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Method for separating and cultivating II-type epithelial cells of people alveolus pulmonis

An epithelial cell, separation and culture technology, applied to artificially induced pluripotent cells, animal cells, vertebrate cells, etc., can solve the problems of high cost, long operation time, and large damage to ATII cells, and achieves lower separation costs and lower costs. Dosage, damage reduction effect

Inactive Publication Date: 2013-04-24
GUANGZHOU INST OF RESPIRATORY DISEASE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In the existing method, the concentration of trypsin and elastase used in the digestion step is too high, which will cause great damage to ATII cells, and the cost is relatively high
The time for differential wall attachment is short, 1.5h, and it also includes the step of removing fibroblasts and macrophages by magnetic bead sorting. The whole experimental operation time is too long, and at least 12h is required; Tissue volume is still high and underutilized; experiments are expensive
[0005] It can be seen that human lung tissue is used as a tissue source for extracting ATII cells. On the one hand, the tissue source is limited, and on the other hand, the phenotype of in vitro culture is maintained for a short time, which greatly limits the in vitro culture of ATII cells. Reports on research in this area

Method used

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  • Method for separating and cultivating II-type epithelial cells of people alveolus pulmonis
  • Method for separating and cultivating II-type epithelial cells of people alveolus pulmonis
  • Method for separating and cultivating II-type epithelial cells of people alveolus pulmonis

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Embodiment 1

[0039] The isolation and culture method of embodiment 1 human alveolar type II epithelial cells

[0040] Referring to the scheme written by rEhhardt et al. (2005), it specifically includes the following steps:

[0041] (1) Cut 5-8g surgically resected marginal lung tissue into pieces of about 1cm in a petri dish 3 size, BSS was washed repeatedly until clarified;

[0042] (2) Cut the lung tissue into 0.6mm size, transfer it to a 100ml beaker, wash with BSS at least 3 times, then filter through a 100-mesh cell sieve, and keep the tissue on the sieve;

[0043] (3) Transfer it to a small Erlenmeyer flask containing 15ml-20ml BSS, add 30-40ml of tissue digestion solution to digest at 37°C for 45min, and add 1ml-2ml of DNase I (10000U / ml) in the last 15 minutes;

[0044] (4) Add an inhibitory solution equal in volume to the interstitial fluid to stop the digestion, blow with a dropper for 10 minutes to obtain a cell suspension;

[0045] (5) Add 300ml BSS to dilute the cell suspen...

Embodiment 2 Embodiment 1

[0052] Example 2 Cell Identification, Purity and Vitality Evaluation of Alveolar Type II Epithelial Cells of Example 1

[0053] 1. Cell identification

[0054] ①Pro-SP-C immunofluorescence staining identification

[0055] SP-C is an alveolar surfactant protein specifically expressed by alveolar type II epithelial cells, while the other three surfactant proteins SP-A, SP-B, and SP-D are also expressed in other cells, so SP among the surfactant proteins was selected. -C can be used to identify ATII cells.

[0056] Mainly referring to the method of Peter F et al., after the ATII cells reached the ideal density on the coverslips, they were transferred to a 24-well plate, and the samples were fixed with 4% paraformaldehyde for 10 minutes; washed with PBS for 5 minutes, treated with 0.1% Triton X-100 for 10 minutes, and increased Permeability of the cell membrane; block with goat serum for 1 h; incubate the primary antibody (pro-SP-C diluted with PBS 1:1000 containing 0.1% Triton ...

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Abstract

The invention discloses a method for separating and cultivating II-type epithelial cells of people alveolus pulmonis. By using pancreatin joint elastase to digest and separate, the II-type epithelial cells of people alveolus pelmonis are obtained through filtering, differential adhension, density gradient centrifugation and the separation and purification of Anti-CD 14 magnetic beads. The invention establishes a set of method of separating, cultivating and identifying II-type epithelial cells of people alveolus pulmonis, and the maintaining situation of the biological characteristics of the cells cultivated by the method is assessed. The capacity of obtaining II-type epithelial cells of people alveolus pulmonis with high purity through the cultivating method is certified, moreover, the biological characteristics of cells can be enduringly maintained, and the method provides possibilities for further deep research of the proliferation, differentiation, liquid transferring and synthetizing secretion of II-type epithelial cells of alveolus pulmonis as well as the functional changes of II-type epithelial cells of alveolus pulmonis in the cases of pathophysiology, such as lung injury, lung repair.

Description

technical field [0001] The invention belongs to the field of in vitro culture of cells, and more specifically, the invention relates to a method for separating and culturing human alveolar type II epithelial cells. Background technique [0002] As a kind of tissue stem cells, alveolar epithelial type II cells (ATII) have very important functions, mainly including five aspects: ① as a permeability barrier to prevent macromolecules in the interstitial space from flowing into the alveolar cavity; ② with Certain tissue stem cell functions, in the process of lung injury repair, in addition to self-proliferation, can also differentiate into alveolar type I epithelial cells and fibroblasts; ③ can secrete alveolar surfactant; ④ can secrete anti-inflammatory and antibacterial substances, in Immunity plays an important role; ⑤ can transport sodium ions across epithelial cells and help clear alveolar fluid. It can be seen that it is very necessary to study its in vitro culture. [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074
Inventor 黎毅敏毛璞傅威吴松林庞晓青张容何为群刘晓青
Owner GUANGZHOU INST OF RESPIRATORY DISEASE
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