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Cryopreservation solution, cryopreservation method and recovery method for peripheral blood mononuclear cells

A cryopreservation method and nuclear cell technology, which is applied in the field of cryopreservation solution for peripheral blood mononuclear cells, can solve the problems of unknown cell viability and reduced cell viability, and achieve the effect of high viability and high application value

Inactive Publication Date: 2019-11-15
GUANGDONG PANGUARD CELL BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Patent Publication No. CN104719282A discloses a cryopreservation scheme using DMSO, dextran-40 and Bomalis A. The recovery rate of PBMC obtained by using this scheme is 84.03% on average, which needs to be improved
Moreover, the patent only records the recovery data of the cell cryopreservation solution (20% DMSO+2% dextran 40+Bomalis A), the dosage of DSMO is still relatively high, and the cell viability of the low dosage of DMSO is unknown, but it can be obtained very quickly. The obvious speculation is that as the amount of DMSO decreases, the cell viability decreases

Method used

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  • Cryopreservation solution, cryopreservation method and recovery method for peripheral blood mononuclear cells
  • Cryopreservation solution, cryopreservation method and recovery method for peripheral blood mononuclear cells

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1 Freezing solution 1

[0024] The cryopreservation solution consists of the following components: 500 μL X-VIVO15 serum-free medium, 200 μL human albumin, 100 μL 0.5 wt% sodium hyaluronate, 100 μL 50 μg / mL ascorbic acid, and 100 μL DMSO.

[0025] In other alternative embodiments, the cryopreservation solution can be composed of the following components: 400 μL X-VIVO15 serum-free medium, 150 μL human albumin, 150 μL 0.5wt% sodium hyaluronate, 150 μL 50 μg / mL ascorbic acid, and 150 μL DMSO ; It can also be composed of the following components: 600 μL X-VIVO15 serum-free medium, 250 μL human albumin, 50 μL 0.5wt% sodium hyaluronate, 50 μL 50 μg / mL ascorbic acid, 50 μL DMSO.

Embodiment 2

[0036] Embodiment 2PBMC separation

[0037] The peripheral blood was collected by anticoagulation, and the upper plasma after centrifugation was set aside. According to the ratio of 1:1, the plasma-removed components in the centrifuge tube were gradually added to the upper layer of the lymphocyte separation medium along the tube wall, and the buffy coat cells were collected after centrifugation, washed with X-VIVO15 serum-free medium for 3 times, and re- Hang count.

Embodiment 3PB

[0038] Embodiment 3 PBMC cryopreservation

[0039] Transfer the counted cell suspension into 15ml centrifuge tubes, discard the supernatant after centrifugation, transfer to 6 cryopreservation tubes and add cryopreservation solutions 1-6, each cryopreservation tube freezes 1.87*10 7 cells, cryopreservation volume 1mL. Seal the cryopreservation tube, place it in a cooling box, and quickly transfer it to a -80°C refrigerator. After overnight, transfer the cryopreservation tube to a liquid nitrogen tank at -196°C.

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Abstract

The invention relates to a cryopreservation solution, a cryopreservation method and a recovery method for peripheral blood mononuclear cells. The cryopreservation solution includes, by volume, 40-60%of a serum-free medium, 15-25% of an impermeable protective agent, 5-15% of a permeable protective agent, 5-15% of sodium hyaluronate and 5-15% of an anti-oxidation substance. The cryopreservation solution composed of the serum-free medium, the impermeable protective agent, the permeable protective agent and the anti-oxidation substance can effectively cryopreserve cells, and makes the cryopreserved cells still have a high cell viability after being recovered, so the cryopreservation solution has high application values, and is worth promoting.

Description

technical field [0001] The invention relates to the technical field of cell cryopreservation, in particular to a cryopreservation solution, a cryopreservation method and a resuscitation method for peripheral blood mononuclear cells. Background technique [0002] Peripheral blood mononuclear cells (PBMC) are cells with a single nucleus in peripheral blood, mainly including lymphocytes (lymphocytes), monocytes (monocytes), dendritic cells (Dendritic Cells, DC) and so on. The cryopreservation of peripheral blood mononuclear cells and the ability to effectively maintain their vitality and function are of great significance for both basic medicine and clinical applications. Frozen storage of peripheral blood mononuclear cells can effectively solve the existing problems of long induction period of immune cells, multiple inductions and multiple blood collections, and can also store the best immune cells in a healthy state , for future needs, such as tumor cell therapy and anti-agi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/078
CPCA01N1/0221C12N5/0634
Inventor 吕品雷何安涛李莉李相鲁
Owner GUANGDONG PANGUARD CELL BIOLOGICAL TECH CO LTD
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