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Method for culturing primary tumor cells

A technology of primary cells and culture methods, which is applied in the field of new and efficient tumor primary cell culture, can solve the problems of easy contamination of samples, low sensitivity, and restrictions on the promotion of drug sensitivity tests, so as to improve the accuracy of drug use and solve the time-consuming problems. Effect

Inactive Publication Date: 2016-06-08
SHENZHEN YOUSHENGKANG MEDICAL LAB CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after the conventional primary tumor cells are separated from the in vivo environment, most of the cells will gradually die and float after the first 24 hours of in vitro culture, and the cells that can adhere to the wall account for a very small proportion. The traditional primary tumor cell culture is time-consuming. Longer, the sample is easily contaminated, and the sensitivity is low, etc.
These shortcomings will interfere with the accuracy of the experimental results, thereby affecting the correct choice of chemotherapy regimen and the final curative effect
To a certain extent, it limits the promotion of drug susceptibility testing, making it fall into a roundabout situation
Primary single-cell culture will become the bottleneck of such methods, restricting the success rate and reliability of such methods

Method used

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  • Method for culturing primary tumor cells
  • Method for culturing primary tumor cells
  • Method for culturing primary tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Isolation and cultivation of primary tumor cells

[0042] (1) Separation and treatment of tumor single cells

[0043] The tumor tissues isolated from patients with solid tumors (breast cancer) are subjected to single cell isolation in a sterile environment within the shortest possible time.

[0044] 1. In order to obtain a sufficient number of viable tumor cells, the part with more tumor cells and not necrotic and liquefied should be excised, and the specimen should not be less than 1.0 g. Immediately after the specimen is taken out, it should be immersed in a sterile vial filled with PBS or RPM11640 culture solution and delivered to the laboratory within 3 hours. The specimens were soaked in the specimen soaking solution for 15 minutes.

[0045] 2. Remove the specimen soaking solution, peel off the connective tissue, fiber and fat on the surface of the tumor specimen, and cut the specimen into 1mm pieces with sterile scissors 3 Then transfer it to the cent...

Embodiment 2

[0056] Example 2 Isolation and cultivation of primary tumor cells

[0057] (1) Separation and treatment of tumor single cells

[0058] The solid tumor comes from a patient with gastric cancer, and the single cell separation and treatment method is the same as in Example 1.

[0059] (2) In vitro culture of primary tumor cells

[0060] After single cell isolation, inoculate together with feeder layer cells into cell culture flasks filled with fresh medium, at 37°C, 5% CO 2 For culturing under environmental conditions, the fresh medium is a standard medium HEPES containing 0.1 mM calcium ions (or 0.8 v / v% serum) and supplemented with Rho kinase inhibitors.

[0061] The feeder layer cells are splenocytes from mice, and after being treated with gamma ray radiation or mitomycin C, their proliferation is inhibited, but their metabolic activity is still maintained. The specific preparation method of feeder layer cells is as follows:

[0062] (1) Gamma-ray radiation: Take the feede...

Embodiment 3

[0067] Example 3 Isolation and Culture of Primary Tumor Cells

[0068] (1) Separation and treatment of tumor single cells

[0069] The solid tumor comes from a patient with non-small cell lung cancer, and the single cell separation and treatment method is the same as in Example 1.

[0070] (2) In vitro culture of primary tumor cells I

[0071] After single-cell isolation, inoculate into cell culture flasks filled with fresh medium, and add feeder layer cell substitutes to the culture flasks, at 37°C, 5% CO 2 The cells were cultured for 2-14 days under ambient conditions, and the cells were collected.

[0072] Described fresh culture medium is to contain 10mM calcium ion (or 30v / v% serum), and adds the standard culture medium D-Hank's of Rho kinase inhibitor, wherein the added Rho kinase inhibitor is TGF-beta, EGFR, cadherin At least one of myosin, G protein, myosin phosphatase inhibitor, vimentin, LIMK, myosin light chain kinase, NHE, sodium / hydrogen exchange protein-1 or myo...

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Abstract

The invention provides a novel efficient method for culturing primary tumor cells. The method comprises steps as follows: the primary tumor cells and / or feeder layer cells are cultured in a standard culture medium as well as feeder layer cells or cell substitutes, and the standard culture medium contains 0.1-10 mM of calcium ions or 0.8-30 v / v % of serum as well as a Rho kinase inhibitor added. According to the method, the problem that the primary tumor cells are difficult to culture in vitro is solved, and the cultured tumor cells can be used for screening novel anti-tumor drugs or detecting sensitivity of the tumor cells to different anti-tumor drugs. With the adoption of drug sensitivity tests, the medication accuracy of a tumor patient can be improved, and scientific basis is provided for a clinician to determine individual chemotherapy regimens and carry out individualized treatment.

Description

technical field [0001] The invention relates to a primary tumor cell culture technology, in particular to a novel and efficient primary tumor cell culture method. Background technique [0002] Malignant tumor is a kind of disease with very high morbidity and mortality, which seriously endangers the life and health of human beings. one. Statistics show that in 2015, there were 4.292 million new cancer cases and 2.814 million cancer deaths in my country in 2015. The incidence rate of tumors is increasing at a rate of 3% and tends to be younger, ranking the highest in the mortality rate of various diseases. [0003] Current treatments for malignant tumors include surgery, radiotherapy, chemotherapy, endocrine therapy, and biological immunotherapy, [0004] Among them, the first three are the main ones. Chemotherapy, as a systemic treatment, plays a very important role in the treatment because it can widely kill tumor cells in the patient's body and improve the patient's surv...

Claims

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Application Information

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IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2500/05C12N2500/14C12N2500/24C12N2500/30C12N2500/32C12N2500/34C12N2500/38C12N2500/40C12N2500/46C12N2500/84C12N2501/15C12N2501/30C12N2501/727C12N2501/998C12N2501/999C12N2502/11C12N2502/1178C12N2502/13
Inventor 喻德华刘佳苏
Owner SHENZHEN YOUSHENGKANG MEDICAL LAB CO LTD
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