PBMC cell cryopreservation method and thawing method

A cryopreservation method and cell technology, applied in the field of cell biology, can solve the problems that scientific researchers are fully concerned about, and achieve the effect of improving the recovery rate of cryopreservation and reducing damage

Inactive Publication Date: 2018-06-01
山东省齐鲁细胞治疗工程技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cooling curve of the programmed cooling instrument, especially for PBMC, has not been paid enough attention by researchers.

Method used

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  • PBMC cell cryopreservation method and thawing method
  • PBMC cell cryopreservation method and thawing method
  • PBMC cell cryopreservation method and thawing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1: the extraction of PBMC cell

[0033] Divide the blood sample evenly into 50ml centrifuge tubes, the volume of each tube should not exceed 45ml, and centrifuge at 2000rpm for 10 minutes; after centrifugation, absorb the upper layer of plasma in the centrifuge tube and place it in a 50ml centrifuge tube to inactivate it for later use, and suck it to 0.5cm from the interface Dilute the blood cells with normal saline at a ratio of 1:1; divide the human lymphocyte separation solution into 50ml centrifuge tubes, each with 15ml, tilt the centrifuge tube containing Ficoll, and slowly add the blood sample diluted in the previous step Centrifuge at 1600rpm for 20 minutes after reaching the surface of Ficoll to make it a clear interface. The ratio of blood sample to Ficoll should not exceed 2:1. Saline to 45ml, mix well, and centrifuge at 1500rpm for 10min; after centrifugation, discard the supernatant, add normal saline to the remaining precipitate to 45ml, mix well...

Embodiment 2

[0034] Example 2: Effects of different cryopreservation densities of PBMC on the recovery rate of cryopreservation

[0035] A 2× freezing solution was prepared at a volume ratio of fetal bovine serum: dimethyl sulfoxide = 4:1 (DMSO was added dropwise to fetal bovine serum and mixed), and placed in a refrigerator at 4°C for cooling.

[0036] Centrifuge the PBMC cells separated from the whole blood to obtain a cell pellet, add fetal bovine serum drop by drop to the pellet to resuspend the cells to make a cell suspension, and then put an equal volume of 2× freezing solution prepared and placed in a refrigerator at 4°C for standby Add dropwise to the cell suspension to maintain the density of the cell suspension at 5×10 6 / ml, 1×10 7 / ml 2×10 7 / ml (this process was performed on ice).

[0037]The cell suspension obtained by mixing the above cells and the cryopreservation solution was dispensed into cryopreservation tubes at 1 ml / tube (this process was carried out on ice). Put ...

Embodiment 3

[0045] Example 3: Effects of Programmable Cooling Apparatus on PBMC Cryopreservation Recovery Recovery Rate

[0046] A 2× freezing solution was prepared at a volume ratio of fetal bovine serum: dimethyl sulfoxide = 4:1 (DMSO was added dropwise to fetal bovine serum and mixed), and placed in a refrigerator at 4°C for cooling.

[0047] Centrifuge the PBMC cells separated from the whole blood to obtain a cell pellet, add fetal bovine serum drop by drop to the pellet to resuspend the cells to make a cell suspension, and then put an equal volume of 2× freezing solution prepared and placed in a refrigerator at 4°C for standby Added dropwise to the cell suspension to maintain the density of the cell suspension at 2×10 7 / ml (this process was performed on ice).

[0048] The cell suspension obtained by mixing the above cells and the cryopreservation solution was dispensed into cryopreservation tubes at 1 ml / tube (this process was carried out on ice).

[0049] Freeze the above-mention...

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PUM

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Abstract

The invention discloses a PBMC cell cryopreservation method, which comprises: (1) centrifuging PBMC cells to obtain a cell precipitation, performing resuspending, adding a 2X cryopreservation liquid drop by drop to maintain the density of a cell suspension at 2*10<7> / ml, and performing split charging to cryopreservation tubes; and (2) putting a program cooler, gradually reducing the temperature to-90 DEG C, and transferring the obtained product to liquid nitrogen for long-term preservation. The invention further discloses a PBMC cell thawing method, which comprises: (1) taking out the cryopreservation tubes with PBMC cells preserved, quickly putting the obtained cryopreservation tubes in a water bath, performing repeated vibration for 5-15 times, and controlling the vibration time in 3 min; and (2) washing the obtained cell suspension with a X-VIVO serum-free medium to maintain the cell density at 1-2*10<6> / ml. According to the cryopreservation method, the cooling rate can be accurately controlled via the program cooler, so that ice crystals are prevented from generation, the cell damage is reduced, and the cryopreservation-thawing rate of PBMC cells after cryopreservation-thawinghas been carried out for 24 h is obviously improved.

Description

technical field [0001] The invention relates to a cryopreservation method and a recovery method of PBMC cells, belonging to the field of cell biology. Background technique [0002] Peripheral blood mononuclear cells (PBMC) are cells with a single nucleus in peripheral blood, including lymphocytes and monocytes. That is: mononuclear hematopoietic stem cells in non-bone marrow blood and hematopoietic stem cells in bone marrow are released into the blood, and then extracted and separated from the blood to obtain hematopoietic stem cells. The stem cells obtained in this way are called peripheral blood mononuclear cells. To detect lymphocytes in vitro, the first step is to separate peripheral blood mononuclear cells. At present, the main separation method is Ficoll-hypaque (dextran-diatrizoate meglumine) density gradient centrifugation. be separated. The density of erythrocytes and granulocytes is greater than that of the layered solution, and at the same time, the erythrocytes...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/078
CPCA01N1/0221A01N1/0284C12N5/0634
Inventor 谭毅张慧慧
Owner 山东省齐鲁细胞治疗工程技术有限公司
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