Culture and application of oligoclonal tumor-infiltrating lymphocytes for liver cancer

A lymphocyte and tumor infiltration technology, applied in the application field of liver cancer immunotherapy, can solve the problems of TIL amplification with high anti-tumor activity, TIL damage, and inability to selectively amplify TIL populations with strong anti-tumor activity

Inactive Publication Date: 2013-11-20
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The anti-tumor efficacy of traditional or improved cultured and amplified TIL is unsatisfactory. The fundamental reasons are as follows: (1) The digestion of tumor tissue by collagenase can damage TIL; (2) The purity of the isolated TIL Not high; (3) There is no selectivity for the amplification of TIL, and it cannot be amplified for TIL with high anti-tumor activity
[0006] Although the current method of preparing and expanding TIL can obtain a large number of cells, there are still defects, such as the inability to selectively amplify TIL populations with strong anti-tumor activity, etc.

Method used

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  • Culture and application of oligoclonal tumor-infiltrating lymphocytes for liver cancer
  • Culture and application of oligoclonal tumor-infiltrating lymphocytes for liver cancer
  • Culture and application of oligoclonal tumor-infiltrating lymphocytes for liver cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. The culture of oligoclonal liver cancer infiltrating lymphocytes, the relationship between TIL dysfunction and Treg, and the distribution characteristics of Treg in liver cancer tumor tissue

[0048] 1. Oligoclonal culture method

[0049] The oligoclonal culture method includes the following steps:

[0050] 1. Take fresh surgical specimens of liver cancer tumor tissue, remove surrounding normal tissue and necrotic tissue, and wash 3 times with PBS.

[0051] 2. Tissue blocks with a diameter of about 2 mm were collected from different parts of the tumor, including the central tissue of the tumor and the junctional area of ​​the tumor (the range of 2 mm inside and outside the border between the tumor and normal tissue).

[0052] 3. Add 2ml of complete medium containing IL-2 (6000IU / ml) to the 24-well plate. Complete medium (pH7.2) is RPMI 1640 medium, also includes: 25mmol / L HEPES (4-hydroxyethylpiperazineethanesulfonic acid), pH7.2, 100U / ml penicillin, 100U / m...

Embodiment 2

[0058] Embodiment 2, conventional culture method cultivates TIL (control example)

[0059] The conventional culture method for culturing TIL includes the following steps: take fresh liver cancer tumor tissue specimens, remove surrounding normal tissue and necrotic tissue, wash with PBS, rinse with RPMI1640 culture solution, move to a sterile vessel and cut the tumor tissue to 1 inch with surgical scissors. -2mm 3 Small pieces were placed in 40ml of RPMI1640 culture solution of digestive enzymes, stirred and mixed at room temperature, and left overnight at 4°C. Remove undigested tissue pieces and use lymphocyte separation medium to make 2×10 6 For a cell suspension of about / ml, after gradient density centrifugation, absorb the white cell layer rich in TIL between 75% and 100% of the separation liquid surface, wash with Hank's solution, centrifuge, and then culture and expand.

Embodiment 3

[0060] Example 3. Isolation, cultivation and identification of tumor infiltrating lymphocytes

[0061]A range of 2 mm inside and outside the boundary between liver cancer tumors and normal tissues was taken, and TILs were cultured as in Example 1 for dynamic observation. During TIL culture, TIL gradually migrated out of the tumor tissue mass and formed a dense lymphocyte population around the tumor tissue mass. Depend on figure 2 It can be seen that on the first day of culture, several lymphocytes can be seen to migrate out of the tumor tissue around the tumor mass; on the 3rd day of culture, more lymphocytes can be seen to migrate out of the tumor tissue and distribute around the tumor tissue; A further increase in lymphocyte density can be seen. On the 14th day of culture, the number of lymphocytes further increased and became almost confluent. With the prolongation of the culture time, the number of lymphocytes in the culture plate continued to increase. The cell densi...

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Abstract

The invention relates to culture and an application of oligoclonal tumor-infiltrating lymphocytes for liver cancer, and provides an oligoclonal cultural method of tumor-infiltrating lymphocytes (TIL), wherein TIL cell population with strong antitumor activity is selectively amplified from liver cancer tissues, and is applied to adoptive immunotherapy of liver cancer.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method capable of effectively extracting, expanding and culturing liver cancer infiltrating lymphocytes and its application in liver cancer immunotherapy. Background technique [0002] Hepatocellular carcinoma (HCC) is one of the five most common malignant tumors in the world, and my country accounts for about 45% of the world. At present, the treatment of liver cancer has developed from a single surgical resection to surgery-based multimodal comprehensive treatment including hepatic arterial chemoembolization, external radiation therapy, local therapy, and immunotherapy[1]. While these methods effectively kill tumors, they inevitably cause varying degrees of damage to the immune function of the body. With the rapid development of immunology and molecular biology, tumor biological therapy has become the fourth mode of tumor treatment after surgery, radiotherapy, and c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/407A61P35/00
Inventor 张小峰施乐华殷正丰张宗勤钱海华阎振林王葵李鹏鹏
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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