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

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...

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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 cannot be reached in a short period of time after culture; the commonly used culture methods at home and abroad need 40-50 days, and the CD3 in TIL cells + CD8 + The killing ability of T cells is not high, so the expected therapeutic effect is often not achieved, and the clinical application is greatly limited. There are not many feasible solutions that can be used in the clinical treatment of tumor patients.

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