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Primary culture method for alveolar epithelial cells of Microhyla ornata

A technology of alveolar epithelium and primary culture, applied in cell dissociation methods, epidermal cells/skin cells, tissue culture and other directions, can solve problems such as blank culture, achieve good growth, simple culture conditions, and less damage.

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

The culture of the alveolar epithelial cells of Rana rugosa is blank at present

Method used

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  • Primary culture method for alveolar epithelial cells of Microhyla ornata
  • Primary culture method for alveolar epithelial cells of Microhyla ornata
  • Primary culture method for alveolar epithelial cells of Microhyla ornata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] The preparation of embodiment 1 solution

[0041] (1) HBSS balanced salt solution (Hanks’balanced salt solution): take 350mL standard HBSS balanced salt solution (HycloneCat.No.SH30030.02), add 150mL pure water, and store at 4°C for half a year.

[0042](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 .

[0043] (3) 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.

[0044] (4) 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°C for half a year.

[0045] (5) Cell complete medium: before use, take 45mL L-15 medium stock solution (see 4), add 5mL fetal bovine serum (FBS) (WISENTC...

Embodiment 2

[0046] Example 2 The primary culture of the alveolar epithelial cells of Rana magnesia

[0047] Go through the following steps:

[0048] (1) Prepare the solution;

[0049] (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;

[0050] (3) the frog is placed in a 3.5 cm diameter dish and dissected;

[0051] (4) Cut the lungs with Venus scissors and microscopic tweezers, put them into a new 3.5 cm diameter petri dish, and wash 3 times with HBSS balanced salt solution;

[0052] (5) Use a Pasteur tube to transfer the cleaned lungs into a 15mL centrifuge 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 5 hours, every 30 minutes Shake the centrifuge tube once;

[0053] (6) Use a Pasteur tube to transfer all the digestive solution (including residual solid lung tissue) i...

Embodiment 3

[0061] Example 3 The primary culture of the alveolar epithelial cells of Rana magnesia

[0062] Go through the following steps:

[0063] (1) Prepare the solution;

[0064] (2) Choose one adult male frog with a body weight of 0.5 grams to freeze and anesthetize, after rinsing with sterile water, wipe the skin with 75% alcohol cotton ball;

[0065] (3) the frog is placed in a 3.5 cm diameter dish and dissected;

[0066] (4) Cut the lungs with Venus scissors and microscopic tweezers, put them into a new 3.5 cm diameter petri dish, and wash 3 times with HBSS balanced salt solution;

[0067] (5) Use a Pasteur tube to transfer the cleaned lungs into a 15mL centrifuge 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 4 hours, every 30 minutes Shake the centrifuge tube once;

[0068] (6) Use a Pasteur tube to transfer all the digestive solution (including residual solid lung tissu...

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Abstract

The invention discloses a primary culture method for alveolar epithelial 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 the lung and carrying out cleaning with a HBSS balanced salt solution; adding a collagenase I solution for digestion; subjecting a digested solution to refrigeration and centrifugation and adding a complete cell culture medium; blowing and beating the fragments of the lung tissue, and then carrying out filtering by using a cell filter screen with a pore diameter of 100 [mu]m so as to obtain a cell suspension; and subjecting the cell suspension to culture at a constant temperature of 27 DEG C and replacing the previous completecell 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 alveolar epithelialcells of Microhyla ornate; needed cell culture equipment is simple and has good operability; and acquired cells have obviously faster growth speed than the alveolar epithelial cells of mammals. The prepared alveolar epithelial cells of the invention significantly supplement existing alveolar epithelial cells of mammals and provide a novel cell material for research on major biological and medicalproblems such as terrestrial adaptability, lung development, pulmonary fibrosis and tuberculosis.

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 alveolar epithelial 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 tiss...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0625C12N2509/00
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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