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Culture medium and organoid culture method for 3D culture of kidney tissue organoids

A culture medium and organoid technology, applied in the field of organoid culture and kidney tissue organoid culture, can solve the problems of unfavorable kidney tissue culture, good operability and repeatability, unfavorable research, etc., to maintain tissue cell specificity, The effect of highly consistent genotyping and highly similar tissue morphology

Active Publication Date: 2019-04-12
ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] The existing technology is mainly a common culture technology for kidney tissue. In the two-dimensional culture process, kidney tissue cells are difficult or unable to fully express the characteristics of kidney tissue, which makes the cultured kidney tissue cells different from living kidney tissue cells. , which is not conducive to the conduct of research
In 3D culture, in the absence of necessary and suitable medium, the culture of kidney tissue is also unfavorable, and it is difficult to fully simulate the physiological characteristics of kidney tissue cells in vivo
[0003] Although a variety of human-derived tissues can successfully culture organoids in vitro under different culture conditions, there are currently no studies and reports on the culture methods of kidney normal tissue and cancer tissue organoids, especially the specific culture conditions, namely the culture medium. The formula has not been tried and reported yet, and the patented formula of the present invention is moderately priced and has good operability and repeatability

Method used

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  • Culture medium and organoid culture method for 3D culture of kidney tissue organoids
  • Culture medium and organoid culture method for 3D culture of kidney tissue organoids
  • Culture medium and organoid culture method for 3D culture of kidney tissue organoids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] A medium for 3D culture of kidney tissue organoids, the medium includes the following components: B27, 50X dilution; N-acetylcysteine, 1.25mM; EGF, 50ng / ml; Noggin, 100ng / ml; R-spondin 1,500ng / ml ml or 30% conditioned medium; A83-01, 500nM; FGF10, 20ng / ml; Nicotinamide, 10mM; Y-27632, 10μM; Prostaglandin E2, 1μM; SB202190, 10μM.

Embodiment 2

[0048] Take normal mouse kidney tissue, cut it into pieces on ice, add 10ml collagenase to resuspend, transfer to 37°C, 220rpm shaker for digestion for 20min, filter the cells with 100μm cell mesh, add 10ml DMEM / F12 to the filtrate to stop digestion, centrifuge (4°C , 200g, 5min), remove the supernatant.

[0049] Take 5ml of erythrocyte lysate and resuspend the cells for 5min. Then centrifuge (4°C, 200g, 5min), remove the supernatant, add 10ml DMEM / F12 to resuspend, centrifuge (4°C, 200g, 5min), remove the supernatant. For cell counting, mix matrigel, 20,000 cells per 40ul, drop in the center of 48 wells, place the culture dish at 37°C, 5% CO 2 In 10min, solidify the martrigel. Add 150 μl of the conditioned medium prepared in Example 1 to each well, 37°C, 5% CO 2 , cell culture incubator. The medium was replaced every 3-4 days, and the conditioned medium prepared in Example 1 was used.

Embodiment 3

[0051] A medium for 3D culture of kidney tissue organoids, the medium includes the following components: cytokine B2750x dilution; N-acetylcysteine ​​1.25mM; EGF 5ng / ml; Noggin 100ng / ml; R-spondin 1 500ng / ml; 01 500nM; FGF10 10ng / ml; Nicotinamide 10mM; Y-2763210uM; Prostaglandin E2 1μM; SB202190 10μM; CHIR99021 5μM; FGF9 200ng / ml;

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Abstract

The invention discloses a culture medium and an organoid culture method for 3D culture of kidney tissue organoids. According to culture and growth characteristics of kidney-tissue-derived cells, the culture medium disclosed by the invention is prepared by blending a plurality of selected cytokine components in certain proportion; so that, signaling pathway regulatory factor content of the cytokines in the blended culture medium is appropriate. Thus, effective formation of organoids by kidney cells and renal cancer cells in 3D environment is allowed.

Description

technical field [0001] The invention relates to the field of organoid culture, and further relates to the field of kidney tissue organoid culture, in particular to a culture medium and an organoid culture method for 3D culture of normal kidney tissue or cancer tissue. Background technique [0002] The existing technology is mainly a common culture technology for kidney tissue. In the two-dimensional culture process, kidney tissue cells are difficult or unable to fully express the characteristics of kidney tissue, which makes the cultured kidney tissue cells different from living kidney tissue cells. , which is not conducive to the conduct of research. In 3D culture, in the absence of necessary and suitable medium, the culture of kidney tissue is also unfavorable, and it is difficult to fully simulate the physiological characteristics of kidney tissue cells in vivo. [0003] Although a variety of human-derived tissues can successfully culture organoids in vitro under differe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2501/00C12N2501/02C12N2501/11C12N2501/119C12N2501/999C12N2513/00
Inventor 郑鸿平黄敏
Owner ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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