Method for culturing adult mouse cardiac fibroblasts

A technology of fibroblasts and culture methods, which is applied in the field of culture of adult mouse cardiac fibroblasts, can solve the problems of cumbersome procedures and cell contamination, and achieve the effect of high efficiency, cost and simple method

Pending Publication Date: 2019-10-22
THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Using Langendoff perfusion device and flow sorting are currently relatively mature methods for extracting adult mouse cardiac fibroblasts, but these methods have high requirements for experimental reagents, instruments and operating techniques, the procedures are cumbersome, and the possibility of cell contamination is also high. Therefore, it is very important to find a simple and quick method for extracting and culturing adult mouse cardiac fibroblasts

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  • Method for culturing adult mouse cardiac fibroblasts
  • Method for culturing adult mouse cardiac fibroblasts
  • Method for culturing adult mouse cardiac fibroblasts

Examples

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Embodiment 1

[0036] An embodiment of the method for culturing adult mouse cardiac fibroblasts of the present invention, the method is used to obtain primary cultured adult mouse cardiac fibroblasts and perform primary cell culture. The method specifically includes the following steps:

[0037] Step (1): SPF grade 8-12-week-old mice were taken, dislocated their cervical vertebrae, and transferred to a clean workbench, soaked in 75% ethanol for 1 min; quickly opened the chest cavity of the mouse with sterile ophthalmic scissors, and took out the Put the heart into a 10cm petri dish filled with pre-cooled calcium-free HBSS, squeeze out the residual blood in the heart, remove the heart atrium, left and right atrial appendages, pericardium, etc., and keep the ventricle;

[0038] Step (2): Use sterile forceps to transfer the heart to a new 10cm Petri dish filled with pre-cooled calcium-free HBSS, and use sterile ophthalmic curved scissors to cut the heart to 1mm 3 Use a 10ml electric pipette to ...

Embodiment 2

[0046] An embodiment of the adult mouse cardiac fibroblast subculture method in the present invention comprises the steps of: when the primary cell density obtained in Example 1 reaches above 90%, take out the primary cell, wash it twice with PBS solution, Add 2mL of 0.25% EDTA-trypsin solution to a 10cm culture dish, digest at 37°C for 45s~60s, when the cells shrink and the gap increases, add 4mL of medium to stop the digestion, blow the cells off with a pipette, and suck the cells The suspension was transferred to a 50mL centrifuge tube, centrifuged at 1000rpm for 3min at room temperature, and passaged at 1:2.

Embodiment 3

[0048] The passaged cells prepared in Example 2 are identified with the fibroblast marker Vimentin (red light) and the nuclear dye DAPI (blue light), and the specific steps of identification are as follows:

[0049] 1. Two to three days after cell subculture, discard the culture medium of the 96-well plate, wash it twice with PBS, add 4% paraformaldehyde to each well and let it stand at room temperature for 15 minutes;

[0050] 2. Discard the paraformaldehyde, rinse with PBS twice;

[0051] 3. Add 50 μl 0.5% TritonX-100 / PBS (permeation solution) to each well and let it stand at room temperature for 10-15 minutes;

[0052] 4. Discard the permeabilization solution and wash twice with PBS;

[0053] 5. Add 50 μl 1% BSA / PBS to each well to block for 1 hour at room temperature;

[0054] 6. Discard the blocking solution, dilute Vimentin primary antibody in 1% BSA / PBS at a ratio of 1:200, add 50 μl to each well, and incubate overnight in a 4°C refrigerator;

[0055] 7. On the secon...

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Abstract

The invention discloses a method for culturing adult mouse cardiac fibroblasts. The method comprises the following steps: taking SPF grade 8-12 weeks-old mice, and sterilizing the material after cervical dislocation; taking out the heart of the mice and placing the mice in a culture dish containing pre-cooled calcium-free HBSS, extruding the residual blood in the heart, removing the heart atrium,left and right auricula, and pericardium, preserving the ventricle, transferring the material to a new culture dish containing the pre-cooled calcium-free HBSS, cutting the heart, transferring the material to a centrifuge tube, and after the cardiac tissue is precipitated, sucking the HBSS by a pipette; adding a collagenase digestion solution in the centrifuge tube for digestion, and then resuspending the material to obtain a cell suspension; spreading the material into a first culture dish and performing standing culture; resuspending the cells, discarding a supernatant by centrifugation, spreading the resuspended cells in the medium to a second culture dish, and adding the original first culture dish to the new medium. The culture method is simple and easy, has good stability, high repeatability and uniform morphology, and the obtained fibroblasts grow well.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a method for culturing adult mouse cardiac fibroblasts. Background technique [0002] Cardiac fibroblasts are often referred to as supporting cells of the cardiac network and perform many fundamental functions that underlie both normal growth of the heart and ventricular remodeling in pathological conditions. Cardiac tissue remodeling is regulated by multiple cell types, including cardiomyocytes, fibroblasts, endothelial cells, smooth muscle cells and hematopoietic cells. [0003] At present, most people choose cardiac fibroblasts of suckling mice (1-3 days old) to study human heart diseases, but since the characteristics of cardiac fibroblasts of suckling mice and adult mice are not necessarily the same, the adult mouse heart It is more meaningful to construct models of fibroblasts to study human heart diseases such as myocardial hypertrophy, ventricular remodeling, myocar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0656C12N2509/00
Inventor 刘世明刘宁宁顾杰蕾周文怡徐如芹
Owner THE SECOND AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIV
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