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Method for culturing Treg cell

A cell culture and cell technology, which is applied in the field of cell culture and immunotherapy, can solve problems such as inability to proceed smoothly in in vitro functional experiments, biological safety needs to be studied, and the number of Tregs is small, and achieve stable functions, low cost, and easy operation.

Inactive Publication Date: 2021-04-09
BEIJING SHUANGYIN BIOTECHNOLOGY CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional CD25+CD4+Treg isolation method not only obtains a small number of cells, but also has low purity, especially in the in vitro expansion process, the Tregs that actually play a suppressive role have not been effectively expanded, so that the number of Tregs in the final cells obtained is very small , the follow-up in vitro functional experiments cannot be carried out smoothly. At the same time, the cost of traditional Tregs in vitro amplification method is high, and the biological safety needs to be studied

Method used

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  • Method for culturing Treg cell
  • Method for culturing Treg cell
  • Method for culturing Treg cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1I

[0031]Example 1IL10 and IL37 fusion genes and recombinant plasmids

[0032](1) Carrier: PCDH-EF1, 3 ug, 37 ° C for 37 ° C, was directly purified. The carrier size is about 7100 bp.

[0033](2) PCR IL10-IL37 fragment raw plasmid sequence 1037 (Zhongmei and synthesis), template 100 ng

[0034]Unempura 55 ° C with EX TAQ

[0035]PRIMER F: EF1-BAMHI-KOZAK-IL10-F, TM = 62 ° C

[0036](Primer sequence cttcccattcaggtgtgtgaggaattggatccccccatgcacagcagcgctctGCT)

[0037]PRIMER R: IL37-EcoRI-WPRE-R, TM = 55 ° C

[0038](Primer sequence acAaatttgtaatccagagaggtgattgtcgacgaattctgtcgctcacctcagaag)

[0039]Glue recovery 1323bp left and right fragments

[0040](3) Recombination with Zhongmei and 2X Mix, carrier and IL10-IL37 fragment

[0041](4) DH5A conversion, chalkiose sequencing

[0042](5) Sequencing primers: EF1 general primer PEF-F

[0043](6) Correct sequencing after storage of strains, large purification granules, plasmid spectrumfigure 1 Indicated.

[0044]The agarose from the above steps from Biowest, DNA Electrophoresis Marke...

Embodiment 2

[0046]Example 2 Preparation of recombinant slow virus carrying IL10-IL37

[0047](1) Removing 1 freezed 293T cells (purchased from ATCC) from liquid nitrogen (purchased from ATCC) to the 37 ° C water bath until ice cubes disappeared, add 15 ml of centrifuge tube containing 5 ml of preheating medium, 1200 rpm centrifuge 3min , Discarded, with a 293T medium (10% FBS + 1 mM pyruvate + 2 mM glutamine + 1% non-essential amino acid + DMEM), 37 ° C, 5% CO2 saturated humidity bring up. During the culture process, when the cell convergence reached 90%, the passage culture was performed, and the old medium was discarded, and 5 ml of sterilized PBS solution was added, gently shake, wash the cells after discarding the PBS solution, add 2mL 0.25% Trypsin EDTA digestive solution, digestion until the cells were completely digestion; the serum-containing medium was added to terminate digestion, and the cell suspension was centrifuged for 3 min, and the cells obtained by centrifugation were resuspended...

Embodiment 3

[0052]Example 3 Preparation of IL10-IL37 modified mesenchymal stem cells

[0053]The mesenchymal stem cells used are umbilical cord metaminite stem cells, using umbilical tissue blocks to climb the slice of mesenchymal stem cells, follow the steps:

[0054]Put the normal delivery of the human vault (containing 200 U / ml penicillin and 200U / ml streptomycin), flushing the huns of the umbilical vein and the umbilical artery with a syringe, and cut umbilical cord tissue with a syringe. Broken into 1mm3The size of the tissue block, then filter the small piece of umbilical cord to filter, collect the umbilical tissue block on the screen, remove too small umbilical tissue block, get tissue blocks having a diameter of 1 to 1.5 mm; direct tissue blocks directly Planted in the culture flask, placed at 37 ° C, 5% CO2In a saturated humidity incubator, stand for 1 to 2 hours; after the wall to be tissue is firm, add 1% of the α-MEM culture solution containing 10% fetal bovine serum, placed at 37 ° ...

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Abstract

The invention provides a mesenchymal stem cell for expressing IL-10 and IL-37, a method for culturing treg cells by using the mesenchymal stem cell, a corresponding cell culture container and application thereof.

Description

Technical field[0001]The present application belongs to the field of cell culture and immunotherapeutic, and in particular, the present application provides a method using mesenchymal stem cell culture TREG cells expressing I ol-10 and IL-37 and corresponding culture tools.Background technique[0002]Regulatory T cells (Treg) is a type of T cell population that controls autoimmune reactivity in vivo, which can inhibit cell populations of the immune response, play in immunopathology, graft tolerance, prevent autoimmune response and maintenance of immune balance. Important role. Although low in peripheral blood cells, only 5% to 10% of normal human peripheral blood, spleen tissue CD4 + T cells, but with tumor, autoimmune disease, graft acceptance, etc. is closely related.[0003]The number of regulatory T cells in the body is very small, accounting for 1-2% of normal human peripheral blood lymphocytes, small number of increases in vitro, low purity, and low cell proliferation ability. The...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/0783C07K14/54
CPCC12N5/0665C12N5/0637C07K14/5428C07K14/54C12N2510/00C12N2501/231C12N2501/23C12N2501/2302C12N2501/15C12N2502/137
Inventor 段海峰薛冰华于婷婷解晶刘丽华庞如梦陆颖张超
Owner BEIJING SHUANGYIN BIOTECHNOLOGY CO LTD
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