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TGamma delta cell derived from tonsil as well as preparation method and application thereof

A tonsil and cell technology, which is applied in the field of Tγδ cell preparation, can solve the problems of poor tumor cell killing ability, short cell survival time, and short expansion cycle, and achieve excellent anti-tumor or virus and excellent anti-tumor effects

Active Publication Date: 2022-01-04
CELARTICS BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is reported that the amplification period of this method is short (generally about 14 days, and the number of amplifications is limited), and the obtained T γδ The survival time of the cells is short (the amplified cells can continue to survive for about 7 days), the cells have poor anti-apoptosis ability, and the ability to kill tumor cells is poor.

Method used

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  • TGamma delta cell derived from tonsil as well as preparation method and application thereof
  • TGamma delta cell derived from tonsil as well as preparation method and application thereof
  • TGamma delta cell derived from tonsil as well as preparation method and application thereof

Examples

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preparation example

[0040] The lentiviral plasmid vector construction and the lentiviral preparation method comprise the following steps:

[0041] (1) Insert the gene fragment encoding the Tax protein of the STLV4 virus into a lentiviral vector, and transform the recombinant plasmid vector into E.coli. After the sequencing is correct and the bacteria are shaken, the recombinant plasmid vector is extracted for use.

[0042](2) Mix the obtained lentiviral plasmid vector of the STLV4 virus with the mixed packaging plasmid (the mixed packaging plasmid includes the expression plasmids of VSV-G, Gag-Pol and Rev), and add a transfection reagent; the transfection reagent can be, phosphoric acid Calcium transfection reagent, liposome transfection reagent or polymer transfection reagent. Subsequently, the lentiviral plasmid vector of the STLV4 virus and the mixed packaging plasmids are co-transfected into host cells for co-cultivation, and the host cells can be 293 cells, 293T cells or 293FT cells.

[004...

Embodiment 1

[0055] A tonsil-derived T γδ A method for preparing cells, comprising the steps of:

[0056] S1. Immune cells isolated from tonsils

[0057] A method for isolating immune cells from tonsils, comprising the following steps:

[0058] S11. Sample transportation and pretreatment: extract fresh tonsils from individuals who underwent conventional tonsillectomy, put fresh tonsils into RPMI1640 medium containing penicillin and streptomycin, and transport them to the laboratory under the condition of cold chain at 2-8°C , and complete the extraction within 1~3h.

[0059] In a biosafety cabinet, using sterile forceps, place a fresh tonsil specimen on a sterile 10 cm first cell culture dish, and keep the tissue moist with 2 mL of antibiotic-containing HANK'S buffer; Cut into small tissue pieces of 3~5mm.

[0060] S12. Prepare immune cell suspension:

[0061] S121. Put the sterilized stainless steel screen (200 mesh) into a sterile 10cm second cell culture dish;

[0062] S122. Add 2...

Embodiment 2

[0082] The difference between this example and Example 1 is that the operation of step S23 is: resuspend the cells with RPMI1640 medium containing 10wt% serum, add 200unit / mL IL2 to continue culturing the cells and change the medium every day for the first 2 weeks. Others are with embodiment 1.

[0083] The experimental results are: compared with Example 1, the culture medium is more likely to turn yellow, suggesting that the cells grow vigorously. However, after 2 weeks, the growth rate of the cells decreased significantly, a large number of dead cells appeared, and the overall state of the cells was worse than that of Example 1, resulting in a lower success rate of final preparation of such DC cells than that of Example 1.

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Abstract

The invention relates to the technical field of preparation of Tgamma delta cells, and particularly discloses a TGamma delta cell derived from tonsil as well as a preparation method and application thereof. The preparation method of the Tgamma delta cell comprises the following steps that S1, immune cells are separated from the tonsil; S2, the immune cells are taken to be in contact with Tax protein of STLV4 virus, and dendritic cells are prepared after cell culture; and S3, mixed culture is performed on the new immune cells prepared in the step S1 and the dendritic cells to prepare the T gamma delta cells existing in a cell culture. According to the TGamma delta cell derived from the tonsil as well as the preparation method and application thereof, the Tgamma delta cell can be used for preparing an anti-tumor or anti-virus cell product; and the Tgamma delta cell still has an excellent anti-tumor effect after being cultured in vitro for 3 weeks.

Description

technical field [0001] This application involves T γδ The technical field of cell preparation, more specifically, it relates to a kind of tonsil-derived T γδ Cells and methods for their preparation and use. Background technique [0002] The tonsils are immunocompetent organs that serve as the immune system's first line of defense against ingested or inhaled foreign pathogens, so tonsils are often congested to aid in the immune response to common ailments such as respiratory viral infections. However, recurrent tonsillitis has seriously affected the quality of life, so tonsillectomy has become the most commonly performed operation in otolaryngology to treat recurrent chronic tonsillitis. It is known that the tonsil mucosa contains a large amount of lymphoid tissue, which is the gathering place of various immune cells. Make full use of the immune cells in the resected tonsils, cultivate and activate them for autologous or allogeneic cancer, or the treatment of viral infecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0636C12N5/0639C07K14/005C12N2740/00022C12N2501/2302C12N2509/00C12N2509/10
Inventor 张欢于洋程铧
Owner CELARTICS BIOPHARMA CO LTD
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