CN110881459A

CN110881459A - Human peripheral blood mononuclear cell cryopreservation method

Info

Publication number: CN110881459A
Application number: CN201911109709.4A
Authority: CN
Inventors: 陈杰; 梁洋泉; 方松刚
Original assignee: Wuhan Jiyuan High-Tech Co Ltd
Current assignee: Wuhan Jiyuan High-Tech Co Ltd
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2020-03-17

Abstract

The invention provides a human Peripheral Blood Mononuclear Cell (PBMC) frozen stock solution and a PBMC frozen stock method, wherein the PBMC frozen stock solution comprises DMSO, dextran, trehalose, a basal medium, human albumin or autologous plasma. The PBMC cryopreservation liquid can effectively protect PBMC from freezing damage, and has high safety and small damage to cells. On the premise of high-density PBMC, the PBMC is cooled through a programmed cooling box, transferred and stored in liquid nitrogen for 1 month, 3 months, 6 months and 12 months at night, and then PMBC is revived to perform induction culture of immune Cells (CIK), and the proliferation rate and the cell phenotype of the PBMC are not obviously different from those of the fresh PBMC.

Description

Human peripheral blood mononuclear cell cryopreservation method

Technical Field

The invention designs a cell cryopreservation technology, and belongs to the field of cell biology research.

Background

The human peripheral blood mononuclear cell can be used for preparing immune cells and carrying out immune cell therapy on blood-taking personnel. The key to influencing immune cell therapy is whether healthy immune cells are available. It is known that aging and background environmental factors are the main causes of the deterioration of the immune system. But with age the immune cells gradually degenerate. Storing human PBMC in early stage, and performing resuscitation induction culture when needed to obtain large amount of healthy immune cells. In the prior art, the density of the frozen cells is generally 5 × 106-1 × 107/ml, the number of the frozen PBMCs is generally 3-10 × 107, and in the scheme, the frozen cells are difficult to popularize, and each cell consumes a large amount of manpower and material resources.

Disclosure of Invention

To ensure that PBMC cells were at 3 x 10⁷-5*10⁷Per ml miWhen the PBMC is frozen at a certain temperature, the cytocidal power and the cell viability are not influenced, and the invention provides a novel PBMC cell freezing method.

The solution of the invention patent is as follows:

peripheral blood from humans was collected and PBMC was isolated by centrifugation using lymphocyte separation medium.

Preparing cell frozen stock solution, and preparing according to 5-10% of DMSO, 1-5% of dextran, 1-5% of trehalose, 30-40% of DMEM (basal medium) and 40-50% of human serum albumin (autoblood plasma).

PBMC was expressed as 3 x 10⁷/ml、5*10⁷/ml、1*10⁸Freezing at a density of one ml, placing in a programmed cooling box, placing in a refrigerator at-80 deg.C overnight, and storing in a liquid nitrogen tank the next day.

Freezing for 1 month, 3 months, half year and 1 year respectively. And calculating the cell survival rate comparison before and after the cryopreservation, simultaneously performing induction culture on the PBMC to obtain immune cells, and calculating the proliferation rate and the phenotypic difference between the unfrozen cells and the cryopreserved cells.

Find 3 x 10⁷/ml、5*10⁷Under a/ml system, the cell survival rate of 1 month, 3 months, half a year and 1 year of frozen storage is higher and reaches more than 90 percent, and the cell proliferation rate and phenotype after recovery and the cells directly induced and cultured by PBMC separation have no obvious difference. 1*10⁸Under a/ml system, the cell survival rate of 1 month, 3 months, half a year and 1 year of frozen storage can only reach 70-80%, and the cell proliferation rate and phenotype after recovery are about 20% lower than that of the direct induction culture of separated PBMC.

TABLE 1 cell viability at different cryopreservation densities and times

TABLE 2 fold increase (CIK) of cell culture for 14 days at different cryopreservation densities and times

TABLE 3 cell phenotype of cells cultured for 14 days at different freezing densities, freezing times (CD 3-CD56 +)

Claims

1. A human peripheral blood mononuclear cell cryopreservation method is characterized by comprising the following steps: separating PBMC in peripheral blood, then using cell freezing medium to resuspend the PBMC to a certain density, controlling the cooling rate by a programmed cooling box, and finally placing liquid nitrogen for long-term storage.

2. The method of claim 1, wherein the cell culture medium comprises 5-10% DMSO, 1-5% dextran, 1-5% trehalose, 30-40% basal medium, 40-50% human serum albumin, or autologous plasma.

3. The cryopreservation method of claim 1 wherein the cell density is from 3 x 107/ml to 5 x 107/ml.

4. The cryopreservation method of claim 1, wherein the controlled cooling mode is that the cryopreservation tube is placed in a programmed cooling box, then placed in a refrigerator at-20 ℃ for 1-2h, then placed in a refrigerator at-80 ℃ for overnight, and placed in a liquid nitrogen tank for long-term storage the next day.