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Economical, simple and convenient method for inducing lung organs and establishment of experimental model

An organoid and simple technology, applied in the field of in vitro induction of mouse lung organoids, can solve the problems of high cost of culture and high technical requirements of lung organoids, and achieve the effect of reducing the cost of culture

Active Publication Date: 2021-07-23
ANHUI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] The purpose of the present invention is to provide an economical and convenient method for culturing lung organoids, which solves the problems of high technical requirements and high cost of cultivating lung organoids, so that it can be widely applied

Method used

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  • Economical, simple and convenient method for inducing lung organs and establishment of experimental model
  • Economical, simple and convenient method for inducing lung organs and establishment of experimental model
  • Economical, simple and convenient method for inducing lung organs and establishment of experimental model

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Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Compound Combination

[0043] 1. Lung mesenchymal cell culture medium

[0044] Carry out the configuration of lung mesenchymal cell culture medium according to the ingredient table shown in Table 1

[0045] Reagent name final concentration DMEM 89% FBS 10% Streptomycin Mixture 1% Primocin TM

100 μg / mL

[0046] Specifically, in one embodiment of the present invention, the DMEM and the penicillin-streptomycin mixed solution are purchased from LifeTechnologies Company, the article numbers are 11330032 and 15140-163 respectively, FBS is purchased from Biowest Company, the article number is S1580-500, Primocin TM Purchased from Invivogen, Cat. No. ant-pm-1. Of course, those skilled in the art can also use reagents produced by other companies with the same efficacy to configure the expansion medium of the present application, which is not specifically limited in the present invention.

[0047] Preferably, the inventor ...

Embodiment 2

[0055] In the present invention, GFP-labeled lung epithelial cells are separated by flow cytometry, co-cultured with lung mesenchymal cells, and natural growth factors and cell components secreted by mesenchymal cells are used to promote lung epithelial cells to form lung organoids. Lung epithelial cell isolation and organoid formation procedures such as figure 1 As shown, specifically:

[0056] 1. Obtainment of transgenic mice (mTmG;;Shh-Cre mice) expressing GFP in lung epithelial cells.

[0057] 1.1 as figure 2 As shown in A, in the present invention, mTmG transgenic mice are used as female parents, Shh-Cre transgenic mice are used as male parents, and offspring (F1) mice are obtained by mating.

[0058] 1.2 as figure 2 As shown in B, the offspring mice were identified by PCR, and the mice with positive mTmG and Shh-Cre PCR amplifications were mTmG;;Shh-Cre genotype mice.

[0059] 1.3 The PCR identification primers of mTmG mice used in the present invention are:

[0...

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Abstract

The invention provides an economic, simple and convenient lung organ culture method and an experimental model, and relates to the technical field of biology. The method comprises the following steps: marking a transgenic mouse with pulmonary epithelial cells by using GFP (Green Fluorescent Protein), sorting the pulmonary epithelial cells and interstitial cells by using a flow cytometry, then co-culturing the sorted lung epithelial cells and mesenchymal cells in an upper-layer small chamber and a lower-layer small chamber divided by Transwell, and promoting the epithelial cells in the upper-layer small chamber to grow and develop into lung organs by using growth factors and biological components secreted by the mesenchymal cells in the lower-layer small chamber. According to the method of the invention, the natural growth environment and mode of in-vivo cells are simulated, and the lung bronchial organ can be obtained under the condition that exogenous growth factors are not added. The mouse lung organ can be used as the experimental model, and can be used for research on lung development process, cell differentiation, lung toxicology, drug screening, organ regeneration and transplantation, etc.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an economical and convenient method for inducing mouse lung organoids in vitro, an experimental model and a combination of medium used. Background technique [0002] With the development of life sciences, in vitro replication and reconstruction of lung organoids has become one of the important research directions in the fields of lung development, disease occurrence, drug screening, organ transplantation, and precision therapy. Lung organoids are small tissues similar to lungs in structure and function formed by inducing differentiation of stem cells or lung progenitor cells with three-dimensional (3D) culture technology in vitro. Long-term culture in vitro. Compared with two-dimensional (2D) cell culture models, 3D cultured lung organoids contain a variety of cell types, breaking through the simple physical contact between cells, promoting the cooperative development of cells and f...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02
CPCC12N5/0688G01N33/5044C12N2509/00C12N2509/10C12N2502/27C12N2513/00C12N2533/90C12N2501/11C12N2500/12C12N2503/04G01N2500/10Y02A50/30
Inventor 李秋伶乔玉龙赵健伍慧晴
Owner ANHUI UNIVERSITY
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