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Improved cryopreservation solution for peripheral blood mononuclear cells

A cryopreservation solution and nuclear cell technology, applied in the biological field, can solve problems such as forgotten steps, cumbersome operation, and physical disadvantages of patients, and achieve the effects of improving safety and economy, improving activity and stability, and maintaining cell activity

Inactive Publication Date: 2019-08-20
方艳秋 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing cell cryopreservation solution mostly uses 10% DMSO and 90% fetal bovine serum, but because this concentration of DMSO still has considerable toxicity, if the DMSO is not removed after cell recovery, it will be harmful to the patient's health
Moreover, the survival rate and cell viability of this kind of cryopreservation solution are low.
And this kind of ready-to-use frozen storage solution needs to be prepared and used immediately, the storage period is short, the batch difference is large, and t

Method used

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  • Improved cryopreservation solution for peripheral blood mononuclear cells
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  • Improved cryopreservation solution for peripheral blood mononuclear cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Configure mother liquor

[0048] Low-molecular-weight dextran mother liquor: purchased from Xi'an Wanlong Pharmaceutical Co., Ltd., with a mass fraction of 10%, that is, every 1 mL contains 40-100 mg of dextran.

[0049] Trehalose mother solution: fully dissolve 10mg trehalose powder in 0.5mL basal medium to fully dissolve it.

[0050] Polyethylene glycol stock solution: Fully dissolve 10mg of polyethylene glycol 6000 powder in 0.5mL of basal medium to fully dissolve it.

[0051] Recombinant human interleukin-2: purchased from Beijing Yuance Pharmaceutical Co., Ltd., with a specification of 1 million IU / bottle. Take 1 tube of IL-2 and add it to 250mL basal medium to prepare recombinant human interleukin-2 dilution.

[0052] Add 2.5mL low-molecular-weight dextran mother solution, 0.5mL trehalose mother solution, 0.5mL polyethylene glycol mother solution, and 0.5mL recombinant human interleukin-2 dilution to 5mL FBS in sequence, and finally 1mL DMSO was slowly added dro...

Embodiment 2

[0054] Configure mother liquor

[0055] Low-molecular-weight dextran mother liquor: purchased from Xi'an Wanlong Pharmaceutical Co., Ltd., with a mass fraction of 10%, that is, every 1 mL contains 40-100 mg of dextran.

[0056] Trehalose mother solution: fully dissolve 20mg trehalose powder in 0.5mL basal medium to fully dissolve it.

[0057] Recombinant human interleukin-2: purchased from Beijing Yuance Pharmaceutical Co., Ltd., with a specification of 1 million IU / bottle. Take 1 tube of IL-2 and add it to 250mL basal medium to prepare recombinant human interleukin-2 dilution.

[0058] Add 3mL low-molecular-weight dextran mother solution, 0.5mL trehalose mother solution, 1mL polyethylene glycol mother solution, 1mL recombinant human interleukin-2 to 4mL FBS in sequence, and finally add 0.5mL DMSO slowly to FBS, and gently blow and mix , placed in a 4°C refrigerator for storage.

Embodiment 3

[0060] Configure mother liquor

[0061] Low-molecular-weight dextran mother liquor: purchased from Xi'an Wanlong Pharmaceutical Co., Ltd., with a mass fraction of 10%, that is, every 1 mL contains 40-100 mg of dextran.

[0062] Trehalose mother solution: Fully dissolve 30mg trehalose powder in 1mL basic medium to fully dissolve it.

[0063] Polyethylene glycol stock solution: fully dissolve 30mg of polyethylene glycol 6000 powder in 1mL of basal medium to fully dissolve it.

[0064] Recombinant human interleukin-2: purchased from Beijing Yuance Pharmaceutical Co., Ltd., with a specification of 1 million IU / bottle. Take 2 tubes of IL-2 and add it to 250mL basal medium to prepare recombinant human interleukin-2 dilution.

[0065] Add 4mL low-molecular-weight dextran mother solution, 1mL trehalose mother solution, 1mL polyethylene glycol mother solution, 1mL recombinant human interleukin-2 to 2.5mL FBS in sequence, and finally add 0.5mL DMSO slowly to FBS, and gently blow and m...

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Abstract

The invention belongs to the field of biotechnology, and particularly relates to an improved cryopreservation solution for peripheral blood mononuclear cells, which comprises the following components:fetal bovine serum, dextran, DMSO, trehalose, polyethylene glycol and recombinant human interleukin-2. Fetal bovine serum and dextran are selected as nutritional support components of the cells to ensure cell nutrients and maintain a good osmotic pressure environment. The combination of permeable and non-permeable protective agents DMSO, trehalose and polyethylene glycol is used to effectively reduce cytotoxicity, maintain cell stability and improve the survival rate of recovery. By adding recombinant human interleukin-2, the activity and stability of cultured immune cells after recovery aresignificantly improved. The experimental results show that the PBMC cells cryopreserved with the improved cryopreservation solution provided by the invention have higher PBMC recovery rate, cell survival rate, stronger cell proliferation ability and high activity to be induced cultured into other immune cells after resuscitation. The cryopreservation solution provided by the invention can be directly frozen at minus 80 DEG C during cryopreservation.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an improved cryopreservation solution formula and a cryopreservation method used when extracting mononuclear cells from peripheral blood for cryopreservation. Background technique [0002] Peripheral blood mononuclear cell (PBMC) refers to cells with a single nucleus in peripheral blood, mainly including lymphocytes (T\B\NK), monocytes, phagocytes, dendritic cells and others Few cell types. The existing technology can cultivate isolated PBMCs into effector cells with different biological functions (such as DC, CIK, NK, CD3AK, γδT and other cells) under the action of different stimulating factors, which not only has good prospects in the treatment of cancer , and from the perspective of health preservation, it is also possible to extract immune cells from the peripheral blood of the patient when he is healthy, and to reinfuse the stored immune cells when the patient devel...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 方艳秋谭岩许淑芬魏海峰
Owner 方艳秋
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