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Method for separating, culturing and in vitro amplifying tumor-infiltrating lymphocytes from primary liver cancer

A primary liver cancer, lymphocyte technology, applied in the field of bioengineering

Pending Publication Date: 2019-07-23
THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

The disadvantage is that the use of human AB serum and donor PBMC these allogeneic blood components will bring potential risks of GVHD (graft versus host disease), HIV and other viral infections to the clinical research of subsequent immunotherapy

Method used

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  • Method for separating, culturing and in vitro amplifying tumor-infiltrating lymphocytes from primary liver cancer
  • Method for separating, culturing and in vitro amplifying tumor-infiltrating lymphocytes from primary liver cancer
  • Method for separating, culturing and in vitro amplifying tumor-infiltrating lymphocytes from primary liver cancer

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Experimental program
Comparison scheme
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Embodiment Construction

[0035] Embodiments of the present invention will be described in detail below.

[0036] 1. Reagents and instruments

[0037] Type Ⅳ collagenase, hyaluronidase and DNase were purchased from Sigma; RPMI1640 medium was purchased from Gibico; phosphate buffer saline PBS powder was purchased from Boster; fetal bovine serum was purchased from Gibico; 40um filter was purchased from Falcon company; recombinant interleukin-2 (rIL-2) for human injection was purchased from Beijing Sihuan Biological Company (trade name: Xinde Lusheng); anti-CD3 monoclonal antibody (OKT3), anti-CD28 monoclonal antibody were purchased from ebioscience; Cell separation medium was purchased from StemCell Company; HBSS liquid was purchased from Corning Company; cocktail flow cytometry antibodies (PerCP-CD3, FITC-CD4, PE-CD8) were purchased from ACEABio Company; six-well cell culture plates were purchased from Thermo Company; CO 2 The cell culture box was purchased from FormaScientific Company of the United St...

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Abstract

The invention discloses a method for separating, culturing and in vitro amplifying tumor-infiltrating lymphocytes from primary liver cancer. The inventor takes sterile fresh tumor nest and para-carcinoma tissues, performs digestion separating through a mixed enzyme solution, and inoculates a co-stimulation culture medium so as to realize the culture and in vitro amplification of lymphocytes. In the aspect of extraction, the method solves the key problem of low TIL extraction quantity and purity; in the aspect of culture, the method enables TIL to be differentiated and amplified to CD8+TIL subtype with immunologic activity; in the aspect of amplification, the method performs in-vitro culture by creatively using peripheral blood plasma of a liver cancer patient and PBMC for breeding, effectively amplifies TIL cells, and solves the problem of immunological rejection caused by autoimmune TIL immunotherapy; and in the aspect of sorting, the method uses a StemCell magnetic bead method to sort out CD8+TIL cells with specific recognition capability, lays a good foundation for the clinical research of the next immunotherapy, and has remarkable application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a method for separating, culturing and in vitro amplifying primary liver cancer tumor infiltrating lymphocytes. Background technique [0002] Primary hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is a highly malignant tumor. The number of deaths from liver cancer in my country exceeds 120,000 every year, accounting for about 44% of the death toll from liver cancer in the world, and its case fatality rate ranks third among all types of tumors. . Although surgical treatment is the first choice for the treatment of liver cancer, the 5-year survival rate of most patients is very low due to the rich blood supply of liver cancer and its easy metastasis and recurrence. The low quality of life and poor prognosis of patients with primary liver cancer after surgery are still very prominent. The current clinical targeted drug Sorafenib can only prolong the survival of patients by 3-4 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2501/2302C12N2501/515C12N2501/51
Inventor 蒋小峰黄镇辉张大伟卢海武温子龙郑强刘颂航杨学伟曹良启彭和平薛平
Owner THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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