Human airway epithelial cell serum-free culture medium with clear chemical components and application of human airway epithelial cell serum-free culture medium with clear chemical components

A technology of serum-free medium and epithelial cells, which is applied in the direction of cell culture active agents, artificial cell constructs, epidermal cells/skin cells, etc., can solve the problems of increasing the cost of cell culture, delaying the research progress of new therapies for trachea and lung-related diseases, The quality of airway epithelial cells cannot meet the standards for clinical treatment, and achieve the effect of clear chemical composition

Pending Publication Date: 2020-11-03
任涛
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] In order to solve the problem in the prior art that experimental researchers use a culture system containing serum or serum substitutes in the culture medium of airway epithelial cells, and rely on trophoblast cells to support in vitro expansion, the qua...

Method used

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  • Human airway epithelial cell serum-free culture medium with clear chemical components and application of human airway epithelial cell serum-free culture medium with clear chemical components
  • Human airway epithelial cell serum-free culture medium with clear chemical components and application of human airway epithelial cell serum-free culture medium with clear chemical components
  • Human airway epithelial cell serum-free culture medium with clear chemical components and application of human airway epithelial cell serum-free culture medium with clear chemical components

Examples

Experimental program
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Embodiment 1

[0050] This example discloses a serum-free medium for in vitro expansion and culture of human airway epithelial cells, the composition of which is shown in Table 1 below.

[0051] The composition and concentration of the serum-free medium of table 1 human airway epithelial cells

[0052]

[0053]

[0054] MCDB153 basal medium can support the colony formation and maintenance growth of human epidermal keratinocytes when used in combination with bovine pituitary gland extract. It does not contain serum or serum replacement components, and contains trace elements, sodium pyruvate glucose, L-glutamine and other nutrient content.

[0055] The experimental design idea of ​​high amino acid composition is as follows: when developing serum-free medium, it is necessary to add a variety of small molecular substances, amino acids, vitamins and trace elements to maintain and regulate the growth of cells and keep cells in a good cell state. When the cells are in a state of active grow...

Embodiment 2

[0080] The combination of small molecules facilitates the cultivation and expansion of airway epithelial cells. The serum-free medium for human airway epithelial cells in Example 1 includes MCDB153 basal medium (Biological Industries, #01-059-1A) and other added components, and is finally prepared as a complete serum-free medium. Serum-free medium can well maintain the growth of human airway epithelial cells, and is close to the effect of commercial KSFM medium in terms of cell morphology, cell viability and expansion fold (such as Figure 1-Figure 4 shown).

[0081] Therefore, in the process of primary culture and passage expansion of airway epithelial cells, the use of serum-free medium combined with the use of small molecule compounds can maintain high cell viability and expansion efficiency, which is beneficial to cell culture and expansion.

Embodiment 3

[0082] Example 3 Optimizing the concentration of rFGF1 (optimization scheme 1) has a significant impact on cell culture and expansion

[0083]When the concentration of rFGF1 in the serum-free medium of human airway epithelial cells was adjusted to 5-10ng / ml, compared with the commercialized KSFM medium, the cell viability and amplification factor were significantly improved ( Figure 5-Figure 7 Optimizing in 1). rFGF1 in the culture medium is a kind of FGF superfamily, mainly involved in receptor tyrosine kinase and RTK-Ras protein signaling pathway, involved in the regulation of cell growth.

[0084] Therefore, there is no particular limitation as long as it is a reagent capable of activating tyrosine kinase and RTK-Ras protein signaling pathway; the concentration of rFGF1 is not particularly limited as long as it can maintain the expansion and growth of airway epithelial cells, for example, 1 ng / ml , 4ng / ml, 6ng / ml, 8ng / ml, 10ng / ml, 12ng / ml, 18ng / ml, 20ng / ml, 22ng / ml, 24ng / ...

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Abstract

The invention belongs to the field of culture media, and particularly relates to a human airway epithelial cell serum-free culture medium with clear chemical components and an application of the humanairway epithelial cell serum-free culture medium. The serum-free culture medium for in-vitro multiplication culture of human airway epithelial cells comprises an MCDB153 basal culture medium, a high-amino-acid composition and a small-molecule composition, wherein the high-amino-acid composition comprises L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-tryptophan and L-tyrosine; the small-molecule composition comprises dibutyryl cyclic adenylate, a recombinant fibroblast growth factor 1, a recombinant epidermal growth factor and a recombinant insulin-like growth factor 1. A serum-free culture medium system disclosed by the invention is clear in chemical components, does not contain serum or serum substitutes, does not need trophoblastic cells for co-culture, does not need to coat a culture vessel with a matrix, and is economical and efficient. Therefore, the culture medium provided by the invention is very suitable for preparation and research of scientific research grade and clinical grade airway epithelial cells.

Description

technical field [0001] The invention belongs to the field of culture medium, and in particular relates to a human airway epithelial cell serum-free culture medium with defined chemical composition and its application. Background technique [0002] Healthy airway epithelium is a pseudostratified epithelium composed of three types of cells: basal cells, goblet cells, and ciliated cells. Human airway epithelial cells are isolated and extracted from respiratory tissue, which belongs to pseudostratified ciliated columnar epithelium, distributed on the inner surface of the respiratory tract, and mainly composed of cells of different shapes such as columnar, cup-shaped, spindle-shaped and cone-shaped. Basal cells line the basement membrane, goblet cells produce mucus to trap inhaled particles and pathogens, and ciliated cells produce the motility to remove them from the airways. [0003] Patients with severe tracheal and pulmonary disease have a poor quality of life, limited treat...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0625C12N2500/92C12N2500/32C12N2500/40C12N2501/113C12N2501/11C12N2501/105
Inventor 任涛张颖单姗辛志强
Owner 任涛
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