Tumor tissue tumor infiltrating lymphocyte (TIL) cell preparation method and dedicated culture medium

A tumor tissue and culture medium technology, applied in the field of cell culture, can solve the problems of low efficiency of inducing TIL in vitro, low killing ability of T cells, and failure to achieve the expected therapeutic effect, so as to reduce the culture cycle, reduce the complexity of culture, The effect of reducing the number of extractions

Active Publication Date: 2017-11-24
CENTURY BIOSTRENGTH BEIJING PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the use of traditional methods, the efficiency of in vitro induction of TIL is too low, the operation process is complicated, the amplification time is long, and the number of amplification is limited, and the proliferation rate is low; there are great differences in the number of cells cultured between different patients, and many patients The number of cells required for clinical treatment c

Method used

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  • Tumor tissue tumor infiltrating lymphocyte (TIL) cell preparation method and dedicated culture medium
  • Tumor tissue tumor infiltrating lymphocyte (TIL) cell preparation method and dedicated culture medium
  • Tumor tissue tumor infiltrating lymphocyte (TIL) cell preparation method and dedicated culture medium

Examples

Experimental program
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Effect test

Embodiment 1

[0033] A method for preparing tumor tissue TIL cells, comprising the steps of:

[0034] 1) Obtain paratumoral tissue under sterile conditions, rinse with sodium chloride injection, and cut off necrotic and connective tissue;

[0035] 2) The paratumoral tissue obtained in step 1) was cut into 1mm 3 Add 0.1% type I collagenase to the fragments overnight at 4°C, add 0.1mg / ml hyaluronidase I and 10ul / ml DNase the next day, digest at 35°C for 5h to obtain a cell suspension, and then The cell suspension is filtered, the filtrate is collected, and the supernatant is discarded by centrifugation to obtain a single cell suspension;

[0036] 3) Add the primary culture medium to the single cell suspension obtained in step 2), mix by pipetting, count the cells, and adjust the cell density to 1×10 6 cells / ml, inoculated in culture flasks, and evenly distributed the cells on the entire bottom surface, placed in a carbon dioxide constant temperature and humidity incubator, and cultivated fo...

Embodiment 2

[0041] A method for preparing tumor tissue TIL cells, comprising the steps of:

[0042] 1) Obtain paratumoral tissue under sterile conditions, rinse with sodium chloride injection, and cut off necrotic and connective tissue;

[0043] 2) The paratumoral tissue obtained in step 1) was cut into 2mm 3 Add 0.1% type I collagenase to the fragments overnight at 4°C, add 0.1mg / ml hyaluronidase I and 10ul / ml DNase the next day, digest at 38°C for 3h to obtain a cell suspension, and then The cell suspension is filtered, the filtrate is collected, and the supernatant is discarded by centrifugation to obtain a single cell suspension;

[0044] 3) Add the primary culture medium to the single cell suspension obtained in step 2), mix by pipetting, count the cells, and adjust the cell density to 2×10 6 cells / ml, inoculated in culture flasks, and evenly distributed the cells on the entire bottom surface, placed in a carbon dioxide constant temperature and humidity incubator, at 37±0.5°C, with...

Embodiment 3

[0049] A method for preparing tumor tissue TIL cells, the difference from Example 1 is that,

[0050] 1) Obtain paratumoral tissue under sterile conditions, rinse with sodium chloride injection, and cut off necrotic and connective tissue;

[0051] 2) The paratumoral tissue obtained in step 1) was cut into 2mm 3 Add 0.1% type I collagenase to the fragments overnight at 4°C, add 0.1mg / ml hyaluronidase I and 10ul / ml DNase the next day, digest at 36°C for 4h to obtain a cell suspension, and then The cell suspension is filtered, the filtrate is collected, and the supernatant is discarded by centrifugation to obtain a single cell suspension;

[0052] 3) Add the primary culture medium to the single cell suspension obtained in step 2), mix by pipetting, count the cells, and adjust the cell density to 2×10 6 cells / ml, inoculated in culture flasks, and evenly distributed the cells on the entire bottom surface, placed in a carbon dioxide constant temperature and humidity incubator, at ...

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Abstract

The invention relates to a tumor tissue tumor infiltrating lymphocyte (TIL) cell preparation method and a dedicated culture medium. The method comprises the following steps of tumor surrounding tissue obtaining, cell digestion, cell primary culture, cell subculture and cell collection, wherein a primary culture medium is based on a RPMI 1640 culture medium and prepared from the following concentration ingredients of 10% volume of human-derived serum, 20 to 45ng/ml of basic fibroblast growth factor (bFGF), 1 to 5mg/ml of riboflavin, 70 to 90ng/ml of cortisol, 10 to 25mg/ml of sodium dihydrogen phosphate monohydrate, 47 to 62ng/ml of recombinant human leukaemia inhibitory factor (LIF) and 500 to 800U/ml of IL-2; a subculture medium is based on the RPMI 1640 culture medium and prepared from the following concentration ingredients of 10% volume of the human-derived serum, 20 to 40mmol/L of HEPES, 1000 to 2000U/ml of the IL-2, 0.03 to 0.07mmol/L of beta-mercaptoethanol and 5 to 15ng/ml of sodium phosphate. According to the preparation method, an existing culture medium is improved, different culture mediums are utilized to culture the TIL cells in pertinence, TIL cell expansion capacity is improved, meanwhile a culture period is reduced, a culture complexity degree is reduced, a use amount of the IL-2 is reduced, and toxic reaction is reduced.

Description

technical field [0001] The invention belongs to the field of cell culture, in particular to a method for preparing tumor tissue TIL cells and a special culture medium. Background technique [0002] Tumor-infiltrating lymphocytes (TIL) are lymphocytes isolated from tumor tissue or pleural fluid of tumor patients. These cells can proliferate in large quantities after being stimulated by interleukin II (IL-2) in vitro. TIL cells are also called "tumor-derived activated cells". In addition to being suitable for patients with solid tumors, TIL therapy is also suitable for cancer patients with various types of advanced pleural and ascites. It has become an effective new immune therapy because of its advantages such as high targeting, strong specificity, low toxicity, and significant curative effect. Therapeutic method provides a more convenient way for adoptive immunotherapy, and provides a new treatment method for advanced cancer patients. [0003] In the 1980s, Professor Rosen...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N5/0783
CPCC12N5/0636C12N5/0693C12N2500/32C12N2500/34C12N2509/00
Inventor 李霞云潘新贺伟刘世红卢家堃
Owner CENTURY BIOSTRENGTH BEIJING PTY LTD
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