Preparation method of high-activity tumor infiltrating lymphocytes

A tumor-infiltrating and lymphocyte technology is applied in the field of preparing tumor-infiltrating lymphocytes, which can solve the problems of cumbersome preparation methods and low yield of tumor-infiltrating lymphocytes, and achieve the advantages of avoiding cell loss, increasing the acquisition rate, and reducing the viscosity. Effect

Pending Publication Date: 2021-06-18
赜誉(上海)生物科技有限公司
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  • Claims
  • Application Information

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Problems solved by technology

[0012] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a method for preparing highly active tumor-infiltrating lymphocytes, which is used to solve the problems of cumbersome preparation methods and low yield of highly active tumor-infiltrating lymphocytes in the prior art. question

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0036] Method for preparing highly active tumor infiltration lymphocytes, including the following steps:

[0037] S1, sterile operation Collecting malignant tumor metastatic pulp lumen, placed in an ice bath at 4 ° C; transfer to the laboratory centrifugation to obtain cell enrich (different serum cavity liquid, Treatment mode);

[0038] S2, adding collagenase IV 0.1 wt%, DNA enzyme Type 1 0.002% by weight, and hyaluronase V 0.01 wt%, and the hyaluroniase V 0.01 wt% is added to the abandonment.

[0039] S3, all of the above-described digestive fluid is placed into a petriper, gently stirred and filtered, and the individual cells were removed twice after HBSS after eluting tissue block.

[0040] S4, the above cells were centrifuged at a density gradient, and the primary tumor infiltration lymphocytes were obtained.

[0041] S5, infiltrating the original tumor infiltrated lymphocytes according to 1.6 × 10 per hole 6 Cells / ML vaccinated to a complete RPMI 1640 medium, half-quoted c...

Embodiment 2

[0050] Method for preparing highly active tumor infiltration lymphocytes, including the following steps:

[0051] S1, the surgical resection lung cancer specimen is immersed in medical grade physiological saline within 2 hours after surgery (2 hours of tissue from the body), removing blood clot and necrotic tissue, fiber connective tissue, and retain tumor tissue as much as possible, with sharp Blade cut 1 × 1 × 1 mm 3 ;

[0052] S2, shredded the tissue small pieces into serum-free RPMI 1640 medium (containing collagenase IV0.1%, DNA enzyme TYPE 1 0.002%, hyaluronic acid enzyme V 0.01%), room temperature digestion 24 h To get digestible liquid;

[0053] S3, all of the above-described digestive fluid is placed into a petriper, gently stirred and filtered, and the individual cells were removed twice after HBSS after eluting tissue block.

[0054] S4, the above cells were centrifuged at a density gradient, and the primary tumor infiltration lymphocytes were obtained.

[0055] S5, inf...

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Abstract

The invention relates to the technical field of cell culture, in particular to a preparation method of high-activity tumor infiltrating lymphocytes which comprises the following steps: step 1, performing density-gradient centrifugation on tumor infiltrating lymphocyte digestive solution treated by collagenase, DNA enzyme and hyaluronidase; seeding the primary tumor infiltrating lymphocytes into a complete RPMI culture medium for proliferation; and seeding the tumor infiltrating lymphocytes at the first passage into a spinner bottle for continuous culture to obtain the high-activity tumor infiltrating lymphocytes. The invention solves the problems of complicated preparation methods and low yield of high-activity tumor infiltrating lymphocytes in the prior art. According to the preparation method, in the preparation process of the tumor infiltrating lymphocyte digestive solution, a comprehensive enzyme digestion method is adopted to digest tissue or cell enrichment, and comprehensive enzyme digestion can ensure the optimal acquisition rate of single cells. The preparation method can be used for preparing clinical tumor infiltrating lymphocytes, and enough lymphocytes can be obtained.

Description

Technical field [0001] The present invention relates to the field of cell culture techniques, and more particularly to a method of preparing highly active tumor infiltration lymphocytes. Background technique [0002] Tumor infiltrating lymphocytes, til / tils refers to lymphocytes separated in tumor tissues, have heterogeneous, mainly CD3 + T cells, including CD4 + CD8 + T cells. Current studies have shown that the number of TILs in tumor tissues has correlations. At the same time, the part of TILS cells can specifically identify and kill the lymphocytes of tumor cells. After the increasing culture is increasing, the tumor cell can be killed, and therefore, the tumor cells can be killed, and therefore there is a function of inhibiting tumor growth and inhibiting metastasis. This method of treating a tumor is called TILS treatment method, which is a method in the treatment of adverse tumors. [0003] In 1986, Rosenberg was isolated from tumor mice to obtain TILS cells. TILS cell p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/00C12N2509/10C12N2500/90C12N2501/515C12N2501/2302C12N2502/11
Inventor 王桂芳孟东朱毅踪家武
Owner 赜誉(上海)生物科技有限公司
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