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Method for cultivating and separating tumor-specific TIL cells

A tumor-specific and separation method technology, applied in the field of cultivation and separation of tumor-specific TIL cells, can solve the problems of instability, hinder research progress, and few reports of biological characteristics, and achieve strong tumor specificity and shortened culture period. , the effect of strong lethality

Active Publication Date: 2022-01-04
ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although it has been proposed to isolate and expand TIL cells extracted from tumor-associated malignant pleural effusion, due to unclear tumor-specific targets, there are few reports on the biological characteristics of tumor-specific TIL obtained by screening and amplifying malignant pleural effusion. Those that have been reported cannot meet the requirements
In addition, the cumbersomeness and instability of the cultivation process are also problems hindering research progress

Method used

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  • Method for cultivating and separating tumor-specific TIL cells
  • Method for cultivating and separating tumor-specific TIL cells
  • Method for cultivating and separating tumor-specific TIL cells

Examples

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Embodiment 1

[0043] This embodiment provides a method for cultivating and isolating tumor-specific TIL cells derived from gastric cancer ascites, comprising the following steps:

[0044] 1. After obtaining gastric cancer ascites, centrifuge at 100 g for 10 minutes at low speed to separate tumor cell clusters and immune cells. The pellet is tumor cell clusters, and the supernatant is immune cells. Pipette the supernatant into a new centrifuge tube, and then centrifuge at 1000g for 10 minutes to obtain immune cell pellets.

[0045] 2. After washing the tumor cell mass obtained in step 1 with HBSS, add an equal amount of Matrigel and mix well.

[0046]3. After the cell liquid is solidified, add tumor organoid medium (according to the composition of the final concentration: Glutamine, 5mM; Nicotinamide, 10mM; A83-01, 0.5μM; Penicillin, 100U / mL; Streptomycin, 100μg / mL; SB -202190, 10μM; EGF, 50ng / mL; L-WRN cell culture supernatant, 50%; solvent is DMEM / F12 medium), at 37 ℃, 5% CO 2 concentrat...

Embodiment 2

[0057] This embodiment provides a method for cultivating and isolating ovarian cancer ascites-derived tumor-specific CD45+ TIL cells, comprising the following steps:

[0058] 1. After obtaining ovarian cancer ascites, centrifuge at a low speed of 100g for 10 minutes to separate tumor cell clusters and immune cells. The pellet is tumor cell clusters, and the supernatant is immune cells. Pipette the supernatant into a new centrifuge tube, and then centrifuge at 1000g for 10 minutes to obtain immune cell pellets.

[0059] 2. After washing the tumor cell mass obtained in step 1 with HBSS, add an equal amount of Matrigel and mix well;

[0060] 3. After the cell liquid is solidified, add tumor organoid medium (Glutamine, 2.5mM; Nicotinamide, 5mM; A83-01, 1μM; Penicillin, 100U / mL; Streptomycin, 100μg / mL; SB-202190, 5μM; EGF, 75ng / mL, Gastrin, 10nM; N-acetylcysteine, 2.5mM; FGF-10, 100ng / mL; L-WRN cell culture supernatant, 40%; solvent is DMEM / F12 medium), at 37℃, 5% CO 2 concentra...

Embodiment 3

[0071] This embodiment provides a method for cultivating and isolating tumor-specific TIL cells derived from lung cancer pleural effusion, comprising the following steps:

[0072] 1. After obtaining lung cancer pleural effusion, centrifuge at 100g for 10 minutes at low speed to separate tumor cell clusters and immune cells. The pellet is tumor cell clusters, and the supernatant is immune cells. Pipette the supernatant into a new centrifuge tube, and then centrifuge at 1000g for 10 minutes to obtain immune cell pellets.

[0073] 2. After washing the tumor cell mass obtained in step 1 with HBSS, add an equal amount of Matrigel and mix well;

[0074] 3. After the cell fluid is solidified, add tumor organoid medium (Glutamine, 8mM; Nicotinamide, 2.5mM; A83-01, 2.5μM; Penicillin, 150U / mL; Streptomycin, 150μg / mL; SB-202190, 2.5μM; EGF, 150ng / mL, Gastrin, 15nM; N-acetylcysteine, 7.5mM; FGF-10, 50ng / mL; L-WRN cell culture supernatant, 30%; solvent is DMEM / F12 medium), at 37°C, 5%CO ...

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Abstract

The invention provides a method for cultivating and separating tumor-specific TIL cells. The method comprises the following steps: step 1, obtaining tumor cell clusters and immune cell sap in pleuroperitoneal fluids; step 2, performing tumor organoid culture on the tumor cell clusters; step 3, performing amplification culture on immune cells; step 4, treating tumor organoid obtained by culture into single cells, and co-culturing the single cells and the immune cells subjected to amplification culture; and step 5, separating a specific immune cell population in the co-cultured cells. According to the method disclosed by the invention, tumor organoids and the tumor-related immune cells are simultaneously separated and cultured by utilizing the sample from the same source, and then the tumor organoids and the immune cells are co-cultured, so that the obtained tumor-specific TIL cells can keep the biological effect of the organoids, the quantity is large, and the culture period is obviously shortened. In addition, the TIL cells separated and cultured by the method are higher in tumor specificity, so that the tumor killing activity is higher.

Description

technical field [0001] The invention relates to the technical field of cell cultivation, in particular to a method for cultivating and separating tumor-specific TIL cells. Background technique [0002] Tumor infiltrating lymphocytes (Tumor infiltrating lymphocytes, TILs) are lymphocytes present in tumor tissues, tumor metastatic lymph nodes, and tumor-associated malignant pleural and peritoneal effusions. Compared with lymphocytes in blood or other organs, TIL cells contain a higher proportion of tumor-specific lymphocytes. This type of lymphocytes has been used as a clinical treatment method for adoptive cellular immunotherapy of tumors after isolation, expansion and activation in vitro, and has been used in malignant melanoma, acute lymphoblastic leukemia (ALL ) and other cancers have achieved remarkable results. However, this adoptive cellular immunotherapy method has little effect on other cancer types. One of the important reasons is how to find accurate TILs that hav...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/0781A61K35/17A61P35/00
CPCC12N5/0635C12N5/0636A61K35/17A61P35/00C12N2509/10C12N2500/32C12N2501/11
Inventor 郑斌朱宇陈泽新兰坚强黄敏
Owner ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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