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A special culture medium and culture method for lung cancer organoids

A culture method and organoid technology, which are applied in the special medium and culture field of lung cancer organoids, can solve the problems of difficulty in the development of lung cancer organoids, and achieve the effects of less impact on personnel operation, tight connection, and simple operation.

Active Publication Date: 2022-04-12
ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, there is a gap in the research technology for establishing lung cancer organoid culture methods in China. The main problem lies in the difficulty in the development of a dedicated culture system for lung cancer organoids.

Method used

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  • A special culture medium and culture method for lung cancer organoids
  • A special culture medium and culture method for lung cancer organoids
  • A special culture medium and culture method for lung cancer organoids

Examples

Experimental program
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Effect test

Embodiment 1

[0038]This example provides a special culture medium and culture method for lung cancer organoids, including basal medium and specific additive factors; the specific additive factors include components at the following final concentrations: GSK429286A, 5 μM; Y27632, 12.5 μM; EGF BMP4, 150ng / ml; Human FGF4, 300ng / ml; Insulin-Transferrin-Selenium, 1.5x; B27, 0.75x; all-trans retinoic acid, 0.5μM; KGF, 200ng / ml; DAPT, 5mM ; GlutaMAX, 1.25x; R-Spondin-1, 300 ng / ml; CHIR99021, 5 μM; 8-Br-cAMP, 150 nM; BSA, 0.5%; 3-isobutyl-1-methylxanthine, 50 nM; Percentages are mass percentages.

[0039] Lung cancer organoid culture methods include:

[0040] (1) Wash the lung cancer tissue taken out with normal saline, cut it into pieces on ice, add 10ml of digestion solution 1 to resuspend, wherein digestion solution 1 includes 500ng / ml of Hydrocortistone and 500U / ml of Collagenase enzyme type IV, transfer to 37 °C, 100rpm shaker for 2 hours, after the digestion is complete, add 5ml of HBSS to...

Embodiment 2

[0045] This example provides a special culture medium and culture method for lung cancer organoids, including basal medium and specific additive factors; the specific additive factors include components at the following final concentrations: GSK429286A, 15 μM; Y27632, 6 μM; EGF, 200ng / ml; BMP4, 50ng / ml; Human FGF4, 550ng / ml; Insulin-Transferrin-Selenium, 0.8x; B27, 1.5x; all-trans retinoic acid, 0.6μM; KGF, 100ng / ml; DAPT, 15mM; GlutaMAX, 0.75x; R-Spondin-1, 600ng / ml; CHIR99021, 8μM; 8-Br-cAMP, 80nM; BSA, 0.3%; 3-isobutyl-1-methylxanthine, 150nM; is the mass percentage. The lung cancer organoid culture method is the same as that in Example 1. Figure 5 The topography of lung cancer organoids obtained after 14 days of culture is provided, and the formed tumor organoid cells are spherical in shape and large in diameter.

Embodiment 3

[0047] This example provides a special culture medium and culture method for lung cancer organoids, including basal medium and specific additive factors; the specific additive factors include components at the following final concentrations: GSK429286A, 2.5 μM; Y27632, 20 μM; EGF BMP4, 30ng / ml; Human FGF4, 300ng / ml; Insulin-Transferrin-Selenium, 2x; B27, 0.5x; all-trans retinoic acid, 1μM; KGF, 10ng / ml; DAPT, 20mM; GlutaMAX , 0.5x; R-Spondin-1, 1000ng / ml; CHIR99021, 1μM; 8-Br-cAMP, 150nM; BSA, 0.1%; 3-isobutyl-1-methylxanthine, 150nM; mass percentage. The lung cancer organoid culture method is the same as that in Example 1. Figure 6 The topography of lung cancer organoids obtained after 14 days of culture is provided. The formed tumor organoid cell spheres are regular in shape, tightly connected between cell spheres, and the size is uniform and controllable.

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Abstract

The present invention provides a special culture medium for lung cancer organoids and a culture method, the culture medium includes a basal medium and specific additive factors; the specific additive factors include: GSK429286A; Y27632; EGF; BMP4; Human FGF4; Insulin‑Transferrin‑Selenium; B27; all‑trans retinoic acid; KGF; DAPT; GlutaMAX; R‑Spondin‑1; CHIR99021; 8‑Br‑cAMP; bovine serum Albumin (BSA); Inhibitor of phosphodiesterases (PDEs). The culture medium of the present invention can realize rapid growth of lung cancer organoids, and can be stably cultured for a long time, and the formed tumor organoid cells are spherical in shape and relatively uniform in size, and can well preserve the heterogeneity of tumor tissues of patients in vitro.

Description

technical field [0001] The invention belongs to the technical field of organoid culture, and in particular relates to a special culture medium and a culture method for lung cancer organoids. Background technique [0002] Lung cancer is one of the malignant tumors that threaten human health and life safety. About 1-3 million people die of lung cancer worldwide every year. In my country, lung cancer has also become the first malignant tumor that threatens human life, with an annual death toll of about 600,000 and an increasing trend year by year. Although in recent years, with the development of molecular biology, researchers have made breakthroughs in the pathogenesis and treatment of lung cancer by using various lung cancer cell lines, but due to the difference in genetic background, the molecular mechanism and treatment of different patients are different. Individualized features. At present, the individualized treatment of lung cancer is based on three levels: clinical, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/09
CPCC12N5/0693C12N5/0688C12N2501/727C12N2501/11C12N2501/155C12N2501/119C12N2500/25C12N2501/117C12N2501/73C12N2501/998C12N2501/01C12N2501/734C12N2501/999C12N2501/415C12N2500/40C12N2509/00C12N2500/38C12N2533/90
Inventor 于言朱宇李刚黄敏
Owner ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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