Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Preparation method and application of human TSCMs (T memory stem cells)

A cell and blood cell separation machine technology, applied in the field of in vitro expansion of human peripheral blood TSCM cells, can solve the problems of not being able to meet one-time reinfusion, and the amount of cultured cells is small

Inactive Publication Date: 2016-05-04
SHENZHEN HORNETCORN BIOTECH
View PDF0 Cites 10 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The existing TSCM culture methods in foreign countries have a small amount of cultured cells, which cannot meet the amount required for one reinfusion

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] In Example 1, PBMCs were stimulated with rhIL-7, rhIL-15, rhIL-2, anti-CD3 monoclonal antibody, and anti-CD28 monoclonal antibody, and IM-12 was used to block stem cell differentiation to obtain TSCM cells. The specific operation includes the following steps:

[0013] 1. Under sterile conditions, collect peripheral venous blood from the patient with a blood cell separator or a 50mL syringe, and obtain mononuclear cells by density gradient centrifugation of Ficoll-diatrizoate glucosamine. The specific steps are: 1500 rpm, centrifuge for 10 minutes, absorb the upper plasma layer, inactivate at 56°C for 30 minutes and centrifuge for later use, dilute the precipitated blood cells with normal saline at a ratio of 1:1, human lymphocyte separation medium and diluted blood at a ratio of 1 Add the ratio of 2 to the centrifuge tube, centrifuge at 2000 rpm for 20 minutes, centrifuge the blood that has been left for a long time for 30 minutes, carefully absorb the buffy coat, wash ...

Embodiment 2

[0016] In Example 2, the morphology, viability and uniformity of the cultured TSCM cells were detected. The specific operation includes the following steps:

[0017] 1. Morphological observation. After 24 hours of cell culture, it can be seen that TSCM cells sink to the bottom of the culture dish and tend to colonize. After 48 hours, the colonies begin to grow larger.

[0018] 2. Viability detection, take the cultured cells, centrifuge at 1500 rpm for 5 minutes, discard the medium, stain with 100ng / mLPI containing 5% fetal bovine serum and 0.1% sodium azide for 15 minutes, 1500 rpm Centrifuge / centrifuge for 5 minutes, discard the supernatant, resuspend in normal saline, and detect with a flow cytometer.

[0019] 3. Homogeneity test, take the cells on day 0, 7, 14 and 21 respectively, wash them twice with normal saline containing 5% fetal bovine serum and 0.1% sodium azide, and adjust the cell density to 1×10 6 cells / mL, add 50 μL cell suspension to each detection tube, add f...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
concentrationaaaaaaaaaa
Login to View More

Abstract

The invention discloses a preparation method of human TSCMs (T memory stem cells), which comprises the following steps: (1) collecting peripheral venous blood with a blood cell separator or injector under aseptic conditions, and carrying out Ficoll-Hypaque density gradient centrifugation to obtain mononuclear cells; (2) putting the separated PBMCs (peripheral blood mononuclear cells) in a culture medium containing irritants and cell factors, regulating the cell density to (0.5-2)*10<6> / mL, transferring into a cell culture plate, culture bottle or culture bag, and culturing in an incubator; (3) after culturing the cells for 48-72 hours, replacing half of the culture solution, supplementing the equivalent culture solution to keep the cell density at (0.5-2)*10<6> / mL; and according to the cell growth state, harvesting the cells in the 10th-21st day. An anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody are utilized as the irritants to activate the TSCMs, the cell factors rh IL-7, rh IL-15 and rh IL-2 are combined for irritation, and IM-12 is utilized to stop the stem cell differentiation, so that the cultured TSCMs have the common characteristics of both memory cells and stem cells, thereby providing a new optional scheme for clinical adoptive immunity cellular therapy.

Description

technical field [0001] The invention belongs to the technical field of cellular immunology, in particular to a method and application for expanding human peripheral blood TSCM cells in vitro. Background technique [0002] In recent years, with the development of immunology technology, non-specific immune cell therapy has been accepted by more and more people in clinic because of its certain effect. At present, the existing immune cell therapy includes CIK, CTL and other cells, which have a short survival time in the body, and the therapeutic effect varies greatly with individual differences and different preparation methods. The ability of immune cells to self-renew, maintain homeostasis, and kill tumors will weaken with differentiation, and finally become terminally differentiated cells, showing a state of aging. Most of the CIK, CTL and other cells on the market tend to be terminally differentiated cells. TSCM cells can exist in the body for a long time through self-re...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0638A61K35/17C12N2501/2302C12N2501/2307C12N2501/2315C12N2501/50C12N2501/515
Inventor 崔博靖付凡饶秀茸马飞王宇环
Owner SHENZHEN HORNETCORN BIOTECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com