A method for culturing human decidual mesenchymal stem cells and its application

Abstract

The present invention relates to a method for culturing human decidual mesenchymal stem cells and its application, and belongs to the field of stem cell technology. The culturing method comprises washing and separation, tissue processing, separation and processing of decidual layers, performing two digestions, inoculation and culture, changing the medium for passage, terminating digestion, and freezing and storing. The present invention provides a suitable enzyme concentration and enzyme combination for overnight digestion of human decidual tissue, treats human decidual tissue by a secondary digestion method, and then uses a special culture medium for culture, eliminating the use of an oscillating shaker, which can not only effectively decompose tissue to promote stem cell release, shorten the culture cycle, reduce cell damage, and improve survival rate and activity, but also the culture medium has no xenogeneic components, is easy to operate, and has a low cost, which can meet the application requirements of cell therapy and clinical related applications.

Description

Technical Field

[0001] The invention belongs to the technical field of stem cells and relates to a method for culturing human decidua mesenchymal stem cells and an application thereof. Background Art

[0002] MSCs were first derived from bone marrow, but their various potentials are greatly affected by age, and their isolation process is invasive, which can easily lead to infection and bleeding. The placenta is an abundant source, does not require invasive operations to obtain, and does not need to worry about ethical issues when using it. It has now become an important source of mesenchymal stem cells (MSCs), among which decidua mesenchymal stem cells are derived from the decidua layer in the placental tissue. Human decidua mesenchymal stem cells (decidua basalis-derived mesenchymal stem cells, DB-MSCs) from the decidua basalis of the placenta belong to one of the categories of mesenchymal stem cells (MSCs). Like mesenchymal stem cells (MSCs), they have the characteristics of undifferentiated cells, can self-renew efficiently, and have the advantages of multidirectional differentiation potential. They have significant effects in beauty, anti-aging, postpartum recovery, women's diseases (such as premature ovarian failure, ovarian cancer), tissue regeneration, immune system diseases, and transplantation to repair damaged organs.

[0003] However, the current methods for isolating and amplifying human decidual mesenchymal stem cells are relatively complex and there is no unified and effective standard, making it difficult to meet many needs of practical applications. For example, human decidual mesenchymal stem cells obtained by existing separation and culture methods have various cell components mixed together during the separation process, and their purity is often difficult to reach an ideal level; in addition, various enzymes directly digest tissues in a short period of time to obtain human decidual mesenchymal stem cells, which will inevitably cause a certain degree of damage to the cells; furthermore, the current culture method makes the cell growth and proliferation multiples low and the culture cycle long, which increases the time cost on the one hand and limits the possibility of large-scale application on the other hand. Although there are patents that use fetal bovine serum in the separation and culture process at this stage, fetal bovine serum has potential pathogen risks and there are still obstacles in clinical application. Summary of the invention

[0004] The purpose of the present invention is to provide a method for culturing human decidual mesenchymal stem cells and its application. The present invention provides a suitable enzyme concentration and enzyme combination for overnight digestion of human decidual tissue, treats the human decidual tissue by secondary digestion, and then uses a special culture medium for culturing, eliminating the use of a shaking table. It can not only effectively decompose tissue to promote stem cell release, shorten the culture cycle, reduce cell damage, and improve survival rate and activity, but also the culture medium has no xenogeneic components, is easy to operate, and has low cost, which can meet the application requirements of cell therapy and clinical related applications.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] A method for culturing human decidual mesenchymal stem cells, the culturing method comprising washing and separation, tissue processing, separation and processing of decidual layers, two digestions, inoculation and culture, fluid replacement and passage, digestion termination, and cryopreservation;

[0007] The two digestions are performed, i.e., after centrifugation, the tissue precipitate is added to a special culture medium, and then type II collagenase with a final concentration of 0.8-1.2 g / L and neutral protease with a final concentration of 0.1-0.3 g / L are added to obtain a tissue suspension, the tissue suspension is added to a culture bottle and placed in an incubator for overnight digestion for 12-28 hours, and then a recombinant enzyme with a final concentration of 0.5-1.0 g / L is added and digestion is carried out for 5-15 minutes to obtain a tissue digestion suspension.

[0008] Furthermore, the washing and separation is to wash the placenta with 0.9% saline and triple antibody for three times and then separate it, put it in a culture dish filled with saline after separation, and use sterile tweezers to remove blood stains adhering to the tissue.

[0009] Furthermore, the tissue processing is to separate the tissue into 2-5 cm 2 Tissue slices of different sizes were spread flat in a culture dish containing 1% tertiary antibody and allowed to stand for sterilization for 5 minutes.

[0010] Furthermore, the separation and treatment of the decidual layer is to wash the tissue slices with physiological saline for 3 times and then separate the decidual layer, put the separated decidual layer into a culture dish containing 1% triple antibody, stand and sterilize for 5 minutes, then wash the decidual layer tissue with physiological saline for 3 times, and cut the tissue into 1-3 mm with new sterile scissors. 3 Mince the tissue at different sizes and centrifuge at 400-600g for 5-15 minutes.

[0011] Furthermore, the volume ratio of tissue precipitation to special culture medium in the two digestion cultures is 2:15-21.

[0012] Furthermore, the inoculation culture is to centrifuge the tissue digestion suspension at 400-600g for 5-15 minutes, remove the supernatant, add the cell tissue suspension, special culture medium, penicillin with a final concentration of 100U / mL, streptomycin with a final concentration of 100U / mL and amphotericin B with a final concentration of 100U / mL into the culture bottle, place it in a 37°C, 5% CO2 incubator for culture, and obtain the P0 generation.

[0013] Furthermore, the medium replacement and subculturing is to replace the medium in full on the 2nd and 5th day of culture, culture until the cell confluence reaches 75-85%, remove the supernatant, wash once with 0.9% saline, digest with 0.125% trypsin for 2-5 minutes, terminate the digestion with a special culture medium, and then filter with a 100μm filter to obtain a cell digestion suspension, count and centrifuge at 200-400g for 5-15 minutes, resuspend the cell suspension with a special culture medium and culture in a T175 culture flask to obtain the P1 generation.

[0014] Furthermore, the digestion is terminated when the cells are cultured until the cell confluence reaches 75-85%, the supernatant is removed, the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 2-5 minutes, and digested with a special culture medium to obtain a cell digestion suspension, which is then counted and centrifuged at 200-400g for 5-15 minutes.

[0015] Furthermore, the freezing solution in the cryopreservation is composed of DMSO, human serum albumin, and special culture medium in a mass ratio of 1:1:8, and the freezing density is 5-8×10 6 cells / ml.

[0016] Furthermore, the special culture medium is based on DMEM high-glucose culture medium, supplemented with 5-15% platelet lysate, 300-360 ng / ml fibronectin, 5-25 g / L serum albumin, 15-25 ng / mL human basic fibroblast growth factor, 4 mmol L-glutamine, 0.5-1.2×10 7 cells / mL human umbilical cord mesenchymal stem cell lysate;

[0017] The preparation method of the human umbilical cord mesenchymal stem cell lysate is as follows: prepare a water bath and set the temperature to 37° C.; prepare liquid nitrogen, take human umbilical cord mesenchymal stem cells, freeze and thaw repeatedly for 3-5 times, centrifuge at 1200-1800 g for 5-15 min, take the supernatant, and obtain the human umbilical cord mesenchymal stem cell lysate.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention provides a suitable enzyme concentration and enzyme combination for overnight digestion of human decidual tissue, eliminating the use of a shaking table. It can not only effectively decompose tissue to promote stem cell release, reduce cell damage and increase survival rate and activity, but also is easy to operate and has low cost.

[0020] (2) The present invention provides a secondary digestion method for treating human decidual tissue, which can deeply decompose residual tissue to improve cell harvest rate, remove impurities and cell fragments, and optimize cell quality.

[0021] (3) The present invention provides a special culture medium, which is prepared by adding platelet lysate, fibronectin, serum albumin, human basic fibroblast growth factor, L-glutamine and human umbilical cord mesenchymal stem cell lysate to the DMEM high-glucose culture medium as the base, wherein the human umbilical cord mesenchymal stem cell lysate contains a variety of active substances, which can interact with decidual mesenchymal stem cells to promote cell proliferation, differentiation and self-renewal, and have a certain positive effect on enhancing the biological function and growth and proliferation of cells; the human platelet lysate is purified and extracted from platelet-rich plasma, and the platelet-derived growth factor promotes cell differentiation and proliferation, and does not contain xenobiotic additives; the fibronectin can bind to the specific membrane on the surface of the cell membrane The combination of the two culture mediums promotes cell adhesion, growth, and cell proliferation by combining with the receptors; serum albumin can maintain the normal physiological state of cells, enhance the cell's antioxidant capacity, and protect against cell damage; human basic fibroblast growth factor, as a multifunctional protein, has an important regulatory effect on cell proliferation, differentiation, and fibroblastic function; L-glutamine provides carbon and nitrogen sources for the synthesis of proteins, nucleic acids, etc., which can promote the growth of stem cells and maintain the life activities of cells; this special culture medium enables cells to grow rapidly by attaching to the wall after crawling out, effectively shortening the culture cycle, and the harvested cells have good activity, strong proliferation ability, and great proliferation potential. In addition, this culture medium does not contain xenobiotic components and can meet the application requirements of cell therapy and clinical-related applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to facilitate understanding by those skilled in the art, the present invention is further described below with reference to the accompanying drawings.

[0023] Figure 1 This is the digestion diagram after the second day of direct centrifugation culture in Experiment 1;

[0024] Figure 2 This is the digestion profile after the second day of secondary digestion with the recombinant enzyme in Experiment 1;

[0025] Figure 3 This is the digestion diagram after 4 days of direct centrifugation culture in Experiment 1;

[0026] Figure 4 This is the digestion profile after 4 days of secondary digestion with the recombinant enzyme in Experiment 1;

[0027] Figure 5 This is the digestion diagram after 7 days of direct centrifugation culture in Experiment 1;

[0028] Figure 6 This is the digestion profile after 7 days of secondary digestion with the recombinant enzyme in Experiment 1;

[0029] Figure 7 This is the digestion diagram after 9 days of direct centrifugation culture in Experiment 1;

[0030] Figure 8 This is the digestion profile after 9 days of secondary digestion with the recombinant enzyme in Experiment 1;

[0031] Fig. 9 This is the digestion diagram after 12 days of direct centrifugation culture in Experiment 1;

[0032] Fig.10 This is the digestion diagram after 12 days of secondary digestion with the recombinant enzyme in Experiment 1. DETAILED DESCRIPTION

[0033] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0034] The three antibodies in all the embodiments and comparative examples of the present invention were purchased directly from the market, all purchased from Feijing Bio PHYGENE, and the item number was PH1757; type II collagenase was purchased directly from the market, all purchased from sigma, and the item number was C0130; neutral proteases were purchased directly from the market, all purchased from sigma, and the item number was D4693; recombinant enzymes were purchased directly from the market, all purchased from Shanghai Yaxin Biotechnology Co., Ltd., and the item number was RPT0204; human albumin was purchased directly from the market, all purchased from Shenzhen Weiguang Biological Products Co., Ltd., and the item number was All of them have the national medicine standard number S10960053; all of the platelet lysates were purchased directly from the market, all of them were purchased from Shenzhen Dakoway Biotechnology Co., Ltd., and the item number was EPA-050; all of the fibronectins were purchased directly from the market, all of them were purchased from Baoriyi Biotechnology (Beijing) Co., Ltd., and the item number was T100B; all of the human basic fibroblast growth factor were purchased directly from the market, all of them were purchased from PEPROTECH, and the item number was 100-18B-100; all of the high-glucose DMEM were purchased directly from the market, all of them were purchased from Gibco, and the item number was 11965092.

[0035] Example 1

[0036] A method for culturing human decidual mesenchymal stem cells, the culturing method of this embodiment comprises washing and separation, tissue processing, separation and processing of the decidual layer, two digestions, inoculation and culture, fluid replacement and passage, terminating digestion, and cryopreservation;

[0037] In this example, digestion was performed twice, i.e., after centrifugation, 2 mL of tissue precipitate was added to 18 mL of special culture medium, and then type II collagenase with a final concentration of 0.8 g / L and neutral protease with a final concentration of 0.1 g / L were added to obtain a tissue suspension. The tissue suspension was added to a T175 culture flask and placed in an incubator for overnight digestion for 20 hours, and then the recombinant enzyme with a final concentration of 0.5 g / L was added and digested for 10 minutes to obtain a tissue digestion suspension.

[0038] In this embodiment, the placenta is washed and separated by washing with 0.9% saline and three antibodies for three times, and then placed in a 90 mm culture dish filled with 35 ml of saline, and blood stains adhering to the tissue are removed with sterile tweezers.

[0039] The tissue processing in this example is to separate the tissue into 3.5 cm 2 Tissue slices of different sizes were spread flat in a 90 mm culture dish containing 1% tertiary antibody and allowed to stand for sterilization for 5 minutes.

[0040] The decidual layer separation and treatment of this embodiment is to wash the tissue slices with physiological saline for 3 times and then separate the decidual layer. The separated decidual layer is placed in a 90 mm culture dish containing 1% triple antibody and sterilized for 5 minutes. The decidual layer tissue is then washed with physiological saline for 3 times and the tissue is cut into 2 mm pieces with new sterile scissors. 3 Mince the tissue into small pieces and centrifuge at 500 g for 10 min.

[0041] In the inoculation culture of this embodiment, the tissue digestion suspension is centrifuged at 500g for 10 minutes, the supernatant is removed, 2 mL of cell tissue suspension, 18 mL of special culture medium, penicillin with a final concentration of 100 U / mL, streptomycin with a final concentration of 100 U / mL, and amphotericin B with a final concentration of 100 U / mL are added to a T175 culture flask, and the flask is placed in a 37°C, 5% CO2 incubator for culture to obtain the P0 generation.

[0042] In the present embodiment, the medium replacement and subculture is to replace the whole medium on the 2nd and 5th day of culture, and when the cell confluence reaches 80% on the 9th day of culture, the supernatant is removed, and the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 3.5 minutes, and digested with a special culture medium, and then filtered with a 100 μm filter to obtain a cell digestion suspension. After counting, the cells are centrifuged at 300 g for 10 minutes, and the cell suspension is resuspended with a special culture medium and cultured in a T175 culture flask to obtain the P1 generation.

[0043] The digestion in this embodiment is terminated when the cell fusion degree reaches 80% on the twelfth day of culture, the supernatant is removed, the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 3.5 minutes, and digested with a special culture medium to obtain a cell digestion suspension, which is counted and centrifuged at 300g for 10 minutes.

[0044] The cryopreservation solution in the cryopreservation of this embodiment is composed of DMSO, human serum albumin, and special culture medium in a mass ratio of 1:1:8. The cryopreservation density of this embodiment is 6.5×10 6 cells / ml.

[0045] The special culture medium of this embodiment is based on DMEM high-glucose medium, supplemented with 5% platelet lysate, 300 ng / ml fibronectin, 10 g / L serum albumin, 15 ng / mL human basic fibroblast growth factor, 4 mmol L-glutamine, 0.6×10 7 cells / mL human umbilical cord mesenchymal stem cell lysate;

[0046] The preparation method of the human umbilical cord mesenchymal stem cell lysate of the present embodiment comprises preparing a water bath and setting the temperature to 37° C.; preparing liquid nitrogen, repeatedly freezing and thawing for 4 times, centrifuging at 1500 g for 10 min, and taking the supernatant to obtain the human umbilical cord mesenchymal stem cell lysate.

[0047] Example 2

[0048] A method for culturing human decidual mesenchymal stem cells, the culturing method of this embodiment comprises washing and separation, tissue processing, separation and processing of the decidual layer, two digestions, inoculation and culture, fluid replacement and passage, terminating digestion, and cryopreservation;

[0049] In this example, digestion was performed twice, that is, after centrifugation, 2 mL of tissue precipitate was added to 18 mL of special culture medium, and then type II collagenase with a final concentration of 0.9 g / L and neutral protease with a final concentration of 0.15 g / L were added to obtain a tissue suspension. The tissue suspension was added to a T175 culture flask and placed in an incubator for overnight digestion for 20 hours, and then the recombinant enzyme with a final concentration of 0.5 g / L was added and digested for 10 minutes to obtain a tissue digestion suspension.

[0050] In this embodiment, the placenta is washed and separated by washing with 0.9% saline and three antibodies for three times, and then placed in a 90 mm culture dish filled with 35 ml of saline, and blood stains adhering to the tissue are removed with sterile tweezers.

[0051] The tissue processing in this example is to separate the tissue into 3.5 cm 2 Tissue slices of different sizes were spread flat in a 90 mm culture dish containing 1% tertiary antibody and allowed to stand for sterilization for 5 minutes.

[0052] The decidual layer separation and treatment of this embodiment is to wash the tissue slices with physiological saline for 3 times and then separate the decidual layer. The separated decidual layer is placed in a 90 mm culture dish containing 1% triple antibody and sterilized for 5 minutes. The decidual layer tissue is then washed with physiological saline for 3 times and the tissue is cut into 2 mm pieces with new sterile scissors. 3 Mince the tissue into small pieces and centrifuge at 500 g for 10 min.

[0053] In the inoculation culture of this embodiment, the tissue digestion suspension is centrifuged at 500g for 10 minutes, the supernatant is removed, 2 mL of cell tissue suspension, 18 mL of special culture medium, penicillin with a final concentration of 100 U / mL, streptomycin with a final concentration of 100 U / mL, and amphotericin B with a final concentration of 100 U / mL are added to a T175 culture flask, and the flask is placed in a 37°C, 5% CO2 incubator for culture to obtain the P0 generation.

[0054] In the present embodiment, the medium replacement and subculture is to replace the whole medium on the 2nd and 5th day of culture, and when the cell confluence reaches 80% on the 9th day of culture, the supernatant is removed, and the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 3.5 minutes, and digested with a special culture medium, and then filtered with a 100 μm filter to obtain a cell digestion suspension. After counting, the cells are centrifuged at 300 g for 10 minutes, and the cell suspension is resuspended with a special culture medium and cultured in a T175 culture flask to obtain the P1 generation.

[0055] The digestion in this embodiment is terminated when the cell fusion degree reaches 80% on the twelfth day of culture, the supernatant is removed, the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 3.5 minutes, and digested with a special culture medium to obtain a cell digestion suspension, which is counted and centrifuged at 300g for 10 minutes.

[0056] The cryopreservation solution in the cryopreservation of this embodiment is composed of DMSO, human serum albumin, and special culture medium in a mass ratio of 1:1:8. The cryopreservation density of this embodiment is 6.5×10 6 cells / ml.

[0057] The special culture medium of this embodiment is based on DMEM high-glucose medium, supplemented with 8% platelet lysate, 320ng / ml fibronectin, 15g / L serum albumin, 18ng / mL human basic fibroblast growth factor, 4mmol L-glutamine, 0.8×10 7 cells / mL human umbilical cord mesenchymal stem cell lysate;

[0058] The preparation method of the human umbilical cord mesenchymal stem cell lysate of the present embodiment comprises preparing a water bath and setting the temperature to 37° C.; preparing liquid nitrogen, repeatedly freezing and thawing for 4 times, centrifuging at 1500 g for 10 min, and taking the supernatant to obtain the human umbilical cord mesenchymal stem cell lysate.

[0059] Example 3

[0060] A method for culturing human decidual mesenchymal stem cells, the culturing method of this embodiment comprises washing and separation, tissue processing, separation and processing of the decidual layer, two digestions, inoculation and culture, fluid replacement and passage, terminating digestion, and cryopreservation;

[0061] In this example, digestion was performed twice, that is, after centrifugation, 2 mL of tissue precipitate was added to 18 mL of special culture medium, and then type II collagenase with a final concentration of 1.0 g / L and neutral protease with a final concentration of 0.2 g / L were added to obtain a tissue suspension. The tissue suspension was added to a T175 culture flask and placed in an incubator for overnight digestion for 20 hours, and then the recombinant enzyme with a final concentration of 0.5 g / L was added and digested for 10 minutes to obtain a tissue digestion suspension.

[0062] In this embodiment, the placenta is washed and separated by washing with 0.9% saline and three antibodies for three times, and then placed in a 90 mm culture dish filled with 35 ml of saline, and blood stains adhering to the tissue are removed with sterile tweezers.

[0063] The tissue processing in this example is to separate the tissue into 3.5 cm 2 Tissue slices of different sizes were spread flat in a 90 mm culture dish containing 1% tertiary antibody and allowed to stand for sterilization for 5 minutes.

[0064] The decidual layer separation and treatment of this embodiment is to wash the tissue slices with physiological saline for 3 times and then separate the decidual layer. The separated decidual layer is placed in a 90 mm culture dish containing 1% triple antibody and sterilized for 5 minutes. The decidual layer tissue is then washed with physiological saline for 3 times and the tissue is cut into 2 mm pieces with new sterile scissors. 3 Mince the tissue into small pieces and centrifuge at 500 g for 10 min.

[0065] In the inoculation culture of this embodiment, the tissue digestion suspension is centrifuged at 500g for 10 minutes, the supernatant is removed, 2 mL of cell tissue suspension, 18 mL of special culture medium, penicillin with a final concentration of 100 U / mL, streptomycin with a final concentration of 100 U / mL, and amphotericin B with a final concentration of 100 U / mL are added to a T175 culture flask, and the flask is placed in a 37°C, 5% CO2 incubator for culture to obtain the P0 generation.

[0066] In the present embodiment, the medium replacement and subculture is to replace the whole medium on the 2nd and 5th day of culture, and when the cell confluence reaches 80% on the 9th day of culture, the supernatant is removed, and the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 3.5 minutes, and digested with a special culture medium, and then filtered with a 100 μm filter to obtain a cell digestion suspension. After counting, the cells are centrifuged at 300 g for 10 minutes, and the cell suspension is resuspended with a special culture medium and cultured in a T175 culture flask to obtain the P1 generation.

[0067] The digestion in this embodiment is terminated when the cell fusion degree reaches 80% on the twelfth day of culture, the supernatant is removed, the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 3.5 minutes, and digested with a special culture medium to obtain a cell digestion suspension, which is counted and centrifuged at 300g for 10 minutes.

[0068] The cryopreservation solution in the present embodiment is composed of DMSO, human serum albumin, and special culture medium in a mass ratio of 1:1:8. The cryopreservation density in the present embodiment is 6.5×10 6 cells / ml.

[0069] The special culture medium of this embodiment is based on DMEM high-glucose medium, supplemented with 10% platelet lysate, 340ng / ml fibronectin, 20g / L serum albumin, 20ng / mL human basic fibroblast growth factor, 4mmol L-glutamine, 1.0×10 7 cells / mL human umbilical cord mesenchymal stem cell lysate;

[0070] The preparation method of the human umbilical cord mesenchymal stem cell lysate of the present embodiment comprises preparing a water bath and setting the temperature to 37° C.; preparing liquid nitrogen, repeatedly freezing and thawing for 4 times, centrifuging at 1500 g for 10 min, and taking the supernatant to obtain the human umbilical cord mesenchymal stem cell lysate.

[0071] Example 4

[0072] A method for culturing human decidual mesenchymal stem cells, the culturing method of this embodiment comprises washing and separation, tissue processing, separation and processing of the decidual layer, two digestions, inoculation and culture, fluid replacement and passage, terminating digestion, and cryopreservation;

[0073] In this example, digestion was performed twice, i.e., after centrifugation, 2 mL of tissue precipitate was added to 18 mL of special culture medium, and then type II collagenase with a final concentration of 1.1 g / L and neutral protease with a final concentration of 0.25 g / L were added to obtain a tissue suspension. The tissue suspension was added to a T175 culture flask and placed in an incubator for overnight digestion for 20 hours, and then the recombinant enzyme with a final concentration of 1.0 g / L was added and digested for 10 minutes to obtain a tissue digestion suspension.

[0074] In this embodiment, the placenta is washed and separated by washing with 0.9% saline and three antibodies for three times, and then placed in a 90 mm culture dish filled with 35 ml of saline, and blood stains adhering to the tissue are removed with sterile tweezers.

[0075] The tissue processing in this example is to separate the tissue into 3.5 cm 2 Tissue slices of different sizes were spread flat in a 90 mm culture dish containing 1% tertiary antibody and allowed to stand for sterilization for 5 minutes.

[0076] The decidual layer separation and treatment of this embodiment is to wash the tissue slices with physiological saline for 3 times and then separate the decidual layer. The separated decidual layer is placed in a 90 mm culture dish containing 1% triple antibody and sterilized for 5 minutes. The decidual layer tissue is then washed with physiological saline for 3 times and the tissue is cut into 2 mm pieces with new sterile scissors. 3 Mince the tissue into small pieces and centrifuge at 500 g for 10 min.

[0077] In the inoculation culture of this embodiment, the tissue digestion suspension is centrifuged at 500g for 10 minutes, the supernatant is removed, 2 mL of cell tissue suspension, 18 mL of special culture medium, penicillin with a final concentration of 100 U / mL, streptomycin with a final concentration of 100 U / mL, and amphotericin B with a final concentration of 100 U / mL are added to a T175 culture flask, and the flask is placed in a 37°C, 5% CO2 incubator for culture to obtain the P0 generation.

[0078] In the present embodiment, the medium replacement and subculture is performed on the second and fifth days of culture. When the cell confluence reaches 80% on the ninth day of culture, the supernatant is removed, the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 3.5 min, digested with a special culture medium, and filtered with a 100 μm filter to obtain a cell digestion suspension. After counting, the cell suspension is centrifuged at 300 g for 10 min, resuspended with a special culture medium, and inoculated into a seed bottle at a ratio of 1:3. A T175 culture flask is used for culture to obtain the P1 generation.

[0079] The digestion in this embodiment is terminated when the cell fusion degree reaches 80% on the twelfth day of culture, the supernatant is removed, the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 3.5 minutes, and digested with a special culture medium to obtain a cell digestion suspension, which is counted and centrifuged at 300g for 10 minutes.

[0080] The cryopreservation solution in the cryopreservation of this embodiment is composed of DMSO, human serum albumin, and special culture medium in a mass ratio of 1:1:8. The cryopreservation density of this embodiment is 6.5×10 6 cells / ml.

[0081] The special culture medium of this embodiment is based on DMEM high-glucose culture medium, supplemented with 12% platelet lysate, 360ng / ml fibronectin, 25g / L serum albumin, 25ng / mL human basic fibroblast growth factor, 4mmol L-glutamine, 1.2×10 7 cells / mL human umbilical cord mesenchymal stem cell lysate;

[0082] The preparation method of the human umbilical cord mesenchymal stem cell lysate of the present embodiment comprises preparing a water bath and setting the temperature to 37° C.; preparing liquid nitrogen, repeatedly freezing and thawing for 4 times, centrifuging at 1500 g for 10 min, and taking the supernatant to obtain the human umbilical cord mesenchymal stem cell lysate.

[0083] Comparative Example 1

[0084] On the basis of Example 3, the step of recombinase digestion was removed, and other conditions were consistent with Example 3.

[0085] Comparative Example 2

[0086] On the basis of Example 3, the human umbilical cord mesenchymal stem cell lysate in the special culture medium was removed, and other conditions were the same as those in Example 3.

[0087] Comparative Example 3

[0088] On the basis of Example 3, the step of recombinase digestion and the human umbilical cord mesenchymal stem cell lysate in the special culture medium were removed, and other conditions were consistent with those in Example 3.

[0089] A routine culture group was set up, in which 1.0 g / L type II collagenase and 0.2 g / L neutral protease were used for enzyme treatment during overnight digestion, and 10% platelet lysate was used for DMEM high-glucose medium treatment.

[0090] 1. Primary culture test

[0091] Example 3 was compared with the conventional culture group in the primary culture test. The test method was to use type II collagenase and neutral protease for overnight digestion, and the digestion was completed within 12-28 hours. After the digestion was completed, direct centrifugation culture and recombinant enzyme secondary digestion were performed for 10 minutes respectively, and then the growth status was continuously photographed on the 2nd, 4th, 7th, 9th and 12th days.

[0092] The results showed that sporadic adherent cells began to appear on the second day of primary culture. Figure 1 It can be seen that the tissues in the overnight digestion of the conventional culture group were not completely digested, and there were obvious tissue clumps. Figure 2 The recombinant enzyme secondary digestion diagram of Example 3 has tiny tissue blocks, but they are much smaller than the tissue blocks of the overnight single digestion of the conventional culture group; small clusters of cells appeared on the 4th day of primary culture, which Figure 3It can be seen that the cells in the overnight digestion group of the conventional culture group have less adherence to the wall and grow in small areas in the form of aggregate growth, while Figure 4 In the second digestion diagram of the recombinant enzyme in Example 3, the adherent cells have a wide range and show diffuse growth; on the 7th day of primary culture, the adherent cells enter a rapid growth and proliferation phase. Figure 5 It can be seen that the cells in the conventional culture group grew obviously in clusters in the overnight digestion diagram, with obvious large and small cell clusters. Figure 6 The recombinant enzyme secondary digestion diagram of Example 3 shows that the cells are evenly distributed and the cell morphology is good; on the 9th day of culture (primary cell passage P1), Figure 7 It can be seen that the cells in the overnight digestion of the conventional culture group grew densely. Due to the agglomerative growth, the cells were densely packed in the center of the growth area, and there were obvious space gaps around the growth area. Figure 8 In the recombinant enzyme secondary digestion diagram of Example 3, the cells grow evenly, the growth space is fully utilized, the cell gap is small, and the cell fusion degree is high compared with the same culture cycle; on the 12th day of culture (harvesting P1 generation cells), the same culture cycle, by Fig. 9 It can be seen that the overnight digestion diagram of the conventional culture group showed obvious intercellular gaps, while Fig.10 The recombinant enzyme secondary digestion diagram of Example 3 shows that the growth gap between cells is small and the cell fusion degree is high.

[0093] According to the above growth process diagram, the cells after overnight one-step digestion showed obvious aggregation during the culture process, which was not conducive to the subsequent cell growth and proliferation. However, the cells after secondary digestion with the recombinant enzyme had a good digestion effect, a low aggregation rate, and good cell morphology during the subsequent growth and proliferation process. The cells could grow evenly distributed until the degree of fusion reached 80-85%.

[0094] 2. Detection of the number of P0 cells on the 8th day of culture and P1 cells on the 11th day of culture: Taking the culture conditions of the conventional group, Examples 1-4 and Comparative Examples 1-3 as samples, on the 8th day of culture, when the cell fusion degree reached about 80%, 0.125% trypsin was used for digestion for about 2-3 minutes to obtain P0 cells. All the obtained P0 cells were inoculated and cultured. After three days, they were digested under the same conditions to obtain P1 cells. The data obtained are shown in Table 1 below.

[0095] Table 1

[0096]

[0097] The P0 generation cells are derived from cells that crawl out of the tissue and adhere to the wall and proliferate. As shown in Table 1, the cell fusion degree under the microscope was the highest on the 8th day of culture in Example 3, and the number of P0 generation cells obtained after digestion was the largest, that is, the cells cultured in Example 3 had the strongest adhesion ability and growth ability. At the same time, all the P0 generation cells obtained from each group of culture were inoculated into culture bottles, and P1 generation cells were obtained by digestion three days later. Similarly, the number of P1 generation cells obtained after digestion on the 11th day of culture in Example 3 was the largest, that is, the cells cultured in Example 3 still had the best growth ability and proliferation ability.

[0098] 3. Comparison of P0-P1 proliferation multiples: Taking the culture conditions of the conventional group, Examples 1-4 and Comparative Examples 1-3 as samples, the number of cells harvested in the P1 generation was divided by the number of cells harvested in the P0 generation to obtain the P0-P1 proliferation multiples, and recorded as shown in Table 2.

[0099] Table 2

[0100]

[0101] As can be seen from Table 2, the optimal ratio of type II collagenase, neutral protease and recombinant enzyme in Example 3 is obtained through Examples 1-4. At the same time, based on Example 3, comparative examples 1-4 are set to change the components and steps. Among the results obtained, the experimental results of Example 3 are the most ideal, among which Example 3 has the highest proliferation multiple of 4.85. Example 3 has the best activity in the cell adhesion stage and the highest proliferation multiple in the cell proliferation stage. The higher the proliferation multiple, the greater the proliferation potential, which means that under the same culture conditions, there will be better amplification performance in subsequent subculture.

[0102] 4. Comparison of P0 and P1 viability and comparison of mean viability: Taking the culture conditions of the conventional group, Examples 1-4 and Comparative Examples 1-3 as samples, the cell viabilities of P0 and P1 generation cells were counted, and the mean viability was calculated and recorded as shown in Table 3.

[0103] Table 3

[0104]

[0105] As can be seen from Table 3, the viability of the cells in Example 3 at the P0 generation was 96.45%, which was the highest value among all groups of cell viabilities; the viability data of all groups at the P1 generation were statistically analyzed, and the viability of each group was good, with the lowest being 96.00%, so the mean viability and the overall data performance were calculated to evaluate the activity of the cells. The mean viability of the cells in Example 3 was 96.94%, which was the highest value among the mean viabilities of all groups. Combined with the overall data, the cells in Example 3 were the only group whose P0 generation viability, P1 generation viability, and mean viability were all greater than 96%. The high viability and high stability of viability indicate that the cells in Example 3 have the best activity.

[0106] 5. Take the human decidual stem cells of the P1 generation cultured in Examples 1-4 and Comparative Examples 1-3 for cell purity detection, measure the positive indicators of decidual stem cell surface markers CD73, CD90, CD105 and negative indicators (CD11b, CD19, CD34, CD45 and HLA-DR), and record the results as shown in Table 4.

[0107] Table 4

[0108]

[0109] As shown in Table 4, each group took P1 generation human decidual stem cells for cell purity detection. The data showed that the cell purity was high, the positive expression rates of human decidual stem cell surface markers CD73, CD90, and CD105 were all above 95%, and the negative index expression rates were all below 2%, among which Example 3 was the best example.

[0110] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for culturing human decidual mesenchymal stem cells, characterized in that: The culture method comprises washing and separation, tissue processing, separation and processing of the decidual layer, two digestions, inoculation and culture, fluid replacement and passage, digestion termination, and cryopreservation; The two digestions are performed, i.e., after centrifugation, 2 mL of tissue precipitate is added to 18 mL of special culture medium, and then type ǁ collagenase with a final concentration of 1.0 g / L and neutral protease with a final concentration of 0.2 g / L are added to obtain a tissue suspension, the tissue suspension is added to a T175 culture flask and placed in an incubator for overnight digestion for 20 hours, and then the recombinant enzyme with a final concentration of 0.5 g / L is added for digestion for 10 minutes to obtain a tissue digestion suspension; The washing and separation is to wash the placenta with 0.9% saline and three antibodies for three times, and then separate it. After separation, put it into a 90mm culture dish filled with 35ml saline, and use sterile tweezers to remove blood stains adhering to the tissue; The tissue was processed by separating the tissue into 3.5 cm 2 Tissue slices of different sizes were spread flat in a 90 mm culture dish containing 1% tertiary antibody and allowed to stand for sterilization for 5 min; The separation and treatment of the decidual layer is to wash the tissue slices with physiological saline for 3 times and then separate the decidual layer, put the separated decidual layer into a 90 mm culture dish containing 1% triple antibody, stand and sterilize for 5 minutes, wash the decidual layer tissue with physiological saline for 3 times, and cut the tissue into 2 mm with new sterile scissors. 3 The tissues were minced and centrifuged at 500 g for 10 min; The inoculation culture is to centrifuge the tissue digestion suspension at 500g for 10 minutes, remove the supernatant, add 2mL of cell tissue suspension, 18mL of special culture medium, penicillin with a final concentration of 100U / mL, streptomycin with a final concentration of 100U / mL and amphotericin B with a final concentration of 100U / mL in a T175 culture bottle, and culture in a 37°C, 5% CO2 incubator to obtain the P0 generation; The medium replacement passaging is to replace the medium in full on the second and fifth days of culture, and observe that the cell confluence reaches 80% on the ninth day of culture, remove the supernatant, wash once with 0.9% physiological saline, digest with 0.125% trypsin for 3.5 minutes, terminate the digestion with a special culture medium, and then filter with a 100 μm filter to obtain a cell digestion suspension, count and centrifuge at 300g for 10 minutes, resuspend the cell suspension with a special culture medium, and culture in a T175 culture flask to obtain the P1 generation; The digestion termination means that on the twelfth day of culture, the cell fusion degree is observed to reach 80%, the supernatant is removed, the cells are washed once with 0.9% saline, digested with 0.125% trypsin for 3.5 minutes, and digested with a special culture medium to obtain a cell digestion suspension, which is counted and centrifuged at 300g for 10 minutes; The cryopreservation solution in the cryopreservation storage is composed of DMSO, human serum albumin, and special culture medium in a mass ratio of 1:1:8, and the cryopreservation density is 6.5×10 6 cells / ml; The special culture medium is based on DMEM high-glucose culture medium, supplemented with 10% platelet lysate, 340 ng / ml fibronectin, 20 g / L serum albumin, 20 ng / mL human basic fibroblast growth factor, 4 mmol L-glutamine, 1.0×10 7 cells / mL human umbilical cord mesenchymal stem cell lysate; The preparation method of the human umbilical cord mesenchymal stem cell lysate is as follows: prepare a water bath, set the temperature to 37° C.; prepare liquid nitrogen, freeze and thaw repeatedly for 4 times, centrifuge at 1500 g for 10 minutes, take the supernatant, and obtain the human umbilical cord mesenchymal stem cell lysate.

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