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Method for reconstructing colorectal cancer organoid immune microenvironment

A colorectal cancer and organoid technology, applied in the field of medical models, can solve unseen problems and achieve the effect of obvious killing ability

Pending Publication Date: 2020-12-29
SHANGHAI TENTH PEOPLES HOSPITAL
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  • Abstract
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  • Claims
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Problems solved by technology

If the immune microenvironment of colorectal cancer organoids can be successfully constructed, it will provide an in vitro experimental basis for clinical autologous T cell reinfusion immunotherapy, but there are no relevant reports so far

Method used

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  • Method for reconstructing colorectal cancer organoid immune microenvironment
  • Method for reconstructing colorectal cancer organoid immune microenvironment

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Embodiment 1

[0047] This embodiment provides a method for remodeling the immune microenvironment of colorectal cancer organoids, comprising the following steps:

[0048] (1) The construction process of PDOs model:

[0049] 1. Place the surgically resected fresh colorectal cancer tissue (within 30 minutes from the body) in a petri dish containing 5ml PBS+1% penicillin / streptomycin and wash it twice;

[0050] 2. After thoroughly cleaning the tumor tissue, cut the tumor tissue into 2-4mm with sterile instruments 3 Fragments of the same size were placed in a 15ml centrifuge tube (containing 10ml of ice-cold PBS buffer), centrifuged at 1500rpm x 3min, and the supernatant was discarded.

[0051] 3. Add 5ml of tissue digestion solution (stemcell, cat: 07174), resuspend the tumor fragment tissue obtained in the second step, shake vigorously, and incubate on ice for 30 minutes.

[0052] 4. Filter the supernatant through a 100 μm filter into a 50 ml centrifuge tube to obtain the filtrate and enrich ...

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Abstract

The invention provides a method for reconstructing a colorectal cancer organoid immune microenvironment. The method comprises the following steps: constructing a colorectal cancer organoid by using fresh colorectal cancer tissue; obtaining peripheral blood, then performing erythrocyte lysis, and separating lymphocytes by adopting a lymphocyte separating medium; enriching CD3 <+> T cells; and digesting the organoids into single cells, co-culturing the single cells and the cultured CD3 <+> T cells according to a ratio of 1: (10-30), and constructing the colorectal cancer patient-derived organoids immune microenvironment according to the steps. According to the method for reconstructing the colorectal cancer organoid immune microenvironment provided by the invention, fresh colorectal cancer tissue from the same individual and CD3 <+> T cells from peripheral blood are co-cultured to obtain the organoid immune microenvironment, it is, from cellular morphology and ATP values, shown that immune cells have an obvious killing effect on organoids of CRC patients, and an in-vitro experimental basis is provided for immunotherapy of clinical autologous T cell transfusion.

Description

technical field [0001] The invention relates to the technical field of medical models, in particular to a method for reconstituting the immune microenvironment of colorectal cancer organoids. Background technique [0002] At present, improving the sensitivity and efficacy of individualized immunotherapy is an urgent problem in the field of tumor immunity. How to simulate the tumor environment in vivo in vitro and enhance the ability of immune cells to kill tumor cells has become the key to solving the problem. [0003] Toshiro Sato et al. constructed human tumor organoids (patient-derived tumor organoids, PDOs) in vitro from colorectal cancer patient samples. This technology can simulate the 3D growth environment in vivo in vitro, which is more realistic than the traditional two-dimensional culture system. It reflects intestinal function, signal transduction and morphology in vivo, and is highly consistent with the gene mutation type of the primary tumor focus 1 . Scientis...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/09
CPCC12N5/0636C12N5/0693C12N2533/90C12N2529/00C12N2513/00C12N2502/00
Inventor 秦环龙蔚青李曼
Owner SHANGHAI TENTH PEOPLES HOSPITAL
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