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Optimized clinical TCM sorting, induction culture and quality control identification kit and application thereof

A kit and clinical technology, applied in tissue culture, biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of long-term survival of immune cells and lack of targeting, and achieve the elimination of residual lesions, prevention of Recurrence and metastasis, the effect of reducing the apoptosis index

Inactive Publication Date: 2019-01-25
路春光
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem of long-term survival of immune cells in the body and lack of targeting in adoptive cell therapy technology; TCM or CD8T+ cells, kits and applications with high TCM induction activation rate, and then transfect tumor-specific or virus-specific TCR genes into autologous TCM, so that immune cell therapy has long-acting and targeted

Method used

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  • Optimized clinical TCM sorting, induction culture and quality control identification kit and application thereof
  • Optimized clinical TCM sorting, induction culture and quality control identification kit and application thereof
  • Optimized clinical TCM sorting, induction culture and quality control identification kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] 1. Peripheral blood collection and dilution:

[0070] Collect 100ml of sodium citrate anticoagulant blood, divide it into two equally, add 16ml buffer CI to dilute, the volume is about 66ml, and prepare to add to CD8 gravity column.

[0071] 2. Recovery of PBMCs after freezing: Take out the PBMCs that have been frozen for 6 months, quickly put them into a 37 ℃ water bath, and keep shaking gently to ensure that the liquid in the tube is 80% dissolved within 1 min, and absorb the cells into a certain amount of DMEM washing solution , and wash the cryovial once, transfer it to a 50 mL centrifuge tube, and centrifuge at 233 g for 5 min. Discard the supernatant, add an appropriate amount of medium, mix gently, and retain 500 microliters for counting and measuring cell viability.

[0072] 3. Cell counting and trypan blue exclusion method to measure cell viability

[0073] The Countstar measures the cell sample and reads the average value of the cells displayed on the screen...

Embodiment 2

[0077] On the basis of Example 1, CD8+ T cells or TCM sorting kits were used for sorting:

[0078] Preparation and use of T cell subset magnetic bead sorting kit

[0079] The Human TCM Isolation Kit includes, Reagent1: CD8 Purification Reagent, consisting of CD8 Fab-Strep and Buffer IS. Reagent2: CD62L+ purification reagent, composed of CD62L+Fab-Strep and Buffer IS. Reagent3: CD45RA-purification reagent, composed of CD45RA-Fab-Strep and Buffer IS. Reagent4: Strep-Tactin Magnetic Microbeads. Reagent5: eluent, composed of D-biotin and Buffer IS.

[0080] 1) Wash CD8 with 8ml Buffer CI + T gravity column.

[0081] 2) Add 1ml of Reagent1 to the gravity column in step 1.

[0082] 3) Add 2ml Buffer CI to wash the gravity column after loading the sample in step 2.

[0083] 4) PBMC according to the density 1x10 6 -1x10 7 / ml on the column; peripheral blood is loaded on the column according to the volume of 66ml.

[0084] 5) Wash the column four times with 40ml Buffer CI, 10m...

Embodiment 3

[0101] Preparation and use of TCM induction kit

[0102] The invention provides a TCM induction culture kit, comprising a container and a serum-free expansion complete culture medium TCM-I for clinical use of TCM cells loaded in the container, TCM cell expansion coating liquid TCM-II and an inducer composition TCM -III:

[0103] 1) The present invention provides a serum-free TCM cell expansion complete culture medium TCM-I for clinical use, which contains carbohydrates, growth-promoting factors and hormones, vitamins, human albumin, amino acids, inorganic salt ions, Trace elements, water and human interleukin-2 and human interleukin-15. The present invention does not exclude the use of media other than the above serum-free complete media for culturing.

[0104] 2) The present invention provides a TCM cell expansion coating solution TCM-II, which contains recombinant human fibrin fragments and PBS, and the working concentration of recombinant human fibrin fragments is 12.5ug / ...

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Abstract

The invention relates to the technical field of immunocyte targeting therapy, in particular to an optimized kit system and application for separating and purifying memory immune cells (TCM) or CD8T+ cells from human peripheral blood or peripheral blood mononuclear cells, a TCM induction and amplification kit and a kit for product quality control and identification. In clinical application, TCM cells were cultured with many reagents, inconsistent standards, as that culture process has the advantages of large workload, long culture time, tedious process, instability, high contamination risk anddifficult control of cell quality standards, it is difficult for many technicians to culture TCM cell with uniform quality in a short period of time, and it is urgent to develop a simple and formattedkit. Meanwhile, the invention further optimizes the inducer composition TCM-III, the raw material is saved and the production cost is reduced; TCM-II-coated culture plate increased the proliferationof TCM cells by 2-3 times, the apoptotic index decreased, and the activity is increased by 10%-20%, the absolute number of effective cells increased by 2-3 times; before TCR gene transfection, TCM-II-coated culture plate can improve the transfection efficiency. The invention improves the process, which has important significance for clinically improving the curative effect of tumor treatment, removing residual lesions, and preventing the recurrence and metastasis of tumor.

Description

technical field [0001] The present invention relates to the technical field of immune cell targeted therapy, in particular to an optimized kit for isolating and purifying memory immune cells (TCM) or CD8 T+ cells from human peripheral blood or peripheral blood mononuclear cells, a TCM induction expansion kit and Kit system and application for product quality control identification. Background technique [0002] Adoptive cell therapy technology provides an effective method for breaking the central and peripheral immune tolerance of tumor patients, restoring or enhancing their anti-tumor immune function. However, there are still many problems to be solved, including: 1) how to obtain a sufficient number of tumor-reactive T cells is the most critical factor in determining the effect of adoptive cell therapy; 2) the current reinfusion of immune cells, especially cloned CD8+ It is difficult for T cells to survive for a long time in the body, which greatly affects the clinical ef...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12Q1/02A61K35/17A61P35/00A61P31/12A61P31/06A61P31/18
CPCA61K35/17A61P31/06A61P31/12A61P31/18A61P35/00C12N5/0636C12N2509/00G01N33/5005
Inventor 路春光
Owner 路春光
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