A culture medium for rat amniotic mesenchymal stem cells and a culture method for rat amniotic mesenchymal stem cells

Abstract

The application belongs to the field of cell culture. The application provides a culture medium of rat amniotic membrane mesenchymal stem cells and a culture method of rat amniotic membrane mesenchymal stem cells. The culture medium is a complete culture medium, and the culture method is as follows: the rat amnion is cut into tissue blocks smaller than 2mm 2 , is uniformly laid in a culture dish with a spacing of 0.3-0.8cm, complete culture medium is slowly added after the tissue blocks are adhered, the culture medium is supplemented after 12h-36h, the primary medium replacement is performed after 48h-72h of culture, the impurity cells are removed, and the cells are subcultured when the cell fusion degree reaches 80%-90%. The light microscope observation and identification result show that the rat amniotic membrane mesenchymal stem cells cultured by the application basically conform to the cell morphology of rat amniotic membrane mesenchymal stem cells, the CD90&DAPI immunofluorescence identification result shows that the cell purity is greater than or equal to 90%, the cells have the characteristics of stem cells, and the culture method is efficient in amplification.

Description

Technical Field

[0001] This invention belongs to the field of cell culture, specifically relating to a culture medium for rat amniotic mesenchymal stem cells and a method for culturing rat amniotic mesenchymal stem cells. Background Technology

[0002] The amniotic membrane, located inside the chorionic villi of the placenta, is a transparent, thin membrane without blood vessels, nerves, lymphatics, or muscle. It plays a protective role for the fetus and is closely connected to the developing fetus. Human amniotic cells begin to form on the 8th day after fertilization and possess multi-differentiation potential, capable of differentiating into cells from all three germ layers. Recent studies have shown that amniotic epithelial cells can differentiate into mature nerve cells and synthesize and release neurotransmitters such as dopamine, acetylcholine, and norepinephrine. Human amniotic cells have low immunogenicity and effective immunosuppressive activity, and no acute rejection reaction occurs after cell transplantation.

[0003] Rat amniotic mesenchymal stem cells (rAMSCs) are a type of stem cell extracted from the rat amnion. They have multi-lineage differentiation and self-renewal capabilities and can differentiate into various tissues such as smooth muscle, endothelium, epithelium, muscle, cartilage, and bone.

[0004] The present invention aims to establish a relatively ideal rat amniotic mesenchymal stem cell culture system, providing a theoretical basis and technical method for the application of rat amniotic mesenchymal stem cells. Summary of the Invention

[0005] To overcome the shortcomings of existing rat amniotic mesenchymal stem cell culture techniques, this invention provides a culture medium and a method for culturing rat amniotic mesenchymal stem cells, which effectively improves the in vitro proliferation rate of rat amniotic mesenchymal stem cells.

[0006] The purpose of this invention is to provide a culture medium for rat amniotic mesenchymal stem cells. The culture medium is a complete culture medium, and its composition and dosage include: 85-95% DMEM high glucose medium (volume percentage), 5-15% FBS (volume percentage), 0.5-2% penicillin-streptomycin solution (volume percentage), 0.5-1.5 mmol / L L-glutamine (volume concentration), 0.5-1.5 μM A-83-01 (volume concentration), 45-75 μg / mL vitamin C (volume concentration), 5.3-11.4 μg / mL transferrin (volume concentration), and 1.2-2.2 ng / mL basic fibroblast growth factor (bFGF) (volume concentration). The penicillin-streptomycin solution contains 5000-20000 U / mL penicillin and 5-20 mg / mL streptomycin.

[0007] In the culture medium of this invention, L-glutamine provides a more abundant energy and nitrogen source, helping to maintain the healthy state of highly metabolically active cells and reduce apoptosis; basic fibroblast growth factor (bFGF) and transferrin are potent mitogens and trophic factors that can significantly stimulate cell division and improve cell proliferation efficiency; A-83-01 is a TGF-β kinase inhibitor that can effectively inhibit the spontaneous differentiation of cells (especially stem cells) and help maintain their pluripotency or progenitor cell state; vitamin C is an important cofactor for collagen synthesis, which can promote the secretion of collagen by cells, which is beneficial to the formation of extracellular matrix and cell adhesion. The culture medium of this invention promotes rapid cell adhesion, fusion and proliferation, and the different components exert a synergistic effect on proliferation, improving the in vitro proliferation rate of rat amniotic mesenchymal stem cells, shortening the cell culture cycle, reducing the unit cell production cost, and possessing industrialization potential.

[0008] Preferably, in the above-mentioned complete culture medium, the volume percentage of DMEM high-glucose medium is 89%, the volume percentage of FBS is 10%, the volume percentage of penicillin-streptomycin solution is 1%, the volume concentration of L-glutamine is 1 mmol / L, the concentration of A-83-01 is 0.75 μM, the concentration of vitamin C is 60 μg / mL, the concentration of transferrin is 8.3 μg / mL, and the concentration of basic fibroblast growth factor (bFGF) is 1.8 ng / mL, and the complete culture medium is obtained by mixing.

[0009] The purpose of this invention is to provide a method for preparing the culture medium for the above-mentioned rat amniotic mesenchymal stem cells, wherein A-83-01, vitamin C, transferrin, basic fibroblast growth factor, FBS, penicillin-streptomycin solution, L-glutamine and DMEM high glucose culture medium are mixed to obtain the culture medium for rat amniotic mesenchymal stem cells.

[0010] The present invention also aims to provide a method for culturing rat amniotic mesenchymal stem cells, comprising the following steps:

[0011] (1) Cut the washed rat amnion into pieces smaller than 2 mm. 2 The tissue blocks were evenly spread in the culture dish, spaced 0.3-0.8 cm apart. The culture dish was inverted and placed in a cell culture incubator at 36-37.5℃ for 10-30 minutes. After the tissue blocks adhered, the above complete culture medium was slowly added until it covered the tissue blocks. The culture medium was replenished after 12-36 hours.

[0012] (2) After culturing for 48-72 hours, change the medium for the first time to remove non-adherent cells and dead cells and other miscellaneous cells. When the cell confluence reaches 80%-90%, passage the cells.

[0013] Furthermore, in step (2), the medium is changed every 2-3 days after the initial medium change to remove contaminating cells.

[0014] In step (2), the passage includes the following steps: removing the tissue block and reattaching the tissue block to the wall using the plating method in step (1), and then digesting and passageing the culture dish with 0.25% trypsin-EDTA.

[0015] Furthermore, the aforementioned rat amniotic mesenchymal stem cells were obtained from SD rats at gestation days 15-20.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] (1) This invention first adds A-83-01, vitamin C, transferrin and basic fibroblast growth factor in a certain proportion to the culture medium and applies it to the in vitro culture system of rat amniotic cells, which promotes the rapid adhesion, fusion and proliferation of cells. Under the culture medium, different components play a synergistic role in proliferation, which improves the in vitro proliferation rate of rat amniotic mesenchymal stem cells, can shorten the cell culture cycle, reduce the unit cell production cost, and has industrialization potential.

[0018] (2) The results of light microscopy observation show that the morphology of rat amniotic mesenchymal stem cells cultured by the culture method and proliferation culture medium used in this invention is basically consistent with the cell morphology of rat amniotic mesenchymal stem cells.

[0019] (3) The CD90 & DAPI immunofluorescence identification results show that the rat amniotic mesenchymal stem cells cultured by the culture method and proliferation medium used in this invention have a cell purity of ≥90%, which is excellent and has the characteristics of stem cells.

[0020] (4) By using the rat amniotic mesenchymal stem cell proliferation culture medium and the rat amniotic mesenchymal stem cell culture operation, rat amniotic mesenchymal stem cells can be cultured efficiently and at low cost, and the resulting rat amniotic mesenchymal stem cells are numerous and have good activity.

[0021] (5) The rat amniotic mesenchymal stem cell culture method used in this invention has high expansion efficiency, simple composition formula, ideal culture effect, and broad application prospects. Attached Figure Description

[0022] Figure 1 Cell morphology diagram of P0 generation rat amniotic mesenchymal stem cells cultured after Day 8;

[0023] Figure 2 A morphological diagram of rat amniotic mesenchymal stem cells cultured to generation P1.

[0024] Figure 3 Cell morphology diagram of rat amniotic mesenchymal stem cells cultured to generation P2;

[0025] Figure 4 Cell morphology diagram of rat amniotic mesenchymal stem cells cultured to generation P3;

[0026] Figure 5 The results of CD90 & DAPI immunofluorescence identification of P3 generation rat amniotic mesenchymal stem cells;

[0027] Figure 6 This is a comparison chart of the proliferation rates of Example 1 and Comparative Example 1. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] This invention establishes a relatively ideal rat amniotic mesenchymal stem cell culture system by isolating, purifying, and expanding rat amniotic mesenchymal stem cells in vitro, and identifying them through morphological observation (light microscopy) and immunofluorescence, thereby providing a theoretical basis and technical method for the application of rat amniotic mesenchymal stem cells.

[0030] To overcome the shortcomings of existing rat amniotic mesenchymal stem cell culture techniques, the present invention aims to provide a method for culturing and expanding rat amniotic mesenchymal stem cells (rAMSCs), which effectively improves the in vitro proliferation rate of rat amniotic mesenchymal stem cells.

[0031] To achieve the above objectives, the present invention provides the following technical solution:

[0032] (1) Preparation of complete culture medium for rAMSCs: 89% DMEM high glucose medium, 10% FBS, 1% penicillin and streptomycin, 1 mmol / L L-glutamine, 0.5-1.5 μM A-83-01, 45-75 μg / mL vitamin C, 5.3-11.4 μg / mL transferrin, and 1.2-2.2 ng / mL basic fibroblast growth factor (bFGF);

[0033] (2) SD rats (gestation day 18) were anesthetized by intraperitoneal injection of 5 ml paraformaldehyde, and then euthanized and soaked in 75% ethanol for 5 min.

[0034] (3) After preparing the abdomen, the rat's abdominal cavity was cut open with sterile tissue scissors. The uterus was found to be a narrow, gourd-shaped structure extending to both sides, with about 7-8 embryos on each side. Each embryo was about 2.5cm*1cm*1cm in size. The uterine layer was mechanically separated, the placenta was peeled off, and the amniotic membrane was separated.

[0035] (4) Soak it in a PBS solution containing 1% penicillin and streptomycin to wash away the blood. Then, briefly soak it in a 75% alcohol solution for 5-10 seconds and quickly remove it. Soak it in a clean PBS solution containing 1% penicillin and streptomycin twice.

[0036] (5) Use sterile ophthalmic scissors to cut the cleaned amniotic membrane into small pieces, about 1 mm in size. 2 Spread the tissue evenly in a 10cm sterile cell culture dish, spacing them about 0.5cm apart. Invert the culture dish and place it in a 37℃ cell culture incubator for 10-30 minutes. After the tissue adheres, slowly add an appropriate amount of complete culture medium, just enough to cover the tissue block. Add more culture medium after 24 hours.

[0037] (6) Change the medium for the first time after 48 hours of culture to remove non-adherent cells and dead cells. Change the medium every 2-3 days thereafter. When the cell confluence reaches 80%, remove the tissue block and re-adhere the tissue block using the plating method in step (5). The culture dish is then digested and passaged with 0.25% trypsin-EDTA.

[0038] (7) After digesting the cells, resuspend the digested cells in cryopreservation solution and freeze the cells in liquid nitrogen at -80°C; the cells can be identified after passage to P3. The culture method provided by this invention yields a large number of rat amniotic mesenchymal stem cells with good activity.

[0039] Preferably, the rat amniotic mesenchymal stem cell proliferation culture medium in step (1) includes: DMEM high glucose medium, FBS, penicillin-streptomycin solution, and L-glutamine; 10% FBS is added to the DMEM high glucose medium, followed by 1% penicillin-streptomycin solution, L-glutamine at a final concentration of 1 mmol / L, A-83-01 at a final concentration of 0.5-1.5 μM, vitamin C at 45-75 μg / mL, transferrin at 5.3-11.4 μg / mL, and basic fibroblast growth factor (bFGF) at 1.2-2.2 ng / mL, and the mixture is thoroughly mixed to obtain the above-mentioned rat amniotic mesenchymal stem cell proliferation culture medium.

[0040] Preferably, in step (6), the medium is changed every 3 days after the step of changing the medium to remove impurities.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] This invention is the first to add A-83-01, vitamin C, transferrin, and basic fibroblast growth factor to a culture medium in a specific ratio and apply it to an in vitro culture system of rat amniotic cells. Studies have found that these culture conditions not only promote rapid cell adhesion, fusion, and proliferation, but also provide a high-quality source of seed cells for subsequent applications in tissue engineering.

[0043] This invention compares the growth efficiency of rat amniotic mesenchymal stem cells (rAMs) cultured under the same culture environment (37°C, 5% CO2) with that cultured under the same environment using rat amniotic mesenchymal stem cell proliferation medium. The conclusion is that, under the same culture environment, rat amniotic mesenchymal stem cells cultured using rat amniotic mesenchymal stem cell proliferation medium have a faster growth rate and higher cell purity.

[0044] This invention provides an improved and efficient method for culturing and expanding rat amniotic mesenchymal stem cells (rAMSCs): Rat amniotic membranes are aseptically collected and expanded using a proliferation medium. Light microscopic observation results show that the morphology of the rat amniotic mesenchymal stem cells cultured using the method and proliferation medium of this invention is basically consistent with that of rat amniotic mesenchymal stem cells. CD90 & DAPI immunofluorescence identification results show that the rat amniotic mesenchymal stem cells cultured using the method and proliferation medium of this invention have a cell purity ≥90%, exhibiting excellent purity and stem cell characteristics. Using the aforementioned rat amniotic mesenchymal stem cell proliferation medium and the aforementioned rat amniotic mesenchymal stem cell culture procedures, efficient and low-cost rat amniotic mesenchymal stem cell culture can be achieved, resulting in a large number of rat amniotic mesenchymal stem cells with good activity. The rat amniotic mesenchymal stem cell culture method of this invention has high expansion efficiency, a simple composition formula, and ideal culture results, showing broad application prospects.

[0045] Material information used in this invention:

[0046] Example 1

[0047] An improved and efficient method for culturing and expanding rat amniotic mesenchymal stem cells (rAMSCs) is described below.

[0048] (1) Preparation of complete culture medium for rAMSCs: 89% DMEM high glucose medium, 10% FBS, 1% penicillin-streptomycin solution (penicillin concentration of 10000 U / mL and streptomycin concentration of 10 mg / mL), 1 mmol / L L-glutamine, 0.75 μM A-83-01, 60 μg / mL vitamin C, 8.3 μg / mL transferrin, and 1.8 ng / mL basic fibroblast growth factor (bFGF) were mixed to obtain the complete culture medium.

[0049] (2) SD rats (gestation day 18) were anesthetized by intraperitoneal injection of 5 ml paraformaldehyde and then euthanized and soaked in 75% ethanol for 5 min.

[0050] (3) After preparing the abdomen, the rat's abdominal cavity was cut open with sterile tissue scissors. The uterus was found to be a narrow gourd shape extending to both sides, with about 7-8 embryos on each side. Each embryo was about 2.5cm*1cm*1cm in size. The uterine layer was mechanically separated, the placenta was peeled off, and the amnion tissue was separated.

[0051] (4) Soak it in a PBS solution containing 1% penicillin and streptomycin to wash away the blood. Then, briefly soak it in a 75% alcohol solution for 5-10 seconds and quickly remove it. Soak it in a clean PBS solution containing 1% penicillin and streptomycin twice.

[0052] (5) Use sterile ophthalmic scissors to cut the cleaned amniotic membrane into small pieces, about 1 mm in size. 2 Spread the tissue evenly in a 10cm sterile cell culture dish, spacing them about 0.5cm apart. Invert the dish and place it in a 37℃ cell culture incubator for 10-30 minutes. After the tissue adheres, slowly add an appropriate amount of complete culture medium, just enough to cover the tissue block. Replenish the culture medium after 24 hours.

[0053] (6) Change the medium for the first time after 48 hours of culture to remove non-adherent cells and dead cells. Change the medium every 2-3 days thereafter. When the cell confluence reaches 80%, remove the tissue block and re-adhere the tissue block using the plating method in step (5). The culture dish is then digested and passaged with 0.25% trypsin-EDTA.

[0054] (7) After digesting the cells, resuspend the digested cells in cryopreservation solution and freeze the cells in liquid nitrogen at -80°C; cell identification can be performed after passage to P3.

[0055] Comparative Example 1

[0056] The complete culture medium was prepared using 89% DMEM high-glucose medium, 10% FBS, and 1% penicillin-streptomycin solution (the penicillin concentration in the penicillin-streptomycin solution was 10000 U / mL, and the streptomycin concentration was 10 mg / mL). Other steps were the same as in Example 1.

[0057] Comparison of proliferation rates between Example 1 and Comparative Example 1

[0058] The growth efficiency of rat amniotic mesenchymal stem cells cultured in Example 1 was compared with that in Comparative Example 1. The comparison results are as follows: Figure 6 As shown. It can be concluded that, under the same culture conditions, rat amniotic mesenchymal stem cells cultured using the proliferation medium of this invention grow faster. The formulation of this invention is more suitable for supporting the long-term culture of rat amniotic mesenchymal stem cells, allowing them to retain better biological functions while expanding. In the medium of this invention, L-glutamine provides a more abundant energy and nitrogen source, helping to maintain the healthy state of highly metabolically active cells and reduce apoptosis. Basic fibroblast growth factor (bFGF) and transferrin are potent mitogens and trophic factors that can significantly stimulate cell division and improve cell expansion efficiency. A-83-01 is a TGF-β kinase inhibitor that can effectively inhibit the spontaneous differentiation of cells (especially stem cells), helping to maintain their pluripotency or progenitor cell state. Vitamin C is an important cofactor for collagen synthesis, promoting cell secretion of collagen, which is beneficial for the formation of the extracellular matrix and cell attachment.

[0059] Example 2: Light microscopic observation and immunofluorescence identification of rat amniotic mesenchymal stem cells

[0060] 1. Observation under a light microscope

[0061] Cell morphology of P0 generation rat amniotic mesenchymal stem cells cultured for 8 days is shown in the figure below. Figure 1 As shown in the image, P0 generation cells emerge from a tissue block. The dark shadows in the image represent the tissue block, which is three-dimensional. This image illustrates the morphology of P0 generation cells emerging from the tissue block.

[0062] Cell morphology diagram of rat amniotic mesenchymal stem cells cultured to generation P1 is shown below. Figure 2 As shown;

[0063] Cell morphology diagram of rat amniotic mesenchymal stem cells cultured to generation P2 is shown below. Figure 3 As shown;

[0064] Cell morphology diagram of rat amniotic mesenchymal stem cells cultured to passage P3 is shown below. Figure 4 As shown.

[0065] The results of light microscopy observation show that the morphology of rat amniotic mesenchymal stem cells cultured using the culture method and proliferation culture medium used in this invention is basically consistent with the cell morphology of rat amniotic mesenchymal stem cells.

[0066] 2. Immunofluorescence identification

[0067] Rat amniotic mesenchymal stem cells at 2×10 4 The cells were seeded at a density of / ml in 6-well plates pre-placed with coverslips. After the cells reached 80%–90% confluence, 4% paraformaldehyde was added for fixation in the dark for 5 minutes. The cells were washed three times with PBS, and CD90 primary fluorescent antibody was added. The cells were incubated overnight at 4°C. The cells were washed three times with PBS, and DAPI was added for incubation in the dark for 10 minutes. After washing three times with PBS, one drop of anti-fluorescence quencher was added to a glass slide. The slide was removed, and the cell-containing side was placed face down on the glass slide. The slide was then observed under a fluorescence microscope and stored at 4°C in the dark.

[0068] The results of CD90 and DAPI immunofluorescence identification of P3 generation rat amniotic mesenchymal stem cells are as follows: Figure 5 As shown. The CD90 & DAPI immunofluorescence identification results indicate that the rat amniotic mesenchymal stem cells cultured using the culture method and proliferation medium used in this invention have a cell purity of ≥90%, which is excellent and exhibits the characteristics of stem cells.

[0069] This invention establishes a relatively ideal rat amniotic mesenchymal stem cell culture system by isolating, purifying, and expanding rat amniotic mesenchymal stem cells in vitro, and identifying them through morphological observation (light microscopy) and immunofluorescence, providing a theoretical basis and technical method for the application of rat amniotic mesenchymal stem cells.

[0070] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the scope of the invention and should be defined by the claims.

Claims

1. A culture medium for rat amniotic mesenchymal stem cells, characterized in that, The culture medium is a complete culture medium, and its composition and dosage include: 85-95% DMEM high glucose medium (volume percentage), 5-15% FBS (volume percentage), 0.5-2% penicillin-streptomycin solution (volume percentage), 0.5-1.5 mmol / L L-glutamine (volume concentration), 0.5-1.5 μM A-83-01 (volume concentration), 45-75 μg / mL vitamin C (volume concentration), 5.3-11.4 μg / mL transferrin (volume concentration), and 1.2-2.2 ng / mL basic fibroblast growth factor (bFGF) (volume concentration). The penicillin-streptomycin solution contains 5000-20000 U / mL penicillin and 5-20 mg / mL streptomycin.

2. The culture medium according to claim 1, characterized in that, The complete culture medium contained 89% DMEM high-glucose medium (by volume), 10% FBS (by volume), 1% penicillin-streptomycin solution (by volume), 1 mmol / L L-glutamine, 0.75 μM A-83-01, 60 μg / mL vitamin C, 8.3 μg / mL transferrin, and 1.8 ng / mL basic fibroblast growth factor (bFGF).

3. A method for culturing rat amniotic mesenchymal stem cells, characterized in that, Includes the following steps, (1) Cut the washed rat amnion into pieces smaller than 2 mm. 2 Tissue blocks are evenly spread in a culture dish with a spacing of 0.3-0.8 cm. The culture dish is inverted and placed in a cell culture incubator at 36-37.5℃ for 10-30 min. After the tissue blocks adhere, the complete culture medium described in claim 1 is slowly added until it covers the tissue blocks. The culture medium is replenished after 12-36 h. (2) Change the medium for the first time after culturing for 48h-72h to remove contaminating cells, and passage the cells when the cell confluence reaches 80%-90%.

4. The cultivation method according to claim 3, characterized in that, In step (2), the medium is changed every 2-3 days after the initial medium change to remove impurities.

5. The cultivation method according to claim 3, characterized in that, In step (2), the passage includes the following steps: removing the tissue block and reattaching the tissue block to the wall using the plating method in step (1), and then digesting and passageing the culture dish with 0.25% trypsin-EDTA.

6. The cultivation method according to claim 3, characterized in that, The amniotic membrane of the rat was obtained from SD rats at 15-20 days of gestation.

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