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Nucleation cryopreservation method of immune cells

A cryopreservation method and technology of immune cells, applied in the field of cell biology, can solve the problems of lack of rapid cryopreservation methods for immune cells, etc., to solve the problem of uncontrollable sample cryopreservation process, good cryopreservation effect, and little damage to cells or human body Effect

Pending Publication Date: 2022-03-11
上海原天生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to solve the defect in the prior art that lacks a high survival rate and high recovery rate immune cell rapid freezing method, a method for nuclear freezing of immune cells, preferably T lymphocytes, is provided

Method used

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  • Nucleation cryopreservation method of immune cells
  • Nucleation cryopreservation method of immune cells
  • Nucleation cryopreservation method of immune cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The specific process of nuclear freezing of human T lymphocytes, the experimental results of 50℃ / min programmed cooling after nucleation at -5℃ and -10℃.

[0059] 1. Preparation of cryopreservation medium for human T lymphocytes

[0060] 13.692g trehalose (Sinopharm Chemical Reagent Co., Ltd.) was dissolved with 56ml physiological saline, and mixed; then slowly added 40ml human albumin injection (GRIFOLS, 10g (20%: 50ml)), 4ml ethylene glycol (Sinopharm Chemical Reagent Co., Ltd.), mix well, and store in a 4°C refrigerator for later use. Therefore, the formula of the 2×cell cryopreservation solution in this embodiment is: 8% HSA, 4% ethylene glycol, and 0.4M trehalose.

[0061] 2. Collection of Human T Lymphocytes and Loading of Protective Agents

[0062] Transfer the expanded and cultured human T lymphocyte suspension to a 50ml centrifuge tube and take 50μl for counting, balance and place in a centrifuge with a centrifugal force of 500g and a centrifugation time of 5...

Embodiment 2

[0070] In this embodiment, the survival rate and recovery rate of the cells are the results when the cells have just recovered. The survival rate is directly obtained from the Cellometer Auto 2000 cell counter (NexcelomBioscience, Lawrence, USA). The formula for calculating the recovery rate is as follows:

[0071] Cell recovery rate = viable cell concentration before cryopreservation / viable cell concentration after cryopreservation.

[0072] Determination of the best range of cooling rate after nuclear setting

[0073] 1. Preparation of cryopreservation medium for human T lymphocytes

[0074] 10.269g trehalose (Sinopharm Chemical Reagent Co., Ltd.) was dissolved with 20ml of normal saline, mixed evenly, and settled to a final volume of 30ml, and prepared into 1M trehalose mother liquor; The ratio is 2× freezing solution, and it is stored in a refrigerator at 4°C for later use.

[0075] 2. Collection of Human T Lymphocytes and Loading of Protective Agents

[0076] Transfer ...

Embodiment 3

[0084] Determination of the optimum range of core temperature

[0085] 1. Preparation of cryopreservation medium for human T lymphocytes

[0086] 10.269g trehalose (Sinopharm Chemical Reagent Co., Ltd.) was dissolved with 20ml of normal saline, mixed evenly, and settled to a final volume of 30ml, and prepared into 1M trehalose mother liquor; The ratio is 2× freezing solution, and it is stored in a refrigerator at 4°C for later use.

[0087] 2. Collection of Human T Lymphocytes and Loading of Protective Agents

[0088] Transfer the expanded and cultured human T lymphocyte suspension to a 50ml centrifuge tube and take 50μl for counting, balance and place in a centrifuge with a centrifugal force of 500g and a centrifugation time of 5min. Resuspend the cells with 1640 medium and determine the amount of 1640 medium to be added according to the counting results, and adjust the cell density to 1×10 7 A / ml or so. After adjusting the cell density, centrifuge again, the centrifugal ...

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Abstract

The invention discloses a cell cryopreservation solution, a nucleus cryopreservation method using the cell cryopreservation solution and application of the cell cryopreservation solution. The cell freezing medium is prepared from 4 to 6 percent of HSA (Human Serum Albumin), 2 to 4 percent of DMSO (Dimethylsulfoxide) or glycerol or ethylene glycol and 0.2 to 0.6 M of trehalose or lactose or cane sugar. The cryopreservation liquid provided by the invention has less harm to cells or human bodies. According to the nucleus placing cryopreservation technology, the technical problem of rapid cryopreservation of the T lymphocytes is solved, a good cryopreservation effect can be achieved when the cooling rate ranges from 15 DEG C / min to 90 DEG C / min, and the probability of production accidents in actual operation is reduced.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a method for setting and freezing immune cells. Background technique [0002] Cellular immunotherapy is currently internationally recognized as one of the most promising methods to overcome cancer. Regulatory T cells (T regulatory cells), λδT cells (gamma delta T cells), dendritic cells (dendritic cells) and genetic engineering technology Modified chimeric antigen receptor T cells (chimeric antigen receptor T cells) are important members of cellular immunotherapy, among which chimeric antigen receptor T cells (CAR-T cells) are effective in the treatment of hematological lymphoma Notably, it has been approved by the FDA in the United States and some European countries as an effective means of treating B-lineage lymphocytic leukemia. The production and preparation of CAR-T cells are based on human T lymphocytes, so long-term deep cryogenic storage of human T lymph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0284A01N1/0221
Inventor 刘威黄智勇杨焕凤何晓文刘宝林
Owner 上海原天生物科技有限公司
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