Preparation method of high-purity, high-multiplication capacity and high-cytotoxin activity CIK (cytokine induced kill) cell
A proliferative and high-purity technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of low cytotoxic activity, low proliferative power, and low purity of CIK cells, and achieve enhanced anti-tumor effect , the effect of increasing the quantity
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Embodiment 1
[0024] Embodiment 1: the preparation of CIK cell
[0025] 1. Preparation of peripheral blood mononuclear cells (PBMC)
[0026] Use a blood cell separator to collect anticoagulant blood from the patient under sterile conditions, centrifuge at 1500rpm / min for 10 minutes to absorb the upper layer of plasma, inactivate at 56°C for 30 minutes and centrifuge for later use, dilute the blood cell pellet with normal saline, and the human lymphocytes with a specific gravity of 1.077 The separated liquid and the diluted blood were added to the centrifuge tube at a ratio of 1:1.5, centrifuged at 2000rpm / min for 15 minutes, carefully extracted the white blood cell layer, washed twice with normal saline, and obtained PBMC after low-speed centrifugation.
[0027] 2. Mini MACS to remove CD4 + CD25 + Treg cells
[0028] Take the PBMC cell suspension to adjust to an appropriate cell concentration, add PE-labeled AntiHuman CD25, incubate at 4°C for 30 minutes, take it out, centrifuge and wash...
Embodiment 2
[0029] Example 2: Properties of CIK cells
[0030] 1. Morphology, cell viability and immunophenotype detection of CIK cells
[0031] After adding PHA and INF-γ, most of the cells were still in a suspended state; after 3 days, the volume of the cells increased, the cells gradually aggregated into clusters, the cells were translucent, and the cytoplasm was abundant; from the 7th day, the cells began to proliferate in large quantities, and the cell shapes were diverse. Cell clusters increased; take 100 μl of cells cultured for 10 days, add 100 μl of 0.4% placenta blue staining solution, live cells will not be stained, and dead cells will be stained blue. The viability of the cells prepared by the invention is greater than 90%. On the 14th day, a small amount of dead cells could be seen. Cell flow detection analysis showed that CIK cells belonged to a heterogeneous cell population. With the prolongation of culture time, CD4 + , CD8 + , CD3 + CD56 + The ratio of T cells was s...
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