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Preparation method of EBV specific cytotoxic T cells

A cytotoxic and specific technology, applied in the field of cell biology and biomedicine, can solve the problems of low efficiency, high cost and long time course of EBV-CTL preparation

Active Publication Date: 2021-04-09
山东省齐鲁细胞治疗工程技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the existing preparation defects such as low production efficiency, high cost and long time course of EBV-CTL, the present invention provides a preparation method of EBV-specific CTL, which can significantly improve the amplification efficiency and killing function of EBV-CTL

Method used

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  • Preparation method of EBV specific cytotoxic T cells
  • Preparation method of EBV specific cytotoxic T cells
  • Preparation method of EBV specific cytotoxic T cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of stimulated cell fz-BLCL

[0032] EBV-transformed human B lymphocytes can be obtained by infecting human B lymphocytes with viruses carrying EBV antigens. The EBV-transformed human B lymphocytes used in the present invention were purchased from the Kunming Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (EBV-Human , No.: KCB 200546M, referred to as BLCL). The initial density of BLCL culture was 5.0×10 5 / mL, the culture medium used is RPMI-1640, add 20% FBS, add 1% Gluta Mix, pass once every two days during the culture process, BLCL culture condition is 37 ℃ incubator, containing 5% CO 2 and saturated water vapor. When the cells are expanded to a sufficient number, the reserved samples are frozen and irradiated:

[0033] The dose of BLCL cryopreservation is 1.0×10 7 / cartridge, the freezing solution is fetal bovine serum FBS containing DMSO at a final concentration of 10%, and the freezing volume is 1mL; ...

Embodiment 2

[0036] Example 2 Preparation, cultivation and identification of EBV-CTL

[0037] 1. Isolation of Mononuclear Cells from Cord Blood or Peripheral Blood

[0038] The source of the mononuclear cells is umbilical cord blood, and the isolation of the mononuclear cells is operated in a biological safety cabinet and obtained by the following methods:

[0039] (1) Divide the umbilical cord blood into 50mL centrifuge tubes, 45mL per tube, rise by 9 and fall by 7, centrifuge at 2000rpm for 10min;

[0040] (2) After centrifugation, remove the upper layer of plasma, dilute the blood cell suspension with normal saline at a ratio of 1:1, mix well, and slowly add the diluted blood cell suspension to the lymphocyte separation chamber at a ratio of 2:1. In a 50mL centrifuge tube of liquid, there is a clear interface layering, up 6 down 4, 400g, centrifuge for 20min;

[0041] (3) After centrifugation, the interface is divided into four layers, from top to bottom: supernatant layer, buffy coat...

Embodiment 3

[0049] Example 3 In vitro killing efficiency of EBV-CTL

[0050] The EBV-CTL cultured to the 21st day in Example 2 was tested for cell-specific killing efficiency by an in vitro killing experiment. Two lymphocyte cell lines, BLCL and K562, were selected as target cells. Among them, the surface of BLCL cells expressed EBV antigen, while the surface of K562 cells did not express EBV antigen. The specificity of EBV-CTL was verified by detecting the apoptosis of BLCL and K562 cells Killing function:

[0051] BLCL and K562 were inoculated in a 96-well plate at a ratio of 1:1, and the T cells cultured to the 21st day, the EBV-CTL of Example 2 were inoculated according to the effect-to-target ratio E:T of 1:3 and 1:1, respectively. Cells were placed in 96-well plates, with 3 replicates per group, placed at 37°C, 5% CO 2 Cultured in an incubator for 24 hours, BLCL cells and K562 cells were detected by flow cytometry (such as Figure 5 shown), the antibodies used were PE anti-human ...

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Abstract

The invention belongs to the technical field of cell biology, and particularly relates to a preparation method of EBV specific cytotoxic T cells. The preparation method comprises the following steps that mononuclear cells derived from umbilical cord blood or peripheral blood are stimulated for 3 weeks by adopting stimulating cells according to different proportions, a cell culture solution is replaced every 3 days in the culture process, the cell density is maintained to be 3.0*10<6> / mL, and co-stimulation culture is carried out for 21 days; the stimulated cells are irradiated EBV transformed B lymphocytes, the mononuclear cells and the stimulated cells are reduced in proportion every week, that is, on the zeroth day, the proportion of the mononuclear cells to the stimulated cells is 40 to 1, the proportion of the mononuclear cells to the stimulated cells is 20 to 1 on the 7 day, the proportion of the mononuclear cells to the stimulated cells is 5 to 1 on the 14 day, and each proportion stimulates for three weeks. According to the preparation method of the EBV specific cytotoxic T cells, the amplification efficiency can be rapidly and effectively improved, in-vitro function experiments show that the EBV-CTL can specifically kill EBV infected cells, and the killing efficiency is 90% or above. In clinic, the preparation method of the EBV specific cytotoxic T cells has important potential significance for EBV immunotherapy of the receptor after allogeneic stem cell transplantation.

Description

technical field [0001] The invention belongs to the field of cell biology and biomedicine, and specifically relates to a preparation method and application of EBV-specific cytotoxic T cells (EBV-CTL). Background technique [0002] EBV (Epstein-Barr virus) is a member of the herpes virus family. EBV-induced lymphoproliferative disease is a serious complication associated with allogeneic stem cell transplantation. In normal hosts, these viruses cause self-limiting disease, and in immunocompromised individuals, especially recipients of allogeneic T-cell depleted (TCD) stem cell transplantation (SCT), EBV can cause significant morbidity and mortality Rate. [0003] The reported incidence of viral reactivation after SCT is 70% to 80%. EBV-induced lymphoproliferative disease (EBV-LPD) can be as high as 20% in patients after TCD-SCT. In these patients, cellular immunity plays a very important role in the prevention and control of viral infection, therefore, restoration of cellu...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/0781C12N13/00
CPCC12N5/0638C12N5/0635C12N13/00C12N2502/1107C12N2501/2302
Inventor 吕晓菲张慧慧谭毅李想
Owner 山东省齐鲁细胞治疗工程技术有限公司
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