Freeze-storage liquid of peripheral blood mononuclear cells and freeze-storage method

A cryopreservation method and technology for nuclear cells, which is applied in the field of cryopreservation of peripheral blood mononuclear cells, can solve problems such as biological damage, changes in cell thermodynamics, chemical and physical environment, etc.

Inactive Publication Date: 2012-09-19
深圳市博泰生物医疗机构管理有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cryopreservation process will significantly change the thermodynamic, chemical and physical environment of cells, accompanied by the risk of biological damage

Method used

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  • Freeze-storage liquid of peripheral blood mononuclear cells and freeze-storage method
  • Freeze-storage liquid of peripheral blood mononuclear cells and freeze-storage method
  • Freeze-storage liquid of peripheral blood mononuclear cells and freeze-storage method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Take 50ml of peripheral blood from healthy volunteers, anticoagulated with heparin, centrifuge at 700g at room temperature for 20min, draw the upper plasma, and bath at 56℃ for 30min; add D-PBS to 50ml for the lower cells, mix well, and slowly add to 2 cells with 20ml human lymph Centrifuge the cell separation solution at 800g for 15 minutes at room temperature in a 50ml centrifuge tube to extract the buffy coat cells, which are peripheral blood mononuclear cells.

[0037] The plasma obtained after the above 700g centrifugation was allowed to stand at 4°C for 15 minutes, and then centrifuged at 900g for 30 minutes. The autologous plasma was taken and the PBMC cryopreserved solution was prepared according to the following volume percentage components:

[0038] 10% DMSO + 12% β-glucan + 10% autologous plasma + 68% saline for injection.

[0039] 2. Freezing storage of PBMC

[0040] (1) Wash the isolated PBMC twice with PBS, then resuspend the cells with RPMI1640 culture medium ...

Embodiment 2

[0073] 1. Take 50ml of peripheral blood from healthy volunteers and separate peripheral blood mononuclear cells. The procedure is the same as in Example 1. The PBMC cryopreservation solution is prepared according to the following volume percentage components:

[0074] 11% DMSO + 14% β-glucan + 9% fetal bovine serum + 66% saline for injection.

[0075] 2. Freezing storage of PBMC

[0076] (1) Wash the isolated PBMC twice with PBS, and then resuspend the cells in RPMI1640 medium to adjust the cell density to 2×10 7 Pcs / ml;

[0077] (2) Mix the above-mentioned cell suspension and PBMC cryopreservation solution gently according to the volume ratio of 1:1, and divide them into cell cryopreservation tubes, 1ml per tube, so that the cell content is 1×10 7 Piece / branch;

[0078] (3) Place the above-mentioned cell cryopreservation tube in the program cryopreservation box, the bottom of the freezing box contains 100% isopropanol, and then put the program cryopreservation box in the -80℃ ultra-low...

Embodiment 3

[0090] 1. Take 50ml of peripheral blood from healthy volunteers and separate peripheral blood mononuclear cells. The procedure is the same as in Example 1. The PBMC cryopreservation solution is prepared according to the following volume percentage components:

[0091] 12% DMSO + 14% β-glucan + 8% calf serum + 66% saline for injection.

[0092] 2. Freezing storage of PBMC

[0093] (1) Wash the isolated PBMC twice with PBS, and then resuspend the cells in RPMI1640 medium to adjust the cell density to 10×10 7 Pcs / ml;

[0094] (2) Mix the above-mentioned cell suspension and PBMC cryopreservation solution gently according to the volume ratio of 1:1, and divide them into cell cryopreservation tubes, 1ml per tube, so that the cell content is 5×10 7 Piece / branch;

[0095] (3) Place the above-mentioned cell cryopreservation tube in the program cryopreservation box, the bottom of the freezing box contains 100% isopropanol, and then put the program cryopreservation box in the -80℃ ultra-low temper...

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Abstract

The invention relates to the technical field of biology and provides freeze-storage liquid of peripheral blood mononuclear cells. The freeze-storage liquid comprises 10 to 12 percent (V / V) of osmotic cryoprotectants, 10 to 14 percent (V / V) of beta-glucan, 8 to 10 percent (V / V) of blood serum or blood plasma and 64 to 72 percent (V / V) of normal saline. The invention also provides a freeze-storage method of the peripheral blood mononuclear cells. The method comprises the following steps that (1) the peripheral blood mononuclear cells are suspended in cell culture liquid to obtain peripheral blood mononuclear cell suspension; (2) the peripheral blood mononuclear cell suspension and the peripheral blood mononuclear cell freeze-storage liquid provided by any one item in the claim 1-5 are uniformly mixed according to a proportion being (0.9-1.1):1 and are respectively contained in cell freeze-storage tubes; and (3) the cell freeze-storage tubes are placed into a program freeze-storage box to be firstly frozen at 80 DEG C below zero, and then, the cell freeze-storage tubes are transferred into liquid nitrogen to be subjected to freeze storage.

Description

Technical field [0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a cryopreservation solution and a cryopreservation method for peripheral blood mononuclear cells. Background technique [0002] Peripheral blood mononuclear cell (PBMC) refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, dendritic cells and other small amounts of cells (hematopoietic stem cells, etc.). PBMC is the seed cell for the preparation of cytokine-induced killer cells (CIK). CIK cells are considered to be the first choice for a new generation of tumor adoptive immunotherapy due to their simultaneous expression of CD3 and CD56. In the process of clinical application, a common problem is that it is difficult to guarantee the provision of sufficient PBMC. To solve this problem, the current common method is to use cryopreservation technology to preserve peripheral blood mononuclear cells, resuscitate and i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 刘韬
Owner 深圳市博泰生物医疗机构管理有限公司
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