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Slow cryopreservation method of human T lymphocytes

A cryopreservation method and technology of lymphocytes, applied in the field of slow cryopreservation of human T lymphocytes, can solve the problems of little attention to cell cryopreservation and recovery methods, and achieve good cryopreservation effect, less damage to cells or human body, and reduced Effects of Probability of Quality Control Bias

Pending Publication Date: 2022-02-22
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

In the currently reported studies on cryopreservation of immune cells, little attention has been paid to the cryopreservation and recovery methods of cells. A few studies have mentioned the use of a programmed cooling device for cooling and rewarming in a water bath at 36-38°C, of ​​which 1 °C / min is the most commonly used cooling rate, but the rate of 1 °C / min is not necessarily suitable for all cell types

Method used

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  • Slow cryopreservation method of human T lymphocytes
  • Slow cryopreservation method of human T lymphocytes
  • Slow cryopreservation method of human T lymphocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Toxic effects of different protective agents on human T cells

[0064] Preparation of 1% DMSO + 0.1M trehalose + 4% HSA for human T lymphocyte cryopreservation: Take 17.115g trehalose (Sigma), dissolve it with physiological saline to a final volume of 50ml, and prepare 1M trehalose stock solution. Use a pipette to draw 0.5ml DMSO (Sigma) and 5ml trehalose mother solution into a 50ml centrifuge tube, add about 20ml of normal saline, then add 10ml of 20% HSA (purchased from GRIFOLS), mix well and add normal saline to a final volume of 50ml and stored in a 4°C refrigerator for later use.

[0065] Preparation of 0.2M trehalose + 4% HSA: pipette 10ml of trehalose mother solution into a 50ml centrifuge tube, add about 20ml of normal saline, then add 10ml of 20% HSA, mix well and add normal saline until the final volume is 50ml and stored in a 4°C refrigerator for later use.

[0066] Preparation of CS10 working solution (2:1 dilution): commercial protective agent ...

Embodiment 2、0

[0070] Example 2, Exploration of the cooling rate of 0.2M trehalose and 4% HSA

[0071] 2.1 Preparation of human T lymphocyte cryopreservation solution 0.2M trehalose and 4% HSA

[0072] Take 17.115g of trehalose (Sigma), dissolve it with physiological saline to a final volume of 50ml, and prepare 1M trehalose mother solution. Use a pipette gun to draw 10ml of trehalose mother liquor into a 50ml centrifuge tube, add about 20ml of normal saline, then add 10ml of 20% HSA (purchased from GRIFOLS), mix well, add normal saline to a final volume of 50ml, and place in a refrigerator at 4°C Save for later.

[0073] 2.2 Collection of human T lymphocytes and loading of protective agents

[0074] Take 50 μl of the expanded cultured human T lymphocyte suspension for counting. Adjust the total number of cells according to the counting results, collect by centrifugation, the centrifugal force is 500g, the centrifugation time is 5min, and the cell density in the freezing solution is adjus...

Embodiment 3

[0082] Example 3, 1%DMSO+0.1M trehalose, 4%HSA cooling rate exploration

[0083] 3.1 Preparation of human T lymphocyte cryopreservation solution 1% DMSO, 0.1M trehalose, 4% HSA

[0084] Take 17.115g of trehalose (Sigma), dissolve it with physiological saline to a final volume of 50ml, and prepare 1M trehalose mother solution. Use a pipette gun to draw 0.5ml DMSO (Sigma) and 5ml trehalose mother solution into a 50ml centrifuge tube, add about 20ml of normal saline, then add 10ml of 20% HSA (purchased from GRIFOLS), mix well and add normal saline until the final volume is 50ml, and store in a 4°C refrigerator for later use.

[0085] 3.2 Collection of human T lymphocytes and loading of protective agents

[0086] Take 50 μl of the expanded cultured human T lymphocyte suspension for counting. Adjust the total number of cells according to the counting results, collect by centrifugation, the centrifugal force is 500g, the centrifugation time is 5min, and the cell density in the fr...

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Abstract

The invention discloses a slow cryopreservation method of human T lymphocytes. The method comprises the steps of (1) putting a cryopreservation tube containing the human T lymphocytes and a cell cryopreservation liquid into a cooling agent for cooling; (2) enabling metal pre-cooled by liquid nitrogen to be in contact with the opposite side of the tube wall of the cryopreservation tube for nucleation; (3) placing the cryopreservation tube in the cooling agent for balancing; and (4) transferring the cryopreservation tube into low-temperature equipment, and cooling to -50 DEG C to -55 DEG C at the speed of 5-50 DEG C / min. According to the method, the degree of supercooling of the cryopreservation liquid in the cooling process is controlled, and the human T lymphocytes are cryopreserved in combination with the cooling rate, so that a better cryopreservation effect can be achieved, and the probability of quality control deviation in actual operation is reduced. The invention also solves the problem of cytotoxicity of the cell cryopreservation liquid, provides a combination formula with low or no DMSO protective agent, has osmotic pressure far lower than that of other cell cryopreservation liquids used in the market, and has less harm to cells or human bodies.

Description

technical field [0001] The invention belongs to the field of cells, and in particular relates to a slow freezing method for human T lymphocytes. Background technique [0002] Cancer is one of the malignant diseases that threaten the safety of human life. Its incidence has not decreased with the improvement of people's living conditions, but has been increasing year by year. With the development of gene carrier technology and gene editing technology, cell therapy has rapidly moved from scientific experiment to clinical application. In 2017, Kymriah (for the treatment of relapsed or refractory acute lymphoblastic leukemia), the first CAR-T therapy product jointly developed by the University of Pennsylvania and Novartis, was officially approved by the US Food and Drug Administration (FDA). Since then, the number of clinical trials of cell therapy worldwide has exploded. Unmodified human T lymphocytes are crucial for the production and preparation of CAR-T cells, and their lon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226A01N1/0284A01N1/0278
Inventor 黄智勇刘威薛素霞刘宝林赵鸿莲李鹰杨焕凤何晓文
Owner 上海原天生物科技有限公司
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