Method and application of sfn combined with IL-15 and IL-21 to prepare memory T cells

An IL-21 and memory technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problem of low differentiation state, achieve long-lasting and high-efficiency enhancement, long survival time in vivo, and high anti-tumor activity Effect

Active Publication Date: 2021-05-11
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a method and application of SFN combined with IL-15 and IL-21 to prepare memory T cells, so as to obtain a large number of T cells with memory characteristics and low differentiation state for the treatment of tumors, so as to solve the existing problems. Technical bottleneck of clinical T cell culture in technology

Method used

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  • Method and application of sfn combined with IL-15 and IL-21 to prepare memory T cells
  • Method and application of sfn combined with IL-15 and IL-21 to prepare memory T cells
  • Method and application of sfn combined with IL-15 and IL-21 to prepare memory T cells

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Embodiment 1

[0030] Collect 20 mL of peripheral blood under sterile conditions and use density gradient centrifugation to obtain PBMCs from human peripheral blood mononuclear cells. The specific steps are as follows: first, centrifuge at 1500 rpm for 10 minutes, and the acceleration and deceleration accelerations are both 9m / s 2 , transfer the serum to a sterile 50mL centrifuge tube, inactivate at 56°C for 25min, then centrifuge the serum at 4000rpm for 20min, the acceleration and deceleration accelerations are both 9m / s 2 . Use PBS buffer to dilute the precipitated blood cells at a ratio of 1:3 to the amount of blood cells, and then slowly add the blood cell dilution solution to the lymph separation solution, and the volume of the lymph separation solution is 1 / 3 of the volume of the blood cell dilution solution . Finally, 2500rpm, 25min centrifugation, its acceleration and deceleration acceleration are 5m / s 2 , carefully absorb the buffy coat layer, wash twice with PBS buffer, centrifu...

Embodiment 2

[0037] Collect 20 mL of peripheral blood under sterile conditions and use density gradient centrifugation to obtain PBMCs from human peripheral blood mononuclear cells. The specific steps are as follows: first, centrifuge at 1500 rpm for 10 minutes, and the acceleration and deceleration accelerations are both 9m / s 2 , transfer the serum to a sterile 50mL centrifuge tube, inactivate at 56°C for 25min, then centrifuge the serum at 4000rpm for 20min, the acceleration and deceleration accelerations are both 9m / s 2 . Use PBS buffer to dilute the precipitated blood cells at a ratio of 1:2 to the amount of blood cells, and then slowly add the blood cell dilution solution to the lymph separation solution, and the volume of the lymph separation solution is 1 / 2 of the volume of the blood cell dilution solution . Finally, 2500rpm, 25min centrifugation, its acceleration and deceleration acceleration are 5m / s 2 , carefully absorb the buffy coat layer, wash twice with PBS buffer, centrifu...

Embodiment 3

[0044] Collect 20 mL of peripheral blood under sterile conditions and use density gradient centrifugation to obtain PBMCs from human peripheral blood mononuclear cells. The specific steps are as follows: first, centrifuge at 1500 rpm for 10 minutes, and the acceleration and deceleration accelerations are both 9m / s 2 , transfer the serum to a sterile 50mL centrifuge tube, inactivate at 56°C for 25min, then centrifuge the serum at 4000rpm for 20min, the acceleration and deceleration accelerations are both 9m / s 2 . Use PBS buffer to dilute the precipitated blood cells at a ratio of 1:4 to the amount of blood cells, and then slowly add the blood cell dilution solution to the lymph separation solution, and the volume of the lymph separation solution is 1 / 4 of the volume of the blood cell dilution solution . Finally, 2500rpm, 25min centrifugation, its acceleration and deceleration acceleration are 5m / s 2 , carefully absorb the buffy coat layer, wash twice with PBS buffer, centrifu...

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Abstract

The present invention relates to the technical field of clinical immune cells, in particular to a method and application of SFN combined with IL-15 and IL-21 to prepare memory T cells, comprising the following steps: (1) collecting human peripheral blood and obtaining it by density gradient centrifugation Human peripheral blood mononuclear cells; (2) using a magnetic sorter to sort CD8 + T cells; (3) the sorted CD8 + T cells were cultured in a medium containing stimulators CD3 / CD28beads, cytokines IL-15, IL-21 and SFN; (4) CD8 cells with a large number of memory precursor cell phenotypes could be obtained after expansion to 7 days + T cells. This method can obtain a large amount of expansion of memory T cells in in vitro culture, solve the bottleneck of current clinical culture technology, enhance the long-lasting and efficient anti-tumor activity of immune cells, and provide a feasible treatment method for further clinical application of cellular immunotherapy.

Description

technical field [0001] The invention relates to the technical field of clinical immune cells, and the specific field is a method for preparing memory T cells. Background technique [0002] As the main force involved in adaptive immunity, T cells play an important role in the body's immune response, and are currently a research hotspot in cancer immunotherapy. As an important part of cell therapy, adoptive immune cell therapy refers to the infusion of immune effector cells cultured in vitro into patients to kill tumor cells in patients. Studies have found that the proliferative ability and anti-tumor activity of memory T cells in vivo are superior to effector memory T cells and effector T cells. In addition, memory T cells play an important role in maintaining the homeostasis and reconstruction of the immune environment. significance. This means that the longer the memory T cells survive in the body, the stronger their anti-tumor clinical effect will be. However, the in vi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCA61K35/17C12N5/0636C12N2501/2315C12N2501/2321C12N2501/405C12N2501/51C12N2501/515
Inventor 李红张震张毅
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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