中每1~3天换液一次。Preferably, the medium is changed every 1 to 3 days during the second culture process.
优选地,所述细胞包含:皮肤成纤维细胞、骨髓间充质干细胞、脂肪间充质干细胞、牙髓间充质干细胞、牙周膜间充质干细胞、脐带间充质干细胞中的至少一种;进一步为皮肤成纤维细胞。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.
优选地,所述基础培养基为DMEM高糖培养基、DMEM-F12培养基、RPMI1640培养基、DMEM低糖培养基中的至少一种;进一步为DMEM高糖培养基。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.
优选地,所述细胞的接种密度为3.5~75万/mL。Preferably, the seeding density of the cells is 3.5 to 750,000/mL.
优选地,所述细胞培养采用普通细胞培养皿培养。Preferably, the cells are cultured using ordinary cell culture dishes.
优选地,所述第二次培养结束后,还包括剥离细胞薄膜的步骤。Preferably, after the second culture is completed, the step of peeling off the cell membrane is also included.
优选地,所述剥离细胞薄膜的步骤为:用移液枪轻轻吹打培养皿底部边缘。Preferably, the step of peeling off the cell film is: gently blowing the bottom edge of the culture dish with a pipette.
本发明的第五个方面,提供一种细胞薄膜,通过本发明第四个方面的方法得到。A fifth aspect of the present invention provides a cell film obtained by the method of the fourth aspect of the present invention.
本发明的第六个方面,提供本发明第一个方面的组合物、本发明第二个方面的试剂盒、本发明第四个方面的方法和/或本发明第五个方面的细胞薄膜在(1)~(3)中任一项中的应用;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)制备修复损伤的组织和/或器官的产品;(1) Preparation of products for repairing damaged tissues and/or organs;
(2)构建三维组织和/或器官;(2) Construct three-dimensional tissues and/or organs;
(3)制备构建三维组织和/或器官的产品。(3) Preparation of products for constructing three-dimensional tissues and/or organs.
本发明的有益效果是:The beneficial effects of the present invention are:
本发明首次公开了一种组合物,包含:ITS细胞培养添加物、地塞米松和维生素C;采用该组合物可通过普通细胞培养皿制备细胞薄膜,相对于传统的利用温度敏感型培养皿制备细胞薄膜,采用该组合物可以大幅地降低制备细胞薄膜的成本,并且增加细胞数量,提高制备细胞薄膜的成功率,有利于更好地运用在组织工程和再生医学领域(修复损伤的组织和/或器官、构建三维组织和/或器官),有望实现商业化的大规模生产。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.
本发明进一步限定组合物还包含:聚蔗糖,从而进一步提高制备细胞薄膜的质量(完整性),以及增加细胞数量。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.
附图说明Description of the drawings
图1是效果实施例1、效果实施例2、效果对比例1、效果对比例2、效果对比例3、效果对比例4、效果对比例5、效果对比例6、效果对比例7、效果对比例8和效果对比例9制备得到的细胞薄膜的直观图:其中,A为效果对比例2制备得到的细胞薄膜的直观图;B为效果对比例3制备得到的细胞薄膜的直观图;C为效果对比例4制备得到的细胞薄膜的直观图;D为效果对比例5制备得到的细胞薄膜的直观图;E为效果对比例6制备得到的细胞薄膜的直观图;F为效果对比例7制备得到的细胞薄膜的直观图;G为效果对比例8制备得到的细胞薄膜的直观图;H为效果对比例9制备得到的细胞薄膜的直观图;I为效果实施例1制备得到的细胞薄膜的直观图;J为效果实施例2制备得到的细胞薄膜的直观图;K为效果对比例1制备得到的细胞薄膜的直观图。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.
图2是效果实施例1、效果实施例2、效果对比例2、效果对比例3、效果对比例4、效果对比例5、效果对比例6、效果对比例7、效果对比例8和效果对比例9的细胞活性检测结果图:*表示p<0.05。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.
具体实施方式Detailed ways
以下通过具体的实施例对本发明的内容作进一步详细的说明。The content of the present invention will be further described in detail below through specific examples.
本实施例中所使用的材料、试剂等,如无特别说明,为从商业途径得到的试剂和材料。The materials, reagents, etc. used in this example are reagents and materials obtained from commercial sources unless otherwise specified.
本实施例中的ITS细胞培养添加物(100×)购自Cyagen,地塞米松购自麦克林,维生素C购自Solarbio Life Science,Ficoll 400购自GE Healthcare,NHDF细胞购自ATCC,直径为35mm普通细胞培养皿购自康宁;直径为35mm温度敏感型培养皿购自CellSeed。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.
实施例1一种培养基Example 1 A culture medium
一种培养基,该培养基为包含ITS细胞培养添加物、地塞米松和维生素C的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,ITS细胞培养添加物在该培养基中的浓度为1v/v%,地塞米松在该培养基中的浓度为0.04μg/mL,维生素C在该培养基中的浓度为50μg/mL。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.
上述培养基的制备方法为:将ITS细胞培养添加物、地塞米松和维生素C添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
实施例2一种培养基Example 2 A culture medium
一种培养基,该培养基为包含ITS细胞培养添加物、地塞米松、Ficoll 400和维生素C的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,ITS细胞培养添加物在该培养基中的浓度为1v/v%,地塞米松在该培养基中的浓度为 0.04μg/mL,维生素C在该培养基中的浓度为50μg/mL,Ficoll 400在该培养基中的浓度为25mg/mL。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.
上述培养基的制备方法为:将ITS细胞培养添加物、地塞米松、Ficoll 400和维生素C添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
实施例3一种培养基Example 3 A culture medium
一种培养基,该培养基为包含ITS细胞培养添加物、地塞米松和维生素C的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,ITS细胞培养添加物在该培养基中的浓度为0.1v/v%,地塞米松在该培养基中的浓度为1ng/mL,维生素C在该培养基中的浓度为1μg/mL。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.
上述培养基的制备方法为:将ITS细胞培养添加物、地塞米松和维生素C添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
实施例4一种培养基Example 4 A culture medium
一种培养基,该培养基为包含ITS细胞培养添加物、地塞米松和维生素C的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,ITS细胞培养添加物在该培养基中的浓度为2v/v%,地塞米松在该培养基中的浓度为0.04μg/mL,维生素C在该培养基中的浓度为500μg/mL。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.
上述培养基的制备方法为:将ITS细胞培养添加物、地塞米松和维生素C添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
实施例5一种培养基Example 5 A culture medium
一种培养基,该培养基为包含ITS细胞培养添加物、地塞米松、Ficoll 400和维生素C的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,ITS细胞培养添加物在该培养基中的浓度为2v/v%,地塞米松在该培养基中的浓度为0.04μg/mL,维生素C在该培养基中的浓度为500μg/mL,Ficoll 400在该培养基中的浓度为100mg/mL。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.
上述培养基的制备方法为:将ITS细胞培养添加物、地塞米松、Ficoll 400和维生素C添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
对比例1一种培养基Comparative Example 1 A culture medium
一种培养基,该培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基。A culture medium, which is a DMEM high-glucose culture medium containing 10v/v% fetal bovine serum and 1v/v% penicillin-streptomycin.
对比例2一种培养基Comparative Example 2 One culture medium
一种培养基,该培养基为包含Ficoll 400的基础培养基,其中,基础培养基为包含10v/v% 胎牛血清、1v/v%青链霉素的DMEM高糖培养基,Ficoll 400在该培养基中的浓度为25mg/mL。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.
上述培养基的制备方法为:将Ficoll 400添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
对比例3一种培养基Comparative Example 3 One culture medium
一种培养基,该培养基为包含维生素C的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,维生素C在该培养基中的浓度为50μg/mL。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.
上述培养基的制备方法为:将维生素C添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
对比例4一种培养基Comparative Example 4 One culture medium
一种培养基,该培养基为包含Ficoll 400和维生素C的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,维生素C在该培养基中的浓度为50μg/mL,Ficoll 400在该培养基中的浓度为25mg/mL。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.
上述培养基的制备方法为:将Ficoll 400和维生素C添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
对比例5一种培养基Comparative Example 5 One culture medium
一种培养基,该培养基为包含ITS细胞培养添加物和维生素C的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,维生素C在该培养基中的浓度为50μg/mL,ITS细胞培养添加物在该培养基中的浓度为1v/v%。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%.
上述培养基的制备方法为:将ITS细胞培养添加物和维生素C添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
对比例6一种培养基Comparative Example 6 One culture medium
一种培养基,该培养基为包含地塞米松和维生素C的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,维生素C在该培养基中的浓度为50μg/mL,地塞米松在该培养基中的浓度为0.04μg/mL。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.
上述培养基的制备方法为:将地塞米松和维生素C添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
对比例7一种培养基Comparative Example 7 One culture medium
一种培养基,该培养基为包含ITS细胞培养添加物的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,ITS细胞培养添加物在该培养基中的浓度为1v/v%。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%.
上述培养基的制备方法为:将ITS细胞培养添加物添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
对比例8一种培养基Comparative Example 8 One culture medium
一种培养基,该培养基为包含地塞米松的基础培养基,其中,基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,地塞米松在该培养基中的浓度为0.04μg/mL。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.
上述培养基的制备方法为:将地塞米松添加至基础培养基中,使用0.22μm无菌滤头(购自密理博)过滤,得到培养基。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.
效果实施例采用不同培养皿和/或培养基制备细胞薄膜Effect Example Preparing cell films using different culture dishes and/or culture media
效果实施例1Effect Example 1
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL实施例1的培养基,置于37℃,5%CO2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中I所示,所得细胞薄膜有轻微破损。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.
效果实施例2Effect Example 2
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL实施例2的培养基,置于37℃,5%CO2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中J所示,所得细胞薄膜完整无缺损。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.
效果实施例3Effect Example 3
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL实施例3的培养基,置于37℃,5%CO2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,所得细胞薄膜有轻微破损。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.
效果实施例4Effect Example 4
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL实施例4的培养基,置于37℃,5%CO2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,所得细胞薄膜有轻微破损。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.
效果实施例5Effect Example 5
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL实施例5的培养基,置于37℃,5%CO2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,所得细胞薄膜有轻微破损。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.
效果对比例1Effect comparison example 1
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm温度敏感型培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,即对比例1中的培养基);将细胞置于37℃,5%CO 2培养箱内培养6天,每2天换液1次,6天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中K所示,所得细胞薄膜缺损严重。 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.
效果对比例2Effect comparison example 2
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基,即对比例1中的培养基);将细胞置于37℃,5%CO 2培养箱内培养6天,每2天换液1次,6天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中A所示,不能获得细胞薄膜。 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.
效果对比例3Effect comparison example 3
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL对比例2的培养基,置于37℃,5%CO2培养箱内培养5天, 每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中B所示,不能获得细胞薄膜。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.
效果对比例4Effect comparison example 4
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL对比例3的培养基,置于37℃,5%CO2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中C所示,不能获得细胞薄膜。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.
效果对比例5Effect comparison example 5
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL对比例4的培养基,置于37℃,5%CO2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中D所示,细胞薄膜破损。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.
效果对比例6Effect comparison example 6
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL对比例5的培养基,置于37℃,5%CO2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中E所示,所得细胞薄膜破损严重。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.
效果对比例7Effect comparison example 7
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL对比例6的培养基,置于37℃,5%CO 2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中F所示,所得细胞薄膜破损严重。 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.
效果对比例8Effect comparison example 8
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL对比例7的培养基,置于37℃,5%CO 2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中G所示,不能获得细胞薄膜。 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.
效果对比例9Effect comparison example 9
1.将NHDF细胞(购自ATCC)传至第6代,接种到直径为35mm普通细胞培养皿上,接种细胞数为100万;加入2mL基础培养基(基础培养基为包含10v/v%胎牛血清、1v/v%青链霉素的DMEM高糖培养基);将细胞置于37℃,5%CO 2培养箱内培养24h; 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. 24h后换液,加入2mL对比例8的培养基,置于37℃,5%CO 2培养箱内培养5天,每2天换液1次,5天后取出细胞,用移液枪吸取适量基础培养基,轻轻吹打培养皿底部边缘,剥离获得NHDF细胞薄膜,如图1中H所示,不能获得细胞薄膜。 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.
根据效果实施例1、效果实施例2、效果对比例1、效果对比例2、效果对比例3、效果对比例4、效果对比例5、效果对比例6、效果对比例7、效果对比例8和效果对比例9的细胞薄膜制备方法,除去11种方法使用的相同耗材,分别计算11种方法制备NHDF细胞薄膜的成本。各耗材的价格如表1所示。11种方法制备NHDF细胞薄膜的成本如表2所示,可以看出效果对比例1获得NHDF细胞薄膜所产生的费用是效果实施例1的114.4倍,是效果实施例2的57.3倍。在获得细胞薄膜的成功率方面(效果实施例1~5和效果对比例1~9分别重复10次,成功获得细胞薄膜的定义是剥离细胞薄膜过程中,细胞薄膜没有损失一部分,效果实施例1的细胞薄膜确实边缘处有破损,但是只是边缘破了或者说裂开了,并没有丢掉一部分碎片。当细胞薄膜在剥离过程中丢掉一部分碎片定义为不能成功获得),效果实施例1和效果实施例2的成功率都达到100%,但是效果实施例2所得细胞薄膜完整性更好(如图1)。效果对比例5和效果对比例7的成功率为67%,效果对比例6的成功率为50%,效果对比例5~7的成功率相对较低,并且效果对比例6和效果对比例7所得细胞薄膜破损严重,完整性远不如效果实施例2;而采用温度敏感型培养皿的效果对比例1成功率都只有33%;而效果对比例2~3、8~9的成功率为0。效果实施例3、4、5的成本、获得细胞薄膜的成功率与效果实施例1、2相似。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.
对效果实施例1、效果实施例2、效果对比例2、效果对比例3、效果对比例4、效果对 比例5、效果对比例6、效果对比例7、效果对比例8和效果对比例9进行细胞活性检测(每种处理重复3次,每个时间点检测重复3次),具体方法参考CCK-8(购自碧云天)试剂检测说明书,分别在加入步骤2)加入特异性培养基后的24h、72h和120h(效果对比例2则是在加入基础培养基后的48h、96h和144h)检测细胞活性。如图2所示,随着时间推移,效果实施例2的细胞活性与对比例之间差异逐渐增大,即在包含ITS、地塞米松、Ficoll 400和维生素C的基础培养基中培养120h时,细胞活性达到峰值,除了效果对比例6以外,效果实施例2的细胞活性显著高于所有效果对比例和效果实施例1。虽然效果实施例2的细胞活性与效果对比例6无统计学差异,但是从剥离的细胞薄膜效果来看(如图1),效果实施例2显然完整性更好、成功率更高。效果实施例3、4、5的细胞活性与效果实施例1、2相似。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.
表1实施例和对比例使用耗材详情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
表2成本及制作细胞薄膜的成功率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
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。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 (10)

  1. 一种组合物,包含:ITS细胞培养添加物、地塞米松和维生素C。A composition includes: ITS cell culture supplement, dexamethasone and vitamin C.
  2. 根据权利要求1所述的组合物,其特征在于:所述组合物还包含:聚蔗糖;The composition according to claim 1, characterized in that: the composition further comprises: polysucrose;
    优选地,所述聚蔗糖为聚蔗糖400、聚蔗糖70中的至少一种。Preferably, the polysucrose is at least one of polysucrose 400 and polysucrose 70.
  3. 一种培养基,包含权利要求1或2所述的组合物。A culture medium comprising the composition of claim 1 or 2.
  4. 根据权利要求3所述的培养基,其特征在于:The culture medium according to claim 3, characterized in that:
    所述ITS细胞培养添加物在所述培养基中的浓度为0.1~2.0v/v%;The concentration of the ITS cell culture additive in the culture medium is 0.1-2.0v/v%;
    优选地,所述地塞米松在所述培养基中的浓度为0.001~0.04μg/mL;Preferably, the concentration of dexamethasone in the culture medium is 0.001 to 0.04 μg/mL;
    优选地,所述维生素C在所述培养基中的浓度为1~500μg/mL。Preferably, the concentration of vitamin C in the culture medium is 1 to 500 μg/mL.
  5. 根据权利要求4所述的培养基,其特征在于:The culture medium according to claim 4, characterized in that:
    所述聚蔗糖在所述培养基中的浓度为0~100mg/mL,不包含0;The concentration of polysucrose in the culture medium is 0 to 100 mg/mL, excluding 0;
    优选地,所述培养基的基础培养基为DMEM高糖培养基、DMEM-F12培养基、RPMI1640培养基、DMEM低糖培养基中的至少一种。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.
  6. 权利要求1~2中任一项所述的组合物和/或权利要求3~5中任一项所述的培养基在(1)~(4)中任一项的应用;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)制备细胞薄膜;(1) Preparation of cell membrane;
    (2)制备用于细胞薄膜制备的产品;(2) Prepare products for cell film preparation;
    (3)促进细胞增殖;(3) Promote cell proliferation;
    (4)制备促进细胞增殖的产品。(4) Prepare products that promote cell proliferation.
  7. 一种制备细胞薄膜的方法,包含:采用权利要求1~2中任一项所述的组合物和/或权利要求3~5中任一项所述的培养基的步骤。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.
  8. 根据权利要求7所述的方法,其特征在于:The method according to claim 7, characterized in that:
    所述方法包括如下步骤:将细胞接种至基础培养基中进行第一次培养,然后,更换包含权利要求1~2中任一项所述的组合物的基础培养基和/或权利要求3~5中任一项所述的组合物进行第二次培养,得到细胞薄膜;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;
    优选地,所述细胞包含:皮肤成纤维细胞、骨髓间充质干细胞、脂肪间充质干细胞、牙髓间充质干细胞、牙周膜间充质干细胞、脐带间充质干细胞中的至少一种。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. .
  9. 一种细胞薄膜,通过权利要求7或8所述的方法制备得到。A cell film prepared by the method described in claim 7 or 8.
  10. (a1)~(a4)中至少一种在(1)~(3)中任一项中的应用;The application of at least one of (a1) to (a4) in any one of (1) to (3);
    (a1)权利要求1~2中任一项所述的组合物;(a1) The composition according to any one of claims 1 to 2;
    (a2)权利要求3~5中任一项所述的试剂盒;(a2) The kit according to any one of claims 3 to 5;
    (a3)权利要求7或8所述的方法;(a3) The method of claim 7 or 8;
    (a4)权利要求9所述的细胞薄膜;(a4) The cell membrane according to claim 9;
    (1)制备修复损伤的组织和/或器官的产品;(1) Preparation of products for repairing damaged tissues and/or organs;
    (2)构建三维组织和/或器官;(2) Construct three-dimensional tissues and/or organs;
    (3)制备构建三维组织和/或器官的产品。(3) Preparation of products for constructing three-dimensional tissues and/or organs.
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