In-vitro amplification method and application of glioma-derived tumor infiltration lymphocytes (TIL)

A technology of in vitro expansion and tumor infiltration, which is applied in the field of in vitro expansion of tumor-infiltrating lymphocytes derived from glioma, can solve the problems of high usage of IL-2, complicated operation procedures, low activity, etc. Tumor cells, simple operation procedures, and short time-consuming effects

Active Publication Date: 2020-10-30
SHENZHEN HOSPITAL OF SOUTHERN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Therefore, the embodiment of the present invention provides an in vitro expansion method and application of glioma-derived tumor infiltrating lymphocytes (TIL) to sol

Method used

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  • In-vitro amplification method and application of glioma-derived tumor infiltration lymphocytes (TIL)
  • In-vitro amplification method and application of glioma-derived tumor infiltration lymphocytes (TIL)
  • In-vitro amplification method and application of glioma-derived tumor infiltration lymphocytes (TIL)

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Effect test

Embodiment 1

[0031] Cultivation of glioma tumor tissue cells

[0032] Take part of the glioma tumor tissue, mill, digest, remove the red blood cells, collect the cells by centrifugation at 800 rpm / min for 5 min, discard the supernatant, and then use the culture solution at 37°C, 5% CO 2 Cell culture is carried out in the incubator. Among them, the composition of the culture medium is as follows: On the basis of DMEM / F12 medium, add 10% FBS, 1% P / S (penicillin / streptomycin), 2mM L-glutamine, 1×MEM-Eagle, 1mM Sodium pyruvate (Sodium pyruvate), 10mM HEPES. After culturing for 1-3 generations, the cells were digested, and the cells were collected by centrifugation at 800 rpm / min for 5 minutes, and the culture medium was added to resuspend and wash the cells before storage or the next experiment. See the results of primary tumor cell culture for glioma figure 1 .

Embodiment 2

[0034] In vitro amplification method of tumor infiltrating lymphocytes (TIL) of glioma

[0035] (1) Pre-culture of TIL cells

[0036] The collection tube containing RMPI 1640 medium containing 1% P / S is placed on ice, and the glioma tissue collected in the operating room is quickly put into the collection tube, which is beneficial to preserve the activity of the tissue during transportation.

[0037] Take out the glioma tissue and wash it 3 times with 1×PBS and cut into 1-3mm 3 The tissue block is inoculated into a 100mm cell culture dish containing 1% P / S RMPI 1640 medium and placed in 5% CO 2 Incubate in a 37°C incubator for 30 minutes to fully release the PBMC in the capillaries of the tissue mass.

[0038] (2) Cultivation and expansion of TIL cells

[0039] The glioma tissue obtained in step (1) was seeded on a 24-well plate containing TIL cell culture medium I, 2 pieces / well, and placed at 37°C, 5% CO 2 Culture in the incubator, change the medium every 2 days, after 8 days of cultu...

Embodiment 3

[0044] TIL cell component analysis

[0045] Flow cytometry was used to analyze CD3 in TIL cells released from the glioma tissue mass obtained by the amplification method of Example 2. + -T, CD4 + -T, CD8 + -T, CD19 + -T, and CD127 + -T for testing, see the results image 3 , Figure 4 And Table 1.

[0046] Table 1 The content of each component of TIL cells and their expansion (including CD4 and CD8)

[0047]

[0048] The results showed that in the in vitro expansion and culture of TIL cells, CD8 + -The proportion of T cells is at a stable high level, and CD8 + -The proportion of T cells increases continuously during the slow expansion period and is at a stable high level during the rapid expansion period.

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Abstract

The invention discloses an in-vitro amplification method and an application of glioma-derived tumor infiltration lymphocytes (TIL), and belongs to the technical field of cell culture. The method comprises the following steps: pre-culturing TIL cells; and culturing and amplifying the TIL cells. According to the method, under the condition that glioma infiltrating lymphocytes are not separated, theTIL cells are directly induced and cultured, then a large number of TIL cells are amplified, the operation procedure is simple, consumed time is short, the TIL cell amplification number and cell activity can be obviously improved, and the method has a good effect of killing glioma tumor cells. In the steps of TIL cell culture and amplification, a low-concentration cytokine composite nutrient medium is adopted, so that the requirements of TIL cell culture and amplification are met, and the potential risk of cytokine storm in the subsequent treatment process is reduced.

Description

Technical field [0001] The embodiment of the present invention relates to the technical field of cell culture, in particular to an in vitro amplification method and application of tumor infiltrating lymphocytes (TIL) derived from glioma. Background technique [0002] Glioma is the most common primary intracranial tumor originating from the neuroepithelium. It is difficult to cure and easy to relapse. At present, the main treatment methods are surgery, radiotherapy and chemotherapy. The 5-year survival period of high-grade gliomas, especially glioblastomas, is less than 10%. [0003] Tumor infiltrating lymphocytes (TIL) are lymphocytes isolated from tumor tissues. The existing TIL cell culture technology uses mechanical treatment and / or enzymatic digestion methods to isolate tumor infiltrating lymphocytes from tumor tissues and add high-dose interleukin-2 (IL-2, 6000U / mL) for in vitro to cultivate. Studies have shown that TIL activated by IL-2 has higher tumor-killing activity a...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCC12N5/0638A61K35/17A61P35/00C12N2509/00C12N2509/10C12N2500/80C12N2500/32C12N2500/30C12N2500/44C12N2501/2302C12N2501/2315C12N2501/2321
Inventor 吴丁兰
Owner SHENZHEN HOSPITAL OF SOUTHERN MEDICAL UNIV
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