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Primary culture method for skeletal muscle cells of Microhyla ornata

A technology for primary culture of skeletal muscle cells, which is applied in the field of primary culture of skeletal muscle cells of Adenola spp., can solve the problems of no reports of skeletal muscle cells that can be subcultured, and achieves benefits for observation, cell survival, and light damage Effect

Inactive Publication Date: 2018-11-09
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No reports of cultured skeletal muscle cells from amphibians

Method used

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  • Primary culture method for skeletal muscle cells of Microhyla ornata
  • Primary culture method for skeletal muscle cells of Microhyla ornata
  • Primary culture method for skeletal muscle cells of Microhyla ornata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The preparation of embodiment 1 solution

[0047] (1) HBSS balanced salt solution (Hanks’balanced salt solution): Take 350mL standard HBSS balanced salt solution (Hyclone Cat.No.SH30030.02), add 150mL pure water, and store at 4°C for half a year.

[0048] (2) Collagenase I solution (collagenase I solution): dissolve 100mg of collagenase I (InvitrogenCat.No.17100-017) in 40mL of HBSS balanced salt solution (see 1), filter through a 0.1μm filter head, and store at -20°C in the dark for one year .

[0049] (3) Trypsin Solution: Take 35 mL of the original solution (Gibco Cat. No. 25200056), add 15 mL of pure water, and store at -20°C for one year.

[0050] (4) Y27632 solution: Dissolve 2mg of Y27632 (MCE Cat.No.HY-10583) in 625μL DMSO (SigmaCat.No.D2650), then add 5.625mL of pure water and store at -80°C for half a year.

[0051] (5) L-15 medium stock solution: take 335mL of standard Leibovitz L-15 medium (HycloneCat.No.SH30525.01), add 165mL of pure water, and store at 4...

Embodiment 2

[0053] The primary culture of the skeletal muscle cells of embodiment 2 Adenia magnesia

[0054] Go through the following steps:

[0055] (1) Prepare the solution;

[0056] (2) Choose one adult female frog with a body weight of 2.0 grams for freezing anesthesia, rinse with sterile water, and wipe the skin with 75% alcohol cotton ball;

[0057] (3) the frog is placed in a 3.5 cm diameter dish and dissected;

[0058] (4) Cut the thigh muscle with scissors and tweezers, and cut it into about 30-40mm 3 Muscle pieces of the same size were put into a new 3.5 cm diameter petri dish and washed 3 times with HBSS balanced salt solution;

[0059] (5) Transfer the cleaned muscle mass into a 15mL centrifuge tube with a Pasteur tube, absorb the residual HBSS balanced salt solution, add 1.5mL collagenase Ⅰ solution, tighten the cap of the centrifuge tube, and digest at 27°C for 90 minutes, every 30 Shake the centrifuge tube once every minute;

[0060] (6) Use a Pasteur tube to transfer ...

Embodiment 3

[0073] Embodiment 3 Primary culture of skeletal muscle cells of Rana aphidatidis

[0074] Go through the following steps:

[0075] (1) Prepare the solution;

[0076] (2) Choose a frozen adult female frog with a body weight of 0.5 grams, wash it with sterile water, and wipe the skin with 75% alcohol cotton ball;

[0077] (3) the frog is placed in a 3.5 cm diameter dish and dissected;

[0078] (4) Cut the thigh muscle with scissors and tweezers, and cut it into about 30-40mm 3 Muscle pieces of the same size were put into a new 3.5 cm diameter petri dish and washed 3 times with HBSS balanced salt solution;

[0079] (5) Transfer the cleaned muscle mass into a 15mL centrifuge tube with a Pasteur tube, absorb the residual HBSS balanced salt solution, add 1.0mL collagenase Ⅰ solution, tighten the cap of the centrifuge tube, and digest at 27°C for 90 minutes, every 30 Shake the centrifuge tube once every minute;

[0080] (6) Use a Pasteur tube to transfer all the digestion soluti...

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Abstract

The invention discloses a primary culture method for skeletal muscle cells of Microhyla ornata. The primary culture method comprises the following steps: subjecting adult Microhyla ornate to surface sterilization; dissecting the adult Microhyla ornate, taking out leg muscle and carrying out cleaning with a HBSS balanced salt solution; successively adding collagenase I and trypsin for digestion; subjecting a tissue suspension to blowing, beating and extruding with a sharpened tip and a non-sharpened tip, and then carrying out filtering by using cell filter screens with pore diameters of 100 [mu]m and 40 [mu]m respectively so as to obtain a cell suspension; and subjecting the cell suspension to culture at a constant temperature of 27 DEG C and replacing a previous complete cell culture medium with a new complete cell culture medium every 3 to 4 d. The primary culture method of the invention can rapidly and repeatedly establish primary culture of the skeletal muscle cells of Microhyla ornate; and needed cell culture equipment is simple and has good operability. The prepared skeletal muscle cells of the invention significantly supplement existing skeletal muscle cells and provide a novel cell material for research on major biological and medical problems such as terrestrial adaptability, development by metamorphosis, muscle development and muscle injury.

Description

technical field [0001] The invention belongs to the technical field of animal cell culture, and in particular relates to a method for primary culture of skeletal muscle cells of Rana adenocarpus. Background technique [0002] In 1907, Harrison used the hanging drop culture method to culture frog embryonic neural tissue in a lymphatic clot for several weeks, and observed that axons grew from the cultured tissue block, thereby creating a cover sheet covering The dimple glass hanging drop culture method has laid the foundation for the in vitro culture of animal tissues. In 1913, Carrel introduced the strict aseptic technique in surgical operation into the in vitro culture of animal cells, enabling the cells to grow in vitro for a long time. In 1916, Rous and Jones applied trypsinization to tissue digestion and cell passage, marking the beginning of real animal cell culture. In 1948, Earle isolated and continuously subcultured the first cell line from the subcutaneous tissue o...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0658C12N2509/00
Inventor 刘炯宇江建平常利明刘露莎
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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