CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cell cryopreservation liquid for direct venous retransfusion and preparation method and application of CAR-T cell cryopreservation liquid

A cryopreservation and cell technology, applied in the field of biotechnology applications and medicine, can solve the problems of cumbersome cryopreservation process and subsequent application procedures, unsuitable for large-scale production, unclear composition of cryopreservation solution, etc., and achieve the maintenance of immune cell activity. The effect is good, the quantity and source of raw materials are not limited, and the effect of ensuring safety and effectiveness

Inactive Publication Date: 2018-09-21
WUHAN BIO RAID BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to overcome the deficiencies in the prior art, such as poor cryopreservation effect, unclear composition of cryopreservation solution, relatively cumbersome cryopreservation process and subsequent application p

Method used

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  • CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cell cryopreservation liquid for direct venous retransfusion and preparation method and application of CAR-T cell cryopreservation liquid
  • CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cell cryopreservation liquid for direct venous retransfusion and preparation method and application of CAR-T cell cryopreservation liquid
  • CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cell cryopreservation liquid for direct venous retransfusion and preparation method and application of CAR-T cell cryopreservation liquid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1- Embodiment 28

[0045] Example 1-Example 28: Preparation of cryopreservation solution

[0046] raw material

[0047] Table 1: Raw material ratio of frozen storage solution of embodiment 1-embodiment 28

[0048]

[0049] Embodiments 1-27 are the orthogonal experimental proportions of the frozen storage liquid raw materials, and the analysis of the orthogonal experimental results shows that embodiment 28 is the best proportion of the frozen storage liquid raw materials.

[0050] 1. Preparation method

[0051] Accurately weigh various raw materials according to the amount in Table 1 to obtain a frozen storage solution.

Embodiment 29

[0052] Example 29: Cryopreservation preparation of CAR-T cells

[0053] 2. Materials

[0054] 1) CAR-T cells, prepared according to the method of Chinese patent application 201610014730.6 (CAR-T cells from other sources can also be used).

[0055] 2) Freeze storage solution, prepared according to the ratio of Example 28.

[0056] 3. Method

[0057] 1) CAR-T cell isolation

[0058] a) Mix the CAR-T cells cultured for 10 days, transfer to a 50ml centrifuge tube, centrifuge at 500g for 6min, and discard the supernatant.

[0059] b) Resuspend the centrifuged cell mass with 10ml of 0.9% sodium chloride injection, confirm that the cell mass is completely broken, add 0.9% sodium chloride injection to 25ml, rest on the magnetic stand for 2min, pass through the magnetic force The magnetism of the frame realizes the separation of cells and magnetic beads.

[0060] c) Transfer the cell suspension to a new 50ml centrifuge tube with a pipette, and repeat the demagnetization operation ...

Embodiment 30

[0067] Example 30: Inspection of cryopreserved preparations of CAR-T cells

[0068] 1. Materials

[0069] The CAR-T cell cryopreservation preparation obtained in Example 30

[0070] Trypan blue staining reagent;

[0071] Other commercial freezing solutions: CTS TM Medium, purchased from Yingwei Jieji (Shanghai) Trading Co., Ltd.;

[0072] Tumor cell line: U266 cell line, purchased from China Center for Type Culture Collection.

[0073] 2. Method

[0074] 1) Cell recovery: Take out the cell cryopreservation bag from the liquid nitrogen tank, and put it into a 37°C water bath to thaw quickly; after the cell mixture in the cryopreservation bag is completely dissolved, slowly add the cell mixture into a certain volume of T In a centrifuge tube of complete cell culture medium, mix well; centrifuge at 500g for 6min, discard the supernatant, and set aside.

[0075] 2) Determination of cell viability

[0076] After the CAR-T cells were prepared in cryopreservation solution, s...

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Abstract

The invention discloses a CAR-T (Chimeric Antigen Receptor T-Cell Immunotherapy) cell cryopreservation liquid for direct venous retransfusion. The CAR-T cell cryopreservation liquid comprises the following components in percentage by volume: 2.0-10.0v/v% of dimethyl sulfoxide, 2.0-20.0v/v% of a inverted sugar solution, 5.0-40.0v/v% of a dextran solution, 1.0-10.0v/v% of a glucose solution, 10.0-40.0v/v% of a human albumin solution and 5.0-25.0v/v% of normal saline. The invention further provides a preparation method and application of the CAR-T cell cryopreservation liquid. According to the CAR-T cell cryopreservation liquid, components which are not suitable for clinical application in common cell cryopreservation liquids are avoided, clinically common medicines are selected and proportioned scientifically to prepare the CAR-T cell cryopreservation liquid, in addition, cryopreservation liquid preparation operation is simplified in a scientific storage mode, operation steps are simplified and cryopreservation effects are improved through optimized cryopreservation methods, and implementation of CAR-T treatment can be basically ensured.

Description

technical field [0001] The invention belongs to the fields of biotechnology application and medical technology, in particular to a CAR-T cell cryopreservation solution for direct intravenous infusion and its preparation method and application. technical background [0002] Immune cell therapy is a new type of therapy after surgery, chemotherapy, radiotherapy and targeted therapy, and is known as the "fifth major therapy" for cancer treatment. Although immunotherapy has been developed intermittently for many years, its research is achieving more and more exciting results, so it was named the first of the top ten scientific breakthroughs in 2013 by the top academic journal "Science". [0003] CAR-T immune cell therapy (Chimeric Antigen Receptor T-Cell Immunotherapy, Chimeric Antigen Receptor T-Cell Immunotherapy), is to use genetic engineering technology to specifically transform the patient's own immune T cells so that they can recognize tumor cells, Thereby achieving the ef...

Claims

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Application Information

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IPC IPC(8): A01N1/02A61K35/17A61K9/08A61K47/20A61K47/26A61K47/36A61K47/42A61K47/02
CPCA01N1/021A01N1/0226A61K9/0019A61K9/08A61K35/17A61K47/02A61K47/20A61K47/26A61K47/36A61K47/42
Inventor 张同存张尚昆顾潮江刘慧宁
Owner WUHAN BIO RAID BIOTECH CO LTD
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