In-vitro amplification method of tumor infiltration lymphocyte (TIL)

A lymphocyte, in vitro expansion technology, applied in the biological field, can solve the problems of low expansion efficiency, limit the use of large amount of TIL reinfusion in tumor patients, and insufficient tumor lethality, so as to improve the lethality and promote the effect of in vitro proliferation

Inactive Publication Date: 2018-11-06
淮安诺康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] First, the amplification efficiency is not high, which limits the use of a large amount of reinfusion of TIL in tumor patients;
[0006] Second, the lethality to tumor cells needs to be improved, and the lethality to tumors after reinfusion into tumor patients is insufficient

Method used

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  • In-vitro amplification method of tumor infiltration lymphocyte (TIL)
  • In-vitro amplification method of tumor infiltration lymphocyte (TIL)
  • In-vitro amplification method of tumor infiltration lymphocyte (TIL)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 1. Experimental materials

[0059] X-VIVO-15 medium was purchased from Lonza company; phosphate buffered saline (PBS), RPMI 1640 medium, CD3 monoclonal antibody, CD28 monoclonal antibody were purchased from Gibco company; human AB serum was provided by Huaian Blood Center; IL-2 It was purchased from Beijing Sihuan Biopharmaceutical Company; collagenase IV was purchased from Sigma Company; lymphocyte separation solution was purchased from Tianjin Meide Pacific Company.

[0060] The human hepatoma cell line HepG2 was cryopreserved by our company. After resuscitation, it was placed in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO. 2 Subculture in an incubator.

[0061] Furan geranone A (CAS number: 1143-45-9), furan geranone B (CAS number: 1143-46-0), nootropic terpene glycol A (CAS number: 363610-30-4), nootropic Arlendiol B (CAS No.: 363610-32-6), Manzonone C (CAS No.: 5574-34-5), Manzonone F (CAS No.: 5090-88-0), Manzonone G (CAS No.: 7715-96-0...

Embodiment 2

[0089] A kind of TIL cell culture medium, by adding 20 μg / mL of furan geranone A, furan geranone B, nozhirendiol A, nozhirendiol B, mansong in X-VIVO-15 medium Ketone C or Mansonone G. When used for culturing TIL cells, it is only necessary to add an appropriate amount of anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody, which is convenient to use.

[0090] In the method provided by the present invention, by adding a small amount of furan geranone A, furan geranone B, nootropic terpene diol A, nootropic terpene diol B, mansonone C or mansonone G in the culture medium, not only can It can significantly promote the proliferation of TIL cells in vitro, and can also significantly improve the lethality of TIL cells to tumor cells. Furanopelargonone A, furanopelargonone B, nootropic perolindiol A, nootropic perolindiol B, mansonone C or mansonone G can be used to prepare TIL cell culture medium.

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Abstract

The invention discloses an in-vitro amplification method of tumor infiltration lymphocyte (TIL). The method includes the steps of S1, separating the TIL, to be more specific, soaking cut-up para-carcinoma tissue into a collagenase solution for digestion, filtering, performing PBS washing, collecting filtrate, centrifuging, abandoning supernate, adding PBS to prepare a cell suspension, using lymphocyte separation liquid to perform discontinuous density gradient centrifuging, collecting the lymphocyte of the lower interface after the centrifuging is finished, performing PBS washing, culturing ina culture medium containing 10% human AB type serum and IL-2 in a suspending manner according to the concentration of 1*10<6>/mL, and changing liquid once every other 3-5 days; S2, culturing and amplifying, to be more specific, when the lymphocyte in holes grows to 1*10<7>, transferring into a culture medium containing anti-CD3 monoclonal antibody, anti-CD28 monoclonal antibody and medicine for incubation, adding IL-2 after 24 hours to perform culture, and changing a fresh culture medium containing IL-2 with the same concentration every other 2-3 days in a half-amount manner to perform culture for 22-25 days.

Description

technical field [0001] The invention belongs to the field of biology, and relates to a method for expanding tumor-infiltrating lymphocyte TIL in vitro. Background technique [0002] Immunotherapy is an emerging tumor treatment method, which has been listed as the fourth treatment method after surgery, radiotherapy, and chemotherapy, and is gradually playing an important role in the comprehensive treatment of tumors. In 2010, the US FDA approved Provenge, the world's first cellular immunotherapy product, to go on the market, fully demonstrating the value of cellular immunotherapy in tumor treatment. [0003] Tumor-infiltrating lymphocytes (TIL) are a heterogeneous population of lymphocytes present in the tumor stroma. Its main component is T lymphocytes present in the tumor stroma, and a small part is MHC non-restricted NK cells, whose common feature is the expression of T cell receptor (TCR). TILs are derived from tumor tissue regions, can specifically recognize autologous...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N5/0646C12N2500/30
Inventor 何英广马思航
Owner 淮安诺康生物科技有限公司
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