Clinical TSCM (T-memory stem cells) induction culture and quality control identification kit and application

A kit and clinical technology, applied in the field of product quality control and identification, can solve problems such as easy differentiation into TEM, and achieve the effects of removing residual lesions, improving proliferation ability, and preventing recurrence and metastasis.

Pending Publication Date: 2020-11-06
路春光
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problem that TCM is easy to differentiate into TEM during in vitro culture; this application provides a method to isolate and purify T cells from fresh or cryopreserved human peripheral blood PBMC and umbilical cord blood mononuclear cells (UBMC). Kits and applications for inducing TSCM with a high induction activation rate; purified T cells can directly induce TSCM compared with PBMC, and the induction efficiency after purification is higher; then transfect tumor-specific or virus-specific TCR genes into autologous TSCM to enable immune cell therapy Long-acting and targeted

Method used

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  • Clinical TSCM (T-memory stem cells) induction culture and quality control identification kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] 1. Isolation of peripheral blood PBMC or umbilical cord blood UBMC and purification of T cells:

[0053] 100ml of sodium citrate anticoagulated blood was collected, PBMC and UBMC were separated by lymphocyte separation medium, T cells were purified, and cryopreserved.

[0054] 2. Recovery of T cells after freezing: Take out the frozen T cells, quickly put them into a 37 ℃ water bath, and shake gently constantly 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.

[0055] 3. Cell counting and trypan blue exclusion method to measure cell viability

[0056] The Countstar measures the cell sample and reads the average value of the cells display...

Embodiment 2

[0060] On the basis of Example 1, use the TSCM induction kit to induce TSCM:

[0061] The invention provides a TSCM induction culture kit, comprising a container and a clinically used TSCM cell serum-free expansion complete culture medium TSCM-I, a TSCM cell expansion coating liquid TSCM-II and an induction factor composition TSCM packed in the container -III:

[0062] 1) The present invention provides a serum-free TSCM cell expansion complete culture medium TSCM-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 7, human interleukin 15 and human interleukin 21. The present invention does not exclude the use of media other than the above serum-free complete media for culturing.

[0063] 2) The present invention provides a TSCM cell expansion coating solution TSCM-II, which contains recombinant human fibrin fragments and PBS, and the wor...

Embodiment 3

[0066] Example 3 Comparison of TSCM induction efficiency between T cells and PBMCs

[0067] On D0 day, TSCM-II was made into 12.5ug / ml working solution, coated into a culture bottle, and placed flat in a 37°C incubator for more than 2 hours. PBMC cells according to the cell density 1×10 6 / mL was resuspended with TSCM-III, in which 8ng / mL human interleukin 7, 8ng / mL human interleukin 15, 80ng / mL CD3 monoclonal antibody, and 80ng / mL CD28 were added. After the next day, TSCM cells were expanded and cultured with TSCM-I culture medium. After 12-15 days of expansion and culture, TSCM cells that reached clinical application standards were harvested, and the endotoxin, activity and phenotype of the cell preparations were tested. , the results of cell identification showed that the induction rate of TSCM directly induced by PBMC cells was lower than that of T cells, see image 3 At the same time, it can be seen that after 22 days of induction culture, CD8TCM accounted for 0.92%, CD...

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Abstract

The invention relates to the technical field of immune cell targeted therapy, and particularly relates to an optimized kit for separating and purifying T cells from fresh or low-temperature cryopreserved human peripheral blood mononuclear cells or umbilical cord blood mononuclear cells and inducing and amplifying TSCM (T-memory stem cells) through the T cells, a product quality control identification kit system and application. The TSCM have higher proliferation, self-updating and survival capability and can be differentiated into TCM (central memory T cell), TEM (effector memory T cell) and effector T cells. Mouse animal experiments show that compared with other differentiated T cell subgroups, the TSCM have higher anti-tumor effect in vivo. In clinical application, more reagents are adopted in the TSCM culture process, the cell quality standard is difficult to control, TSCM with uniform quality are difficult to culture in a short time, and a simple and formatted kit is urgently needed to be developed and has important significance for clinical application.

Description

technical field [0001] The present invention relates to the technical field of immune cell targeted therapy, in particular to an optimized isolation and purification of T cells from fresh or cryopreserved human peripheral blood mononuclear cells or umbilical cord blood mononuclear cells, and T cell induction and expansion of stem cell-like memory Kits for T cells (TSCM) and kit systems and applications 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, the currently reinfused immune cells are based on terminally differentiated effector T cells, which are difficult to survive in the body for a long time, which greatly affects the clinical efficacy of adoptive cell therapy. [0003] Gattinoni et al. found a class of antigen-specific CD8+ T cells with se...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00A61P31/12A61P31/06A61P31/18
CPCC12N5/0636A61K35/17A61P35/00A61P31/12A61P31/06A61P31/18C12N2500/34C12N2501/10C12N2501/30C12N2500/38C12N2501/998C12N2500/32C12N2500/05C12N2501/2307C12N2501/2315C12N2501/2321
Inventor 路春光
Owner 路春光
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