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Preparation and amplification method and application of gamma delta T cell

A cell, in vitro amplification technology, applied in the field of cell biology, can solve the problem of unable to generate a sufficient number of cells, and achieve the effect of contributing to functional cure, strong proliferation ability, and strong killing of tumor cells

Pending Publication Date: 2022-06-07
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, traditional γδT expansion protocols cannot generate sufficient numbers of cells to qualify as commercially viable allogeneic products

Method used

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  • Preparation and amplification method and application of gamma delta T cell
  • Preparation and amplification method and application of gamma delta T cell
  • Preparation and amplification method and application of gamma delta T cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Isolation of γδ T cells

[0079] Take the anticoagulated peripheral blood sample, after balancing, centrifuge at 20°C, 800×g for 20 minutes; transfer the upper plasma to a new sterile centrifuge tube. Use sterile phosphate buffered saline (PBS) 100ml to resuspend and dilute the cell pellet, and slowly attach the cell suspension to the human lymphocyte separation solution in stages (the volume ratio of cell suspension to human lymphocyte separation solution is 1:1: 1); Centrifuge at 20°C, 800×g for 20 minutes (1 increase, 0 decrease). Use a 10ml pipette to gently insert 0.5cm above the middle layer of thin cloud-like mononuclear cells, aspirate the layer of cells along the tube wall, transfer the middle layer of cells to a new 50ml sterile centrifuge tube, use 30ml sterile PBS was centrifuged at 20°C, 800 xg, for 20 minutes (1 9, 9 lower), and washed twice. Discard the supernatant, resuspend the cell pellet in 4ml x-vivo 15 medium, harvest PBMC, store at 4°C ...

Embodiment 2

[0084] Example 2 γδT amplification

[0085] Use 25ml of the prepared γδT cell culture medium to resuspend the γδT cells obtained by magnetic bead sorting, transfer the cell suspension to a 75cm2 culture flask, and place the culture flask in a saturated humidity, 37°C, 5.0% CO2 incubator to cultivate. The growth of cells in the flasks was observed by microscope every other day. According to the cell growth state, observe the cell density in time and control it at 0.5-2×10 6 cells / ml, when the density exceeds this range, the medium is replaced with γδT cell culture medium, and the cell density is adjusted to within this range. After 14-16 days of continuous culture, the γδT cells were harvested, and the aseptic operation was observed during the entire cell culture process. Cell culture supernatants were taken at 0, 3, 7, 14, and 16 days of cell culture, and counted under a microscope to determine cell viability according to the above method; The total number and expansion fo...

Embodiment 3

[0087] Example 3 In vitro construction of a chimeric antigen receptor expression vector targeting HIV-1 gp120

[0088] Using the pCDH-CMV-MCS-EF1α-Puro plasmid as the backbone, the MCS-EF1α-Puro fragment was removed by double digestion with EcoRI and SalI endonucleases. Then, using the pTRPE-3BNC117-G4H-BBz plasmid as a template, using onestep-3BNC117-F, onestep-3BNC117-R primers to PCR amplify ScFv, IgG4 hinge, CD8 molecules containing the variable region of HIV broadly neutralizing antibody Transmembrane region, 3BNC117 CAR fragment of 4-1BB. Finally, the double-enzyme digested product and the PCR product were gel recovered and then connected by Onestep homologous recombination. The ligated product was transformed in DH5α competent and then coated on Amp+ plate to screen positive clones. After the positive clones were expanded and cultured, the plasmids were extracted and sequenced for verification. The positive plasmid pCDH-CMV-3BNC117 ScFv-IgG4-CD8Tm-4-1BB was obtained an...

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Abstract

The invention belongs to the technical field of cell biology, and relates to a separation and amplification method of peripheral blood source gamma delta T cells. The invention provides an in-vitro method for separating, amplifying and modifying in-vitro amplified gamma delta T cells. The in-vitro method comprises the following steps: acquiring or separating gamma delta T cells; and / or expanding the isolated [gamma] [delta] T cells in a culture medium in the presence of a culture medium containing zoledronic acid, human recombinant interleukin 2 (IL-2) and human recombinant interleukin 7 (IL-7). The invention further provides the gamma delta T cell modified by the chimeric antigen receptor and application of the gamma delta T cell to preparation of anti-tumor and anti-AIDS products.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and relates to the separation, preparation, expansion and application of γδT cells. The invention also relates to a method for separating and amplifying γδT cells derived from peripheral blood, a method for in vitro gene modification of γδT cells, and the application of modified γδT cells in the field of anti-tumor. Background technique [0002] At present, T-cell immunotherapy for cancer continues to develop and achieve good clinical efficacy. However, barriers to allogeneic therapy remain. γδT cells do not require specific antigen stimulation when they exert their killing activity, and are not restricted by major histocompatibility complex (MHC). A powerful tool for healing. [0003] According to the different T cell receptors (TCRs), human T lymphocytes are divided into αβ T cells and γδ T cells. αβT cells are the main immune T cells in the body, and have the cytotoxic function of spe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/10C12N15/867C12N15/62A61K39/00A61K35/17A61P31/18A61P35/00
CPCC12N5/0636C07K16/1063C07K14/7051C12N15/86A61K39/001111A61K39/0005A61K35/17A61P35/00A61P31/18C12N2501/2302C12N2501/2307C12N2500/30C12N2740/15043C12N2510/00C12N2800/107C07K2317/622C07K2319/03C07K2319/33A61K2039/5158
Inventor 朱焕章姜正涛梁玥梁卉彤
Owner FUDAN UNIV
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