Cell membrane for rapid amplification of NK cells and application thereof

A NK cell and cell membrane technology, applied in the field of biomedicine, can solve the problem of low NK cell expansion efficiency, reduce the loss and pollution probability, promote in vitro proliferation, and improve the effect of expansion efficiency

Active Publication Date: 2021-03-23
SHANGHAI NAT ENG RES CENT FORNANOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cells currently used for NK cell therapy are mainly derived from autologous NK cells. In vitro, cytokines (IL-15, 4-1BBL) and feeder cells are used to promote the rapid expansion of NK cells in about 2 weeks to obtain high purity. NK cells, but the expansion efficiency of NK cells using traditional methods is low

Method used

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  • Cell membrane for rapid amplification of NK cells and application thereof
  • Cell membrane for rapid amplification of NK cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Synthesis and construction of IL-15 and 4-1BBL expression plasmids:

[0040] The nucleic acid and amino acid sequences of IL-15 and 4-1BBL were obtained from the NCBI database, and IL-15 and 4-1BBL molecules were synthesized by the method of total gene synthesis, and then the obtained IL-15 sequences and 4-1BBL molecules were respectively Constructed into pDNA3.1 molecule by double enzyme digestion method, the transfection-positive strains were expanded and cultured, and the pDNA3.1-IL-15 and 4-1BBL plasmids were extracted using the endotoxin-free plasmid extraction kit.

[0041] Nucleic acid sequence: (IL-15+ linker +transmembrane protein)

[0042]gaattcgccgccaccATGGAGTTCGGACTCAGTTGGCTGTTCCTGGTGGCCATCCTGAAGGGTGTGCAGTGATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGTTGCAAAGT...

Embodiment 2

[0051] Cultured transfection of K562 cells:

[0052] Recovery and culture of K562 cells:

[0053] The original cell of K562 is a malignant tumor cell of the hematopoietic system with multilineage differentiation potential, which can spontaneously differentiate into identifiable progenitor cells of erythroid, myeloid and monocytic. Culture conditions: 1640 medium 10% FBS, subculture method: maintain cell density at 10 5 -10 6 Each / ml, change the medium 2-3 times a week.

[0054] Electroporation:

[0055] Use BIO-RAD's electroporator to electroporate the plasmids of pDNA3.1-IL-15 and 4-1BBL, the steps are as follows:

[0056] For a 0.2 cm electroporation cup, the cell density is 10x10^6 cells / ml, the amount of DNA is 2 μg, the volume of electrotransfer solution is 100 μl, the voltage is 130 V, and the capacitance is 950 μF. After electroporation, 1640 medium was quickly added, and the cells were transferred to culture flasks to resume culture.

Embodiment 3

[0058] Preparation of cell membranes for NK cell expansion

[0059] Grow at K562 cell density up to 10 9 -10 10 cells / ml, collect the cells, wash the cells with PBS, add 10mM tris hydrochloric acid solution, stir at 4°C for 2 hours to break the cells, then centrifuge at low temperature for 30 minutes, collect the precipitate, wash 3 times with 10mM tris hydrochloric acid solution, After centrifugation at low temperature and high speed for 10 minutes, the pellet was resuspended with PBS to obtain the material of K562 cell membrane.

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Abstract

The invention belongs to the field of biological medicine, and relates to a cell membrane for rapid amplification of NK cells and application thereof. The invention also relates to sorting and amplification processes of the NK cells. The invention discloses the cell membrane. 41BBL and IL15 are expressed on the surface of the cell membrane. The invention further relates to a preparation method ofthe cell membrane. According to the invention, a set of NK cell separation and rapid efficient amplification method is established, PBMCs (or the NK cells separated by magnetic beads) are used as an original material, the NK cells are specifically amplified in vitro through stimulation of specific cytokines, and the NK cells are obtained and used for treating malignant tumors.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to a cell membrane for rapid expansion of NK cells and an application thereof. The invention also includes the process of sorting and expanding the NK cells. Background technique [0002] NK cells (natural killer cells, Natural killer cells), as an important innate immune effector cell, are the body's first line of defense against tumors. NK cells are the third type of lymphocytes other than T and B cells, and exist in lymphoid organs and peripheral tissues. CD56+ and CD16+ lymphoid cells are clinically identified as NK cells. NK cells are different from T cells and are non-specific immune cells. They do not need antigen pre-sensitization, and they can directly kill target cells when they meet tumor cells or virus-infected cells for the first time, so they are called natural killer cells. [0003] There are several ways for NK cells to kill target cells: Natural cytotoxicity: NK cells rel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/85C12N15/12C12N15/62C12N5/0783
CPCC12N5/0694C12N15/85C07K14/70575C07K14/5443C12N5/0646C12N2510/00C12N2800/107C07K2319/03C12N2509/00C12N2533/90C12N2501/2315C12N2501/599
Inventor 崔大祥梁辉倪健田静李雪玲沈琦高昂
Owner SHANGHAI NAT ENG RES CENT FORNANOTECH
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