Method for culturing mesenchymal stem cells based on in-vitro simulated microenvironment

Abstract

The invention discloses a culture method for culturing mesenchymal stem cells based on an in-vitro simulated microenvironment, which comprises the following steps: treating an umbilical cord to prepare umbilical cord mesenchymal stem cells, and performing morphological and sterile detection and flow cytometry quality inspection; performing in-vitro culture on the stem cells by adopting a traditional 2D culture mode and a bone marrow-derived extracellular matrix culture medium of an in-vitro simulated microenvironment respectively, and comparing cell amplification and cell surface marker conditions under a light microscope after the stem cells and the bone marrow-derived extracellular matrix culture medium are cultured; performing quality control on the stem cells cultured by the bone marrow-derived extracellular matrix culture medium of the in-vitro simulated microenvironment through biological characteristics and three-line differentiation detection; the characteristics of the stem cells can be better maintained and the amplification amount can be improved by simulating in-vivo microenvironment culture conditions of the stem cells, a large number of researches at present show that a culture system method based on an in-vitro simulated microenvironment can promote proliferation, adhesion and differentiation of the cells, and compared with a traditional 2D culture mode, the efficiency is higher, and the cost is lower.

Description

Technical Field

[0001] The present invention relates to the technical field of mesenchymal stem cell culture, and in particular to a culture method for mesenchymal stem cells based on an in vitro simulated microenvironment. Background Art

[0002] Stem cells are a type of multipotent cell with the ability to self-replicate. Under specific induction conditions in vivo or in vitro, they can differentiate into a variety of tissue cells, including fat, bone, cartilage, muscle, myocardium, ligaments, nerves, liver, myocardium, and endothelium. They retain this multidirectional differentiation potential even after continuous subculture and cryopreservation, earning them the medical nickname "universal cells." They possess a robust proliferation capacity and multidirectional differentiation potential. Under appropriate in vivo or in vitro conditions, they can differentiate not only into hematopoietic cells, but also into myocytes, hepatocytes, osteoblasts, chondrocytes, stromal cells, and other cell types.

[0003] Commonly used stem cells in clinical practice include mesenchymal stem cells, embryonic stem cells, and hematopoietic stem cells. Embryonic stem cells are limited by immune rejection, while hematopoietic stem cells have limited in vitro proliferation capacity, so the application of mesenchymal stem cells to treat diseases is the most widely studied. Mesenchymal stem cells are mainly concentrated in human bone marrow and umbilical cord blood. Based on their source, mesenchymal stem cells can be divided into bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, and adipose mesenchymal stem cells. Because they do not express MHC class II antigens and rarely express MHC class I antigens, their immunogenicity is very low. Therefore, mesenchymal stem cell transplantation has a high safety profile. UC-mesenchymal stem cells have a high success rate in obtaining, rich tissue sources, low treatment risks, multiple differentiation potentials, no risk of teratogenicity or fetal tumors, and low immunogenicity, making them an ideal source for cell therapy.

[0004] The technical problems existing in the current culture methods of mesenchymal stem cells are:

[0005] 1. The traditional 2D in vitro culture model has a low efficiency in stem cell expansion (multiplication factor <10^5), which is difficult to meet the demand for clinical-grade cell dosage;

[0006] 2. The difference between the in vitro culture environment and the in vivo microenvironment leads to genomic instability of mesenchymal stem cells (the incidence of abnormal karyotype is >15%) and functional decline (the loss rate of differentiation potential is >40%).

[0007] Therefore, it is necessary to provide a culture method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment. Summary of the Invention

[0008] In response to the problems existing in the above-mentioned prior art, the present invention provides a culture method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment. Through this patented technology, the number of stem cells cultured by the bone marrow-derived extracellular matrix culture medium method can be significantly better than that of the traditional 2D culture model, and the characteristics of stem cells can be maintained; and the bone marrow-derived extracellular matrix culture medium method using an in vitro simulated microenvironment can significantly improve the efficiency of stem cell expansion and maintain its characteristics.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solutions: a method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment, wherein the steps of the mesenchymal stem cell culture system are as follows:

[0010] Step 1: Human umbilical cord-derived mesenchymal stem cells are used to resuscitate and prepare for use;

[0011] Step 2: Inoculation using traditional 2D plastic culture model:

[0012] Preparation of 2D culture medium: Add laminin, EGF, bFGF, soybean trypsin inhibitor, insulin, bovine serum albumin, and non-essential amino acids to the basal medium DMEM-F12.

[0013] Stem cell inoculation: The stem cells recovered in step 1 were plated at a rate of 1x10 4 cells / mL were inoculated into a T75 culture flask containing 10 mL of standard culture medium and cultured in a cell culture incubator at 5% CO2 concentration and 37°C.

[0014] Step 3: Inoculation using the extracellular matrix culture system:

[0015] Preparation of bone marrow-derived culture medium: Use freshly isolated bone marrow-derived cells at a concentration of 2 × 10 5 / cm 2 The cells were seeded into 2D culture medium at a density of 100 μg / mL and non-adherent cells were removed after one week of culture.

[0016] Stem cell inoculation: The stem cells recovered in step 1 were plated at a rate of 1x10 4 cells / mL were inoculated into a T75 culture flask containing 10 mL of bone marrow-derived culture medium and cultured in a cell culture incubator at 5% CO2 concentration and 37°C.

[0017] Step 4: Samples were taken from the stem cells seeded in the traditional 2D plastic culture model in Step 2 and the stem cells seeded in the extracellular matrix culture system in Step 3 after 24, 48, and 72 hours of culture, and the growth of the stem cells in the traditional 2D plastic culture model and the extracellular matrix culture system were observed under an inverted microscope.

[0018] Step 5: Quality control and differentiation capacity testing of stem cells inoculated into the extracellular matrix culture system

[0019] 1. Quality control of mesenchymal stem cells inoculated into the extracellular matrix culture system:

[0020] Cell morphology detection: The cell morphology of human umbilical cord mesenchymal stem cells in the extracellular matrix culture system was observed under an inverted microscope.

[0021] Chromosome karyotype detection: G-banding analysis was used to detect chromosome karyotypes. P2 cells were passaged into culture flasks. 0.05 μg / mL colchicine was added as appropriate depending on the cell adherence and mitosis. Culture was continued for 4 h. The cells were scraped off the bottom of the flask and centrifuged at 2000 rpm for 15 min. The supernatant was removed, and the slides were treated with hypotonic solution at 37°C for 5 min. The slides were fixed twice with fixative and baked at 50°C overnight. The slides were digested with 0.025% trypsin for 30 s and stained with Giemsa. Fifty metaphases were counted per slide, and 10 karyotypes were analyzed. Chromosome karyotype analysis was then performed using VideoTest-Karyo software from the United States.

[0022] 2. Detect the differentiation ability of mesenchymal stem cells inoculated in the extracellular matrix culture system:

[0023] (1) Osteogenesis induction of mesenchymal stem cells:

[0024] Osteogenesis induction step: prepare a six-well plate, add 2 mL of complete culture medium to each well, and culture P3 cells at a rate of 3×10 4 Cells were seeded at 1 mL / well and the medium was completely replaced every 72 hours. When the cells reached 80% confluency, 2 mL of complete osteogenic differentiation medium was replaced and culture continued. This step was repeated five times. After 7 days of osteogenic induction, cells were harvested and centrifuged at 1000 rpm for 8 minutes. The supernatant was discarded and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde and stained with 0.1% Alizarin Red S for 20 minutes. The formation of intracellular calcium nodules was observed under a microscope.

[0025] (2) Adipogenesis induction of mesenchymal stem cells:

[0026] Adipogenic induction step: prepare a six-well plate, add 2 mL of complete culture medium to each well, and culture P3 cells at a rate of 2 × 10 4 Cells were seeded at 1 mL / well and the medium was completely replaced every 72 hours. When the cells reached 80% confluency, 2 mL of complete adipogenic differentiation medium was replaced and culture continued. This step was repeated five times. After 7 days of adipogenic induction, cells were harvested and centrifuged at 1000 rpm for 8 minutes. The supernatant was discarded and the cells were washed three times with PBS. The cells were fixed with 4% paraformic acid for 20 minutes, stained with 2% Oil Red O for 30 minutes, and rinsed with 70% ethanol. Lipid droplet formation was observed under a microscope.

[0027] (3) Mesenchymal stem cells chondrogenic induction:

[0028] Chondrogenic induction step: prepare a six-well plate, add 2 mL of complete culture medium to each well, and culture P3 cells at a rate of 2 × 10 4 Cells were seeded at 1 mL / well and the medium was completely replaced every 72 hours. When the cells reached 80% confluency, 2 mL of new complete adipogenic differentiation medium was replaced and culture continued. This step was repeated five times. After 7 days of chondrogenic induction, the cells were centrifuged at 1000 rpm for 8 minutes, the supernatant was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% polyformic acid for 20 minutes, washed three times with pure water, and stained with Alcian blue for 30 minutes. The cells were washed twice with pure water and the staining was observed under an inverted microscope.

[0029] Step 6: Compare the stem cell expansion efficiency of traditional 2D plastic culture model seeding and extracellular matrix culture system seeding;

[0030] Step 7: It is concluded that the extracellular matrix culture system has a significant efficiency in the expansion of stem cell culture.

[0031] Preferably, the steps of preparing mesenchymal stem cells for recovery include: isolation and culture of mesenchymal stem cells, subculture, identification, cryopreservation and recovery;

[0032] Isolation and culture: Take an umbilical cord of 20 cm or longer and place it in 30 mL of 0.9% sodium chloride injection. After removing the ligatures at both ends, cut the umbilical cord into 2-3 cm segments. Wash the surface of the umbilical cord repeatedly until no blood or other impurities are visible to the naked eye. Transfer the cord to a new culture dish to separate Wharton's jelly for later use. Cut the Wharton's jelly into 1-4 mm pieces. 2 , inoculate 3 bottles of T75 culture bottles, place them in a carbon dioxide incubator, and culture them at 5% CO2 concentration and 37℃;

[0033] Subculture: When the cell fusion rate is greater than 50%, collect the cells for subculture. After adding an appropriate amount of physiological saline for rinsing, add 3mL of 0.25% trypsin to digest for 3min until most of the cells fall off, immediately add 5mL of serum-free culture medium to stop digestion, and collect the cell suspension into a 50mL centrifuge tube; centrifuge the cell suspension at 1300r / min for 6min to remove the supernatant, take the precipitate and dissolve it in 40mL of 0.9% sodium chloride injection, filter it with a 70μm filter, repeat the above centrifugation step to remove the supernatant, add serum-free culture medium to the collected cells and place them in a cell culture incubator, and culture them at 5% CO2 concentration and 37°C;

[0034] Identification: P1 human umbilical cord mesenchymal stem cells were obtained, digested with 0.25% trypsin, and washed twice with 0.9% sodium chloride injection to prepare a cell suspension. Flow cytometry was used to sort and identify the mesenchymal stem cells to improve their purity.

[0035] Cryopreservation: Use a pipette to take serum-free freezing solution and add it to the cell suspension prepared in the previous step. Resuspend the cells, pipette evenly, cool the solution to a low temperature, and then transfer to a liquid nitrogen tank for storage.

[0036] Thawing: Remove the cryovial from the liquid nitrogen tank and immediately place it in a 37°C water bath. Gently shake to thaw within 1-2 minutes. If the sealing film falls off, do not allow the water level to approach or exceed the bottom edge of the cryovial cap during thawing to avoid contamination. Once completely thawed, use a 10mL pipette to mix the cell suspension in the cryovial and store in a cool, dry place.

[0037] Preferably, the culture system method of mesenchymal stem cells further comprises the identification of cell surface markers of mesenchymal stem cells: the P3 cells in step 4 are made into 2×10 5 L -1 Concentrated cell suspension, take 40 μL of anti-human CD73, CD90, CD105, CD45, CD34, CD11b, CD19, CD79a, HLA-DR and mix with the cells, incubate at 4 ° C in the dark for 20 min, wash twice with flow cytometry washing buffer, add appropriate amount of fixative and fix it for flow cytometer analysis, and detect the expression of cell surface markers of human umbilical cord mesenchymal stem cells in traditional 2D plastic culture model and extracellular matrix culture system by flow cytometry.

[0038] Preferably, in step 2, the amounts of the additive components in the basal culture medium DMEM-F12 are calculated based on the final concentrations: laminin 0.02 ul / mL, EGF 0.04 ul / mL, bFGF 0.10 ul / mL, soybean trypsin inhibitor 14 ug / mL, insulin 15 ug / mL, bovine serum albumin 0.3 g / mL, and non-essential amino acids 10 ul / mL.

[0039] Preferably, in step 3, the adherent cells in the 2D culture medium were digested with 0.25% trypsin and then plated at 1×10 4 / cm 2 After 15 days, the cells were inoculated and cultured at a density of 100 μg / mL. After 15 days, they were treated with 0.5% TritonX-100 and 100 U / mL DNase and stored at 4°C for use in the next step of stem cell inoculation.

[0040] In summary, the present invention provides a method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment, which achieves the following beneficial effects:

[0041] 1. The traditional 2D culture model cannot fully reproduce the growth conditions of stem cells in the in vivo microenvironment, which in turn has a negative impact on the proliferation and differentiation process of stem cells in vitro. The present invention establishes a culture method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment to culture stem cells, highly restore the microenvironment of stem cell proliferation in vivo, and ensure the stability of their expansion.

[0042] 2. The traditional 2D culture model has low expansion efficiency and cannot meet the cell dosage required for clinical research, which wastes resources and increases scientific research costs. The present invention establishes a culture method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment to culture stem cells, maximize the number of stem cells used for research, and reduce scientific research costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a schematic diagram of the separation and culture of mesenchymal stem cells of the present invention;

[0044] Figure 2 Schematic diagram of mesenchymal stem cell passage according to the present invention;

[0045] Figure 3 Schematic diagram of cryopreservation of mesenchymal stem cells according to the present invention;

[0046] Figure 4 is a schematic diagram of mesenchymal stem cell recovery according to the present invention;

[0047] Figure 5 Schematic diagram of the growth status of mesenchymal stem cells in 2D culture medium for 24 hours, 48 hours, and 72 hours under a light microscope of the present invention;

[0048] Figure 6 Schematic diagram of the growth status of mesenchymal stem cells of the present invention in bone marrow-derived culture medium at 24 h, 48 h, and 72 h under a light microscope;

[0049] Figure 7 Schematic diagram of changes in expression of stem cell surface markers of mesenchymal stem cells of the present invention in a 2D culture medium;

[0050] Figure 8 Schematic diagram of changes in expression of stem cell surface markers when the mesenchymal stem cells of the present invention are cultured in a bone marrow-derived culture medium;

[0051] Figure 9 Schematic diagram of changes in cell surface marker expression of mesenchymal stem cells of the present invention in 2D culture medium and bone marrow-derived culture medium;

[0052] Figure 10 This is a schematic diagram of cell morphology under a light microscope in the extracellular matrix culture system of the present invention;

[0053] Figure 11This is a schematic diagram of chromosome karyotype detection in an extracellular matrix culture system according to the present invention;

[0054] Figure 12 Schematic diagram of the detection of osteogenic differentiation ability of mesenchymal stem cells of the present invention;

[0055] Figure 13 This is a schematic diagram of the detection of adipogenic differentiation ability of mesenchymal stem cells of the present invention;

[0056] Figure 14 This is a schematic diagram of the detection of the cartilage-inducing differentiation ability of mesenchymal stem cells of the present invention; DETAILED DESCRIPTION

[0057] The present invention will be further described below with reference to the accompanying drawings.

[0058] like Figures 1 to 14 As shown:

[0059] At present, the in vitro culture method of stem cells is mainly based on DMEM / F12 culture medium, supplemented with FBS and some other nutrients required by the cells. Although studies have shown that the biological characteristics of stem cells remain stable when cultured in vitro for 15 generations, cells cultured using traditional in vitro culture methods generally cannot avoid replicative senescence after the sixth generation. Domestic and foreign studies have shown that phenomena such as genomic abnormalities or functional decline during in vitro culture of stem cells are closely related to the lack of a microenvironment (Niche) in the body. Simulating the culture conditions of the in vivo microenvironment of stem cells can better maintain the characteristics of stem cells and provide expansion capacity. A large number of studies have shown that the culture system method based on the simulated in vitro microenvironment can promote cell proliferation, adhesion and differentiation, which is more efficient and less costly than the traditional 2D culture model.

[0060] Therefore, the present invention adopts a method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment. The steps of the mesenchymal stem cell culture system are as follows:

[0061] Step 1: Human umbilical cord-derived mesenchymal stem cells are used to resuscitate and prepare for use;

[0062] The method for preparing mesenchymal stem cell recovery includes: isolation and culture of mesenchymal stem cells, subculture, identification, cryopreservation and recovery;

[0063] Isolation and culture: Take an umbilical cord of 20 cm or longer and place it in 30 mL of 0.9% sodium chloride injection. After removing the ligatures at both ends, cut the umbilical cord into 2-3 cm segments. Wash the surface of the umbilical cord repeatedly until no blood or other impurities are visible to the naked eye. Transfer the cord to a new culture dish to separate Wharton's jelly for later use. Cut the Wharton's jelly into 1-4 mm pieces. 2, inoculate 3 bottles of T75 culture bottles, place them in a carbon dioxide incubator, and culture them at 5% CO2 concentration and 37℃;

[0064] Subculture: When the cell fusion rate is greater than 50%, collect the cells for subculture. After adding an appropriate amount of physiological saline for rinsing, add 3mL of 0.25% trypsin to digest for 3min until most of the cells fall off, immediately add 5mL of serum-free culture medium to stop digestion, and collect the cell suspension into a 50mL centrifuge tube; centrifuge the cell suspension at 1300r / min for 6min to remove the supernatant, take the precipitate and dissolve it in 40mL of 0.9% sodium chloride injection, filter it with a 70μm filter, repeat the above centrifugation step to remove the supernatant, add serum-free culture medium to the collected cells and place them in a cell culture incubator, and culture them at 5% CO2 concentration and 37°C;

[0065] Identification: P1 human umbilical cord mesenchymal stem cells were obtained, digested with 0.25% trypsin, and washed twice with 0.9% sodium chloride injection to prepare a cell suspension. Flow cytometry was used to sort and identify the mesenchymal stem cells to improve their purity.

[0066] Cryopreservation: Use a pipette to take serum-free freezing solution and add it to the cell suspension prepared in the previous step. Resuspend the cells, pipette evenly, cool the solution to a low temperature, and then transfer to a liquid nitrogen tank for storage.

[0067] Thawing: Remove the cryovial from the liquid nitrogen tank and immediately place it in a 37°C water bath. Gently shake to thaw within 1-2 minutes. If the sealing film falls off, do not allow the water level to approach or exceed the bottom edge of the cryovial cap during thawing to avoid contamination. Once completely thawed, use a 10mL pipette to mix the cell suspension in the cryovial and store in a cool, dry place.

[0068] Step 2: Inoculation using traditional 2D plastic culture model:

[0069] Preparation of 2D culture medium: Add laminin, EGF, bFGF, soybean trypsin inhibitor, insulin, bovine serum albumin, and non-essential amino acids to the basal medium DMEM-F12.

[0070] Stem cell inoculation: The stem cells recovered in step 1 were plated at a rate of 1x10 4 cells / mL were inoculated into a T75 culture flask containing 10 mL of standard culture medium and cultured in a cell culture incubator at 5% CO2 concentration and 37°C.

[0071] Step 3: Inoculation using the extracellular matrix culture system:

[0072] Preparation of bone marrow-derived culture medium: Use freshly isolated bone marrow-derived cells at a concentration of 2 × 10 5 / cm 2The cells were seeded into 2D culture medium at a density of 100 μg / mL and non-adherent cells were removed after one week of culture.

[0073] Stem cell inoculation: The stem cells recovered in step 1 were plated at a rate of 1x10 4 cells / mL were inoculated into a T75 culture flask containing 10 mL of bone marrow-derived culture medium and cultured in a cell culture incubator at 5% CO2 concentration and 37°C.

[0074] Step 4: Samples were taken from the stem cells seeded in the traditional 2D plastic culture model in Step 2 and the stem cells seeded in the extracellular matrix culture system in Step 3 after 24, 48, and 72 hours of culture, and the growth of the stem cells in the traditional 2D plastic culture model and the extracellular matrix culture system were observed under an inverted microscope.

[0075] Step 5: Quality control and differentiation capacity testing of stem cells inoculated into the extracellular matrix culture system

[0076] 1. Quality control of mesenchymal stem cells inoculated into the extracellular matrix culture system:

[0077] Cell morphology detection: The cell morphology of human umbilical cord mesenchymal stem cells in the extracellular matrix culture system was observed under an inverted microscope.

[0078] Chromosome karyotype detection: G-banding analysis was used to detect chromosome karyotypes. P2 cells were passaged into culture flasks. 0.05 μg / mL colchicine was added as appropriate depending on the cell adherence and mitosis. Culture was continued for 4 h. The cells were scraped off the bottom of the flask and centrifuged at 2000 rpm for 15 min. The supernatant was removed, and the slides were treated with hypotonic solution at 37°C for 5 min. The slides were fixed twice with fixative and baked at 50°C overnight. The slides were digested with 0.025% trypsin for 30 s and stained with Giemsa. Fifty metaphases were counted per slide, and 10 karyotypes were analyzed. Chromosome karyotype analysis was then performed using VideoTest-Karyo software from the United States.

[0079] 2. Detect the differentiation ability of mesenchymal stem cells inoculated in the extracellular matrix culture system:

[0080] (1) Osteogenesis induction of mesenchymal stem cells:

[0081] Osteogenesis induction step: prepare a six-well plate, add 2 mL of complete culture medium to each well, and culture P3 cells at a rate of 3×10 4Cells were seeded at 1 mL / well and the medium was completely replaced every 72 hours. When the cells reached 80% confluency, 2 mL of complete osteogenic differentiation medium was replaced and culture continued. This step was repeated five times. After 7 days of osteogenic induction, cells were harvested and centrifuged at 1000 rpm for 8 minutes. The supernatant was discarded and the cells were washed three times with PBS. The cells were fixed with 4% paraformaldehyde and stained with 0.1% Alizarin Red S for 20 minutes. The formation of intracellular calcium nodules was observed under a microscope.

[0082] (2) Adipogenesis induction of mesenchymal stem cells:

[0083] Adipogenic induction step: prepare a six-well plate, add 2 mL of complete culture medium to each well, and culture P3 cells at a rate of 2 × 10 4 Cells were seeded at 1 mL / well and the medium was completely replaced every 72 hours. When the cells reached 80% confluency, 2 mL of complete adipogenic differentiation medium was replaced and culture continued. This step was repeated five times. After 7 days of adipogenic induction, cells were harvested and centrifuged at 1000 rpm for 8 minutes. The supernatant was discarded and the cells were washed three times with PBS. The cells were fixed with 4% paraformic acid for 20 minutes, stained with 2% Oil Red O for 30 minutes, and rinsed with 70% ethanol. Lipid droplet formation was observed under a microscope.

[0084] (3) Mesenchymal stem cells chondrogenic induction:

[0085] Chondrogenic induction step: prepare a six-well plate, add 2 mL of complete culture medium to each well, and culture P3 cells at a rate of 2 × 10 4 Cells were seeded at 1 mL / well and the medium was completely replaced every 72 hours. When the cells reached 80% confluency, 2 mL of new complete adipogenic differentiation medium was replaced and culture continued. This step was repeated five times. After 7 days of chondrogenic induction, the cells were centrifuged at 1000 rpm for 8 minutes, the supernatant was discarded, and the cells were washed three times with PBS. The cells were fixed with 4% polyformic acid for 20 minutes, washed three times with pure water, and stained with Alcian blue for 30 minutes. The cells were washed twice with pure water and the staining was observed under an inverted microscope.

[0086] Step 6: Compare the stem cell expansion efficiency of traditional 2D plastic culture model seeding and extracellular matrix culture system seeding;

[0087] Step 7: It is concluded that the extracellular matrix culture system has a significant efficiency in the expansion of stem cell culture.

[0088] Furthermore, the culture system method of mesenchymal stem cells also includes the identification of cell surface markers of mesenchymal stem cells: the P3 cells in step 4 are made into 2×10 5 L -1Concentrated cell suspension, take 40 μL of anti-human CD73, CD90, CD105, CD45, CD34, CD11b, CD19, CD79a, HLA-DR and mix with the cells, incubate at 4 ° C in the dark for 20 min, wash twice with flow cytometry washing buffer, add appropriate amount of fixative and fix it for flow cytometer analysis, and detect the expression of cell surface markers of human umbilical cord mesenchymal stem cells in traditional 2D plastic culture model and extracellular matrix culture system by flow cytometry.

[0089] Furthermore, in step 2, the amounts of the additive components in the basal culture medium DMEM-F12 are calculated based on the final concentrations: laminin 0.02 ul / mL, EGF 0.04 ul / mL, bFGF 0.10 ul / mL, soybean trypsin inhibitor 14 ug / mL, insulin 15 ug / mL, bovine serum albumin 0.3 g / mL, and non-essential amino acids 10 ul / mL.

[0090] Furthermore, in step 3, the adherent cells in the 2D culture medium were digested with 0.25% trypsin and plated at 1×10 4 / cm 2 After 15 days, the cells were inoculated and cultured at a density of 100 μg / mL. After 15 days, they were treated with 0.5% TritonX-100 and 100 U / mL DNase and stored at 4°C for use in the next step of stem cell inoculation.

[0091] The purpose of this patent is to increase the stem cell proliferation rate and ensure its stability.

[0092] The patented technical method for achieving stem cell expansion rate is as follows: umbilical cord mesenchymal stem cells are prepared by processing the umbilical cord, and after morphological, sterility and flow cytometric quality inspections, the stem cells are cultured in vitro using a traditional 2D culture model and a bone marrow-derived extracellular matrix culture medium that simulates the in vitro environment, respectively. The cell expansion and cell surface markers under the light microscope after culture of the two models are compared; the stem cells cultured in the bone marrow-derived extracellular matrix culture medium that simulates the in vitro microenvironment are quality controlled through biological characteristics and tri-lineage differentiation detection.

[0093] The effect achieved by this patent: The number of stem cells cultured by the bone marrow-derived extracellular matrix culture medium method is significantly better than that of the traditional 2D culture model, and it can maintain the characteristics of stem cells.

[0094] The conclusion achieved by this patent: The bone marrow-derived extracellular matrix culture medium method that simulates the microenvironment in vitro can significantly improve the efficiency of stem cell expansion and maintain its characteristics.

[0095] The embodiments of the present invention are only used to illustrate the present invention and do not limit the scope of the claims. Other substantially equivalent alternatives that can be thought of by those skilled in the art are all within the scope of protection of the present invention.

Claims

1. A method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment, characterized in that: The culture system method and steps of mesenchymal stem cells are as follows: Step 1: Human umbilical cord-derived mesenchymal stem cells are used to resuscitate and prepare for use; Step 2: Inoculation using traditional 2D plastic culture model: Preparation of 2D culture medium: Add laminin, EGF, bFGF, soybean trypsin inhibitor, insulin, bovine serum albumin, and non-essential amino acids to the basal medium DMEM-F12. Stem cell inoculation: The stem cells recovered in step 1 were plated at a rate of 1x10 4 cells / mL were inoculated into a T75 culture flask containing 10 mL of standard culture medium and cultured in a cell culture incubator at 5% CO2 concentration and 37°C. Step 3: Inoculation using the extracellular matrix culture system: Preparation of bone marrow-derived culture medium: Use freshly isolated bone marrow-derived cells at a concentration of 2 × 10 5 / cm 2 The cells were seeded into 2D culture medium at a density of 100 μg / mL and non-adherent cells were removed after one week of culture. Stem cell inoculation: The stem cells recovered in step 1 were plated at a rate of 1x10 4 cells / mL were inoculated into a T75 culture flask containing 10 mL of bone marrow-derived culture medium and cultured in a cell culture incubator at 5% CO2 concentration and 37°C. Step 4: Samples were taken from the stem cells seeded in the traditional 2D plastic culture model in Step 2 and the stem cells seeded in the extracellular matrix culture system in Step 3 after 24, 48, and 72 hours of culture, and the growth of the stem cells in the traditional 2D plastic culture model and the extracellular matrix culture system were observed under an inverted microscope. Step 5: Quality control and differentiation capacity testing of stem cells inoculated into the extracellular matrix culture system 1. Quality control of mesenchymal stem cells inoculated into the extracellular matrix culture system: Cell morphology detection: The cell morphology of human umbilical cord mesenchymal stem cells in the extracellular matrix culture system was observed under an inverted microscope. Chromosome karyotype detection: G-banding analysis was used to detect chromosome karyotype. P2 cells were passaged into culture flasks. 0.05 μg / mL colchicine was added as appropriate depending on the cell adherence and mitosis. Culture was continued for 4 h. The cells were scraped off the bottom of the flask and centrifuged at 2000 rpm for 15 min. The supernatant was removed and the cells were treated with hypotonic solution at 37°C for 5 min. The cells were fixed twice with fixative and the slides were baked at 50°C overnight. The slides were digested with 0.025% trypsin for 30 seconds and stained with Giemsa. Fifty metaphases were counted and 10 karyotypes were analyzed per slide. Karyotype analysis was then performed using the American VideoTest-Karyo software.

2. Detect the differentiation ability of mesenchymal stem cells inoculated in the extracellular matrix culture system: (1) Osteogenesis induction of mesenchymal stem cells: Osteogenesis induction step: prepare a six-well plate, add 2 mL of complete culture medium to each well, and culture P3 cells at a rate of 3×10 4 Cells were seeded per well and the medium was completely replaced every 72 hours. When the cell fusion reached 80%, 2 mL of new osteogenic differentiation complete medium was replaced and culture was continued. This step was repeated 5 times. After 7 days of osteogenic induction, the cells were collected and centrifuged at 1000 rpm for 8 minutes, the supernatant was discarded, and the cells were washed 3 times with PBS. The cells were fixed with 4% paraformaldehyde and stained with 0.1% Alizarin Red S for 20 minutes. The formation of intracellular calcium nodules was observed under a microscope. (2) Adipogenesis induction of mesenchymal stem cells: Adipogenic induction step: prepare a six-well plate, add 2 mL of complete culture medium to each well, and culture P3 cells at a rate of 2 × 10 4 Cells were seeded per well and the medium was completely replaced every 72 hours. When the cell fusion reached 80%, 2 mL of new complete adipogenic differentiation medium was replaced and culture was continued. This step was repeated 5 times. After 7 days of adipogenic induction, the cells were collected and centrifuged at 1000 rpm for 8 minutes, the supernatant was discarded, and the cells were washed 3 times with PBS. The cells were fixed with 4% polyformic acid for 20 minutes, stained with 2% Oil Red O for 30 minutes, and rinsed with 70% ethanol. The lipid droplet formation was observed under a microscope. (3) Mesenchymal stem cells chondrogenic induction: Chondrogenic induction step: prepare a six-well plate, add 2 mL of complete culture medium to each well, and culture P3 cells at a rate of 2 × 10 4 Cells were seeded per well and the medium was completely replaced every 72 hours. When the cells reached 80% confluency, 2 mL of new complete adipogenic differentiation medium was replaced and culture was continued. This step was repeated 5 times. After 7 days of chondrogenic induction, the cells were centrifuged at 1000 rpm for 8 minutes, the supernatant was discarded, and the cells were washed 3 times with PBS, fixed with 4% polyformic acid for 20 minutes, washed 3 times with pure water, stained with Alcian blue for 30 minutes, washed twice with pure water, and the cells were observed under an inverted microscope for staining. Step 6: Compare the stem cell expansion efficiency of traditional 2D plastic culture model seeding and extracellular matrix culture system seeding; Step 7: It is concluded that the extracellular matrix culture system has a significant efficiency in the expansion of stem cell culture.

2. A method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment, characterized in that: The steps for preparing mesenchymal stem cells for recovery include: isolation and culture of mesenchymal stem cells, subculture, identification, cryopreservation and recovery; Isolation and culture: Take an umbilical cord of 20 cm or longer and place it in 30 mL of 0.9% sodium chloride injection. After removing the ligatures at both ends, cut the umbilical cord into 2-3 cm segments. Wash the surface of the umbilical cord repeatedly until no blood or other impurities are visible to the naked eye. Transfer the cord to a new culture dish to separate Wharton's jelly for later use. Cut the Wharton's jelly into 1-4 mm pieces. 2 , inoculate 3 bottles of T75 culture flasks, place them in a carbon dioxide incubator, and culture them at 5% CO2 concentration and 37℃; Subculture: When the cell fusion rate is greater than 50%, collect the cells for subculture. After adding an appropriate amount of physiological saline for rinsing, add 3mL of 0.25% trypsin to digest for 3min until most of the cells fall off, immediately add 5mL of serum-free culture medium to stop digestion, and collect the cell suspension into a 50mL centrifuge tube; centrifuge the cell suspension at 1300r / min for 6min to remove the supernatant, take the precipitate and dissolve it in 40mL of 0.9% sodium chloride injection, filter it with a 70μm filter, repeat the above centrifugation step to remove the supernatant, add serum-free culture medium to the collected cells and place them in a cell culture incubator, and culture them at 5% CO2 concentration and 37°C; Identification: P1 human umbilical cord mesenchymal stem cells were obtained, digested with 0.25% trypsin, and washed twice with 0.9% sodium chloride injection to prepare a cell suspension. Flow cytometry was used to sort and identify the mesenchymal stem cells to improve their purity. Cryopreservation: Use a pipette to take serum-free freezing solution and add it to the cell suspension prepared in the previous step. Resuspend the cells, pipette evenly, cool the solution to a low temperature, and then transfer to a liquid nitrogen tank for storage. Thawing: Remove the cryovial from the liquid nitrogen tank and immediately place it in a 37°C water bath. Gently shake to thaw within 1-2 minutes. If the sealing film falls off, do not allow the water level to approach or exceed the bottom edge of the cryovial cap during thawing to avoid contamination. Once completely thawed, use a 10mL pipette to mix the cell suspension in the cryovial and store in a cool, dry place.

3. The method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment according to claim 1, characterized in that: The culture system method of mesenchymal stem cells also includes the identification of cell surface markers of mesenchymal stem cells: the P3 cells in step 4 are made into 2×10 5 L -1 Concentrated cell suspension, take 40 μL of anti-human CD73, CD90, CD105, CD45, CD34, CD11b, CD19, CD79a, HLA-DR and mix with the cells, incubate at 4 ° C in the dark for 20 min, wash twice with flow cytometry washing buffer, add appropriate amount of fixative and fix it for flow cytometer analysis, and detect the expression of cell surface markers of human umbilical cord mesenchymal stem cells in traditional 2D plastic culture model and extracellular matrix culture system by flow cytometry.

4. The method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment according to claim 1, characterized in that: In step 2, the amounts of the additives in the basal culture medium DMEM-F12 are calculated based on the final concentrations: laminin 0.02 ul / mL, EGF 0.04 ul / mL, bFGF 0.10 ul / mL, soybean trypsin inhibitor 14 ug / mL, insulin 15 ug / mL, bovine serum albumin 0.3 g / mL, and non-essential amino acids 10 ul / mL.

5. The method for culturing mesenchymal stem cells based on an in vitro simulated microenvironment according to claim 1, characterized in that: In step 3, adherent cells in 2D culture were digested with 0.25% trypsin and plated at 1×10 4 / cm 2 After 15 days, the cells were inoculated and cultured at a density of 100 μg / mL. After 15 days, they were treated with 0.5% TritonX-100 and 100 U / mL DNase and stored at 4°C for use in the next step of stem cell inoculation.

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