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Primary gastrointestinal stromal tumor cell culture medium, culture method and application thereof

A technology of gastrointestinal stromal tumors and culture methods, which is applied in the field of cell efficacy evaluation and screening of drugs, can solve the problems of low culture success rate and interference of non-gastrointestinal stromal tumor cells, and achieve high uniformity and low cost. controllable effect

Pending Publication Date: 2022-07-19
PRECEDO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no recognized effective method for culturing patient-derived primary gastrointestinal stromal tumor cells in vitro.
Most of the reported methods add serum to the medium (Liu et al., Am JPathol, 183(6):1862-1870, 2013; Liu et al., Ther Adv Med Oncol, 11:1-15, 2019; Fukuda, Oncology Reports 30 :71-78,2013; Zhu et al., Journal of Central South University, 35(11):1138-1144, 2010), there are common problems such as low culture success rate and interference by non-gastrointestinal stromal tumor cells such as fibroblasts

Method used

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  • Primary gastrointestinal stromal tumor cell culture medium, culture method and application thereof
  • Primary gastrointestinal stromal tumor cell culture medium, culture method and application thereof
  • Primary gastrointestinal stromal tumor cell culture medium, culture method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Isolation of primary human gastrointestinal stromal tumor cells and optimization of culture medium for gastrointestinal stromal tumor cells

[0047] (1) Isolation of primary human gastrointestinal stromal tumor cells

[0048] A commercially available penicillin-streptomycin dual antibody solution (manufactured by Corning, containing 10,000 U / ml penicillin and 10 mg / ml streptomycin) was added to DMEM / F12 medium (manufactured by Corning) at a volume ratio of 2%. For transport and cleaning of samples. Hereinafter referred to as "transport fluid".

[0049] Gastrointestinal stromal tumor tissue samples were derived from surgically resected cancer tissue samples from four described and consented gastrointestinal stromal tumor patients, GIST-1, GIST-2, GIST-3, and GIST-4. One of the samples (GIST-1) is described below. The above-mentioned tissue samples were collected within half an hour of the patient's surgical resection. More specifically, in a sterile environment, a ti...

Embodiment 2

[0089] Culture of human primary gastrointestinal stromal tumor cells and comparison with existing culture methods

[0090] (1) Culture of primary gastrointestinal stromal tumor cells derived from human gastrointestinal stromal tumor tissue

[0091] Primary gastrointestinal stromal tumor cells (GIST-1, GIST-2, GIST-3) were isolated from the cancer tissues of three patients with gastrointestinal stromal tumor using the same method as in (1) of Example 1, respectively. Next, the isolated gastrointestinal stromal tumor cells were counted with a hemocytometer, and then counted at 5 × 10. 4 The cells / well density was inoculated into a 12-well plate coated with Collagen I (manufactured by Corning). The coating method is as follows: Collagen I is diluted with ultrapure water at a ratio of 1:50 to prepare a collagen dilution solution, and 1 ml / well of collagen dilution solution is added to a 12-well culture plate to completely cover the culture plate holes. After standing in a 37°C i...

Embodiment 3

[0109] Identification of immune markers in gastrointestinal stromal tumor cells

[0110] (1) The complete medium CM in (5) of Example 1 and the gastrointestinal stromal tumor cell medium F-media in (2) of Example 2 and the known formulation 1 were used.

[0111] (2) Take out a cancer tissue about the size of a soybean grain from a clinical surgical resection sample of a patient with gastrointestinal stromal tumor, and separate and obtain primary gastrointestinal stromal tumor cells (GIST) using the same method as in (1) of Example 1. -1). Primary gastrointestinal stromal tumor cells (GIST-1) were cultured using the culture methods A, B, and C in (3) of Example 2, respectively.

[0112] (3) Immunofluorescence assay was used to detect the expression of important cancer-related biomarkers on human gastrointestinal stromal tumor cells.

[0113] The primary antibodies used in this experiment were CKIT (CD117) (manufactured by Cell Signaling Technology) and α-SMA (manufactured by ...

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Abstract

The invention provides a cell culture medium for culturing primary gastrointestinal stromal tumor cells. The cell culture medium contains gastrin, N2, insulin, a receptor tyrosine kinase ligand and a Rock kinase inhibitor. The invention also provides a method for culturing gastrointestinal stromal tumor cells by adopting the cell culture medium, and application and a method of an amplified cell population obtained by adopting the method in curative effect evaluation or screening.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a culture medium and a culture method for culturing or expanding gastrointestinal stromal tumor cells in vitro, and also relates to a method for evaluating and screening the efficacy of drugs by the cultured cells. application. Background technique [0002] Gastrointestinal stromal tumor (GIST) is a type of tumor originating from the mesenchymal tissue of the gastrointestinal tract, accounting for most of the gastrointestinal mesenchymal tumors, mostly occurring in the stomach (50-70%). Since this tumor is not sensitive to conventional radiotherapy, surgical resection is an effective treatment for GIST. For patients who lost the opportunity for surgery due to tumor progression, metastasis or postoperative recurrence, the median survival time of GIST was only 6-18 months, and the 5-year survival rate was less than 10%. Over the past 10 years, with the wide applicati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/02
CPCC12N5/0693C12N5/0679G01N33/5011C12N2501/345C12N2501/33C12N2501/727C12N2501/135C12N2501/125C12N2501/105C12N2501/115C12N2501/119C12N2500/90C12N2533/54C12N2503/02G01N2500/10C12N2500/46C12N2500/84C12N2500/25C12N2501/39C12N2501/392C12N2501/11C12N2501/113C12N2501/2306C12N2501/2322
Inventor 刘青松刘飞扬李晓雨梅沪生王文超任涛王黎
Owner PRECEDO PHARMA CO LTD
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