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Method for transfecting immune cells by lentivirus

A technology of lentiviral transfection and immune cells, applied in blood/immune system cells, viruses, animal cells, etc., can solve the problems of inaccessibility and high transfection efficiency

Active Publication Date: 2021-07-16
HEBEI SENLANG BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, traditional transfection aids such as Polybrene and PS (Protamine Sulfate) cannot achieve high transfection efficiency on immune cells, so it is necessary to find more effective methods for transfection

Method used

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  • Method for transfecting immune cells by lentivirus
  • Method for transfecting immune cells by lentivirus
  • Method for transfecting immune cells by lentivirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Materials and Methods

[0066] 1. Obtaining of PBMC (peripheral blood mononuclear cells) and CBMC (cord blood mononuclear cells):

[0067] Take peripheral blood or umbilical cord blood from a healthy person, add an equal volume of normal saline to dilute at room temperature, and slowly add it to the lymphocyte separation solution along the wall of the centrifuge tube, so that the ratio of diluted blood to lymphocyte separation solution is 2:1. 2000rpm (equivalent to 872g), centrifuged for 20min, and divided into four layers from the bottom of the tube to the liquid surface after centrifugation, followed by erythrocyte and granulocyte layer, liquid separation layer, mononuclear cell layer, and plasma layer. Aspirate the mononuclear cell layer into a new centrifuge tube, add physiological saline not less than 3 times the volume of the mononuclear cell layer to wash the cells, 2000rpm, 5min, twice, and count the cells. Obtain PBMC or CBMC.

[0068] 2. Sorting and culturi...

Embodiment 2

[0128] Example 2 lentivirus transfection of αβT cells derived from peripheral blood

[0129] The peripheral blood-derived αβT cells cultured for 2 days in Example 1 were used and divided into PS group, PGE2 group, P407 group and Stau group. According to 3×10 5 Inoculate each well in a 24-well plate, add serum-free medium (KBM581+200IU / ml IL-2) to each well, add the virus concentrate U6-SR8-MND1904 prepared in Example 1 according to the amount of MOI=2, PS Group, PGE2 group, P407 group and Stau group were added different transfection aids according to the concentration of PS 8μg / ml, PGE21μM, P407100μg / mL, Stau 200nM, and the final volume of the system was 500ul / well. Centrifuge at 2000rpm at 35°C for 2h, take it out and put it in 37°C, 5% CO 2 For culture, the medium is KBM581+200IU / ml IL-2, and tested after 96h.

[0130] The transfection efficiency was detected by flow cytometry, and the specific antibodies used were EGFR-APC, CD3-APC-cy7, CD4-FITC, CD8-PB, 7AAD, and SA-PE....

Embodiment 3

[0133] Example 3 lentivirus transfection of peripheral blood-derived γδT cells

[0134] The peripheral blood-derived γδT cells cultured for 6 days in Example 1 were used and divided into PS group, PGE2 group, P407 group and Stau group. According to 1×10 6 Cells were inoculated at a concentration of 1 / mL, the medium was serum-free medium (KBM581+1000IU / mlIL-2), and the virus concentrate U6-SR8-MND1904 prepared in Example 1 was added according to the dosage of MOI=50, PS group, PGE2 group, P407 group and Stau group were added with different transfection-assisted reagents according to the concentrations of PS 8 μg / ml, PGE2 1 μM, P407 100 μg / mL, and Stau 200 nM, and the final volume of the system was 500 μl / well. The incubation was continued after centrifugation at 35°C for 2 hours. The next day, the medium was replaced with the expansion medium described in Example 1. 96h after transfection, samples were taken for detection.

[0135] The transfection efficiency was detected b...

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Abstract

The invention discloses a method for transfecting immune cells by lentivirus. The method comprises the step of transfecting immune cells in a transfection system containing prostaglandin E2 by adopting lentivirus, wherein the lentivirus comprises chimeric antigen receptor gene transcribed RNA. The method can be applied to transfection of alpha beta T cells, gamma delta T cells and NK cells, the transfection efficiency is high, and the influence of transfection on immune cells can be reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for lentivirus transfection of immune cells. Background technique [0002] CAR-T, the full name is Chimeric Antigen Receptor T-Cell Immunotherapy, Chimeric Antigen Receptor T-cell Immunotherapy, refers to the genetic material (CAR) with specific antigen recognition domain and T cell activation signal through gene modification technology ) into T lymphocytes, so that T lymphocytes can be directly combined with specific antigens on the surface of tumor cells to be activated. After T lymphocytes are activated, on the one hand, they directly kill tumor cells by releasing perforin and granzyme B; . At the same time, immune memory T cells can also be formed to obtain a specific long-term anti-tumor mechanism. CAR-T therapy has significant efficacy in the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered to be one of the most promising tumor treatments. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
CPCC12N15/86C12N5/0636C12N5/0646C12N2740/15043C12N2800/107C12N2510/00C12N2501/02C12N2501/06C12N2501/24C12N2501/2302C12N2501/2315C12N2501/2321
Inventor 李建强牛星邢思捷
Owner HEBEI SENLANG BIOTECH CO LTD
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