Efficient amplification method of TILs serving as sources of cancerous pleural effusion

A cell, thoracic and abdominal technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problems of affecting the effect of TIL cells and the low killing ability of T cells, to increase the number of cells and killing activity, enhance The effect of high killing ability and cell activity

Inactive Publication Date: 2013-12-25
青岛麦迪赛斯医疗技术有限公司
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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 expansion of TIL in vitro is limited, usually in about 20 days, the number of cells is only expanded by

Method used

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Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0033] 1) Coating of the culture flask: Take 100 micrograms of laminin and fibronectin respectively, dissolve them fully in 20 ml of phosphate buffer, filter the liquid with a 0.22 micron sterile filter membrane, and add it to the bottom with an area of ​​175 square centimeter culture flask at room temperature for 4 hours.

[0034] 2) Cell inoculation: collect 600 ml of cancerous ascites, centrifuge at 1500 rpm for 10 minutes. Resuspend the cell pellet with 30ml of serum-free medium. Slowly add the cell suspension to the centrifuge tube to which 15ml of lymphocyte separation medium has been added. Centrifuge at 2000 rpm for 15 minutes. Take the white PBMC cell layer in the middle, wash it twice with normal saline, resuspend it with 60 ml of serum-free medium, take a sample and count it, and calculate the cell number as 2.6×10 7 indivual. The cell suspension was inoculated into the culture flask without the coating solution, and 60000 units of IL-2 was added. The component...

experiment example 2

[0044] 1) Coating of the culture flask: Take 100 micrograms of laminin and fibronectin respectively, dissolve them fully in 20 ml of phosphate buffer, filter the liquid with a 0.22 micron sterile filter membrane, and add it to the bottom with an area of ​​175 square centimeter culture flask at room temperature for 5 hours.

[0045] 2) Cell inoculation: collect 400 ml of cancerous pleural effusion, centrifuge at 1500 rpm for 10 minutes. Resuspend the cell pellet with 30ml of serum-free medium. Slowly add the cell suspension to the centrifuge tube to which 15ml of lymphocyte separation medium has been added. Centrifuge at 2000 rpm for 15 minutes. Take the white PBMC cell layer in the middle, wash it twice with normal saline, resuspend it with 80 ml of serum-free medium, take a sample and count it, and calculate the cell number as 3.2×10 7 indivual. The cell suspension was inoculated into the culture flask without the coating solution, and 80000 units of IL-2 was added.

[0...

experiment example 3

[0050] 1) Coating of the culture flask: Take 100 micrograms of laminin and fibronectin respectively, dissolve them fully in 20 ml of phosphate buffer, filter the liquid with a 0.22 micron sterile filter membrane, and add it to the bottom with an area of ​​175 square centimeter culture flask at room temperature for 3 hours.

[0051] 2) Cell inoculation: collect 900 ml of cancerous ascites, centrifuge at 1500 rpm for 10 minutes. Resuspend the cell pellet with 30ml of serum-free medium. Slowly add the cell suspension to the centrifuge tube to which 15ml of lymphocyte separation medium has been added. Centrifuge at 2000 rpm for 15 minutes. Take the white PBMC cell layer in the middle, wash it twice with normal saline, resuspend it with 80 ml of serum-free medium, take a sample and count it, and calculate the cell number as 3.6×10 7 indivual. The cell suspension was inoculated into the culture flask without the coating solution, and 80000 units of IL-2 was added.

[0052] 3) T...

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PUM

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Abstract

The invention aims to provide an efficient amplification method of TILs serving as sources of cancerous pleural effusion. The TILs prepared with the method have the advantages of being high in multiplication speed, large in number, high in cell activity and the like. The method is mainly characterized in that by utilizing a cultivation bottle coated by laminin protein and fibronectin, the TILs are cultivated. The adsorption effect of the two kinds of protein can make cells in half-adherence state, therefore, the cells are favorable for being stimulated by various factors, and the two kinds of protein has the multiplication promoting effect on the TILs; by using a resistant OX-40 monoclone antibody, co-stimulatory signals of CD3+CD8+T cell subgroups in the TILs can be stimulated, cell activation is promoted, the expression of cell perforin and granular enzyme is improved, and the killing capacity of the TILs is enhanced. So far, the study report that the laminin protein, the fibronectin and the resistant OX-40 monoclone antibody are combined and used for preparing the TILs has not been given, and the method for increasing the cell number and improving the killing activity by adding the three factors in preparation of the TILs is put forward for the first time.

Description

technical field [0001] The invention belongs to the field of cell culture, and in particular relates to the culture of tumor infiltrating lymphocytes (Tumor Infiltrating lymphocytes, TILs), that is, the combination of various cytokines is used to stimulate lymphocytes derived from pleural and ascites, so that they can be efficiently expanded. Background technique [0002] Tumor infiltrating lymphocytes (Tumor Infiltrating lymphocytes, TIL) refer to lymphocytes that infiltrate into the surrounding tumor tissue, and the cell phenotype is CD3 + CD8 + T and CD3 + CD4 + T cells are predominant, and there are also a small amount of NKT cells and NK cells. Since most TIL cells have been in contact with tumor cells, they can theoretically have a specific killing effect on tumor cells. However, due to the presence of strong immunosuppressive factors in the tumor microenvironment, the function of TIL cells is actually greatly inhibited and cannot effectively kill tumor cells. [...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 徐矫健孙威
Owner 青岛麦迪赛斯医疗技术有限公司
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