A kind of feeder layer cell, culture method and application
A feeder layer cell and in vitro culture technology, applied in feeder layer cells, culture methods and application fields, can solve the problems of high uncertainty and instability, and achieve avoidance of uncertainty and instability, long life cycle, expansion A powerful effect
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Embodiment 1
[0023] Example 1: Isolation and culture of cBM-MSC
[0024] (1) Obtaining chicken bone marrow:
[0025] 1-7 day-old chickens were killed by dislocation, separated chicken thighs and chicken shanks, soaked in 75% ethanol for 15 minutes after separation; stripped muscle and connective tissue under aseptic conditions, completely separated femur and tibia, cut off the dry end, and Expose the bone marrow cavity; use a syringe to draw serum-free KO-DMEM medium and wash the bone marrow cavity, and collect it with a centrifuge tube added with anticoagulant.
[0026] (2) Primary culture of cBM-MSCs
[0027] Pass the washed bone marrow cells through a sieve to make a single-cell suspension, centrifuge at 1000rpm / min for 5min, discard impurities such as fat in the upper layer; collect the bottom cells and wash 3 times with PBS, then resuspend and inoculate into cBM-MSC-containing medium In, the cBM-MSC medium comprises KO-DMEM medium, 7.5% fetal bovine serum, 2mM L-glutamine, 2mM gluta...
Embodiment 2
[0031] Embodiment 2: The drawing of growth curve of cBM-MSC and CEFs
[0032] The cBM-MSCs and CEFs of the third passage were counted, and the growth curve was drawn according to the obtained data, with a scale of 1×10 4 Inoculate into a 24-well plate, take three wells every 24 hours, and record the average number. have to figure 1 Comparison of growth curves shown.
Embodiment 3
[0033] Example 3: Immunofluorescence Identification of cBM-MSCs
[0034] (1) The cBM-MSCs of the third generation were inoculated on a gelatin-coated 24-well plate, and when 70% confluence was reached, the culture medium was discarded and washed twice with PBS;
[0035] (2) Add 3.7% paraformaldehyde solution to the 24-well plate to fix for 30 minutes, and wash the cells three times with PBS containing 5% FBS (10 minutes each time);
[0036] (3) Add permeabilization agent (0.1% TritonX-100, PBS) to the 24-well plate, discard after incubating at room temperature for 15 minutes, wash with PBS three times, 5 minutes each time;
[0037] (4) Add blocking solution (10% FBS:PBS) to the 24-well plate, block at 37°C for 1 hour, discard it, wash with PBS three times, 5 minutes each time;
[0038] (5) Incubate overnight at 4°C with primary antibodies against CD29 and CD44 (1:200, Santa Cruz, CA), after removing the primary antibodies, wash the cells three times with PBS containing 5% FBS...
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