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Method for cultivating adult distal arteria pulmonalis smooth muscle cells

A technology of smooth muscle cells and culture methods, applied in animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of cell biological characteristics affecting experimental results, expensive cell lines, and unsatisfactory experiments, and achieve rich materials. source, good growth, good reproducibility

Inactive Publication Date: 2010-10-06
广州医学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is not ideal to use the main pulmonary artery to study the mechanism of pulmonary hypertension.
In addition to using animals as materials (mostly rats), foreign scholars also purchased adult pulmonary artery smooth muscle cell lines for experiments, but the cell lines are expensive and not primary cultured cells, and the biological characteristics of the cells have changed. Experimental results; while a small number of scholars have also cultivated adult distal pulmonary artery smooth muscle cells, but the culture medium they use is smooth muscle cell basal medium (SMBM), which is expensive and difficult for domestic general laboratories to accept

Method used

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  • Method for cultivating adult distal arteria pulmonalis smooth muscle cells
  • Method for cultivating adult distal arteria pulmonalis smooth muscle cells
  • Method for cultivating adult distal arteria pulmonalis smooth muscle cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Specific steps:

[0037] 1. Main experimental materials

[0038] (1) Reagent preparation:

[0039]1. HBSS: containing 130mol / L sodium chloride, 5mol / L potassium chloride, 1.2mol / L magnesium chloride, 10mol / L hydroxyethylpiperazine ethylsulfuric acid (HEPES), 20μmol / L calcium chloride, 10mol / L Anhydrous glucose, 100U / ml penicillin and 100mg / ml streptomycin aqueous solution, the pH value of this solution is 7.4.

[0040] 2. Calcium-free HBSS: containing 130mol / L sodium chloride, 5mol / L potassium chloride, 1.2mol / L magnesium chloride, 10mol / L hydroxyethylpiperazine ethylsulfuric acid (HEPES), 10mol / L anhydrous glucose, 100U / ml penicillin and 100mg / ml streptomycin aqueous solution, the pH value of this solution is 7.4.

[0041] 3. Pre-digestion solution: containing 130mol / L sodium chloride, 5mol / L potassium chloride, 1.2mol / L magnesium chloride, 10mol / L hydroxyethylpiperazine ethylsulfuric acid (HEPES), 10mol / L anhydrous glucose, 1.5 mg / ml type I collagenase, 100 U / ml p...

Embodiment 2

[0066] Specific steps:

[0067] 1. Main experimental materials

[0068] (1) Reagent preparation:

[0069] 1. HBSS: containing 125mol / L sodium chloride, 6mol / L potassium chloride, 1mol / L magnesium chloride, 5mol / L HEPES, 15μmol / L calcium chloride, 15mol / L anhydrous glucose, 105U / ml penicillin and 105mg / ml Streptomycin aqueous solution, the pH value of this solution is 7.4.

[0070] 2. Calcium-free HBSS: containing 125mol / L sodium chloride, 6mol / L potassium chloride, 1mol / L magnesium chloride, 5mol / L HEPES, 15mol / L anhydrous glucose, 105U / ml penicillin and 105mg / ml streptomycin aqueous solution , the pH of the solution is 7.4.

[0071] 3. Pre-digestion solution: containing 125mol / L sodium chloride, 6mol / L potassium chloride, 1mol / L magnesium chloride, 5mol / L HEPES, 15mol / L anhydrous glucose, 1.3mg / ml type I collagenase, 105U / ml penicillin and 105mg / ml streptomycin aqueous solution, the pH value of this solution is 7.4.

[0072] 4. Digestive juice: containing 125mol / L sodium...

Embodiment 3

[0093] Specific steps:

[0094] 1. Main experimental materials

[0095] (1) Reagent preparation:

[0096] 1. HBSS: containing 135mol / L sodium chloride, 3mol / L potassium chloride, 1.5mol / L magnesium chloride, 15mol / L HEPES, 25μmol / L calcium chloride, 5mol / L anhydrous glucose, 90U / ml penicillin and 90mg / L ml streptomycin aqueous solution, the pH value of this solution is 7.4.

[0097] 2. Calcium-free HBSS: containing 135mol / L sodium chloride, 3mol / L potassium chloride, 1.5mol / L magnesium chloride, 15mol / L HEPES, 5mol / L anhydrous glucose, 90U / ml penicillin and 90mg / ml streptomycin Aqueous solution, the pH value of the solution is 7.4.

[0098] 3. Pre-digestion solution: containing 135mol / L sodium chloride, 3mol / L potassium chloride, 1.5mol / L magnesium chloride, 15mol / L HEPES, 5mol / L anhydrous glucose, 1.0mg / ml type I collagenase, 90U / ml Penicillin and 90mg / ml streptomycin aqueous solution, the pH value of this solution is 7.4.

[0099] 4. Digestive juice: containing 135mol / L...

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Abstract

The invention discloses a method for culturing adult distal end pulmonary artery smooth muscle cells, which comprises following steps: firstly, separating pulmonary artery smooth muscle cells from an adult distal end pulmonary artery smooth muscle layer which is obtained and culturing primary cells, secondly, obtaining the adult distal end pulmonary artery smooth muscle cells which are subcultured, taking out the primary cells and cleaning with PBS solution after the primary cells in the first step grow to the logarithmic growth phase, adding trypsin solution 0.3-0.5ml 0.1%-0.2%, digesting for 2-3 minutes under the temperature of 35-37 DEG C, gettering the trypsin solution when cells appear retraction and the intercellular space is enlarged, adding complete culture medium 3-5ml to enable the trypsin solution to form cell suspension, inoculating in a new culture bottle, changing medium each three days, then, finishing culturing subculture cells, and obtaining the culturing adult distalend pulmonary artery smooth muscle cells which are subcultured. The method for culturing has stable technique and good repeatability, and the cells which are cultured have uniform forms with good growth and have the forms and characteristics of typical smooth muscle cells.

Description

technical field [0001] The invention relates to a cell culture method, in particular to a culture method for adult distal pulmonary artery smooth muscle cells, and belongs to the field of biotechnology. Background technique [0002] Adult distal pulmonary artery smooth muscle cells are important cell materials for studying the pathogenesis of pulmonary circulation-related diseases, especially pulmonary hypertension and other diseases. The current methods for culturing pulmonary artery smooth muscle cells include enzymatic digestion and tissue block patching. Most of the animal sources are rats, and the differences between different species of rats and humans are relatively large, so the significance of using adult pulmonary artery smooth muscle cells to study the pathogenesis of pulmonary hypertension and related research is significantly greater than using animal cells. However, due to the difficulty in obtaining materials, and human beings are highly evolved organisms, it...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/08C12N5/071
Inventor 王健洪城冉丕鑫李冰卢文菊彭公永胡锦兴李晓岩
Owner 广州医学院
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