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A storage method of immune cells and cell cryopreservation solution

A storage method and immune cell technology, applied in the field of cell preservation, can solve the problems of immune cell sample and data information mismatch, sample data information is easy to lose, increase the risk of infectious diseases, etc., to maintain physiological functions and biological characteristics, The storage method is simple and effective, and the effect of protecting from freezing damage

Inactive Publication Date: 2019-02-12
DONGGUAN BOALAI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing technology makes the resuscitated cells less active and the cell survival rate is low, and most of the cell cryopreservation fluids currently used for freezing cells contain animal serum, that is, they contain foreign proteins compared to the human body, which increases the risk of infectious diseases; There are technologies to manage and track the cell bank for storing immune cells, and there are disadvantages such as easy loss and confusion of sample data information, which may easily cause a mismatch between immune cell samples and data information, and there are certain security risks

Method used

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  • A storage method of immune cells and cell cryopreservation solution
  • A storage method of immune cells and cell cryopreservation solution
  • A storage method of immune cells and cell cryopreservation solution

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Blood is collected from the peripheral blood of healthy subjects, and the preparation of autologous plasma includes the following steps: Centrifuge the peripheral blood of the human body for 20 minutes, take out the upper layer of plasma and put it in a container, inactivate it at 56°C for 30 minutes, and place it at -20°C Freeze for 10 minutes; then centrifuge at 2500 rpm for 15 minutes at 4°C, and take the supernatant to obtain autologous plasma for cell cryopreservation.

[0054] Short centrifugation time or too low centrifugal force of human peripheral blood will result in less plasma volume obtained from peripheral blood plasma separation. Long centrifugation time or too high centrifugal force will reduce the viability of lower blood cells, resulting in less immune cells obtained from the lower layer of PBMC induction culture. The centrifugation time, centrifugation speed and centrifugation temperature of this embodiment can not only obtain a relatively large amount...

Embodiment 2

[0103] The difference between the present embodiment and the storage method of embodiment 1 is:

[0104] Take 2 mL of resuspension of peripheral blood mononuclear cells after resuscitation, and use 10 mL of CIK cell culture medium (that is, 1000 mL of AIM-V medium containing IL-2 at a concentration of 1000 IU / mL and gentamicin sulfate at a concentration of 160 IU / mL) Resuspend and add to the culture container that has been pre-coated with anti-CD3 monoclonal antibody and retronectin, where the final concentration of anti-CD3 monoclonal antibody in the culture container is 5 μg / mL, the final concentration of retronectin is 12.5 μg / mL, and IFN-γ factor encapsulated by microcapsules, the final concentration of IFN-γ factor is 1000IU / mL, and then placed at 37°C, 5% CO 2 cultured in an incubator. Depending on the growth of the cells, replenish liquid, expand the culture, and count; co-culture for 17 days.

Embodiment 3

[0106] The difference between the present embodiment and the storage method of embodiment 1 is:

[0107] In this example, the preparation of autologous plasma includes the following steps: centrifuge human peripheral blood, take out the upper plasma and put it in a container, inactivate it at 55°C for 32 minutes, and freeze it at -22°C; Centrifuge at ℃, take the supernatant to obtain autologous plasma for cell freezing.

[0108] In this example, the step (3) is: divide the cell resuspension into cryopreservation bags, then put the cryopreservation bags into the cryopreservation bag box, perform program cooling to -78°C and then transfer to - Freeze in a liquid nitrogen tank at 196°C. After passing the test, a storage qualification report will be issued.

[0109] Further, the material of the sustained-release microcapsules is polylactic acid-polyethylene glycol.

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Abstract

The invention relates to the technical field of cell preservation, in particular to a storage method of immune cells and a cell freezing medium. The method includes: separating and collecting peripheral blood mononuclear cells, and adding the cell freezing medium to store the cells in a frozen manner, wherein the cell freezing medium comprises, by weight percentage, 0-62% of serum-free liquid culture medium, 0.5-1.5% of penicillin-streptomycin solution, 5-10% of dimethyl sulfoxide and 30-92% of autologous plasma. The storage method of the immune cells has the advantages that the method is simple and effective, the infectious disease risks of exogenous serum are avoided, the survival rate of the revived freezing-stored cells after induction culture is above 90%, the various biological indexes of the cells satisfy the requirements, and a good storage effect is achieved; the method can monitor and trace cell bank samples in real time, accurately record information such as cell storage positions, temperature and cell sources, each cell has a unique identifier, and the method is convenient to use and safe and reliable.

Description

technical field [0001] The invention relates to the technical field of cell preservation, in particular to a method for storing immune cells and a cell cryopreservation solution. Background technique [0002] Immune cells refer to cells involved in or related to immune responses, including lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. In recent years, tumor and virus immunotherapy techniques have been applied clinically, and the immunity of patients can be improved by stimulating, culturing, expanding and reinfusing autologous immune cells in vitro. Immune cell therapy mainly comes from the patient's own peripheral blood, and peripheral blood mononuclear cells are obtained through separation and collection. But generally speaking, the groups receiving immune cell therapy are mainly tumor or virus-infected groups. The function of immune cells in such groups is already very low, and the activity of isolated immune cells is low. Therefore...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
CPCA01N1/0215A01N1/0221A01N1/0226
Inventor 罗天恩杨志华刘虎赵含梅
Owner DONGGUAN BOALAI BIOLOGICAL TECH CO LTD
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