CN105820996A - Human primary airway epithelial cell culture method - Google Patents

Abstract

The present invention provides a culture method of human primary airway epithelial cells, by removing airway fat and fibrous tissue, cutting into small pieces and pasting them on a 24-well culture plate pre-coated with IV placental collagen, adding 150 μl of BMGM medium, When the epithelial cells crawl out from the edge of the tissue block and the density reaches 70%, move the tissue block to a new hole pre-coated with IV collagen to continue culturing, repeat 5-8 times, and when the cell proliferation reaches 80-90% abundance, use Digest with trypsin-EDTA; remove fibroblasts by differential adherence method, add BEGM medium to resuspend epithelial cells, inoculate in new culture dishes pre-coated with IV placental collagen, the passage ratio is not more than 1:3, every 3 The medium was changed once a day to complete the cell culture. The culture method of the invention has reasonable design, stable results, good repeatability, and cultured cells have uniform shape and good growth, and have the shape and characteristics of typical airway epithelial cells.

Description

A kind of human primary airway epithelial cell culture method

technical field

The invention belongs to the field of biological technology, and relates to a method for culturing primary cells, in particular to a method for culturing airway epithelial cells.

Background technique

Asthma and chronic obstructive pulmonary disease are the most common chronic airway diseases. China is a large country with chronic airway diseases, with more than 100 million patients estimated. The airway epithelium, as the first line of defense of lung innate immunity, plays an important role in the pathogenesis of chronic airway diseases. The main functions of normal human bronchial epithelial cells: (1) The epithelial cells on the bronchial surface constitute the basal columnar structure and remove mucus and cilia. (2) The physical barrier is composed of ciliated, non-ciliated and mucus-secreting cells. (3) Produce and secrete a large number of chemical mediators and cytokines to form a highly complex host defense system. Therefore, airway epithelial cells are important materials for studying the pathogenesis of chronic airway diseases.

At present, research on airway epithelial cells mainly uses immortalized epithelial cell lines, while primary culture mostly adopts enzyme digestion method, and the animal source is mainly mice. However, cell lines and mouse airway epithelial cells are quite different from human airway epithelial cells in vivo, so if primary human airway epithelial cells can be used for research on chronic airway diseases, the significance is obviously better than cell lines and animal cells. However, due to the difficulty in obtaining materials, and human beings are highly evolved organisms, it is far more difficult to successfully cultivate cells in vitro than lower organisms. At the same time, because the incubation period of adult cells in vitro growth is significantly longer than that of rodents, successful in vitro cultivation is even more difficult. Existing studies use enzymatic digestion to isolate and culture human airway epithelial cells, but this method requires large tissues and strict digestion conditions. Often due to differences in digestive enzymes from different manufacturers or even different batches of the same manufacturer, the culture conditions need to be re-optimized , poor culture stability. Some scholars have also purchased primary human airway epithelial cell lines from overseas for experiments, but the cell lines are expensive and cannot be passaged multiple times, and it is difficult for ordinary domestic laboratories to use them in large quantities. The state of the cell line is poor, and the survival rate is extremely low. In summary, finding an efficient, simple, and low-cost method for primary culture of human airway epithelial cells has become the key to the success of the experiment.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and provide a method for culturing human airway epithelial cells. The culture method is stable in technique and good in repeatability, and the cultured cells are uniform in shape, grow well, and have the shape and characteristics of typical epithelial cells.

A culture method of human primary airway epithelial cells of the present invention is realized through the following technical solutions:

(1) Collect lung tissue samples with relatively normal airways, and store the tissue in pre-cooled sterile PBS solution containing double antibodies;

The pre-cooling condition is preferably pre-cooling on ice; the formula of the sterile PBS solution containing the double antibody is 100mg/ml penicillin and 100mg/ml streptomycin, and the storage time of the lung tissue samples in the PBS solution is within 12h.

(2) Pre-spread type IV placental collagen acetic acid solution in 24-well cell culture plates: dissolve type IV placental collagen (commercial preparation: Sigma-alorich C5533) with acetic acid solution, the final concentration of the solution is 0.05mg/ml, each 24 Add 500 μl to the orifice plate, the collagen concentration in each well is 13.2 μg/cm ² , and place it in an ultra-clean bench to air dry overnight;

Wherein the acetic acid solution is 10ml of sterile deionized water + 200ul of glacial acetic acid, and the storage condition of the pre-coated collagen plate is -20°C for 1 month.

(3) Put the tissue sample from step (1) into a sterile petri dish containing pre-cooled PBS, separate the airway with a diameter of about 1 cm with a cartilage ring under the microscope, and carefully remove the fat and fibers in the outer layer of the airway under the microscope For tissue, wash the airway endocrine with pre-cooled sterile PBS solution containing double antibody, cut the tissue sample into tissue blocks with a size of about 3×3mm, and paste them on the culture plate prepared in step (2), and the direction of the block is The tracheal epithelium is attached to the board;

(4) Add 150 μl of BEGM medium to each culture well, and put it into an incubator for culture;

(5) When the epithelial cells climbed out from the edge of the tissue block and the density reached 70%, the tissue block was moved to a new well pre-laid with IV placental collagen to continue culturing, thereby obtaining primary airway epithelial cells;

(6) When the primary airway epithelial cells are amplified to 80-90% abundance, after washing with room temperature sterile PBS solution, add 0.3-0.5ml 0.1%-0.2% trypsin solution to digest at 37°C for 1-2 Minutes, when the cells retract and the intercellular space increases, add 1ml of DMEM complete medium to stop digestion and centrifuge. After the epithelial cells are purified by the differential attachment method, the cells are resuspended in BEGM medium for sedimentation, and inoculated on pre-coated IV placenta In the collagen culture dish, the medium was changed once every 3 days to complete subculture cell culture and obtain subcultured human airway epithelial cells. The preferred passage ratio is not greater than 1:3.

Differential adherence method: centrifuge to remove the supernatant, resuspend the cell pellet with preheated DMEM complete medium, inoculate in a culture dish without pre-coated IV placental collagen, and carefully absorb the supernatant after standing in the incubator for 10 minutes, then centrifuge again Remove the supernatant and take the precipitate. The centrifugation condition was 1000 rpm for 1 minute. The preferred condition for preheating the complete DMEM medium is a 37°C water bath.

The BEGM is BEBM (Bronchial Epithelial Basal Medium) 500ml adding epidermal growth factor (hEGF, 0.5ml), bovine insulin (Insulin, 0.5ml), transferrin (Transferrin, 0.5ml), triiodothyronine (Triiodothyronine ,0.5ml), hydrocortisone (Hydrocortisone,0.5ml), retinoic acid (Retinoic Acid,0.5ml), epinephrine (Epinephrine,0.5ml), calf pituitary extract (BPE,2.0ml), GA, 0.5ml.

The culture conditions in the steps (4) and (6) are 37° C., 5% CO ₂ .

In the step (5), the cell crawling time is about 3-5 days, and the tissue block can be pasted 5-8 times repeatedly.

The trypsin solution in the described step (6) contains 0.1%-0.2% EDTA (ethylenediaminetetraacetic acid), and the addition amount of each well is 0.3-0.5ml, and the digestion time is 1-2 minutes; the DMEM complete medium is Add 10% fetal bovine serum to DMEM medium.

In the method of the present invention, the patient's age, sex and primary disease are not limited.

Compared with the prior art, the present invention has the following advantages and effects:

(1) Fibroblast contamination is one of the defects of existing methods for culturing airway epithelial cells. This method removes the fibrous tissue of the outer layer of the airway with the help of fine tools such as a microscope; uses a specific medium for epithelial cells; and utilizes the difference in the attachment time of epithelial cells and fibroblasts during passage to maximize the purification of epithelial cells during culture and passage.

(2) The specimens required for the culture method are abundant, and all specimens with airways with a diameter of 1 cm can be used as tissue sources.

(3) The culture method is low in cost, stable in technology, good in repeatability, and the cultured cells have uniform morphology and good growth.

(4) The present invention can cultivate airway epithelial cells under normal and pathological conditions, which can be used for the study of physiology and pathophysiology of epithelial cells. The pathogenesis of chronic airway diseases such as chronic obstructive pulmonary disease lays the groundwork and provides a rich source of material.

Description of drawings

Figure 1 is a photograph of epithelial cells gradually crawling out from the periphery of the tissue block 3-5 days after the tissue block was attached under an inverted phase-contrast microscope (100X).

Figure 2 is a photograph of human airway epithelial cells 3-5 days after the fifth patch culture of the tissue under an inverted phase-contrast microscope (400X).

Figure 3 is the airway epithelial cells isolated and cultured for 7 days by enzyme digestion under an inverted phase-contrast microscope (100X).

Fig. 4 is a photograph of epithelial cells grown to 90% abundance after tissue patching under an inverted phase-contrast microscope (400X).

Fig. 5 is a photograph of human airway epithelial cells subcultured for 3 days under an inverted phase-contrast microscope (400X).

detailed description

The present invention will be described in further detail below in conjunction with the examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. Under the premise of technical thinking, other modifications, substitutions or changes can also be made in various forms.

Example 1: Isolation and culture of human primary airway epithelial cells

Specific steps:

(1) Surgically resected adjacent normal tissues were collected. The lung tissue was stored at 4°C in a sterile PBS solution containing double-antibody formulations of 100 mg/ml penicillin and 100 mg/ml streptomycin.

(2) Dissolve 5 mg of type IV placental collagen with 10 ml of sterile deionized water + 200 ul of glacial acetic acid to a final concentration of 0.05 mg/ml, add 500 μl to each 24-well plate, and place it in an ultra-clean bench to air-dry overnight for later use.

(3) Put the tissue sample into a sterile petri dish containing pre-cooled PBS, separate the airway with a diameter of about 1 cm with a cartilage ring with sterilized instruments, and carefully remove the fat and fibrous tissue in the outer layer of the airway under a microscope. Flush airway secretions with pre-cooled sterile PBS solution containing double antibody. The tracheal tissue was cut into tissue blocks with a size of about 3×3 mm, and the airway epithelial surface was attached to a culture plate pre-coated with IV placental collagen.

(4) Add 150 μl of BEGM medium to each culture well, BEGM is BEBM (Bronchial Epithelial Basal Medium) 500ml, add epidermal growth factor (hEGF, 0.5ml), bovine insulin (Insulin, 0.5ml), transferrin (Transferrin, 0.5ml), triiodothyronine (0.5ml), hydrocortisone (Hydrocortisone, 0.5ml), retinoic acid (0.5ml), epinephrine (Epinephrine, 0.5ml), calf pituitary extract (BPE, 2.0ml), GA, 0.5ml. The culture plate was placed in an incubator, and the culture conditions were 37° C., 5% CO ₂ .

(5) After 3-5 days, the epithelial cells crawl out from the edge of the tissue block (Figure 1). When the density reaches 70%, move the tissue block to a new hole pre-coated with IV placental collagen to continue culturing. The tissue block can be pasted repeatedly. 5-8 times (Figure 2).

(6) During the same period of the experiment, airway epithelial cells were separated and cultured by the most commonly used enzymatic digestion method. A large amount of fibroblast contamination was seen in the epithelial cells cultured by the digestion method (Figure 3), and the purity of the cells was poor.

Example 2: Isolation and culture of human primary airway epithelial cells

Specific steps:

(1) Under the premise of the informed consent of the patient or the patient's authorized person, collect the paracancerous normal tissues of the patients with lung cancer after surgical resection. The lung tissue was stored at 4°C in a sterile PBS solution containing double-antibody formulations of 100 mg/ml penicillin and 100 mg/ml streptomycin.

(2) Dissolve 5 mg of type IV placental collagen with 10 ml of sterile deionized water + 200 ul of glacial acetic acid to a final concentration of 0.05 mg/ml, add 1000 μl to each 12-well plate, and place it in an ultra-clean bench for overnight air-drying.

(3) Put the tissue sample into a sterile petri dish containing pre-cooled PBS, separate the airway with a diameter of about 1 cm with a cartilage ring with a sterilized instrument, and remove the fat and fibrous tissue in the outer layer of the airway under a microscope. Flush airway secretions with pre-cooled sterile PBS solution containing double antibody. The tracheal tissue was cut into tissue blocks with a size of about 3×3 mm, and the airway epithelial surface was attached to a culture plate pre-coated with IV placental collagen.

(4) Add 150 μl of BEGM medium to each culture well, BEGM is BEBM (Bronchial Epithelial Basal Medium) 500ml, add epidermal growth factor (hEGF, 0.5ml), bovine insulin (Insulin, 0.5ml), transferrin (Transferrin, 0.5ml), triiodothyronine (0.5ml), hydrocortisone (Hydrocortisone, 0.5ml), retinoic acid (0.5ml), epinephrine (Epinephrine, 0.5ml), calf pituitary extract (BPE, 2.0ml), GA, 0.5ml. The culture plate was placed in an incubator, and the culture conditions were 37° C., 5% CO ₂ .

(5) After 3-5 days, the epithelial cells crawl out from the edge of the tissue block, and when the density reaches 70%, move the tissue block to a new hole pre-coated with IV placenta collagen to continue culturing. The tissue block can be pasted 5-8 times repeatedly .

Example 3: Isolation and culture of primary human airway epithelial cells

Specific steps:

(1) Surgically resected adjacent normal tissues were collected. The lung tissue was stored at 4°C in a sterile PBS solution containing double-antibody formulations of 100 mg/ml penicillin and 100 mg/ml streptomycin.

(2) Dissolve 5 mg of type IV placental collagen with 5 ml of sterile deionized water + 200 ul of glacial acetic acid to a final concentration of 0.1 mg/ml, add 250 μl to each 24-well plate, and place it in an ultra-clean bench to air-dry overnight for later use.

(3) Put the tissue sample into a sterile petri dish containing pre-cooled PBS, separate the airway with a diameter of about 1 cm with a cartilage ring with a sterilized instrument, and carefully remove the fat and fibrous tissue in the outer layer of the airway under a microscope . Flush airway secretions with pre-cooled sterile PBS solution containing double antibody. The tracheal tissue was cut into tissue blocks with a size of about 3×3 mm, and the airway epithelial surface was attached to a culture plate pre-coated with IV placental collagen.

(4) Add 150 μl of BEGM medium to each culture well, BEGM is BEBM (Bronchial Epithelial Basal Medium) 500ml, add epidermal growth factor (hEGF, 0.5ml), bovine insulin (Insulin, 0.5ml), transferrin (Transferrin, 0.5ml), triiodothyronine (0.5ml), hydrocortisone (Hydrocortisone, 0.5ml), retinoic acid (0.5ml), epinephrine (Epinephrine, 0.5ml), calf pituitary extract (BPE, 2.0ml), GA, 0.5ml. The culture plate was placed in an incubator, and the culture conditions were 37° C., 5% CO ₂ .

(5) After 3-5 days, the epithelial cells crawl out from the edge of the tissue block, and when the density reaches 70%, move the tissue block to a new hole pre-coated with IV placenta collagen to continue culturing. The tissue block can be pasted 5-8 times repeatedly .

Example 4: Subculture of primary human airway epithelial cells

Specific steps:

(1) When human airway epithelial cells expand to 80-90% (Figure 4), wash with room temperature sterile PBS solution, add trypsin solution containing 0.1% EDTA, add 0.2-0.3ml per well, when the cells appear When retraction and increased intercellular space, DMEM containing 10% fetal bovine serum is added to neutralize the digestion reaction to form a cell suspension.

(2) After centrifuging at 1000rpm for 1 minute, remove the supernatant, resuspend the cell pellet in preheated DMEM medium in a 37°C water bath, and inoculate it in an ordinary culture dish.

(3) After standing in a 37°C, 5% _CO2 incubator for 10 minutes, carefully draw the supernatant, centrifuge again at 1000rpm for 1 minute, and remove the supernatant. In the culture dish, the passage ratio was 1:2 (Figure 5).

Claims (9)

1. people's primary human airway epithelial cells cultural method, it is characterised in that: realized by following steps:

(1) collect with the lung tissue sample of air flue, tissue sample is stored in pre-cooling containing in dual anti-aseptic PBS solution；

(2) in 24 porocyte culture plates, overlay IV type placental collagen acetum, overnight air-dry standby in being placed in super-clean bench；

(3) step (1) tissue sample is put in the sterile petri dish containing pre-cooling PBS, isolate diameter about 1cm under the microscope Air flue, with after the rinsing containing dual anti-aseptic PBS solution of pre-cooling, stripping and slicing is affixed in culture plate prepared by step (2)；

(4) each culture hole adds the BEGM culture medium of 150 μ l, inserts incubator and cultivates；

(5) treat that epithelial cell climbs out of from piece of tissue edge, piece of tissue is moved in the new hole overlaying IV collagen when density reaches 70% Continue to cultivate, be derived from primary human airway epithelial cells；

(6) when primary human airway epithelial cells is expanded to 80-90% abundance, 0.3-0.5ml 0.1%-0.2% trypsin solution is added 37 DEG C digest 1-2 minute, add 1ml DMEM complete medium and terminate digestion and be centrifuged, use difference adherent method purification epithelium After cell, with BEGM culture medium re-suspended cell, it is inoculated in the culture dish overlaying IV placental collagen, within every 3 days, changes liquid 1 time, I.e. complete passage cell to cultivate, it is thus achieved that the human airway epithelial cells of Secondary Culture.

A kind of people's primary human airway epithelial cells cultural method the most according to claim 1, it is characterised in that: step (2) institute Stating the IV final concentration of 0.05mg/ml of placental collagen solution, IV placental collagen bed board concentration is 13.2 μ g/cm², IV placental collagen Plate condition of storage is-20 DEG C, 1 month.

A kind of people's primary human airway epithelial cells cultural method the most according to claim 1, it is characterised in that: step (3) institute Stating piece of tissue is 3 × 3mm.

A kind of people's primary human airway epithelial cells cultural method the most according to claim 1, it is characterised in that: in step (5) Piece of tissue repeatable paste block 5-8 time.

A kind of people's primary human airway epithelial cells cultural method the most according to claim 1, it is characterised in that: in step (6) Containing 0.1%-0.2% ethylenediaminetetraacetic acid in trypsin solution, described cell-seeding-density is 2-3 × 10⁵Individual/ml, described DMEM complete medium be: DMEM and the mixed solution of hyclone, described hyclone accounts for culture medium cumulative volume 10%, centrifugal condition is 1000rpm, 1 minute.

A kind of people's primary human airway epithelial cells cultural method the most according to claim 1, it is characterised in that: in step (6) Described difference adherent method is by the DMEM complete medium re-suspended cell precipitation of preheating, is inoculated in and does not overlay IV Placenta Hominis glue In former culture dish, after standing 10 minutes, draw culture medium recentrifuge.

A kind of people's primary human airway epithelial cells cultural method the most according to claim 1, it is characterised in that: in step (6) The ratio of passing on is not more than 1:3.

A kind of people's primary human airway epithelial cells cultural method the most according to claim 1, it is characterised in that: step (4) and (6) the BEGM culture medium described in is: in BEBM500ml add 0.5ml epidermal growth factor, 0.5ml bovine insulin, 0.5ml transferrins, 0.5ml Lithyronine, 0.5ml hydrocortisone, 0.5ml retionic acid, 0.5ml epinephrine, 2.0ml calf pituitary extract, 0.5ml GA.

A kind of people's primary human airway epithelial cells cultural method the most according to claim 1, it is characterised in that: in step (4) and (6) Condition of culture is 37 DEG C, 5%CO₂。