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Human Tscm cells and preparation method and application thereof

A cell and nuclear cell technology, applied in the field of biological immunology, can solve the problems of limited acquisition of initial cells, unsuitable maintenance of Tscm cells, and small number of Tscm cells, so as to enhance anti-tumor ability, enhance long-lasting and high-efficiency, and enrich the source Effect

Active Publication Date: 2018-06-12
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the methods of cultivating and expanding Tscm cells at home and abroad cannot meet the actual clinical needs. On the one hand, most of the blood sources come from the peripheral blood of patients or relatives of patients, which greatly limits the acquisition of initial cells; on the other hand, Tscm cells are cultured in vitro It is not suitable for stable maintenance, and it is easy to further differentiate, so that the number of cultured Tscm cells is small

Method used

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  • Human Tscm cells and preparation method and application thereof
  • Human Tscm cells and preparation method and application thereof
  • Human Tscm cells and preparation method and application thereof

Examples

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Embodiment 1

[0036] Collect 50 mL of umbilical cord blood under sterile conditions and use density gradient centrifugation to obtain CBMCs from human umbilical cord 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, ...

Embodiment 2

[0041] Collect 50 mL of umbilical cord blood under sterile conditions and use density gradient centrifugation to obtain CBMCs from human umbilical cord 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 in a ratio of 1:2 to the amount of blood cells, and then slowly add the blood cell dilution solution to the lymphatic separation solution, and the volume of the lymphatic 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 ...

Embodiment 3

[0046] Collect 50 mL of umbilical cord blood under sterile conditions and use density gradient centrifugation to obtain CBMCs from human umbilical cord 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 / 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, ...

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Abstract

The invention belongs to the field of biological immunology, and particularly relates to human Tscm cells and a preparation method and application thereof. The preparation method comprises the following steps of: (1) acquiring human umbilical cord blood and obtaining human umbilical cord blood mononuclear cells by adopting a density gradient centrifugation method; (2) sorting CD8+CD45RA +CCR7+initial T cell subsets by a flow cell sorter; (3) adding the sorted initial T cell subsets into a medium containing the stimulant CD3 / CD28 beads, the cytokine IL-2 and metformin for culture; and (4) conducting medium change and cytokine IL-2 supplement every 2 days, so that CD8+T cells with a Tscm phenotype can be obtained after 14 days of amplification. By the adoption of the method, a large number of T cells with a memory characteristic and a low differentiation state can be obtained for the treatment of tumors.

Description

technical field [0001] The invention belongs to the field of biological immunology, and in particular relates to a human Tscm cell and its preparation method and application. Background technique [0002] With the continuous deepening of research in the field of biomedicine and the rapid development of immunological theory and technology, immunotherapy has become a new hope for human beings to defeat tumors. As an important part of immunotherapy, adoptive immunotherapy of immunocompetent cell infusion refers to the infusion of activated immune effector cells in vitro to patients to kill tumor cells in patients. However, while this therapy is giving birth to hope, it also faces huge challenges: the immune cells infused into patients by adoptive cellular immunotherapy are in a terminally differentiated state, have a short survival time in the body, and have limited anti-tumor effects. How to obtain more youthful, durable and stable anti-tumor cells is a hot spot in current im...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCA61K35/17C12N5/0638C12N2501/105C12N2501/2302C12N2501/51C12N2501/515
Inventor 张毅张震张超奇
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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