Tiger adipose-derived mesenchymal stem cell culture solution and tiger adipose-derived mesenchymal stem cell culture method
A technology of mesenchymal stem cells and culture methods, applied in the field of tiger adipose-derived mesenchymal stem cell culture medium, can solve the problems of few reports on tiger adipose-derived mesenchymal stem cells
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Embodiment 1
[0060] Example 1. Isolation of tiger adipose-derived mesenchymal stem cells by collagenase method and culturing them
[0061] (1) Collection of Tiger Adipose Tissue
[0062] Obtain a small amount of adipose tissue around the surgical wound of a tiger that needs surgical treatment, and immediately put the adipose tissue into a PBS buffer solution containing 10% double-antibody solution, wherein the double-antibody solution contains 0.1U / L penicillin and 0.1 μg / L streptomycin.
[0063] (2) Cleaning and shredding the tissue pieces
[0064] 1) Soak the separated adipose tissue in PBS buffer solution containing 20% double antibody solution for 5 minutes, clean the blood clot, and take it out with tweezers;
[0065] 2) Soak the adipose tissue taken out in step 1) in PBS buffer solution containing 10% double antibody for 5 minutes, clean the blood clot, and take it out with tweezers;
[0066] 3) Soak the adipose tissue taken out in step 2) in PBS buffer solution containing 5% do...
Embodiment 2
[0079] Embodiment 2, drawing cell growth curve
[0080] (1) Take the well-grown third-generation tiger mesenchymal stem cells from Example 1, digest them with 0.25% trypsin and make a cell suspension;
[0081] (2) Dilute tiger mesenchymal stem cells to 5×10 6 / mL inoculated into 96-well plate, 0.1mL per well, placed in 5% CO 2 , cultured in a 37°C incubator;
[0082] (3) After 24 hours, 5 wells of tiger mesenchymal stem cells were randomly selected at the same time every day, the supernatant was discarded, and after washing once or twice with PBS, the prepared CCK8 working solution was added to each well, and then placed at 37°C, 5% CO 2 The incubator was incubated for 1h, then the microplate reader was used to measure and record the corresponding OD value, and the average value was measured three times in each hole (4) for 9 days. The plotting of the growth curve, the abscissa represents the number of days, and the ordinate represents the OD value, and the drawing is as fo...
Embodiment 3
[0083] Embodiment 3, common PCR identification
[0084] The cell surface markers identified in this embodiment include CD105, CD29, CD44, CD90, CD34, and the steps include:
[0085] 1. Trizol method to extract cellular RNA
[0086] (1) Select the well-grown third-generation mesenchymal stem cells from Example 1, inoculate them on a six-well plate coated with gelatin, make the cells grow to the full of the well plate, discard the medium, and use DEPC-H 2 O (being DEPC water) washes 2 times;
[0087] (2) Add 1ml Trizol solution, let stand at room temperature for 5min, draw the liquid into a 1.5mL EP tube, 4°C, 12000rpm, 5min, centrifuge and absorb the supernatant;
[0088] (3) Add 200 μL of chloroform, mix well, let stand at room temperature for 5 minutes, 4°C, 12000 rcf, 15 minutes, absorb the upper water layer into a new 1.5mL EP tube after centrifugation;
[0089] (4) Add 0.5ml of isopropanol, mix well, let stand at room temperature for 5 minutes, 4°C, 12000rcf, 10min, dis...
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