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Culture medium for 3D culture of lung tissue

A culture medium and lung tissue technology, applied in the field of biomedicine, can solve the problems of lack of targeted culture medium and poor activity of lung tissue cells, and achieve the effect of maintaining tissue cell specificity, high consistency and consistency

Active Publication Date: 2017-06-13
WEST CHINA HOSPITAL SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to overcome the problems of the poor activity of lung tissue cells in the two-dimensional culture technology in the prior art, and the lack of targeted culture medium for 3D organoid culture of lung / lung cancer tissue cells in vitro, and provide a 3D organoid culture medium for human lung / lung cancer tissue. media for organ culture

Method used

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  • Culture medium for 3D culture of lung tissue
  • Culture medium for 3D culture of lung tissue
  • Culture medium for 3D culture of lung tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] conditioned medium

[0041] The (conditional) medium for 3D culture of lung / lung cancer tissue organoids has the following formula: cytokine B2750X dilution; N-acetylcysteine1mM; EGF50ng / ml; Noggin100ng / ml; R-spondin1250ng / ml; A83-01200nM; ; Nicotinamide 10 mM; Y-27632* 10 uM; WNT3a 25 ng / ml; Glutamax 100X dilution; N2 100X dilution; Gastrin 1 nM.

Embodiment 2

[0043] conditioned medium

[0044] The (conditional) medium for lung tissue organoid 3D culture has the following formula: cytokine B27 50X dilution; N-acetylcysteine ​​1mM; EGF 55ng / ml; Noggin 110ng / ml; R-spondin 1 30% conditioned medium; A83- 01200nM; FGF10 500ng / ml; Nicotinamide 10mM; WNT3a or 10% conditioned medium; Glutamax 100X dilution; N2 100X dilution.

Embodiment 3

[0046] Normal lung tissue organoid culture

[0047] as per figure 1The process sequence shown is for 3D culture of lung tissue / lung cancer tissue organoids. Take the lung tissue and chop it on ice, add 10ml collagenase to resuspend, and run Human Lung program 1 in gentleMACS C tube collagenase. Transfer to 37°C, 220rpm shaker for digestion for 20min. Human Lung program 2 was run using gentalMACS. Transfer to 37°C, 220rpm shaker for digestion for 10min, filter the cells with a 100μm cell mesh, add 10ml DMEM / F12 to the filtrate to stop digestion, centrifuge (4°C, 200g, 5min), and remove the supernatant.

[0048] Take 5ml of erythrocyte lysate to resuspend, and lyse the erythrocytes on ice for 5min. In a centrifuge at 4°C, centrifuge at 200g for 5min, and remove the supernatant. Add 10ml DMEM / F12 to resuspend, centrifuge at 200°C for 5min at 4°C, and remove the supernatant.

[0049] For cell counting, mix Martrigel, 20,000 cells per 40 μL, drop in the center of a 48-well...

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Abstract

The invention discloses a culture medium for 3D culture of lung tissue. The culture medium comprises the following components: B27, N-acetylcysteine, an EGF, Noggin, R-spondin 1, A83-01, FGF10, Nicotinamide, Y-27632*, WNT3a, Glutamax, N2 and Gastin. The culture medium disclosed by the invention is appropriate in nutrient component, tissue cell specificity and stem cell specificity can be effectively maintained for lung tissue and lung cancer tissue, then lung tissue / lung cancer tissue organs with stable inheritance and biological functions can be formed, and scientific study requirements can be met.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to a culture medium for 3D culture of human lung tissue organoids, and belongs to a culture medium material for cell culture. Background technique [0002] Cell culture technology is a technology in which one or a small number of cells are cultured in large quantities to become simple single cells or rarely differentiated multicellular technologies. In the in vivo environment, cells are supplied with energy and raw materials for survival and replication by the body fluid circulation system. When cultured in vitro, it is often necessary to prepare targeted nutrient substrates according to the growth characteristics of different cells. \ [0003] Human-derived tissue organoids (Organoids) culture technology is a new method for recent oncology research. Under in vitro 3D culture conditions, mature human cells are used to cultivate human tissue organoids. This organoid almost retains the sam...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/0688C12N5/0693C12N2500/32C12N2500/38C12N2501/11C12N2501/119C12N2501/15C12N2501/155C12N2501/345C12N2501/415C12N2501/727C12N2501/734C12N2513/00
Inventor 陈崇纳飞飞
Owner WEST CHINA HOSPITAL SICHUAN UNIV
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