CN110358724B

CN110358724B - Isolation and culture method of primary bronchial epithelial cells for COPD patients

Info

Publication number: CN110358724B
Application number: CN201910660315.1A
Authority: CN
Inventors: 费广鹤; 季爽; 张仁泉; 张大卫; 吴旭; 任祥春; 张文英
Original assignee: First Affiliated Hospital of Anhui Medical University
Current assignee: First Affiliated Hospital of Anhui Medical University
Priority date: 2019-07-22
Filing date: 2019-07-22
Publication date: 2020-12-25
Anticipated expiration: 2039-07-22
Also published as: CN110358724A

Abstract

The invention provides a separation culture method of primary bronchial epithelial cells of a COPD patient, which is characterized in that the cells are named DHBE-001 by classification, and the preservation number is CCTCC NO: 18169. the method comprises the steps of obtaining tissues, removing redundant connective tissues, dissecting respiratory tracts, cutting into segments, cleaning, digesting the tissues in a centrifugal tube, and shaking overnight; after digestion is finished, pouring the tissue into a tissue culture dish, adding fetal calf serum to a final concentration of 10%, and stopping digestion; washing with PBS buffer solution, collecting in a centrifuge tube, and centrifuging; adding the tissue digestive juice II, and standing until the tissue blocks are separated; adding FBS, centrifuging the solution, removing supernatant, adding DMEM/F12 to perform basic suspension, and then counting cells; placing the centrifugally precipitated cells in a culture dish containing BEGM culture medium to culture so as to complete separation; the method has the advantages of high cell separation rate, high purity and good activity, and the successfully separated primary bronchial epithelial cells can maintain good cell morphology and activity within 5-10 generations of in vitro culture.

Description

Isolation and culture method of primary bronchial epithelial cells for COPD patients

Technical Field

The invention belongs to the technical field of cell biology, and particularly relates to a separation culture method of human primary bronchial epithelial cells.

Background

In clinical scientific research work, examination and analysis aiming at specific cell functions and biological characteristics often involve a cell separation technology, the separation of a single bronchial epithelial cell from a human tracheal bronchus is an important pretreatment step for in vitro immunological and cytological experimental research, and the quality and quantity of the separated target cell are important links for ensuring the reliability of subsequent experiments.

Bronchial Epithelial Cells (HBECs) are composed of a continuous layer of epithelial cells, and play an important role not only in protecting tissues from external invasion and the first physiological barrier against infection by external pathogens, but also in airway inflammatory responses. Epithelial cells have been thought to be a simple physical barrier to prevent external irritants from entering airway tissues, but it is now thought that they have the role of mediating innate and adaptive immune-related cellular communication and regulating cellular activation as an important component of the immune system.

At present, bronchial epithelial cell strain (BEAS-2B) is generally adopted as a research object, but the experimental conclusion shows that the cell strain has no convincing effect on primary bronchial epithelial cells, and the primary bronchial epithelial cells are closer to the physiological environment of human airways and can better meet the experimental requirements aiming at different research objects (such as patients with chronic obstructive pulmonary disease and asthma).

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides an isolated culture method of primary bronchial epithelial cells of COPD patients.

The purpose of the invention is realized by the following technical scheme:

the classification naming of the primary bronchial epithelial cells DHBE-001 of the COPD patients is that the preservation number is CCTCC NO: 18169. the preservation unit: china general microbiological culture Collection center; and (4) storage address: beijing, China; the preservation date is 2019-06-27.

Preferably, the method for separating and culturing primary bronchial epithelial cells of COPD patients comprises the following steps:

s1, placing the obtained tissue in a culture dish filled with PBS buffer solution; the tissue is lung tissue after a lung lobe resection;

s2, removing redundant connective tissues, dissecting respiratory tracts, and cutting into segments;

s3, preparing a tissue washing solution for washing three times;

s4, transferring the tissue into a centrifuge tube, digesting the tissue by tissue digestive juice I, and shaking the centrifuge tube on a shaker at 4 ℃ overnight;

s5, finishing digestion, pouring the tissue in the centrifugal tube into a tissue culture dish, adding fetal calf serum to a final concentration of 10%, and finishing digestion;

s6, washing the mixture by PBS buffer solution, collecting the mixture in a centrifuge tube, and centrifuging the mixture for 5min at 4 ℃ by adopting a centrifugal force of 500 Xg;

s7, adding the tissue digestive juice II, placing the mixture in a dressing box at 37 ℃, and standing the mixture until the tissue blocks are visually separated;

s8, adding FBS to a final concentration of 10%, transferring the solution to a centrifuge tube, and centrifuging the centrifuge tube in a centrifuge with a centrifugal force of 500 Xg at 4 ℃ for 5 min; discarding the supernatant, adding DMEM/F12 culture medium to perform basic suspension, and then counting cells;

s9, placing the centrifuged and precipitated cells into a culture dish containing BEGM culture medium at 37 ℃ and 5% CO₂Culturing in a box, changing the liquid after 24h, and treating the changed liquid every 2-3 days to complete the separation.

Preferably, also included are cell culture methods comprising,

s10, cell culture:

preparing a culture dish coated with collagen for recovering and culturing P0 and freshly frozen cells and a culture dish which is not coated with collagen for culturing subculture cells;

p0 cells: the plating effect was assessed 24h after plating the P0 generation cells on the plates, if there were clumps or floating cells, washed out with PBS and replaced with BEGM containing the antibiotic; the suspended cell pellet was taken up in a conical tube, centrifuged at 500 Xg for 5min, and repeatedly added with tissue digest II for separation.

Passaged primary cells: the cells were passaged when 70-90% of the primary cells had grown, the cells that were difficult to separate were harvested with minimal pancreatin, washed with PBS, added to the dish pancreatin/EDTA, placed at 37 ℃ for 5-10min, the dish was gently tapped, digestion was stopped by adding 10% FBS, the cells were harvested and centrifuged at 4 ℃ at 500 Xg for 5min, the supernatant was discarded, the cells were resuspended in culture medium and counted.

Preferably, the tissue digestion solution I required for tissue digestion in S4 is a mixture of J-MEM, 1% pronase, and 0.01% bovine pancreatic deoxyribonuclease I.

Preferably, the tissue digestive juice II in the S7 consists of PBS, EDTA, DTT, protease XIV, CaCl₂、Mgcl₂And DNase I.

Preferably, the BEGM medium is serum-free BEGM medium supplemented with 4% (v/v) bovine pituitary extract, 0.25% hydrocortisone, 1% insulin, 1% human vascular endothelial growth factor, 1% epinephrine, 1% transferrin, 1% retinoic acid, 1% triiodothyronine, 1% gentamicin/amphotericin B by volume in serum-free BEBM medium and filtered through a 0.22 μm pore size filter.

Preferably, the collagen in the culture dish in S11 is diluted by deionized water at a ratio of 1:75 when added, and is placed at 37 ℃ for 2-24 hours, the excessive liquid is discarded, and the culture dish is placed under ultraviolet rays for 30 min.

The invention has the beneficial effects that: the method for separating the bronchial epithelial cells has the advantages of high cell separation rate, high purity and good activity, and the primary bronchial epithelial cells successfully separated can maintain good cell morphology and activity within 5-10 generations of in vitro culture in the culture process.

Drawings

FIG. 1: flow chart of isolated culture process.

FIG. 2: the cell growth status of primary bronchial epithelial cells of COPD patients isolated by the isolation method of the present invention was observed under an inverted microscope (within 14 days).

FIG. 3: the immunofluorescence of the invention identifies the bronchial epithelial cell marker protein cytokeratin CK 17/19.

FIG. 4: the immunohistochemical identification of the bronchial epithelial cell marker protein CK17/19 is a schematic diagram.

Detailed Description

The technical scheme of the invention is specifically described below by combining with an embodiment, and the invention discloses a separation culture method of human primary bronchial epithelial cells. The primary cells are generally separated by adopting a tissue adherence method or an enzyme digestion method, the enzyme digestion method is adopted in the invention, and meanwhile, in order to achieve higher yield, the embodiment adopts a fresh operation specimen, and after the operation specimen is cleaned, the operation specimen is slowly and gradually digested by adopting a plurality of digestive enzymes. The compound formula adopting a plurality of digestive enzymes has the advantages of relatively mild digestion capability and reduced damage to cells.

The culture conditions are as follows: the bronchial epithelial cells are different from other types of primary cells, and a serum-free culture medium is adopted during culture, so that the proliferation of other types of cells is inhibited under the serum-free growth environment.

The preparation method of the human primary bronchial epithelial cells is shown in the combined figure 1, and specifically comprises the following steps:

s1, taking out the fresh tissue, transporting the fresh tissue to a laboratory clean bench at a low temperature, and placing the tissue in a culture dish filled with PBS buffer solution;

s2, removing redundant connective tissues, dissecting respiratory tracts, cutting into 0.5-1cm fragments, and longitudinally cutting into 0.1-1.0cm parts;

s3, preparing 300ml of tissue washing liquid (Joklik-Minimum Essential Medium Eagle, J-MEM), and washing three times;

s4, transferring the tissue into a 50ml centrifuge tube, digesting the tissue by tissue digestive juice I (the component of the tissue digestive juice I is 30ml J-MEM +4ml 1% pronase +4ml 0.01% bovine pancreatic Deoxyribonuclease I (DNase I), placing the tissue digestive juice I from bovine pancreatic polynucleotides I, DNase I on a 4 ℃ shaker for overnight shaking, generally 4-24 h;

s5, finishing digestion, pouring the tissues in a 50ml centrifuge tube into a tissue culture dish with the thickness of 150mm, adding Fetal Bovine Serum (FBS) to the final concentration of 10%, and finishing digestion;

s6, washing the mixture by PBS buffer solution, collecting the mixture into a 50ml centrifuge tube, and centrifuging the centrifuge tube for 5min at the temperature of 500 Xg and 4 ℃;

s7, adding the tissue digestive juice II, placing the mixture in a dressing box at 37 ℃, and placing the mixture for 15min to 1h until the tissue blocks can be visually seen to be separated;

the tissue digestive juice II comprises the following components:

100ml	PBS
		58.44mg	EDTA
5mg	DTT
		25mg	protease XIV
75mg	Cacl₂
		100mg	Mgcl₂
1mg	DNase I

. S8, adding FBS to a final concentration of 10%, transferring the solution to a 50ml centrifuge tube, and centrifuging the centrifuge tube at a centrifugal force of 500 Xg for 5min at 4 ℃; discarding the supernatant, adding DMEM/F12 culture medium to perform basic suspension, and then counting cells;

s9, placing the cells subjected to centrifugal precipitation into a culture dish containing BEGM culture medium, culturing in a 5% CO2 dressing box at 37 ℃, changing the liquid after 24 hours, and treating the changed liquid every 2-3 days to complete the separation.

S10, preparing a culture dish coated with collagen for resuscitating and culturing P0 and fresh and frozen cells and a culture dish uncoated with collagen for subculturing cells; collagen treatment: adding 3.0ml collagen diluted with deionized water 1:75 into 100mm culture dish, standing at 37 deg.C for 2-24 hr, removing excessive liquid, standing under ultraviolet for 30 min, and standing at 4 deg.C for 8 weeks.

S11, cell culture:

1. p0 cells: the plating effect was assessed 24h after plating the P0 generation cells on the plates, if there were a small cell clump or some floating cells, washed out with PBS and replaced with BEGM containing the antibiotic. The suspended cell pellet was taken up in a 50ml conical tube, centrifuged at 500 Xg for 5min and the step S7 was repeated, i.e.tissue digest II was added again for separation.

2. Passaged primary cells: the cells were passaged when the primary cells grew to 70-90% and the difficult to isolate cells were harvested with a minimum amount of pancreatin. The cells were washed with PBS, 3ml of trypsin/EDTA per 100mm dish was added and digested at 37 ℃ for 5-10 min. After gently tapping the cell culture dish and separating the cells from the culture dish, the digestion was terminated by adding 10% FBS, the cells were collected in a 50ml conical tube and centrifuged at 500 Xg for 5min at 4 ℃. The supernatant was discarded, and the cells were resuspended in medium and counted.

The cell plating density in the above cell culture needs to be noted: human primary airway epithelial cells are not immortalized and require appropriate cell culture density. In general, the culture density of P0 passage cells: (>1.5×10⁵ cells/cm²) Required to obtain consistent, full, well-differentiated cells. Although it is very tempting to expand the culture of primary cells, excessive growth should be avoided, and the most suitable seeding density for P0 generation cells is 2-6X 10⁶Per 100mm collagen-coated cell culture dish. Under these conditions, the cells grew to 70% confluence between 5 and 7 days. If a longer time is required, this should be because the subsequent culture is disrupted. When the cell density is between 70% and 95%, the cells should be digested and then passaged to a new dish, or passaged, seeded at a density of about 100mm in a dish>1.0×10⁶And (4) cells.

At day 1 of isolation, cells were suspended in bulk in a petri dish, observed under a 200-fold microscope, as shown in fig. 2; on day 3 of isolation, some adherent clumping growth of cell clumps could be observed; on day 7, the cells began to gradually become round and spread or irregular in shape, and gathered together; on day 10, the cells entered the fast growth phase and were arranged in a cobblestone-like fashion.

To further confirm that the isolation was successful, epithelial cells were identified using immunofluorescence and immunohistochemistry for identification of epithelial marker proteins, and in conjunction with the results shown in FIGS. 3 and 4, cytokeratin CK17/19, the expression of CK17/19 was observed under a fluorescence microscope, indicating successful isolation and culture of airway epithelial cells.

There are, of course, many other specific embodiments of the invention and these are not to be considered as limiting. All technical solutions formed by using equivalent substitutions or equivalent transformations fall within the scope of the claimed invention.

Claims

1. A method for separating and culturing primary bronchial epithelial cells of COPD patients is characterized in that: the isolated culture method comprises the following steps:

s1, placing the obtained tissue in a culture dish filled with PBS buffer solution;

s3, preparing a tissue washing solution for washing three times;

s4, transferring the tissue into a centrifuge tube, digesting the tissue by tissue digestive juice I, and shaking the centrifuge tube on a shaking table at 4 ℃ overnight, wherein the tissue digestive juice I comprises the components of J-MEM, 1% of pronase and 0.01% of bovine pancreatic deoxyribonuclease I mixed liquid;

s7, adding tissue digestive juice II, placing in a 37 deg.C dressing box, standing until tissue block separation is seen visually, wherein the tissue digestive juice II comprises PBS, EDTA, DTT, protease XIV, CaCl₂、MgCl₂And DNase I;

s9, placing the cells subjected to centrifugal precipitation into a culture dish containing BEGM culture medium for culture, changing the culture solution after 24 hours, and treating the changed culture solution every 2-3 days to complete the separation.

2. The method for isolated culture of primary bronchial epithelial cells in COPD patients according to claim 1, wherein: also comprises a method for culturing the cells,

s10, preparing a culture dish coated with collagen for resuscitating and culturing P0 and fresh and frozen cells and a culture dish uncoated with collagen for subculturing cells;

s11, cell culture: p0 cells: the plating effect was assessed 24h after plating the P0 generation cells on the plates, and the cell pellet or the floating cells were washed out with PBS and replaced with BEGM containing an antibiotic; collecting the suspended cell mass into a conical tube, centrifuging at 500 Xg for 5min, repeatedly adding tissue digestive juice II for separation, wherein the tissue digestive juice II comprises PBS, EDTA, DTT, protease XIV, CaCl₂、MgCl₂And DNase I; passaged primary cells: the cells were passaged when 70-90% of the primary cells had grown, the cells that were difficult to separate were harvested with minimal pancreatin, washed with PBS, added to the dish pancreatin/EDTA, placed at 37 ℃ for 5-10min, the dish was gently tapped, digestion was stopped by adding 10% FBS, the cells were harvested and centrifuged at 4 ℃ at 500 Xg for 5min, the supernatant was discarded, the cells were resuspended in culture medium and counted.

3. The method for isolated culture of primary bronchial epithelial cells in COPD patients according to claim 1, wherein: the BEGM medium was serum-free BEGM medium supplemented with 4% v/v bovine pituitary extract, 0.25% hydrocortisone, 1% insulin, 1% human vascular endothelial growth factor, 1% epinephrine, 1% transferrin, 1% retinoic acid, 1% triiodothyronine and 1% gentamicin/amphotericin B by volume in serum-free BEBM medium and filtered through a 0.22 μm pore size filter.

4. The method for isolated culture of primary bronchial epithelial cells in COPD patients according to claim 2, wherein: and diluting the collagen in the culture dish in the S11 by using deionized water at a ratio of 1:75 when adding the collagen, placing the culture dish at 37 ℃ for 2-24 hours, removing redundant liquid, and placing the culture dish under ultraviolet rays for 30 min.