Amniotic membrane stromal cell culture system, cell culture method and application

Abstract

The present application provides an amniotic stromal cell culture system, a cell culture method and an application, which belong to the field of tissue repair technology. The amniotic stromal cell culture system includes DMEM / F12 culture medium, fetal bovine serum, double antibody and uridine. Fetal bovine serum can provide the necessary nutrients for the growth of amniotic stromal cells, hormones to maintain cell exponential growth, provide binding proteins, etc., and promote the proliferation of amniotic stromal cells. Uridine has anti-inflammatory and antioxidant effects, can reduce the damage of reactive oxygen species to cells, and also promotes cell proliferation and activity by activating signal pathways such as PI3K / Akt, so that the cultured cells can accelerate wound healing and are beneficial to tissue repair. The amniotic stromal cells cultured in this amniotic stromal cell culture system can significantly improve the repair effect of intravenously infused amniotic stromal cells on intestinal damage, and can be used to prepare drugs for treating intestinal damage.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of tissue repair, and relates to an amniotic membrane stromal cell culture system, a cell culture method and application. BACKGROUND

[0002] Placental amnion is composed of amniotic membrane stromal cells (hAMSCs), amniotic mesenchymal stem cells and collagen-rich extracellular matrix, has low immunogenicity, anti-inflammatory, repair-promoting and other characteristics, and shows great potential in clinical application.

[0003] In the field of tissue repair and regeneration, placental amnion is made into dressings or scaffolds due to its natural three-dimensional structure and biocompatibility, and is used for the treatment of burns, chronic ulcers and corneal injuries. Amniotic ECM can release growth factors such as EGF and bFGF, accelerate epithelialization and reduce scar formation. In addition, hAMSCs in placental amnion have multilineage differentiation potential and can differentiate into nerve, cartilage, pancreatic beta cells and the like, and show repair effects in animal models of spinal cord injury, osteoarthritis and diabetes. In terms of immune regulation and anti-fibrosis, amniotic cells inhibit excessive inflammatory response by secreting cytokines such as IL-10 and TGF-β, and are suitable for the treatment of autoimmune diseases such as rheumatoid arthritis or organ fibrosis such as pulmonary fibrosis.

[0004] hAMSCs, as multipotent stem cells in amniotic tissue, have broad application prospects in the field of tissue repair due to their strong proliferation ability, multilineage differentiation potential and low immunogenicity. These cells are derived from the ectoderm in the early stage of embryonic development, are rich in growth factors such as EGF and bFGF and extracellular matrix components, can effectively promote cell migration, angiogenesis and anti-fibrosis, and significantly accelerate wound healing. In skin injury repair, hAMSCs regulate inflammatory response through paracrine effect and reduce scar formation, and have been successfully applied to the treatment of chronic ulcers and burns. Compared with other cells, hAMSCs have the advantages of no ethical controversy and easy access, and their immune regulation characteristics can reduce the risk of transplantation rejection. At present, the three-dimensional culture technology of hAMSCs combined with biological scaffolds further improves the repair efficiency, and in the future, it will become an important tool for regenerative medicine. SUMMARY

[0005] Based on the above purpose, the application provides an amniotic membrane stromal cell culture system, a cell culture method and application.

[0006] To achieve the above purpose, the following technical solutions are adopted in the application:

[0007] The application provides an amniotic membrane stromal cell culture system, which comprises DMEM / F12 culture medium, fetal bovine serum, double antibodies and uridine; wherein the final concentrations of the fetal bovine serum, the double antibodies and the uridine in the culture system are 5-20%, 0.5-2% and 10-50 ng / mL respectively.

[0008] The DMEM / F12 culture medium is a commonly used culture medium, which is used as a basic culture medium in the application. The fetal bovine serum contains rich nutrient components necessary for cell growth, and can also provide hormones for maintaining the exponential growth of cells, provide binding proteins and the like, thereby promoting cell proliferation.

[0009] Uridine is an important nucleoside compound, which is combined by uracil and ribose, and widely exists in organisms and has multiple biological activities. Uridine is involved in the synthesis of uridine triphosphate (UTP), affects glycogen synthesis and cell energy supply, and is crucial for maintaining liver and muscle functions. Uridine is a precursor of cytidine triphosphate (CTP), promotes phospholipid metabolism and cell membrane repair, especially can enhance synaptic plasticity in neural tissues, improve cognitive function, and has potential application in the treatment of neurodegenerative diseases such as Alzheimer's disease. In addition, uridine also has anti-inflammatory and antioxidant effects, can reduce the damage of reactive oxygen species (ROS) to cells, and protects myocardial and neural cells. At the same time, uridine promotes cell proliferation by activating PI3K / Akt and other signaling pathways, accelerates wound healing, and is beneficial to tissue repair. The cells obtained by adding uridine in the amniotic membrane stromal cell culture system can show significant repair ability to small intestinal tissues.

[0010] In the application, more preferably, the final concentrations of the fetal bovine serum, the double antibodies and the uridine in the culture system are 10%, 1% and 30 ng / mL respectively.

[0011] The application provides an amniotic membrane stromal cell culture method, which comprises the following steps:

[0012] S01: culturing amniotic membrane stromal cells in DMEM / F12 culture medium containing fetal bovine serum and double antibodies to the 3rd generation, when the amniotic membrane stromal cells grow to 60%, adding uridine into the DMEM / F12 culture medium, and continuing to culture for 36-48 h.

[0013] Specifically, the placenta of healthy full-term cesarean section women is transported to the laboratory, and the amnion of the placenta is peeled off in a sterile environment. The amnion tissue is washed with sterile normal saline containing 3% double antibodies for 5 times to prevent bacterial contamination; wherein the double antibodies are penicillin + streptomycin. The amnion tissue is cut into 3-5 cm 2The fragments were digested with 0.25% trypsin for 60 and 30 minutes, respectively. The single-cell suspension obtained in the first 60 minutes was discarded, and the amniotic stromal cell suspension was isolated in the last 30 minutes. The amniotic stromal cell suspension was filtered through a 100 μm mesh and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. The pellet was resuspended in PBS, passed through a 40 μm mesh, and centrifuged again to obtain primary amniotic stromal cells.

[0014] Primary amniotic stromal cells were seeded at a density of 2×10 5 pieces / cm 2 The cells were seeded into DMEM / F12 medium containing fetal bovine serum (FBS) at final concentrations of 5-20% and 0.5-2% respectively, and cultured to the third generation. When the amniotic stromal cells grew to cover 60% of the culture device surface, uridine at a final concentration of 10-50 ng / mL was added to the DMEM / F12 medium to enhance the tissue repair ability of hAMSCs, and the culture was continued for 36-48 hours.

[0015] S02: After the culture period, add 10-100 mM trehalose 2 hours before the cells are released. This stabilizes the cell membranes of the AMS cells, improves their resistance to stress upon reintroduction, and prolongs their survival in vivo. Following the culture period, the AMS cells are harvested using standard trypsin digestion.

[0016] In addition, the amniotic stromal cells cultured in the present application can repair intestinal damage caused by chemotherapy and can be used to prepare drugs for treating intestinal damage.

[0017] The present invention has the following beneficial effects:

[0018] The present application provides an amniotic stromal cell culture system, which includes DMEM / F12 culture medium, fetal bovine serum, double antibody and uridine. Fetal bovine serum can provide the necessary nutrients for the growth of amniotic stromal cells, hormones to maintain cell exponential growth, provide binding proteins, etc., and promote the proliferation of amniotic stromal cells. Uridine has anti-inflammatory and antioxidant effects, can reduce the damage of reactive oxygen species to cells, and also promotes cell proliferation by activating signaling pathways such as PI3K / Akt, accelerates wound healing, and is beneficial to tissue repair. The amniotic stromal cells obtained by culturing the membrane stromal cell culture system can significantly improve the repair effect of intravenously infused amniotic stromal cells on intestinal damage, and can be used to prepare drugs for treating intestinal damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 These are morphological images of amniotic stromal cells obtained by culture in the amniotic stromal cell culture system at different magnifications according to Example 1 of the present application at different days; the left image and the right image are morphological images of the cells on the fourth and seventh days of culture, respectively;

[0020] Figure 2 The small intestinal villus injury of the normal group, model group, hAMSC treatment group and experimental group mice of the application is shown in A-H, which are normal group (×40), normal group (×100), model group (×40), model group (×100), hAMSC treatment group (×40), hAMSC treatment group (×100), experimental group (×40) and experimental group (×100), respectively. DETAILED DESCRIPTION

[0021] The technical solutions of the application are further explained and described below through specific examples.

[0022] Example 1

[0023] The embodiment of the application provides an amniotic membrane stromal cell culture system, which comprises DMEM / F12 culture medium, fetal bovine serum, double antibiotics and uridine; wherein the final concentrations of the fetal bovine serum, double antibiotics and uridine in the culture system are 10%, 1% and 30 ng / mL respectively.

[0024] The embodiment of the application also provides an amniotic membrane stromal cell culture method, which comprises:

[0025] S01: The placenta of a healthy full-term cesarean section woman is transported to a laboratory, and the amnion of the placenta is peeled off in a sterile environment. The amnion tissue is washed with sterile physiological saline containing 3% double antibiotics for 5 times to prevent bacterial contamination; wherein the double antibiotics are penicillin + streptomycin. The amnion tissue is cut into pieces of 3-5 cm 2 , and the pieces are digested with trypsin with a concentration of 0.25% for 60 min and 30 min respectively. The single cell suspension obtained in the first 60 min is discarded, and the amniotic membrane stromal cell suspension is separated in the last 30 min. The amniotic membrane stromal cell suspension is filtered with a 100 μm sieve, and centrifuged at 1000 rpm for 5 minutes, and the supernatant is discarded. The precipitate is resuspended with PBS, filtered through a 40 μm sieve, and centrifuged again to obtain primary amniotic membrane stromal cells.

[0026] The primary amniotic membrane stromal cells are inoculated into DMEM / F12 culture medium containing fetal bovine serum with a final concentration of 1% and 1% and double antibiotics at a seeding density of 2×10 5 / cm 2 , and subcultured to the 3rd generation. When the amniotic membrane stromal cells grow to 60%, uridine with a final concentration of 30 ng / mL is added to the DMEM / F12 culture medium to increase the tissue repair capacity of the hAMSCs, and the culture is continued for 36-48 h.

[0027] S02: After the end of culture, 2h before the cells are taken out, 50mM trehalose is added to stabilize the cell membrane state of amniotic membrane matrix cells, improve the anti-reverse ability of amniotic membrane matrix cells when input into the body, and prolong the survival time of amniotic membrane matrix cells in the body. After the end of culture, amniotic membrane matrix cells are collected by trypsin digestion.

[0028] Example 2

[0029] The embodiment of the present application provides an amniotic membrane matrix cell culture system, which comprises DMEM / F12 culture medium, fetal bovine serum, double antibodies and uridine; wherein the final concentrations of the fetal bovine serum, the double antibodies and the uridine in the culture system are 5%, 2% and 10ng / mL respectively.

[0030] The embodiment of the present application also provides an amniotic membrane matrix cell culture method, which is the same as the embodiment 1.

[0031] Example 3

[0032] The embodiment of the present application provides an amniotic membrane matrix cell culture system, which comprises DMEM / F12 culture medium, fetal bovine serum, double antibodies and uridine; wherein the final concentrations of the fetal bovine serum, the double antibodies and the uridine in the culture system are 20%, 0.5% and 50ng / mL respectively.

[0033] The embodiment of the present application also provides an amniotic membrane matrix cell culture method, which is the same as the embodiment 1.

[0034] Example 4

[0035] The embodiment of the present application provides an amniotic membrane matrix cell culture system, which comprises DMEM / F12 culture medium, fetal bovine serum, double antibodies and uridine; wherein the final concentrations of the fetal bovine serum, the double antibodies and the uridine in the culture system are 15%, 1.5% and 40ng / mL respectively.

[0036] The embodiment of the present application also provides an amniotic membrane matrix cell culture method, which is the same as the embodiment 1.

[0037] The embodiment of the present application carries out SEM detection on the amniotic membrane matrix cells cultured in the embodiment 1 under different magnifications, and obtains the following Figure 1 It can be seen from the following Figure 1 that the amniotic membrane matrix cells cultured in the embodiment 1 are columnar, the cytoplasm is rich, and the nucleus is large and round, which is consistent with the characteristics of amniotic membrane matrix cells.

[0038] This application also studies the repair ability of amniotic stromal cells on intestinal damage. Specifically, 16 C57BL / 6 mice were randomly divided into 4 groups: normal group, model group, hAMSC treatment group and experimental group. Among them, the model group, hAMSC treatment group and experimental group were injected with 10mg / kg cisplatin solution to create an acute intestinal injury model in mice. The normal group was not treated. Two days after the injection of cisplatin solution, the mice in the hAMSC treatment group and the experimental group were treated with 1X10 6 hAMSCs and hAMSCs cultured in Example 1 were intravenously injected at a concentration of 10 cells / mouse. The model group mice were injected with an equal volume of normal saline, and the normal group was not treated. The mice were killed after 3 days of treatment, and the small intestinal tissues of the mice were taken for pathological sections to observe the damage of the small intestinal villi. Figure 2 .

[0039] By the attached Figure 2 The normal group showed intact intestinal wall structure, with evenly distributed mucosal folds and villi. The villi were densely arranged, leaf-like, and of roughly uniform height. The model group showed significant intestinal villi damage, with fewer, shorter, and detached villi, irregular surfaces, fewer glands, and multiple apoptotic cells. The submucosal layer was edematous and widened, consistent with chemotherapy-related intestinal mucosal damage. The hAMSC-treated group showed some repair of the villi, with more and longer villi and more glands than the model group, indicating a restored villus structure. The experimental group showed further repair of the villi, with an increased number of villi and glands, and a more complete structure. This indicates that intestinal damage was further repaired after hAMSC-treated cells were cultured with uridine.

[0040] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A method for culturing amniotic stromal cells, characterized in that: include: The amniotic stromal cells were cultured in DMEM / F12 medium containing fetal bovine serum and double antibody to the third generation. When the amniotic stromal cells grew to cover 60% of the surface of the culture device, uridine was added to the DMEM / F12 medium at a final volume concentration of 10-50 ng / mL and the culture was continued for 36-48 hours. After the culture was completed, trehalose was added 2 hours before the cells were released from the storage, and the amniotic stromal cells were collected by trypsin digestion.

2. The method for culturing amniotic stromal cells according to claim 1, wherein The concentration of trehalose is 10-100 mM.

3. The method for culturing amniotic stromal cells according to claim 1, wherein The final volume concentrations of the fetal bovine serum and the dual antibody are 5-20% and 0.5-2%, respectively.

4. The amniotic stromal cells cultured according to any one of claims 1 to 3 are used to prepare a drug for treating intestinal damage caused by chemotherapy.

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