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Human pulmonary artery smooth muscle cell separation and culturing method and application of same

A technology of smooth muscle cells and culture methods, which is applied in the direction of animal cells, vertebrate cells, artificial cell constructs, etc., can solve the problems of long culture period and uneconomical original cell quantity, and achieve good growth, rich specificity, The effect of mild damage

Inactive Publication Date: 2011-08-31
BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only a few scholars have obtained human pulmonary artery smooth muscle cells from the specimens of patients after PTE. The methods for culturing human pulmonary artery smooth muscle cells include the composite collagenase method or the patch method, but the simple patch method has a long culture cycle and complex The collagenase method uses too many enzymes, which is not only uneconomical but also obtains a small number of viable protocells

Method used

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  • Human pulmonary artery smooth muscle cell separation and culturing method and application of same
  • Human pulmonary artery smooth muscle cell separation and culturing method and application of same
  • Human pulmonary artery smooth muscle cell separation and culturing method and application of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] 1. Main experimental materials

[0042] (1) Specimen source: the dissection obtained during pulmonary thromboendarterectomy (PTE) in patients with thromboembolic pulmonary hypertension.

[0043] (2) HBSS balanced salt solution: add 8.0g sodium chloride, 0.4g potassium chloride, 0.1g magnesium chloride, 1.0g glucose, 0.14g calcium chloride, 0.06g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 0.35 g sodium bicarbonate, 0.09 g disodium hydrogen phosphate and 2.08 g HEPES, prepared 24 hours before the experiment and filter-sterilized or autoclaved at pH 7.4.

[0044] (3) Digestive solution: prepared by adding 25mg type II collagenase to 10ml HBSS balanced salt solution.

[0045] (4) The complete culture medium is: 46.5ml of M231 basal medium (SMBS), 2.5ml of medium supplement (SMGS), 100U / ml of penicillin, 100mg / ml of streptomycin antibiotics.

[0046] 2. Operation steps

[0047] (1) Obtain the stripped material in pulmonary thromboendarterectomy (PTE) in patient...

Embodiment 2

[0062] 1. Main experimental materials

[0063] (1) Specimen source: the dissection obtained during pulmonary thromboendarterectomy (PTE) in patients with thromboembolic pulmonary hypertension.

[0064] (2) HBSS balanced salt solution: add 7.01g sodium chloride, 0.37g potassium chloride, 0.06g magnesium chloride, 0.79g glucose, 0.11g calcium chloride, 0.03g potassium dihydrogen phosphate, and 0.05g magnesium sulfate to 1000ml triple distilled water. 0.29g of sodium bicarbonate, 0.04g of disodium hydrogen phosphate and 1.19g of HEPES were prepared 24 hours before the experiment and sterilized by filtration or autoclaved at pH 7.2.

[0065] (3) Digestive solution: prepared by adding 30mg type II collagenase to 8ml HBSS balanced salt solution.

[0066] (4) The complete culture solution is: 90ml of M231 basal medium (SMBS), 1ml of medium supplement (SMGS), 80U / ml of penicillin, 80mg / ml of streptomycin antibiotics.

[0067] 2. Operation steps

[0068] (1) Obtain the stripped mate...

Embodiment 3

[0083] 1. Main experimental materials

[0084] (1) Specimen source: stripped objects obtained from pulmonary thromboendarterectomy (PTE) in patients with thromboembolic pulmonary hypertension.

[0085] (2) HBSS balanced salt solution: add 8.77g sodium chloride, 0.45g potassium chloride, 0.2g magnesium chloride, 1.39g glucose, 0.17g calcium chloride, 0.136g potassium dihydrogen phosphate, and 0.25g magnesium sulfate to 1000ml triple distilled water. 0.38g of sodium bicarbonate, 0.18g of disodium hydrogen phosphate and 2.38g of HEPES were prepared 24 hours before the experiment and sterilized by filtration or autoclaved at pH 7.4.

[0086] (3) Digestive solution: prepared by adding 20mg type II collagenase to 12ml HBSS balanced salt solution.

[0087] (4) The complete culture medium is: prepared from 100ml of M231 basal medium, 5ml of medium supplement SMGS, 90U / ml of penicillin, and 90mg / ml of streptomycin antibiotics.

[0088] 2. Operation steps

[0089] (1) Obtain the stri...

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Abstract

The invention relates to a method of separating and culturing human pulmonary artery smooth muscle cells from strips of PTE and the cells' application in studies of diseases related to lung circulation. The method comprises treating cell membranes by single enzymic digestion method to decrease damage degree of the cells, thereby benefiting survival of the cells. The culturing method has stable technology and good repeatability, the cultured cells have uniform forms, and the growth of the cells is good. At the same time, the cells obtained provide rich material source with strong singularity for further study on lung circulation diseases, particularly for experiments on Chronic Thromboembolic Pulmonary Hypertension patients' pathogenesis, diseases generation and development processes and PTE prognosis.

Description

technical field [0001] The present invention relates to a method for isolating and cultivating human pulmonary artery smooth muscle cells from strips of human pulmonary artery thromboendarterectomy (PTE), the smooth muscle cells obtained by the method, and the application of the cells in the research of pulmonary circulation-related diseases. It belongs to the field of biotechnology. Background technique [0002] Human pulmonary artery smooth muscle cells are important cell materials for studying the pathogenesis of pulmonary circulation-related diseases, especially pulmonary hypertension and other diseases. At present, most of the sources of pulmonary artery smooth muscle cells used in experimental research at home and abroad are from animals, mainly from rats. However, due to different species and large differences between cells, the significance of using mouse-derived pulmonary artery smooth muscle cells in the study of human pulmonary hypertension and its pathogenesis i...

Claims

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Application Information

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IPC IPC(8): C12N5/071A61K35/42A61P11/00A61P9/12A61P7/02
Inventor 王辰王军刘杰张知非李积凤李晶缪冉郭丽娟刘岩顾松
Owner BEIJING CHAOYANG HOSPITAL CAPITAL MEDICAL UNIV
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