Isolation and culture of stem cells
Isolation and culture of stem cells
Summary
The human endometrium is a highly regenerative tissue, and this great regenerative capacity is thought to be directly related to human endometrial stem cells. Endometrial stem cells are categorized in a more diverse manner, including endometrial cell side population (SP), endometrial mesenchymal stem cells, and menstrual blood-derived stem cells. Endometrial mesenchymal stem cells (enMSCs) are a new class of adult stem cells with biological properties such as self-renewal ability, differentiation potential, low immunogenicity, and low tumorigenicity.
Appliance
Isolated endometrial MSCs can be used to clinically treat menstrual disorders, endometrial diseases, infertility, and organ damage.
Operation method
Isolation and culture of endometrial mesenchymal stem cells
Materials and Instruments
DMEM/F12 (1:1) medium, fetal bovine serum Move I. Isolation and culture of endometrial MSCs 1. The whole endometrium and myometrium (5 mm) were surgically excised and quickly put into a sterile bottle containing 0 ℃ D-Hanks. 2. In the ultra-cleaning table, wash with sterile D-Hanks (containing penicillin) for two times to remove blood and other debris on the surface. 3. After being washed well, the tissue is cut to about 1 mm3 with ophthalmic scissors. After that, the tissues were quickly placed into a 15 mL centrifuge tube containing 300 mg/mL Collagenase III and 40 mg/mL DNase I, and the tissues were kept immersed in digestive enzymes at all times during the process. 4. Shock the digested tissue at 37 ℃ for 45 min, and gently blow bubbles into the bottom of the centrifuge tube with a pipette (to maintain continuous bubbling) during the process. 5、Pick up the digestive solution carefully, keep the tissue fragments, the digestive solution is filtered through a 200-mesh filter, and the digested individual cells form the filtrate. 6、Centrifuge the digested solution obtained in step (5) at 1000 r/min for 10 min, and discard the supernatant. 7. Wash the cells twice with D-Hanks at 0 ℃. 8. Add DMEM-F12 medium (containing 10% fetal bovine serum) and incubate the cells at 37 ℃ in an incubator with 5% CO2. 9. The first time of fluid exchange is 4~5 d of culture, which can remove most of the non-adherent cells and residual erythrocytes, and observe the morphology and arrangement of the cells, and take pictures. During the period of 2~3 d, the liquid was changed once, and when the cell fusion reached about 80%, the cells were digested and passed on with 2.5% trypsin in the ratio of 1:1~1:2. 10. After 2~3 generations of culture and purification, human endometrial MSCs with high purity can be isolated. The cells of the 3rd to 6th generation can be used for subsequent experiments.
D-Hanks buffer, collagenase III, DNase I
Penicillin
CD29, CD45, CD90, CD34, CD73 and CD133 antibodies
2.5% trypsin
1. Select a well-grown cell colony, collect the cells by centrifugation, discard the supernatant, wash the cells twice with D.Hanks' solution, make a single-cell suspension with a concentration of 1×106 L-1 , and then add 10 μL of antibodies against CD29, CD45, CD90, CD34, CD73, and CD133, and incubate at 4 ℃ and away from light for 30 min.
After incubation, the cells were washed twice with D.Hanks solution to remove the unbound antibody, and finally resuspended with 500 μL of D.Hanks solution, and the expression rates of CD29, CD45, CD90, CD34, CD73 and CDl33 were detected by flow cytometry. The results showed that CD29, CD90, CD73 were positive, and CD34, CD45, CD133 were negative.
Caveat
(1) Aseptic operation is used to avoid cell contamination and exogenous microbial infection.(2) Tissue handling needs to be fast, accurate and gentle to avoid cell death or damage.(3) Tissue digestion time needs to be strictly controlled, too short or too long may affect cell separation and culture.(4) Cell culture conditions need to be strictly controlled to maintain a good culture environment, including temperature, humidity, and concentration of nutrient solution.(5) The purity and quality of endometrial MSCs need to be characterized to ensure their application effect and safety.(6) Suitable methods should be used for cell preservation to ensure their long-term preservation and use. Common Problems (1) Unsatisfactory cell separation effect: it may be that the time of tissue digestion is too short or too long, or the tissue is not handled properly, and it is necessary to pay attention to the operation specifications and skills. (2) Unsatisfactory cell culture effect: it may be that the culture environment is not suitable, such as temperature, humidity, concentration of nutrient solution, etc., need to adjust the culture conditions. (3) Slow cell growth: it may be that the cell density is too low, or the cell quality is not good, need to pay attention to the control of cell density and quality. (4) Cell contamination: it may be that the operation is not strict, or the culture environment is contaminated, need to pay attention to aseptic operation and environmental hygiene. (5) Cell death: it may be improper handling of tissues, or inappropriate culture environment, need to pay attention to operation specifications and adjust the culture conditions. (6) Inaccurate cell identification: the identification method may be inaccurate, or the cell quality is not good, need to pay attention to the identification method and cell quality control. (7) Improper cell preservation: it may be inappropriate preservation conditions, such as temperature, humidity, preservation containers, etc., need to pay attention to preservation conditions.
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