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Evaluation method for therapeutic effect of CAR-T immune cells and device thereof

A technology of immune cell therapy and evaluation method, which is applied in the field of evaluation method and device of CAR-T immune cell therapy effect, which can solve the problems of inaccurate prediction, low success rate, high cost, etc., and achieve low cost and high clinical prediction rate , the effect of short modeling cycle

Active Publication Date: 2019-08-16
SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

The study of the killing effect of CAR-T on solid tumors lacks efficient and accurate tumor models. Existing tumor models mainly include 2D tumor cell models and PDX models. The 2D cell tumor cell model lacks the microenvironment of tumor tissue, and the effect of anticancer drugs The prediction accuracy is low, and the PDX mouse model cycle is too long, the cost is too high, the success rate is too low, and there are species differences that lead to inaccurate predictions. Therefore, the tumor organoids generated by this patent can reduce the modeling cycle while improving Forecast accuracy

Method used

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  • Evaluation method for therapeutic effect of CAR-T immune cells and device thereof
  • Evaluation method for therapeutic effect of CAR-T immune cells and device thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0043] 1. Separation of human peripheral blood lymphocytes: extract 50ml of fresh peripheral blood from the patient, add it to 30ml of Ficoll lymphocyte separation medium, centrifuge at 800g for 20 minutes, take buffy coat cells and mix them with 50ml of D-PBS(-), centrifuge at 500g for 10 minutes , add 25ml 5% human serum albumin to resuspend the cells, pass through a 70μm sieve, and count the cells.

[0044] 2. Screen CD4 / CD8 positive T cells: Add 10 7 Cells were incubated at 4°C for 15min, then 2ml of sorting buffer was added, 250g, 4°C, centrifuged for 10 minutes, 5ml of sorting buffer was added to resuspend the cells, CD4 / CD8 cells were separated using a magnetic frame and LS separation column, and AIM- Resuspended cells in V medium, 2×10 5 ~1×10 6 U / L IFN-γ factor.

[0045] 3. T cell activation: 1 x 10 3 ug / L~1×10 4 ug / L CD3 monoclonal antibody and 1×10 3 ug / L~1×10 4 ug / L CD28 monoclonal antibody was added to the 24-well plate, sealed with parafilm, coated overnig...

Embodiment 2

[0050] 1. Use the primary cell extraction method to extract primary cells and count the cells;

[0051] 2. Put 2.0×10 in 4℃ environment 6 Mix one cell with 100μl Matrigel, inject it into a 1ml syringe, and inject fluorine oil into another syringe, put the fluorine oil and gel into two syringe pumps respectively and put them in a 4°C refrigerator

[0052] 3. The syringes are respectively connected to polytetrafluoroethylene (PTFE) tubes with a diameter of 800 μm, and the two tubes are merged into a polydimethylsiloxane (PMDS) tee, and then the tee is connected to another PTFE tube with a diameter of 800 μm. The entire platform was placed in a 4°C refrigerator.

[0053] 4. The flow rate of the injection pump containing glue is 20 μl / min, and the flow rate of the fluorine-containing oil injection pump is 50 μl / min to generate uniform tumor organoid spheroids, and place the generated spheroids at 37°C with 5% CO 2 The incubator freezes.

[0054] 5. The solidified spheres were t...

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Abstract

The invention relates to an evaluation method for therapeutic effect of CAR-T immune cells and a device thereof. Specifically, the method comprises the steps of: 1) preparation of CAR-T cell: constructing a CAR-T plasmid and packaging to lentivirus; obtaining a T cell of the subject, amplifying the cell after activation; and infecting the activated T cell with lentivirus, obtaining the CAR-T cellexpressing an antibody of the target gene; 2) preparation of 3D tumor-like organs: obtaining tumor tissue cells of the same subject as in step 1), obtaining primary cells, mixing primary cells with matrigel, and introducing matrigel and fluorocarbon oil containing primary cells into a three-way device respectively, and obtaining an organ-like sphere and culturing the material; 3) co-culturing the3D tumor-like organs with the CAR-T cells obtained in the step 1); and 4) measuring the survival rate of the tumor cells in the tumor-like organ spheres, and obtaining the therapeutic effect of the CAR-T cells. The method of the invention is simple, the preparation is fast, and the degree of reduction is high.

Description

technical field [0001] The invention relates to the field of pharmaceutical devices, and specifically discloses a method for evaluating the therapeutic effect of CAR-T immune cells and a device thereof. Background technique [0002] Malignant tumors rank second in the global mortality rate, and are second only to cardiovascular diseases as a serious threat to human health and social development. According to the data released by the International Cancer Research Center, in 2012, there were about 14.09 million new cases of malignant tumors in the world, and about 8.2 million deaths [1] . According to the data of National Cancer Center of China in 2014, there were about 3.804 million new cancer cases nationwide, and the incidence rate was 278.07 / 100,000. There were about 2.296 million cancer deaths in my country in 2014, and the mortality rate was 1.6789 / 100,000. The number of malignant tumor cases in China accounts for 25.49% of the world's malignant tumor cases, ranking f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02C12N5/09
CPCG01N33/5011G01N33/505G01N33/5082C12N5/0693C12N2513/00C12N2503/00Y02A50/30
Inventor 黄来强蒋盛威马少华刘昱宏
Owner SHENZHEN GRADUATE SCHOOL TSINGHUA UNIV
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