Method for separating endothelial progenitor cells from cryopreserved cord blood
A technology of endothelial progenitor cells and umbilical cord blood, applied in the field of separation of endothelial progenitor cells, can solve the problem of less than 10% utilization rate and achieve the effect of efficient method, improved separation efficiency, and convenient cell adjustment
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Embodiment 1
[0022] Example 1 Isolation and culture of frozen umbilical cord blood EPCs
[0023] Frozen umbilical cord blood is taken from the cord blood hematopoietic stem cell bank of Shandong Province, and the umbilical cord blood that has passed the test has been officially put into the bank.
[0024] (1) Quickly take out a portion (30-50ml) of frozen cord blood from the liquid nitrogen tank, place it in a 37°C water bath, shake the frozen storage bag while incubating, and quickly recover, transfer the thawed cord blood into In a 250ml centrifuge tube, add 100ml of pre-cooled PBS buffer solution containing 0.5% human serum albumin, mix thoroughly, centrifuge at 1500rpm / min at room temperature for 10min, increase speed 9, decrease speed 7, discard the supernatant, and obtain total nuclear cell (TNCs) pellet;
[0025] (2) Resuspend the TNCs pellet in step (1) with 100ml pre-cooled PBS buffer containing 0.5% human serum albumin, centrifuge at 1000rpm / min at room temperature for 5min, inc...
Embodiment 2
[0030] Example 2 Comparative example: Isolation and culture of frozen cord blood EPCs in the prior art
[0031] Frozen umbilical cord blood is taken from the cord blood hematopoietic stem cell bank of Shandong Province, and the umbilical cord blood that has passed the test has been officially put into the bank.
[0032]Quickly take out a portion of frozen cord blood from the liquid nitrogen tank, recover quickly in a 37°C water bath, shake the frozen bag while incubating, mix the thawed cord blood with PBS 1:1, and take the mixture and Ficoll separation solution After 1:1 mixing, gradient density centrifugation (2000rpm / min, speed up 1, speed down 1, centrifuge 20min), absorb the buffy coat (mononuclear cells).
[0033] The mononuclear cell (MNCs) pellet was resuspended in PBS buffer, centrifuged at 1000rpm / min for 5min, the speed was increased by 9, the speed was decreased by 9, and the supernatant was discarded, twice in total. Finally, mononuclear MNCs were obtained, resus...
Embodiment example 3
[0036] Implementation Case 3 Flow Cytometry Detection of Surface Antigens of Endothelial Progenitor Cells
[0037] Phenotype analysis was performed on the first-generation endothelial progenitor cells (P1-EPCs) cultured in Example 1 by flow cytometry. Fitc-labeled anti-CD31, PE-labeled anti-CD144 and anti-CD309, and those showing stem cell characteristics Anti-CD34 binds to markers on the cell surface, the results are as follows figure 2 The results showed that the positive rates of CD31, CD34, CD144, and CD309 in the isolated cells were 93.88%, 65.03%, 88.69%, and 70.77%, respectively. The isolated cells were determined to be endothelial progenitor cells with stem cell characteristics.
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