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Human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes thereof

A bronchial epithelium, isolation and culture technology, applied in the field of cell biology, can solve the problem of high market price

Active Publication Date: 2013-12-18
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They are also often used as "normal cell" controls in cancer research, and they are very expensive on the market
At this stage, there has never been a "normal cell" that can be used in basic and clinical medical research at home and abroad

Method used

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  • Human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes thereof
  • Human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes thereof
  • Human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Primary isolation and culture of primary human normal bronchial epithelial cells

[0048] (1) With the informed consent of the patient or the patient guardian, collect the paracancerous normal tissue samples of surgically resected patients with lung cancer.

[0049] (2) Preparation of digestive solution: HL medium containing 0.2 mg / mL of collagenase and dispase; among them, HL medium is: DMEM (GIBCO # 11965-092) and serum-free medium SFM (GIBCO # 10744- 019) By volume ratio 1 : 3 mix, add 5% (v / v) fetal bovine serum at the same time, and 0.4 μg / mL cortisol (hydrocortisone), 5 μg / mL insulin (insulin), 8.4 ng / mL cholera toxin (cholera toxin), 10 ng / mL epidermal growth factor (epithelial growth factor (EGF)), 24 μg / mL adenine (adenine), 100 U / mL penicillin (penicillin), 100 μg / mL streptomycin (streptomycin), 0.25 μg / mL amphotericin B (Fungizone), 30 μM Fasudil (Fasudil), the above medium needs to be filtered through a 0.22 μm pore size filter).

[0050] (3) ...

Embodiment 2

[0059] Example 2 Subculture of human normal bronchial epithelial cells

[0060] (1) When human normal bronchial epithelial cells cultured in T25 or T75 culture flasks proliferate to 70-90% abundance, wash the cells twice with 1×PBS (0.01M, pH 7.4), and then wash with 0.05% ( Mass volume ratio) trypsin-EDTA digest monolayer cells for 2-5 minutes.

[0061] (2) Add 10 mL of complete DMEM to neutralize the digestion reaction for 1-2 minutes.

[0062] (3) Centrifuge at 1000 rpm for 5 minutes, remove the supernatant, resuspend the cell pellet and inoculate in 10 mL HL medium.

[0063] (4) If necessary, 1×10 6 Epithelial cells were resuspended in 1-2 mL of cell freezing medium (90% fetal bovine serum and 10% DMSO, v / v), and stored in liquid nitrogen for later use.

[0064] Subculture human normal bronchial epithelial cells according to the above-mentioned method, and the cell growth curve of the culture establishment line is as follows: figure 2 , continuous subculture for 55 da...

Embodiment 3

[0065] Example 3 Karyotype analysis and identification of human normal bronchial epithelial cells

[0066] (1) When human normal bronchial epithelial cells (1×10 6 ) in the exponential growth phase, add colchicine at a final concentration of 0.2 μg / mL, and continue to culture for 3.5 hours.

[0067] (2) Repeatedly blow the cells to make them fall off, and centrifuge at 2000 rpm for 5 minutes to harvest the cells.

[0068] (3) Discard the supernatant, add 8 mL of 0.075 mol / L KCl solution pre-warmed at 37°C, gently blow and beat the cell mass to mix, and place at 37°C for hypotonic treatment for 25 minutes.

[0069] (4) Add 1 mL of freshly prepared fixative (methanol : Glacial acetic acid=3 : 1, v / v), carefully pipette, mix, and centrifuge at 2000 rpm for 5 minutes.

[0070] (5) Discard the supernatant, add 8 mL of fixative, pipette to make a cell suspension, and fix at room temperature for 20 minutes.

[0071] (6) Centrifuge at 2000 rpm for 5 minutes, discard the superna...

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Abstract

The invention discloses a human normal bronchial epithelial cell and preliminary isolated culture and subculture methods and purposes of the human normal bronchial epithelial cell. The cell is named as the human normal bronchial epithelial cell HNBEC / HL-001, the preservation number of the cell is CCTCCNO: C201311. The preliminary isolated culture method includes the steps that fat in a para-carcinoma tissue sample excised from a patient with lung cancer through an operation is removed, dispase and DNasel are added to the fat for action after the fat is digested, the cell is collected through filtering and centrifugal operations, and the cell is suspended again with an HL culture medium for inoculated culture. The subculture method includes the steps that when the cell proliferates to 70-90% abundance, pancreatin-EDTA is used for digestion, and then DMEM is used for neutralization; the cell is collected through a centrifugal operation, and the cell is suspended again with an HL culture medium for inoculated culture. The human normal bronchial epithelial cell can be used for physiological research of human normal cells, drug toxicity research and detection of external normal cells, and research on pathogenesis of bronchia and lung diseases including bronchogenic carcinoma.

Description

[0001] technical field [0002] The invention belongs to the field of cell biology, and relates to a human normal bronchial epithelial cell and its primary isolation culture and subculture method and application. Background technique [0003] Vital organs such as lung, kidney, liver, pancreas and skin are composed of organ-specifically differentiated epithelial cells. These specifically differentiated epithelial cells are directly related to the specific functions of different organs, such as the gas exchange function of the lungs, the filtering function of the kidneys, the detoxification and neutralization functions of the liver, the production of insulin by pancreatic cells, and the protection of the skin from the external environment. s damage. Once these vital organs become diseased or degenerate, human health will be threatened, because these vital organs are difficult to be replaced, and specific cells of different organs cannot replace cells of other organs. These s...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12Q1/02
Inventor 李晖刘红亚冯文强王孝力陈凯
Owner WUHAN UNIV
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