Inactivated cell film as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical engineering, discloses an inactivated cell film as well as a preparation method and application thereof, and particularly discloses a preparation method of the inactivated cell film, which comprises the following steps: preparing a cell film; and inactivating the cell film at-30 to 35 DEG C for 15-30 hours to obtain the inactivated cell film. The invention aims to realize standardized production of cell film products, and provides a preparation method of an inactivated cell film, and the prepared inactivated cell film stored at normal temperature has a wound repair effect similar to that of a fresh cell film and an inactivated cell film stored in a freezing manner; compared with a normal human fibroblast inactivation film, the embryonic gingival-derived mesenchymal stem cell inactivation film shows more remarkable cell proliferation promoting capacity after being stored for 4 weeks; the inactivated cell film prepared by the scheme can keep biological activity after being stored at normal temperature, and is convenient for product transportation and storage.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical engineering, and in particular relates to an inactivated cell film and a preparation method and application thereof. Background Art

[0002] "Cell membrane" is a sheet-like cell aggregate composed of cells and the extracellular matrix (ECM) they secrete. It can greatly improve the survival rate and colonization ability of stem cells. It is a relatively successful regenerative medicine technology. Currently, there are 4 products on the market overseas for tissue repair or function improvement of the heart, cornea and skin.

[0003] However, current cell membrane products mainly use the patient's own cells and are formed into membranes through in vitro culture. Not only is the production cost high, but the timeliness is poor, which limits its commercial application. For example, although autologous epidermal cell membrane is an effective method for treating burn wounds, the production process of this method takes 3 to 4 weeks, which makes it impossible to use it in the early stage of burns that are life-threatening. In contrast, allogeneic epidermal cell membrane is a standardized "shelf-type" product that makes up for the lack of immediacy of autologous cell membranes, but in order to ensure the activity of the cells, strict ultra-low temperature storage conditions are required and the product has a short shelf life.

[0004] Inactivated cell membrane is a new type of regenerative medicine technology that has emerged in recent years. It inactivates cells in the cell membrane under controlled temperature and humidity conditions, but retains the active substances in the cells and the integrity of the cell membrane. Regarding the preparation of inactivated cell membranes, the existing methods include repeated freezing and thawing, water osmotic inactivation, and drying inactivation. Researchers decellularize cell membranes by repeated freezing and thawing. This operation can make the prepared film stable for storage and reduce immune rejection reactions. However, after repeated freezing and thawing, the integrity of the cell membrane will be destroyed, and very few biologically active substances will be retained. The water osmotic method mainly inactivates cells by changing the permeability of the cell membrane. It usually uses a high osmotic pressure solution or pure water treatment to cause cell inactivation. Compared with the repeated freezing and thawing method, the water osmotic method is milder and will not cause excessive mechanical damage to the internal structure of the cell. It can ensure the integrity of the cell membrane, but the effect of the water osmotic method is affected by factors such as temperature, processing time, and cell type. It is necessary to precisely control the processing conditions to ensure the inactivation effect and the availability of cell samples. The drying inactivation method refers to placing cells in a dry environment to make them lose water, thereby destroying the structure of the cell membrane and then inactivating the cells. This method usually inactivates the cells more thoroughly and allows the release of intracellular substances when they are rehydrated. The existing autologous cultured epidermis is a "customized" service. Its preparation process is cumbersome and time-sensitive. It cannot achieve immediate use of the product, and its packaging, transportation and storage processes require the use of ultra-low temperature freezing equipment, which has high storage and transportation costs. Although the emergence of allogeneic cultured epidermis has overcome the timeliness problem, in order to ensure the cell activity of allogeneic cultured epidermis, ultra-low temperature freezing equipment is also required during the packaging, transportation and storage process, and the product has a short shelf life.

[0005] Research results show that in the treatment of chronic skin ulcers, inactivated cell membranes are as effective as ultra-low temperature live cell membranes, and have the advantages of easy preparation and convenient storage. Therefore, inactivated cell membranes are a new regenerative medicine strategy that is expected to be commercialized. Summary of the invention

[0006] The first aspect of the present invention aims to provide a method for preparing an inactivated cell film.

[0007] The second aspect of the present invention aims to provide an inactivated cell membrane.

[0008] The third aspect of the present invention aims to provide a leachate.

[0009] The purpose of the fourth aspect of the present invention is to provide the preparation method of the first aspect of the present invention, the application of the inactivated cell film of the second aspect of the present invention or the leachate of the third aspect of the present invention.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides a method for preparing an inactivated cell film, comprising the following steps:

[0012] Prepare cell membranes and dry them;

[0013] The cell membrane is placed at -30 to 35°C and inactivated for 15 to 30 hours to obtain an inactivated cell membrane.

[0014] In some embodiments of the present invention, the cells include: at least one of human 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.

[0015] In some embodiments of the present invention, the cells include at least one of human fibroblasts, dental pulp mesenchymal stem cells, gingival mesenchymal stem cells, embryonic gingival-derived mesenchymal stem cells, and periodontal ligament mesenchymal stem cells; preferably, embryonic gingival-derived mesenchymal stem cells / human fibroblasts.

[0016] Oral gingival / mucosal wounds heal faster than skin wounds and form less scars, which may be due to the low infiltration of inflammatory cells and the expression of proinflammatory factors in the gingiva. Gingival mesenchymal stem cells can secrete more extracellular vesicles expressing interleukin-1 receptor antagonist (IL-1RA) to accelerate wound healing. Embryonic stem cells are isolated from fetal tissues. Compared with adult stem cells, embryonic stem cells have stronger self-renewal and differentiation potential and can differentiate into various tissues under certain conditions. Human fetal gingival mesenchymal stem cells (HF-GMSC) have the ability to spontaneously differentiate in vivo, which may be related to their higher progenitor cell content, thus facilitating regeneration and repair. Therefore, HF-GMSC has a certain promoting effect on wound repair, and compared with the commonly used normal human dermal fibroblasts (NHDF), the repair effect is more significant and more conducive to reducing scar formation.

[0017] The present invention selects embryonic gingival-derived mesenchymal stem cells with low immunogenicity to prepare cell membranes, and selecting HF-GMSC cells can theoretically reduce scar formation to a greater extent.

[0018] In some embodiments of the present invention, the inactivation condition is -20 to 4°C for 10 to 24 hours.

[0019] In some embodiments of the present invention, the inactivation condition is -20°C for 10 to 24 hours.

[0020] In some embodiments of the present invention, the method for preparing the cell film comprises the following steps: inoculating cells into a basal culture medium for a first culture, and then replacing the culture medium containing vitamin C, ITS, and dexamethasone for a second culture to obtain a cell film.

[0021] In some embodiments of the present invention, the method for preparing the cell film comprises the following steps: inoculating cells into a basal culture medium for a first culture, and then replacing the culture medium containing vitamin C, ITS, and dexamethasone for a second culture to obtain a cell film.

[0022] In some embodiments of the present invention, the first culture is carried out at 33-39° C. and 3%-7% CO 2 for 18-30 hours.

[0023] In some embodiments of the present invention, the first culture is carried out at 36-39° C. and 4%-6% CO 2 for 20-24 hours.

[0024] In some embodiments of the present invention, the second culture is carried out at 33-39° C. and 3%-7% CO 2 for 6-10 days.

[0025] In some embodiments of the present invention, the second culture is carried out at 36-39° C. and 4%-6% CO 2 for 6-8 days.

[0026] In some embodiments of the present invention, the medium is replaced every 1 to 3 days during the second culture.

[0027] In some embodiments of the present invention, the culture medium containing vitamin C, ITS and dexamethasone contains 1-500 μg / mL vitamin C, 0.001-0.05 μg / mL dexamethasone and 0.1-2.0 v / v % ITS. Figure 2 It can be seen that the culture medium with added vitamin C, ITS and dexamethasone is more conducive to the formation of cell membrane than the culture medium with only any one or two of them.

[0028] In some embodiments of the present invention, the culture medium containing vitamin C, ITS and dexamethasone contains 100-200 μg / mL vitamin C, 0.01-0.04 μg / mL dexamethasone and 0.1-2.0 v / v% ITS.

[0029] In some embodiments of the present invention, the culture medium containing vitamin C, ITS and dexamethasone also contains antibiotics and fetal bovine serum.

[0030] In some embodiments of the present invention, the antibiotic comprises at least one of penicillin, gentamicin, amphotericin B, kanamycin, tetracycline, and streptomycin; further comprises at least one of penicillin and streptomycin; and further comprises penicillin and streptomycin.

[0031] In some embodiments of the present invention, the concentration of the antibiotic in the culture medium is 0.5-1.5 v / v %.

[0032] In some embodiments of the present invention, the concentration of the fetal bovine serum in the culture medium is 8-12 v / v%.

[0033] In some embodiments of the present invention, after the second culture is completed, a step of peeling off the cell membrane is also included.

[0034] In some embodiments of the present invention, the step of peeling off the cell membrane is: without discarding the original culture medium, the original culture medium is sucked up by a pipette and the bottom edge of the culture dish is gently blown. PBS is not used to rinse the cells before harvesting the cell membrane.

[0035] In some embodiments of the present invention, the cells are cultured in a common cell culture dish.

[0036] In some embodiments of the present invention, the drying comprises air-drying.

[0037] In some embodiments of the present invention, the air drying comprises the following steps: placing the cell membrane on a workbench and air drying for 15 to 30 minutes, the air drying temperature is 38 to 32° C., the humidity is 36 to 42%, and the wind speed is 0.1 to 0.4 m / s.

[0038] The existing autologous cultured epidermis is a "customized" service, and its preparation process is cumbersome and time-effective, and the product cannot be used immediately. In addition, its packaging, transportation and storage process require the use of ultra-low temperature freezing equipment, and the storage and transportation costs are high. Although the emergence of allogeneic cultured epidermis has overcome the time-effectiveness problem, in order to ensure the cell activity of allogeneic cultured epidermis, ultra-low temperature freezing equipment is also required during the packaging, transportation and storage process, and the product has a short shelf life. In comparison, the preparation method of the inactivated cell film of the present invention is simple and easy to operate, and can realize the "standardized" production of cell film products. The transportation and storage conditions of the prepared inactivated cell film are low, and its repair effect can be guaranteed when stored at room temperature.

[0039] The second aspect of the present invention provides an inactivated cell membrane prepared by the preparation method of the first aspect of the present invention.

[0040] Compared with traditional cell suspension injection, preparing cells into cell films increases the cell loading capacity, is more conducive to cell colonization, and helps promote wound repair.

[0041] The third aspect of the present invention provides an extract obtained by extracting the inactivated cell film of the second aspect of the present invention in a liquid; the liquid includes a culture medium.

[0042] In some embodiments of the present invention, the culture medium includes at least one of DMEM high glucose medium, DMEM-F12 medium, RPMI1640 medium, and Alpha MEM medium; preferably Alpha MEM medium.

[0043] In some embodiments of the present invention, the extraction conditions are leaching at 3-6° C. for 30-40 hours.

[0044] In some embodiments of the present invention, the extraction conditions are leaching at 3-5° C. for 35-40 hours.

[0045] The fourth aspect of the present invention provides the use of the preparation method of the first aspect of the present invention, the inactivated cell film of the second aspect of the present invention, or the leaching solution of the third aspect of the present invention in any one of (1) to (3):

[0046] (1) Preparation of products for repairing damaged tissues and / or organs;

[0047] (2) Promote cell proliferation and / or migration;

[0048] (3) Prepare products that promote cell proliferation and / or migration.

[0049] In some embodiments of the present invention, the cells include at least one of human fibroblasts, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, periodontal ligament mesenchymal stem cells, embryonic gingival-derived mesenchymal stem cells, and umbilical cord mesenchymal stem cells; preferably, embryonic gingival-derived mesenchymal stem cells.

[0050] The beneficial effects of the present invention are:

[0051] The present invention is committed to achieving "standardized" production of cell membrane products and provides a method for preparing an inactivated cell membrane. The prepared inactivated cell membrane has the same wound repair effect as a fresh cell membrane, and the inactivated cell membrane can be stored at room temperature to ensure the repair effect, which is convenient for product transportation and storage.

[0052] The present invention provides a method for preparing an inactivated cell film. The preparation method is simple and convenient to store. Compared with commonly used inactivated cell films, the film has a better effect of promoting tissue repair and can be used immediately. In addition, the packaging, transportation and storage processes do not require the use of ultra-low temperature freezing equipment, which greatly reduces storage and transportation costs and has potential commercial value.

[0053] The inactivated cell film (extract) provided by the present invention can significantly promote cell proliferation and cell migration, and can be used as a potential wound repair material; the inactivated cell film can support storage at room temperature, and can better maintain its effectiveness when stored at -20°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 It is a schematic diagram of the technical solution flow of the present invention.

[0055] Figure 2 These are examples of membrane formation of NHDF and GMSC cells cultured in different culture medium formulas.

[0056] Figure 3 Macroscopic images of cell membranes prepared using VID culture medium recipes in Examples 1 to 8

[0057] Figure 4 The laser confocal images of Calcein / PI staining of the inactivated cell membranes prepared in Example 3 and Example 4 are used to detect the inactivation of cells in the cell membranes.

[0058] Figure 5 The inactivated cell film extracts prepared in Examples 3 to 8 were subjected to CCK-8 to detect the cell proliferation ability of the inactivated cell films prepared at different inactivation temperatures; wherein (a) is the comparison result of Examples 5 and 7, (b) is the comparison result of Examples 6 and 8, (c) is the comparison result of Examples 3 and 4, and (d) is the comparison result of Examples 3, 5 and 7.

[0059] Figure 6 The inactivated cell film extracts prepared in Examples 3 to 8 were used for a scratch test to detect cell migration.

[0060] Figure 7 The migration area of ​​the inactivated cell films prepared in Examples 3 to 8 was quantified in the scratch test of the extract.

[0061] Figure 8 The inactivated cell films prepared in Examples 3 to 8 were stored for 4 weeks and then the extracts were subjected to CCK-8 assay to detect the cell proliferation ability of the inactivated cell films prepared at different inactivation temperatures; wherein (a) is the comparison result of Examples 5 and 7, (b) is the comparison result of Examples 6 and 8, (c) is the comparison result of Examples 3 and 4, and (d) is the comparison result of Examples 3, 5 and 7.

[0062] Fig. 9 The following is a comparison of the in vivo repair effects of the fresh cell membranes of Examples 1 and 2 and the inactivated cell membranes of Examples 3, 4, 5, and 6 on the SD rat scald model. DETAILED DESCRIPTION

[0063] The present invention is further described in detail below through specific examples.

[0064] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0065] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0066] MEM Alpha medium was purchased from Gbico with the catalog number C12571500BT; DMEM low glucose medium was purchased from Gbico with the catalog number C11885500BT; FBS was purchased from Gbico with the catalog number 10099-141; bispecific antibody was purchased from Gbico with the catalog number 15140-122; vitamin C was purchased from Solarbio with the catalog number 1105G033, dexamethasone was purchased from McLean with the catalog number C12937381; ITS was purchased from Cyagen with the catalog number ITSS-10201; Calcein-AM / PIDouble Staining Kit was purchased from Meilunbio with the catalog number MAO361-Oct-31H; Cell Counting Kit-8 was purchased from New Saimei Biotechnology Co., Ltd. with the catalog number C6005.

[0067] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0068] Example 1

[0069] This embodiment provides a method for preparing a HF-GMSC cell film.

[0070] A method for preparing a cell membrane comprises the following steps:

[0071] (1) Human fetal gingival mesenchymal stem cells (HF-GMSCs) were subcultured (within 7 generations) and seeded on a 35 mm diameter ordinary cell culture dish in Alpha MEM medium with a seeding cell number of 1 million to 1.5 million. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0072] (2) using the membrane-forming medium formula, preparing vitamin C / ITS / dexamethasone medium (VID medium), wherein the vitamin C / ITS / dexamethasone medium contains 150 μg / mL vitamin C, 1% ITS (100×), 0.04 μg / mL dexamethasone, 10% fetal bovine serum, 1% penicillin-streptomycin, and Alpha MEM; filtering the vitamin C / ITS / dexamethasone medium using a 0.22 μm sterile filter head and setting aside;

[0073] (3) After 24 h of cell culture, the medium was changed and vitamin C / ITS / dexamethasone medium was added to the culture dish. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium changed every 2 days.

[0074] (4) After 7 days, the HF-GMSCs were removed from the incubator, and an appropriate amount of culture medium was taken out using a pipette and gently blown along the edge of the dish to peel off the HF-GMSC cell film.

[0075] Example 2

[0076] This embodiment provides a method for preparing NHDF cell film.

[0077] A method for preparing a cell membrane comprises the following steps:

[0078] (1) NHDF cells (normal human dermal fibroblasts) were subcultured (within 7 generations) and seeded on a 35 mm diameter ordinary cell culture dish. The culture medium was low-glucose MEM. The number of cells seeded was 1 million to 1.5 million. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0079] (2) using the membrane-forming medium formula, preparing a vitamin C / ITS / dexamethasone medium, wherein the vitamin C / ITS / dexamethasone medium contains 150 μg / mL vitamin C, 1% ITS (100×), 0.04 μg / mL dexamethasone, 10% fetal bovine serum, 1% penicillin-streptomycin, and low-glucose DMEM; filtering the vitamin C / ITS / dexamethasone medium using a 0.22 μm sterile filter head and setting aside;

[0080] (3) After 24 h of cell culture, the medium was changed and vitamin C / ITS / dexamethasone medium was added to the culture dish. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium changed every 2 days.

[0081] (4) After 7 days, remove the NHDF from the incubator, use a pipette to take an appropriate amount of culture medium, and gently blow along the edge of the dish to peel off the NHDF cell film.

[0082] Example 3

[0083] This example provides an inactivated cell membrane obtained by inactivating HF-GMSC cell membrane at -20°C. Figure 1 ).

[0084] A method for preparing an inactivated cell film comprises the following steps:

[0085] (1) Human fetal gingival mesenchymal stem cells (HF-GMSCs) were subcultured (within 7 generations) and seeded on a 35 mm diameter ordinary cell culture dish in Alpha MEM medium with a seeding cell number of 1 million to 1.5 million. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0086] (2) using the membrane-forming medium formula, preparing a vitamin C / ITS / dexamethasone medium, wherein the vitamin C / ITS / dexamethasone medium contains 150 μg / mL vitamin C, 1% ITS (100×), 0.04 μg / mL dexamethasone, 10% fetal bovine serum, 1% penicillin-streptomycin, and Alpha MEM; filtering the vitamin C / ITS / dexamethasone medium using a 0.22 μm sterile filter head and setting aside;

[0087] (3) After 24 h of cell culture, the medium was changed and vitamin C / ITS / dexamethasone medium was added to the culture dish. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium changed every 2 days.

[0088] (4) After 7 days, the HF-GMSCs were removed from the incubator, and an appropriate amount of culture medium was taken out using a pipette and gently pipetted along the edge of the dish to peel off the HF-GMSC cell membrane;

[0089] (5) Spread the cell film evenly, dry the culture medium in the culture dish, and place the peeled HF-GMSC cell film in a clean bench (temperature 30.7°C, humidity 39.6%, wind speed 0.1-0.4 m / s) to air dry (20 min);

[0090] (6) Seal the culture dish containing the HF-GMSC cell film with a sealing film and place it in a -20°C refrigerator. The cells will be completely inactivated the next day, and the HF-GMSC cell film inactivated at -20°C is obtained, which is continuously stored in a -20°C refrigerator.

[0091] Example 4

[0092] This example provides an inactivated cell membrane obtained by inactivating NHDF cell membrane at -20°C.

[0093] A method for preparing an inactivated cell film comprises the following steps:

[0094] (1) NHDF cells (normal human dermal fibroblasts) were subcultured (within 7 generations) and seeded on a 35 mm diameter ordinary cell culture dish. The culture medium was low-glucose MEM. The number of cells seeded was 1 million to 1.5 million. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0095] (2) using the membrane-forming medium formula, preparing a vitamin C / ITS / dexamethasone medium, wherein the vitamin C / ITS / dexamethasone medium contains 150 μg / mL vitamin C, 1% ITS (100×), 0.04 μg / mL dexamethasone, 10% fetal bovine serum, 1% penicillin-streptomycin, and low-glucose DMEM; filtering the vitamin C / ITS / dexamethasone medium using a 0.22 μm sterile filter head and setting aside;

[0096] (3) After 24 h of cell culture, the medium was changed and vitamin C / ITS / dexamethasone medium was added to the culture dish. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium changed every 2 days.

[0097] (4) After 7 days, remove the NHDF from the incubator, use a pipette to take an appropriate amount of culture medium, and gently blow along the edge of the dish to peel off the NHDF cell membrane;

[0098] (5) Spread the cell film evenly, dry the culture medium in the culture dish, and place the peeled NHDF cell film in a clean bench (temperature 30.7°C, humidity 39.6%, wind speed 0.1-0.4 m / s) to air dry (20 min);

[0099] (6) Seal the culture dish containing the NHDF cell film with a sealing film and place it in a -20°C refrigerator. The cells will be completely inactivated the next day, and the NHDF cell film inactivated at -20°C will be obtained, which should be kept in a -20°C refrigerator.

[0100] Example 5

[0101] This embodiment provides an inactivated cell membrane obtained by inactivating the HF-GMSC cell membrane at room temperature (27.6-31.4° C.).

[0102] A method for preparing an inactivated cell film comprises the following steps:

[0103] (1) HF-GMSC cells were subcultured (within 7 generations) and seeded on a 35 mm diameter ordinary cell culture dish. The culture medium was Alpha MEM. The number of cells seeded was 1 million to 1.5 million. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0104] (2) using the membrane-forming medium formula, preparing a vitamin C / ITS / dexamethasone medium, wherein the vitamin C / ITS / dexamethasone medium contains 150 μg / mL vitamin C, 1% ITS (100×), 0.04 μg / mL dexamethasone, 10% fetal bovine serum, 1% penicillin-streptomycin, and low-glucose MEM; filtering the vitamin C / ITS / dexamethasone medium using a 0.22 μm sterile filter head and setting aside;

[0105] (3) After 24 h of cell culture, the medium was changed and vitamin C / ITS / dexamethasone medium was added to the culture dish. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium changed every 2 days.

[0106] (4) After 7 days, the HF-GMSCs were removed from the incubator, and an appropriate amount of culture medium was taken out using a pipette and gently pipetted along the edge of the dish to peel off the HF-GMSC cell membrane;

[0107] (5) Spread the cell membrane evenly, dry the culture medium in the culture dish, and place the peeled HF-GMSC cell membrane in a clean bench (temperature 30.7°C, humidity 39.6%, wind speed 0.1-0.4 m / s) to air dry (20 min), then place it at room temperature to inactivate it, and then store it at room temperature.

[0108] Example 6

[0109] This embodiment provides an inactivated cell membrane obtained by inactivating the NHDF cell membrane at room temperature (27.6-31.4°C).

[0110] A method for preparing an inactivated cell film comprises the following steps:

[0111] (1) NHDF cells were subcultured (within 7 generations) and inoculated on a 35 mm diameter ordinary cell culture dish in low-glucose DMEM medium with a cell number of 1 million to 1.5 million. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0112] (2) using the membrane-forming medium formula, preparing a vitamin C / ITS / dexamethasone medium, wherein the vitamin C / ITS / dexamethasone medium contains 150 μg / mL vitamin C, 1% ITS (100×), 0.04 μg / mL dexamethasone, 10% fetal bovine serum, 1% penicillin-streptomycin, and low-glucose DMEM; filtering the vitamin C / ITS / dexamethasone medium using a 0.22 μm sterile filter head and setting aside;

[0113] (3) After 24 h of cell culture, the medium was changed and vitamin C / ITS / dexamethasone medium was added to the culture dish. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium changed every 2 days.

[0114] (3) After 24 h of cell culture, the medium was changed and vitamin C / ITS / dexamethasone medium was added to the culture dish. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium changed every 2 days.

[0115] (4) After 7 days, remove the NHDF from the incubator, use a pipette to take an appropriate amount of culture medium, and gently blow along the edge of the dish to peel off the NHDF cell membrane;

[0116] (5) Spread the cell film evenly, dry the culture medium in the culture dish, and place the peeled NHDF cell film in a clean bench (temperature 30.7°C, humidity 39.6%, wind speed 0.1-0.4 m / s) to air dry (20 min), then place it at room temperature to inactivate it, and then store it at room temperature.

[0117] Example 7

[0118] This example provides a method of obtaining an inactivated cell membrane by inactivating a HF-GMSC cell membrane at 4°C.

[0119] A method for preparing an inactivated cell film comprises the following steps:

[0120] (1) HF-GMSC cells were subcultured (within 7 generations) and seeded on a 35 mm diameter ordinary cell culture dish. The culture medium was Alpha MEM. The number of cells seeded was 1 million to 1.5 million. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0121] (2) using the membrane-forming medium formula, preparing a vitamin C / ITS / dexamethasone medium, wherein the vitamin C / ITS / dexamethasone medium contains 150 μg / mL vitamin C, 1% ITS (100×), 0.04 μg / mL dexamethasone, 10% fetal bovine serum, 1% penicillin-streptomycin, and low-glucose DMEM; filtering the vitamin C / ITS / dexamethasone medium using a 0.22 μm sterile filter head and setting aside;

[0122] (3) After 24 h of cell culture, the medium was changed and vitamin C / ITS / dexamethasone medium was added to the culture dish. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium changed every 2 days.

[0123] (4) After 7 days, the HF-GMSCs were removed from the incubator, and an appropriate amount of culture medium was taken out using a pipette and gently pipetted along the edge of the dish to peel off the HF-GMSC cell membrane;

[0124] (5) Spread the cell film evenly, dry the culture medium in the culture dish, and place the peeled HF-GMSC cell film in a clean bench (temperature 30.7°C, humidity 39.6%, wind speed 0.1-0.4 m / s) to air dry (20 min);

[0125] (6) Seal the culture dish containing the HF-GMSC cell film with a sealing film and place it in a 4°C refrigerator. The cells will be completely inactivated the next day, and the HF-GMSC cell film inactivated at 4°C is obtained and stored in a 4°C refrigerator.

[0126] Example 8

[0127] This example provides an inactivated cell membrane obtained by inactivating NHDF cell membrane at 4°C.

[0128] A method for preparing an inactivated cell film comprises the following steps:

[0129] (1) NHDF cells were subcultured (within 7 generations) and inoculated on a 35 mm diameter ordinary cell culture dish. The culture medium was low-glucose MEM. The number of cells inoculated was 1 million to 1.5 million. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0130] (2) using the membrane-forming medium formula, preparing a vitamin C / ITS / dexamethasone medium, wherein the vitamin C / ITS / dexamethasone medium contains 150 μg / mL vitamin C, 1% ITS (100×), 0.04 μg / mL dexamethasone, 10% fetal bovine serum, 1% penicillin-streptomycin, and low-glucose DMEM; filtering the vitamin C / ITS / dexamethasone medium using a 0.22 μm sterile filter head and setting aside;

[0131] (3) After 24 h of cell culture, the medium was changed and vitamin C / ITS / dexamethasone medium was added to the culture dish. The cells were cultured in a 37°C, 5% CO2 incubator for 7 days, with the medium changed every 2 days.

[0132] (4) After 7 days, remove the NHDF from the incubator, use a pipette to take an appropriate amount of culture medium, and gently blow along the edge of the dish to peel off the NHDF cell membrane;

[0133] (5) Spread the cell film evenly, dry the culture medium in the culture dish, and place the peeled HF-GMSC cell film in a clean bench (temperature 30.7°C, humidity 39.6%, wind speed 0.1-0.4 m / s) to air dry (20 min);

[0134] (6) Seal the culture dish containing the NHDF cell film with a sealing film and place it in a 4°C refrigerator. The cells will be completely inactivated the next day, and the NHDF cell film inactivated at 4°C will be obtained and stored in a 4°C refrigerator.

[0135] Effect Example

[0136] 1. Appearance test

[0137] The appearance of the fresh cell membranes of Examples 1 and 2 was compared with the appearance of the inactivated cell membranes of Examples 3 to 8. Figure 3 As shown. Figure 3 It can be seen that all cell membranes are gelatinous, have good tissue compliance and adhesion properties, and do not require suturing in practical applications.

[0138] 2. Cell Viability Test

[0139] The cell activities of the HF-GMSC inactivated film in Example 3 and the NHDF inactivated cell film in Example 4 were tested respectively. The specific testing method was as follows: the inactivated cell films prepared in Example 3 and Example 4 were placed in 35 mm culture dishes respectively and fully spread, the working solution was prepared according to the proportion in the instruction manual of the Calcein-AM / PIDouble Staining Kit, 1 mL of the working solution was added to the inactivated cell film and wrapped with tin foil, and incubated in the dark at 37° C., 5% CO2 environment for 20 min. After the incubation, the working solution was discarded, and the cells were washed 3 times with PBS, and then a three-dimensional image of the cell film was taken using an ultra-high resolution biological confocal microscope (Zeiss; LSM980+Airyscan2, GRE).

[0140] The test results are as follows Figure 4 As shown, the staining indicated that the cells in the HF-GMSC cell film prepared in Example 3 and the NHDF cell film prepared in Example 4 were completely inactivated, which means that the inactivated cell film was successfully prepared.

[0141] 3. Inactivation temperature exploration

[0142] CCK-8 test (Cell Counting Kit-8) is a commonly used experimental method for measuring cell proliferation. In this experiment, CCK-8 reagent was used to evaluate the proliferation of NHDF cells treated with the conditioned medium of the inactivated cell film prepared in Examples 3 to 8, and all groups were repeated three times.

[0143] Preparation method of conditioned medium: The specification of the inactivated cell membranes is R = 1.5 ± 0.2 mm. The inactivated cell membranes of Examples 3 to 8 are placed in centrifuge tubes respectively, and 2 mL of low-glucose DMEM medium is added to each centrifuge tube. The tubes are soaked at 4 ° C for 36 hours, and the supernatant is taken. The supernatant is then diluted 10 times with low-glucose DMEM medium as conditioned medium for standby use.

[0144] The specific experimental method is as follows: first, NHDF cells were revived, passaged and cultured to a stable state, NHDF cells were resuspended in low-glucose DMEM and plated, NHDF cells were seeded in a 96-well plate at a density of 3000 cells / well, and the 96-well plate was placed at 37°C and cultured in a 5% CO2 environment for 24 hours to allow the cells to fully adhere to the wall. According to the instructions of the CCK-8 kit, CCK-8 reagent was added to each well, incubated in a dark incubator for 1 hour, and the OD was measured using an enzyme reader. 450 , the OD value measured at this time is recorded as the OD value of Day 0. Then the culture medium in the 96-well plate was replaced, wherein the blank group continued to be cultured with 100 μL / well low-glucose DMEM, and the groups of Examples 3 to 8 were added with 100 μL / well of the corresponding conditioned medium. After administration, the 96-well plate was placed at 37°C and 5% CO2 environment for 24, 48, and 72 hours, and CCK-8 reagent was added to each well, incubated in the incubator in the dark for 1 hour, and the OD was measured using an enzyme reader. 450 The OD values ​​measured at this time are recorded as the OD values ​​of Day 1, Day 2, and Day 3.

[0145] Specific test results are as follows Figure 5 As shown, the conditioned medium of the inactivated cell films of Examples 3 to 8 all showed significant cell proliferation promoting effects, and the cell proliferation promoting effects of the HF-GMSC inactivated cell film and the NHDF inactivated cell film were not significantly different. Therefore, the inactivated cell film prepared by the present invention can be stored at room temperature during practical applications.

[0146] 4. Cell Migration Test

[0147] The scratch test is a commonly used experimental method for measuring cell migration. In this experiment, the scratch test was used to evaluate the cell migration of HUVEC cells treated with the conditioned medium of the inactivated films of Examples 3 to 8, which were immersed for 36 hours.

[0148] The method for extracting the conditioned medium is as follows: the inactivated cell films of Examples 3 to 8 are placed in centrifuge tubes respectively, 2 mL of ECM culture medium is added to each centrifuge tube containing the inactivated cell films, the membranes are soaked for 36 hours at 4°C, the supernatant is taken as the 36-hour leaching solution of the inactivated films of Examples 3 and 4, and then the leaching solution is diluted 10 times with ECM culture medium as the conditioned medium for standby use.

[0149] The specific experimental method is as follows: HUVEC cells were plated at 100,000 / well in a 12-well plate. When the HUVEC cell density reached more than 80%, a 200μL pipette tip was used to scratch the area. After positioning, the scratch area was photographed using an optical microscope and recorded as the 0h image. The culture medium in the well was then replaced. Specifically, 1mL of ECM culture medium was added to each well of the blank group, and 1mL of conditioned culture medium was added to each well of the experimental group. The 12-well plate was then placed at 37°C and 5% CO2 for further culture. The scratch area at the location was photographed at 6h, 12h, and 24h. All groups were repeated three times.

[0150] Specific test results are as follows Figure 6 As shown, compared with the blank group, the conditioned medium prepared from the inactivated cell films of Examples 3 to 8 all have the effect of promoting cell migration, among which the inactivated films of Examples 3, 5, and 7 have a slightly better effect of promoting cell migration than those of Examples 4, 6, and 8. This indicates that the original growth factors or beneficial proteins in the inactivated films of Examples 3, 5, and 7 are more than those in the inactivated films of Examples 4, 6, and 8, and the cell migration promoting effect is better.

[0151] 5. Cell Migration Test Quantification

[0152] Image J software was used to count the cell migration area and calculate the cell migration rate. The specific experimental results are as follows: Figure 7 As shown, compared with the blank group, the conditioned medium prepared from the inactivated cell films of Examples 3 to 8 all had a significant effect of promoting cell migration, among which the inactivated films of Examples 3, 5, and 7 had a slightly better effect of promoting cell migration.

[0153] 6. Shelf life exploration

[0154] The inactivated cell films of Examples 3 to 7 were stored at their specific temperature for 4 weeks, and then the proliferation of NHDF cells treated with conditioned medium in each group was measured. All groups were repeated three times.

[0155] The method for extracting the conditioned medium is as follows: the inactivated cell films of Examples 3 to 7 are placed in centrifuge tubes respectively, 2 mL of low-glucose DMEM medium is added to each centrifuge tube of the inactivated cell film, and the supernatant is extracted by soaking at 4°C for 36 hours, and then the supernatant is diluted 10 times with low-glucose DMEM medium as the conditioned medium for standby use.

[0156] The specific experimental method is as follows: first, NHDF cells were revived, passaged and cultured to a stable state, NHDF cells were resuspended in low-glucose DMEM and plated, NHDF cells were seeded in a 96-well plate at a density of 3000 cells / well, and the 96-well plate was placed at 37°C and cultured in a 5% CO2 environment for 24 hours to allow the cells to fully adhere to the wall. According to the instructions of the CCK-8 kit, CCK-8 reagent was added to each well, incubated in a dark incubator for 1 hour, and the OD was measured using an enzyme reader. 450 , the OD value measured at this time is recorded as the OD value of Day 0. Then the culture medium in the 96-well plate was replaced, wherein the blank group continued to be cultured with 100 μL / well low-glucose DMEM, and the groups of Examples 3 to 8 were added with 100 μL / well of the corresponding conditioned medium. After administration, the 96-well plate was placed at 37°C and 5% CO2 environment for 24, 48, and 72 hours, and CCK-8 reagent was added to each well, incubated in the incubator in the dark for 1 hour, and the OD was measured using an enzyme reader. 450 The OD values ​​measured at this time are recorded as the OD values ​​of Day 1, Day 2, and Day 3.

[0157] Specific test results are as follows Figure 8 As shown, the conditioned medium of the inactivated cell films of Examples 3 to 7 all showed significant cell proliferation promoting effects, and the cell proliferation promoting effect of the HF-GMSC inactivated cell film was significantly higher than that of the NHDF inactivated cell film. In the exploration of inactivation temperature, there was no significant difference in the cell proliferation promoting effect of the inactivated films prepared at 25°C, 4°C, and -20°C. Therefore, the inactivated cell film prepared by the present invention can support room temperature storage during practical application.

[0158] 7. Evaluation of the repair effect of cell membrane in vivo using SD rat scald model

[0159] The effects of the fresh cell films of Examples 1 to 2 and the inactivated films of Examples 3 to 6 on wound healing were evaluated by using full-thickness burn wounds.

[0160] Eighteen male Wistar rats were selected for this part of the experiment and randomly divided into six groups (fresh cell membranes of Examples 1 to 2, inactivated cell membranes of Examples 3 to 6, and a control group). First, the rats were anesthetized by inhalation of isoflurane. After the rats fainted, the hair on the back was removed and the skin surface was wiped with 75% alcohol. Then, four wounds were inflicted on each mouse. Four circles of wounds on the back were cauterized at 100°C with a 10mm copper block. After 4 hours, a circular wound with a length of 12 mm was cut along the burn site. The wounds of the control group were not treated after surgery, and the wounds of the experimental group were attached with cell membranes of Examples 1 to 6. Each mouse was fed individually to prevent them from contacting each other's wounds. The wounds were monitored on days 0, 3, 7, and 14 after surgery. The rats were highly anesthetized on days 3, 7, and 14 after surgery, and the wounds were taken. Each photo was normalized using a 10mm diameter measurement.

[0161] In vivo experimental results Fig. 9 As shown, on the 3rd and 7th days after surgery, the wound healing rates of the cell film groups of Examples 1 to 6 were significantly higher than those of the control group. In addition, the wound healing promoting effect of Examples 1, 3, and 5 was better than that of Examples 2, 4, and 6. The wound size of each group decreased with time. According to the results on the 14th day, compared with the control group, the cell sheet accelerated the wound healing process after treatment. In vivo experiments confirmed that the cell sheet has good wound healing ability, and there is no significant difference between the therapeutic effect of the inactivated cell sheet and the therapeutic effect of the fresh cell sheet.

[0162] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for preparing an inactivated cell film, comprising the following steps: Prepare cell membranes and dry them; The cell membrane is placed at -30 to 35°C and inactivated for 15 to 30 hours to obtain an inactivated cell membrane.

2. The preparation method according to claim 1, characterized in that: The cells include at least one of human fibroblasts, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, gingival mesenchymal stem cells, periodontal ligament mesenchymal stem cells, and umbilical cord mesenchymal stem cells.

3. The preparation method according to claim 1, characterized in that: The method for preparing the cell membrane comprises the following steps: inoculating cells into a basic culture medium for a first culture, and then replacing the culture medium containing vitamin C, ITS and dexamethasone for a second culture to obtain the cell membrane.

4. The preparation method according to claim 3, characterized in that: The first culture condition is 33-39° C., 3%-7% CO2 for 18-30 hours; and / or, the second culture condition is 33-39° C., 3%-7% CO2 for 6-10 days; Preferably, during the second culture, the medium is replaced every 1 to 3 days.

5. The preparation method according to claim 3, characterized in that: 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 membrane is: without discarding the original culture medium, the original culture medium is sucked up with a pipette and gently blows the bottom edge of the culture dish.

6. The preparation method according to any one of claims 3 to 5, characterized in that: The culture medium containing vitamin C, ITS and dexamethasone contains 1-500 μg / mL vitamin C, 0.001-0.05 μg / mL dexamethasone and 0.1-2.0 v / v% ITS.

7. The preparation method according to any one of claims 1 to 5, characterized in that The inactivation condition is -20 to 4°C for 10 to 24 hours.

8. An inactivated cell film prepared by the preparation method according to any one of claims 1 to 7.

9. An extract obtained by extracting the inactivated cell film according to claim 8 in a liquid; the liquid comprises a culture medium.

10. Use of the preparation method according to any one of claims 1 to 7, the inactivated cell film according to claim 8, or the leaching solution according to claim 9 in any one of (1) to (3): (1) Preparation of products for repairing damaged human tissues and / or organs; (2) Promote cell proliferation and / or migration; (3) Prepare products that promote cell proliferation and / or migration.

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