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Resuscitation fluid and method for cryopreserved human PBMC

A recovery solution and cryopreservation technology, applied in biochemical equipment and methods, blood/immune system cells, microorganisms, etc., can solve the problems of difficulty in cell induction, low cell viability, and quantity loss, and improve cell viability, The effect of increasing vitality and increasing stability

Inactive Publication Date: 2020-09-18
珠海贝索细胞科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this way, the cells after cryopreservation and recovery will be affected by the preparation of the cryopreservation solution and the freezing time. Although there is no obvious difference in shape, there will be a large loss in number, and the cell viability is low, which will affect the future. Difficulty in inducing cells

Method used

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  • Resuscitation fluid and method for cryopreserved human PBMC
  • Resuscitation fluid and method for cryopreserved human PBMC
  • Resuscitation fluid and method for cryopreserved human PBMC

Examples

Experimental program
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Effect test

Embodiment 1

[0041] This example is used to introduce a frozen human PBMC resuscitation solution of the present invention and its preparation and reagent quality testing. The resuscitation fluid and its resuscitation method provided by the present invention do not contain DMSO, and can neutralize the toxic effect of DMSO on cells, decompose cell metabolites, improve cell viability, increase cell membrane stability, and increase cell viability after resuscitation , so that the cells can be induced and activated to the greatest extent; the ingredients used in the resuscitation solution of the present invention do not contain animal serum and heterogeneous proteins, and also avoid cell contamination by viruses, mycoplasma, fungi, etc., and the influence of serum batch differences.

[0042]A cryopreserved human PBMC resuscitation solution, the main components of which include recombinant human serum albumin, transferrin, glutamine, insulin, vitamin B, dextran, sodium chloride, and calcium chlor...

Embodiment example 1

[0078] Implementation case 1 result:

[0079]

[0080] As another realization, a cryopreserved human PBMC resuscitation solution, the main components include recombinant human serum albumin at a concentration of 1g / ml, transferrin at a concentration of 5g / ml, and glutamine at a concentration of 0.2ug / ml , insulin with a concentration of 3ug / ml, vitamin B with a concentration of 0.5mg / ml, dextran with a concentration of 0.2g / ml, sodium chloride with a concentration of 1mg / ml, and calcium chloride with a concentration of 0.1mg / ml. The preparation scheme is the same as before.

[0081] As yet another realization, a cryopreserved human PBMC resuscitation solution, the main components of the resuscitation solution include recombinant human serum albumin with a concentration of 20g / ml, transferrin with a concentration of 10g / ml, and a concentration of 0.5ug / ml glutamine, 10ug / ml insulin, 1mg / ml vitamin B, 0.5g / ml dextran, 10mg / ml sodium chloride, 0.5mg / ml calcium chloride. The ...

Embodiment 2

[0083] Collect the peripheral blood of many healthy volunteers, separate the peripheral blood mononuclear cells (human peripheral blood mononuclear cells), use a new type of special resuscitation solution for frozen human peripheral blood PBMC of the utility model to resuscitate the cells, and observe the cells at different time points Post-thaw viability and cell morphology.

[0084] 1. Preparation of human peripheral blood PBMC

[0085] (1) Collect 40ml of fresh peripheral blood from healthy volunteers and put it into a blood collection tube containing heparin sodium anticoagulant;

[0086] (2) On the 15ml lymphocyte separation medium, pour 20-30ml blood carefully;

[0087] (3) Centrifuge at 800xg for 20 minutes at room temperature, slowly rise and fall slowly. If the blood is stored for more than 2 hours, please increase the centrifugation time to 30 minutes;

[0088] (4) After centrifugation, the blood is divided into 4 layers, which are composed of plasma (upper layer),...

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Abstract

The present invention relates to a resuscitation fluid and method for a cryopreserved human PBMC. The resuscitation fluid includes the main components: recombinant human albumin with the concentrationof 5 g / ml, transferring with the concentration of 1 g / ml, glutamine with the concentration of 0.365 ug / ml, insulin with the concentration of 3.5 ug / ml, vitamin B with the concentration of 0.68 mg / ml,dextranum with the concentration of 0.3 g / ml, sodium chloride with the concentration of 6.9 mg / ml and calcium chloride with the concentration of 0.12 mg / ml. The resuscitation fluid contains no plasmas, the toxic effect of DMSO to cells may be effectively neutralized, cellular metabolites may be decomposed, the viability of the cells may be improved, the stability of cell membranes may be enhanced, the survival rate of the resuscitated cells may be increased, the bioactivity achieved before cryopreservation is recovered to the maximum extent, and thus, an immune cell may be effectively inducedto be activated to form a target cell.

Description

technical field [0001] The invention belongs to the technical field of immune cell therapy, and relates to a cryopreserved human PBMC resuscitation solution and a resuscitation method. [0002] technical background [0003] Peripheral blood mononuclear cells (PBMC) are a group of large granular lymphocytes, which are the body's first line of defense against pathogens and tumor cells, and are also used to prepare cytokine-induced killer cells (CIK) and LAK cells in vitro , natural killer cells (NK), NKT cells, CTL and γδT cells and other seed cells. [0004] When cancer patients are treated with cell therapy, it is generally used for treatment after surgical resection or together with radiotherapy and chemotherapy. However, after surgical resection or chemoradiotherapy, the immune function of the patient is low, and blood drawing is not used for cell culture in vitro. Therefore, for patients who need cell therapy, we need to draw blood before surgery or chemoradiotherapy. bl...

Claims

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Application Information

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IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2500/12C12N2500/14C12N2500/25C12N2500/32C12N2500/34C12N2500/38C12N2501/998
Inventor 孔伟圣蓝欣何娟娟陈智妍
Owner 珠海贝索细胞科学技术有限公司
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