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Basic culture medium and culture method for circulating tumor cells

A technology of basal medium and tumor cells, applied in the fields of medicine and biology, basal medium and culture of circulating tumor cells, can solve the problems of difficult cell enrichment and improper culture conditions, and achieve the effect of improving the accuracy of medication

Active Publication Date: 2021-03-16
SUREXAM BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are very limited literature reports on the culture of CTCs in vitro. The reason may be that the cells are difficult to enrich and the culture conditions are not appropriate. Therefore, it is urgent to develop an efficient and gentle method for the enrichment of CTCs. Rapid expansion in vitro, enabling comprehensive molecular and functional studies

Method used

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  • Basic culture medium and culture method for circulating tumor cells
  • Basic culture medium and culture method for circulating tumor cells
  • Basic culture medium and culture method for circulating tumor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation of lyophilized powder of Streptomyces roseus fermentation broth

[0050] 1. The source of bacteria

[0051] Purchased from China Industrial Microorganism Culture Collection and Management Center: Streptomyces roseofuscus (Strain collection number: ACCC40123 (Platform number: bio-52663), hereinafter referred to as laboratory abbreviation SR), it is known that it can be used to inhibit Gram Gram-positive bacteria, Gram-negative bacteria and filamentous fungi.

[0052] 2. Preparation of culture medium

[0053] 1) Seed medium (g / L):

[0054] Glucose 20.0g, peptone 6.0g, NaCl 10.0g, pH 7.0.

[0055] 2) ISP2 medium (medium proposed by the International Streptomyces Planning Conference):

[0056] Yeast extract 4.0g, malt extract 10.0g, glucose 4.0g, after dissolving, adjust the pH and dilute to 1.0L, sterilize by high pressure (1.034×105Pa) at 115°C for 30 minutes (glucose will be decomposed into For the cultivation of harmful microorganisms of sugar...

Embodiment 2

[0059] Example 2 Cytotoxicity test of lyophilized powder of Streptomyces roseus fermentation broth

[0060] After HEK293 cells (human embryonic kidney cells) were inoculated in culture flasks, RPMI1640 medium (Gibco) containing 10% fetal bovine serum was used at 37°C, 5% CO. 2 Cultivated in the environment for 12-72h. Take the cells in the logarithmic growth phase, resuspend the cell suspension in medium, and adjust the density to 1×10 5 1 / mL, add 100 μL PBS to the peripheral wells of the 96-well culture plate using a multi-channel pipette, and add 100 μL to the remaining wells at a density of 1×10 5 cells / mL of cell suspension. Cells at 37°C, 5% CO 2 Incubate in the incubator for 24h, aspirate the medium, add 100μL of 0μg / ml (control group), 0.3125μg / ml, 0.625μg / ml, 1.25μg / ml, 2.5μg / ml, 5μg / ml, 10μg / ml to each well ml, 15μg / ml, 20μg / ml concentration of SR fermentation broth lyophilized powder medium. After being placed in the incubator for 24 hours, the medium was remove...

Embodiment 3

[0068] Example 3 Composition and use steps of the kit

[0069] The kit of the invention mainly includes a circulating tumor cell basal medium, which specifically includes the following components: RPMI1640 / F12 medium, 8ng / mL basic fiber growth factor FGF2, 6μg / mL human recombinant insulin, 1mM pyruvate Sodium (antioxidant), 11mg / L transferrin, 0.7mM N-acetylcysteine, 9mM nicotinamide (vitamin B3 derivative), 0.6mg / mL taurine, 50U / mL penicillin, 50μg / mL streptomycin, 5μg / ml Streptomyces roseus (SR) fermentation broth (lyophilized powder); wherein RPMI1640 / F12 medium is F12 medium (Gibco, Item No.: 11765062) and RPMI1640 medium (Gibco, Item No.: A1049101 ) were prepared 1:1.

[0070] The basal medium can also be used to prepare a complete medium. The complete medium can be composed of basal medium and serum, wherein the serum can be obtained after coagulation and centrifugation of whole blood, or commercial fetal bovine serum.

[0071] The present invention can be applied to...

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Abstract

The invention relates to a basic culture medium and a culture method for circulating tumor cells. The culture medium is added with a plurality of compound components, including inactivated streptomyces roseus fermentation liquor. Secondary metabolites in the streptomyces roseus strain fermentation liquor can be used as nutritional ingredients for cell growth to promote cell proliferation, and meanwhile, antibacterial components generated by the secondary metabolites can effectively inhibit bacteria in the cell culture medium, so that use of antibiotics in the cell culture medium is reduced, and therefore, cell growth state is good without additionally adding serum; and while culture cost is reduced, the possible culture difference problem caused by using different batches of serum is reduced, and therefore, application scene is economically and efficiently expanded. Meanwhile, primary tumor cells can be subjected to subculture in vitro in a great number and infinitely, so that a greatnumber of cells are provided for development of a tumor drug sensitive test, and therefore, possibility is created for establishing a cell model.

Description

technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to a basic culture medium and a culture method of circulating tumor cells. Background technique [0002] In 1869, the Australian doctor Ashworth first proposed the concept of circulating tumor cells (Circulating Tumor Cell, referred to as CTC). In 1976, Nowell revised the definition of CTC as: tumor cells originating from the primary tumor or metastatic tumor, gaining the ability to escape from the basement membrane and invade blood vessels through the tissue matrix. At present, CTC refers to the collective name of various tumor cells existing in peripheral blood. [0003] CTCs can be used for cancer diagnosis and monitoring of cancer status, and are the key to tracking tumor cell metastasis. Clinical studies have demonstrated that CTCs are associated with the disease progression of various cancers. Therefore, the detection of CTCs plays an ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N2500/72C12N2501/115C12N2500/25C12N2500/32C12N2500/30C12N2500/38C12N2500/33C12N2513/00
Inventor 吴诗扬刘志明许嘉森黄洁芬曾杰
Owner SUREXAM BIO TECH
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