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Fibroblasts derived from multiple tissue sources of native dog through primary isolated culture and immortalization construction method of fibroblasts

A technology for fibroblasts and lung fibroblasts, applied in the field of cell culture, can solve the problem of low activity of fibroblasts

Pending Publication Date: 2021-03-30
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no standard construction method for the primary isolation and culture of multi-tissue-derived fibroblasts, and the isolated fibroblasts still have the problem of low activity

Method used

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  • Fibroblasts derived from multiple tissue sources of native dog through primary isolated culture and immortalization construction method of fibroblasts
  • Fibroblasts derived from multiple tissue sources of native dog through primary isolated culture and immortalization construction method of fibroblasts
  • Fibroblasts derived from multiple tissue sources of native dog through primary isolated culture and immortalization construction method of fibroblasts

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] 1. The reagents are prepared as follows:

[0064] 0.2% Collagenase II: Weigh 0.06g collagenase II, dissolve in 30ml DMEM, pass through a 0.22μM filter, store at -20°C, and set aside.

[0065] 100ml DMEM complete culture medium: 89ml DMEM basal medium + 10ml fetal bovine serum + 1ml double antibody.

[0066] 1ml cell freezing solution: 100μl DMSO+900μl FBS.

[0067] DMEM medium, FBS, trypsin, and double antibodies were purchased from Gibco Company; collagenase II and DMSO were purchased from Sigma Company; PBS was purchased from Solebol Company.

[0068] 2. Primary isolation and culture of native dog fibroblasts

[0069] (1) Native dog tissue collection:

[0070] The local dog was killed in the cutting room, and about 1-2g of heart tissue, ventricle part, lung apex part, and leg muscle tissue were cut off with sterile scissors, soaked in 70% or 75% alcohol for 5-10s, and then added with 2% bismuth Wash 3 times with anti-1x PBS.

[0071] (2) Pre-treatment of tissue: ...

Embodiment 2

[0080] Puromycin screening concentration test

[0081] The soil dog cardiac fibroblasts, lung fibroblasts, and myofibroblasts F2 generation cells prepared in Example 1 were divided into 1 × 10 4 / well layout in 24-well plate, gradient screening with 0, 0.5 μg / ml, 1 μg / ml, 1.5 μg / ml, 2 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml puromycin, each The wells were replicated three times at each concentration, and the culture medium containing different concentrations of puromycin was replaced every day. Continuous screening was performed for 7 days, observed under an inverted microscope, and the lowest concentration of puromycin that killed all cells within 7 days was finally used as the optimal screening concentration.

[0082] The results showed that the best selection concentration of puromycin for cardiac fibroblasts was 1.5 μg / ml, the best selection concentration of puromycin for lung fibroblasts was 1 μg / ml, and the best selection concentration of puromycin for myofibroblasts was 2 μ...

Embodiment 3

[0084] SV40 T virus liquid prepared under different 293T cell states

[0085] 293T cells that were newly recovered, passaged three times after recovery, and passed more than 20 times in a row were plated in 100mm culture dishes, and the virus transfection was carried out when the cell density reached 50% to 60%. The ratio of transfection plasmid to transfection reagent was 1:4, the volume ratio of virus liquid and cell culture liquid is 1:5.

[0086] The results showed that the virus liquid obtained from the 293T cells passaged three times after resuscitation had the strongest ability to infect the F2 generation fibroblasts in Example 1, and formed cell clusters after puromycin selection.

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Abstract

The invention provides fibroblasts derived from multiple tissue sources of a native dog through primary isolated culture and an immortalization construction method of the fibroblasts, and belongs to the technical field of cell culture. Heart tissue, lung tip muscle tissue or leg muscle tissue of the native dog are used as materials and subjected to enzymolysis and separation of pancreatin and / or collagenase II, and myocardial fibroblasts, lung fibroblasts and myofibroblasts with typical fibroblast morphological characteristics are obtained. The myocardial fibroblasts, the pulmonary fibroblastsand the myofibroblasts which are subjected to primary isolated culture are transfected by SV40T virus liquid, and then puromycin screening culture and passage are performed to obtain immortalized myocardial fibroblasts, pulmonary fibroblasts and myofibroblasts. Compared with primary culture cells, the immortalized constructed cells are higher in growth speed and remarkably improved in activity, and a material basis is provided for dogs in the aspects of scientific research, disease research and the like.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a primary isolation and culture of fibroblasts derived from multiple tissues of a native dog and a construction method for immortalization thereof. Background technique [0002] Fibroblasts are the most common cells in connective tissue, and their cell shapes are mostly spindle-like, polygonal-like, and fibroblast-like. Fibroblasts are involved in the formation of collagen fibers, in wound repair, and also have the function of synthesizing and secreting proteins. Fibroblasts have a strong ability to divide and proliferate, and are highly adaptable. They are relatively easy to culture animal cell types. Fibroblasts are usually obtained by enzymatic digestion or tissue patch method in primary culture. Common fibroblasts include cardiac fibroblasts (CFs), lung fibroblasts (LFs) and myofibroblasts (MFs). Cardiac fibroblasts are an important part of heart tissue, a...

Claims

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Application Information

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IPC IPC(8): C12N5/077C12N5/071C12N5/10
CPCC12N5/0657C12N5/0656C12N5/0658C12N5/0688C12N2509/00C12N2509/10
Inventor 张树润张亚平高云尹婷婷李锦秀朱春玲
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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