Method for culturing human airway epithelial cells

Abstract

The invention discloses a method for culturing human airway epithelial cells. The method comprises the following steps of: (1) acquiring human airway epithelial cells which are subjected to primary culture; and (2) acquiring human airway epithelial cells which are subjected to subculture, namely cleaning by using a phosphoric acid buffer solution when the fusion density of primary cells reaches 70 to 90 percent, adding a 0.25 percent trypsin solution, digesting at room temperature for 5 to 10 minutes, collecting cell suspension when cells are retracted and suspended, adding an equal amount of solution containing a protease inhibitor, performing centrifugal collection on the cells, inoculating in a novel culture dish containing a complete medium at the concentration of between 1 and 6*10<6> cells / ml, culturing, and thus obtaining the human airway epithelial cells which are subjected to subculture when the cells grow to a logarithmic growth phase after 3 to 5 days. The method for culturing the human airway epithelial cells has a stable technology and is high in repeatability; and the cultured cells have high purity and uniform morphology, grow well and can be subjected to continuous passage.

Description

technical field [0001] The invention relates to a method for culturing human airway epithelial cells, which belongs to the field of biotechnology. Background technique [0002] The trachea or bronchi are an important part of the lower respiratory tract, and airway epithelial cells are an important defense barrier of the respiratory tract. Studies have found that repeated respiratory infections are closely related to mucosal epithelial dysfunction. It is an important research material for the pathogenesis of common diseases of the respiratory system. The biological characteristics of airway epithelial cells have not changed in vitro, which can reflect the state in vivo to a certain extent, and show the morphological characteristics and cell biological characteristics of parental tissues. Therefore, the primary culture and cell biology of airway epithelial cells in vitro The establishment of strains is an effective means to study the biological characteristics of parental tis...

AI Technical Summary

Problems solved by technology

[0003] At present, the culture methods of airway epithelial cells mainly include: trypsin and / or protease digestion combined with mechanical peeling mucosa method and / or mechanical scraping method, these methods have the following disadvantages: (1) Mechanical peeling mucosa method / mechanical scraping method The method is to use tweezers and other instruments to perform blunt separation on the trachea or bronchi.

(2) Trypsin digestion / protease digestion method is to use trypsin / protease solution to digest the trachea or bronchi to obtain epithelial cells, but trypsin / protease has a strong digestive power, and the digestion time is not easy to grasp, which is easy to damage the epithelium Cell loss, low cell gain, low cell viability and poor viability

In addition, there are still the following reasons that lead to the limitation of cell culture based on human airway epithelial cells: 1. The lung is an open organ, so the obtained airway is prone to pollution during the cell culture process. The airway contains a large amount of mucin and other dregs, which increases the difficulty of cell isolation; 2. The specimens that can be used to isolate airway epithelial cells are all surgically resected parts with lesions. The number of specimens is limited, and the normal trachea in the specimens Or there are very few bronchi. If the general airway epithelial cell culture method is used, the cell damage is large, the acquisition rate is low, and the cell attachment ability is poor, and the number of surviving cells is very small; 3. Primary culture of airway epithelial cells Among them, it is most likely to introduce fibroblast contamination. Fibroblasts grow fast, have good vitality, and have a growth advantage, which eventually leads to the failure of primary airway epithelial cell culture.

Images