CN105820996A - Human primary airway epithelial cell culture method - Google Patents
Human primary airway epithelial cell culture method Download PDFInfo
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- CN105820996A CN105820996A CN201610247201.0A CN201610247201A CN105820996A CN 105820996 A CN105820996 A CN 105820996A CN 201610247201 A CN201610247201 A CN 201610247201A CN 105820996 A CN105820996 A CN 105820996A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0688—Cells from the lungs or the respiratory tract
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Abstract
The invention provides a human primary airway epithelial cell culture method. The method comprises the following steps: removing airway fat and fiber tissues, dicing, attaching the dices into a 24-hole culture plate on which IV placenta collagen is spread, adding into a 150 mu l BMGM culture medium, transferring the tissue dices into new holes with prespread IV collagen when the epithelial cells climb out of the edge of the tissue dices and the density reaches 70%, continuing culture, repeating 5-8 times until the cell proliferation abundance is 80-90%, and carrying out pancreatin-EDTA (ethylene diamine tetraacetic acid) digestion; and removing fibroblasts by a different adherent culture process, adding the BMGM culture medium resuspended epithelial cells, inoculating into a new culture dish with prespread IV placenta collagen, and exchanging the solution every three days (the subculture ratio is not greater than 1:3), thereby completing the cell culture. The culture method has the advantages of reasonable design, stable result, favorable repetitiveness, uniform cultured cell morphology and favorable growth, and has the morphology and characteristics of typical airway epithelial cells.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of primitive cell culture method, the cultivation of a kind of human airway epithelial cells
Method.
Background technology
Bronchial asthma and chronic obstructive pulmonary disease are modal chronic airways disease.China is that a chronic airways disease is big
State, it is contemplated that have more than 100,000,000 patients.Airway epithelia as the first line of defence of lung inherent immunity in the morbidity of chronic airways disease
Play an important role.Normal human bronchial's epithelial cell major function: the epithelial cell of (1) bronchial surface constitutes substrate post
Shape structure, removes mucociliary.(2) physical barriers is formed by cilium, fibre-less and cell slimy etc..(3) produce
With the host defense system that a large amount of chemical mediators of secretion and cytokine form high complexity.Therefore human airway epithelial cells is that research is slow
The property pathogenetic important materials of airway disorders.
Currently for the main epithelial cell line using immortalization of research of human airway epithelial cells, and original cuiture many employings enzymic digestion
Method, animal origin is mainly mice.But cell line and airway of mice epithelial cell are big with in body human airway epithelial cells difference,
If therefore the meaning that primary human airway epithelial cells can be used to carry out chronic airways disease correlational study is substantially better than cell line and animal is thin
Born of the same parents.But because of difficulty of drawing materials, and the mankind are that its cell of organism of high evolution is the most successfully cultivated and is difficult to far away unicellular lower eukaryote,
Simultaneously as the incubation period of adult cell's growth in vitro is considerably longer than rodent, successfully cultivate difficulty further so external.Existing
Some research uses enzyme digestion separation and Culture human airway epithelial cells, but the method needs tissue relatively big, and digestion condition requires tight
Lattice, often because of different manufacturers, the difference of the digestive enzyme of the most same producer different batches, need re-optimization condition of culture, training
Support poor stability.Some scholars is also tested from off-shore purchase primary human airway epithelial cells strain, but cell strain is expensive and
Cannot repeatedly pass on, domestic general laboratory is difficult to use in a large number；Biological product importation customs examination and approval procedures are loaded down with trivial details simultaneously, week
Phase is long, and the cell strain state difference frequently resulted in, survival rate is extremely low.In sum, find a kind of efficiently, easy, low cost
The method of human airway epithelial cells original cuiture becomes the key of Success in Experiment.
Summary of the invention
It is an object of the invention to the shortcoming overcoming prior art with not enough, it is provided that the cultural method of a kind of human airway epithelial cells.
This cultural method is consistent, reproducible, and the cellular morphology of cultivation is homogeneous, well-grown, and has typical epithelial cell
Form and feature.
The cultural method of the primary human airway epithelial cells of a kind of people of the present invention, is achieved through the following technical solutions:
(1) collect with the relatively normal lung tissue sample of air flue, by molten containing dual anti-aseptic PBS in pre-cooling of tissue storage
In liquid；
The most pre-cool condition is preferably in pre-cooling on ice；Containing dual anti-aseptic PBS solution formula be 100mg/ml penicillin and
100mg/ml streptomycin, lung tissue sample stores within the time is 12h in PBS solution.
(2) in 24 porocyte culture plates, IV type placental collagen acetum is overlay: dissolve IV type placental collagen with acetum
(commercial formulations: Sigma-alorich C5533), the final concentration of 0.05mg/ml of solution, each 24 orifice plates add 500 μ l,
Every hole collagen concentration is 13.2 μ g/cm2, overnight air-dry standby in being placed in super-clean bench；
Wherein said acetum is sterile deionized water 10ml+ glacial acetic acid 200ul, overlays collagen plate condition of storage and is-20 DEG C, 1
Individual month.
(3) step (1) tissue sample is put in the sterile petri dish containing pre-cooling PBS, isolate diameter under the microscope about
The air flue of 1cm band cartilaginous ring, the most carefully removes the fat of air flue outer layer and fibrous tissue, with pre-cooling containing dual anti-
Aseptic PBS solution rinse air flue endocrine, tissue sample is cut into the piece of tissue of size about 3 × 3mm, is affixed on step (2)
In the culture plate of preparation, paste block direction is that tracheal epithelium face is affixed on plate；
(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, when density reaches 70%, piece of tissue is moved to the new IV placental collagen that overlays
Continue in hole to cultivate, be derived from primary human airway epithelial cells；
(6), when primary human airway epithelial cells is expanded to 80-90% abundance, after cleaning by room-temperature sterile PBS solution, add
0.3-0.5ml 0.1%-0.2% trypsin solution 37 DEG C digests 1-2 minute, when cell occurs that retraction, intercellular substance increase,
Adding 1ml DMEM complete medium terminate digestion and be centrifuged, after using difference adherent method purification epithelial cell, precipitation is used
BEGM culture medium re-suspended cell, is inoculated in the culture dish overlaying IV placental collagen, within every 3 days, changes liquid 1 time, i.e. completes to pass
Cultivate for cell, it is thus achieved that the human airway epithelial cells of Secondary Culture.The preferably ratio of passing on is not more than 1:3.
Difference adherent method: be centrifuged and remove supernatant, by preheating DMEM complete medium re-suspended cell precipitation, is inoculated in and does not overlay
In the culture dish of IV placental collagen, carefully drawing supernatant after standing 10min in incubator, recentrifuge removes supernatant, takes precipitation.
Centrifugal condition is that 1000rpm is centrifuged 1 minute.Wherein the optimum condition of the preheating of DMEM complete medium is 37 DEG C of water-baths.
Described BEGM is addition epidermal growth factor in BEBM (Bronchial Epithelial Basal Medium) 500ml
Son (hEGF, 0.5ml), bovine insulin (Insulin, 0.5ml), transferrins (Transferrin, 0.5ml), triiodo thyroid
Element (Triiodothyronine, 0.5ml), hydrocortisone (Hydrocortisone, 0.5ml), retionic acid (Retinoic Acid, 0.5
Ml), epinephrine (Epinephrine, 0.5ml), calf pituitary extract (BPE, 2.0ml), GA, 0.5ml.
Condition of culture in described step (4) and (6) is 37 DEG C, 5%CO2。
In described step (5), cell climbs out of the time and is about 3-5 days, piece of tissue repeatable paste block 5-8 time.
Containing 0.1%-0.2%EDTA (ethylenediaminetetraacetic acid) in the described trypsin solution in step (6), every hole dosage
0.3-0.5ml, digestion time is 1-2 minute；DMEM complete medium is the DMEM culture medium adding 10% hyclone.
In the inventive method, patient age, sex and protopathy do not limit.
The present invention has the advantage that relative to prior art and effect:
(1) fibroblast pollution is one of defect of existing method cultivation human airway epithelial cells.This method is by essences such as microscopes
Fine workmanship has removal air flue outer layer fiber tissue；Use epithelial cell special media；Epithelial cell and fibroblast is utilized again when passing on
The time difference opposite sex of dimension cell attachment, is cultivating and purification epithelial cell to greatest extent in passing on.
(2) needed for cultural method, Specimen origin enriches, and all specimen with 1cm diameter air flue all can be as tissue-derived.
(3) cultural method low cost, consistent, reproducible, the cellular morphology of cultivation is homogeneous, well-grown.
(4) present invention can cultivate normal and pathological state downtake epithelial cell, for physiology and the pathophysiology of epithelial cell
Research, isolated cells characteristic closer to morbid state, for further further investigation broncho-pulmonary disease, especially asthma and slow
The pathogenesis of the chronic airways disease such as property obstructive pulmonary disease has established condition, it is provided that abundant material source.
Accompanying drawing explanation
Fig. 1 is that 3-5 days epithelial cells gradually climb out of from piece of tissue periphery after inverted phase contrast microscope (100X) undertissue paste block
Photo.
Fig. 2 is the photo of 3-5 days human airway epithelial cells after inverted phase contrast microscope (400X) undertissue the 5th paste block is cultivated.
Fig. 3 is the employing enzyme digestion separation and Culture human airway epithelial cells of 7 days under inverted phase contrast microscope (100X).
Fig. 4 is the photo that inverted phase contrast microscope (400X) undertissue paste block epithelium posterius cell grows to 90% abundance.
Fig. 5 is the photo of 3 days human airway epithelial cells of Secondary Culture under inverted phase contrast microscope (400X).
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this,
According to the foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, above-mentioned substantially without departing from the present invention
On the premise of technological thought, it is also possible to make the amendment of other various ways, replace or change.
Embodiment 1: the separation and Culture of the primary human airway epithelial cells of people
Concrete steps:
(1) Carcinoma side normal tissue of excision is collected.Lung tissue is stored in 4 DEG C and contains in dual anti-aseptic PBS solution,
Dual anti-formula is 100mg/ml penicillin and 100mg/ml streptomycin.
(2) with sterile deionized water 10ml+ glacial acetic acid 200ul dissolving 5mg IV type placental collagen, final concentration of 0.05mg/ml,
Each 24 orifice plates add 500 μ l, overnight air-dry standby in being placed in super-clean bench.
(3) tissue sample is put in the sterile petri dish containing pre-cooling PBS, isolate diameter about 1cm band with disinfection and sterilization equipment
The air flue of cartilaginous ring, the most carefully removes fat and the fibrous tissue of air flue outer layer.With pre-cooling containing dual anti-aseptic
PBS solution rinses air flue endocrine.Tracheal tissue is cut into the piece of tissue of size about 3 × 3mm, and airway epithelia face is close to
It is covered with in advance on the culture plate of IV placental collagen.
(4) each culture hole adds the BEGM culture medium of 150 μ l, and BEGM is BEBM (Bronchial Epithelial Basal
Medium) 500ml adds epidermal growth factor (hEGF, 0.5ml), bovine insulin (Insulin, 0.5ml), transferrins
(Transferrin, 0.5ml), Lithyronine (Triiodothyronine, 0.5ml), hydrocortisone (Hydrocortisone,
0.5ml), retionic acid (Retinoic Acid, 0.5ml), epinephrine (Epinephrine, 0.5ml), calf pituitary extract (BPE,
2.0ml)、GA,0.5ml.Culture plate inserts incubator, and condition of culture is 37 DEG C, 5%CO2。
(5) treat that 3-5 days epithelial cells climb out of (Fig. 1) from piece of tissue edge, when density reaches 70%, piece of tissue is moved to new overlaying
Continue in the hole of IV placental collagen to cultivate, piece of tissue repeatable paste block 5-8 time (Fig. 2).
(6) experiment uses the most the most frequently used enzyme digestion separation and Culture human airway epithelial cells the same period, and the epithelium that digestion method is cultivated is thin
In born of the same parents, visible a large amount of fibroblasts pollute (Fig. 3), and cell purity is poor.
Embodiment 2: the separation and Culture of the primary human airway epithelial cells of people
Concrete steps:
(1) on the premise of patient or patient donor's informed consent, the Carcinoma side normal tissue of Operation for Lung Cancer excision is collected.
Lung tissue being stored in 4 DEG C contain in dual anti-aseptic PBS solution, dual anti-formula is 100mg/ml penicillin and 100mg/ml
Streptomycin.
(2) with sterile deionized water 10ml+ glacial acetic acid 200ul dissolving 5mg IV type placental collagen, final concentration of 0.05mg/ml,
Each 12 orifice plates add 1000 μ l, overnight air-dry standby in being placed in super-clean bench.
(3) tissue sample is put in the sterile petri dish containing pre-cooling PBS, isolate diameter about 1cm band with disinfection and sterilization equipment
The air flue of cartilaginous ring, removes fat and the fibrous tissue of air flue outer layer under the microscope.With pre-cooling containing dual anti-aseptic PBS
Solution rinses air flue endocrine.Tracheal tissue is cut into the piece of tissue of size about 3 × 3mm, and airway epithelia face is close to overlay
Have on the culture plate of IV placental collagen.
(4) each culture hole adds the BEGM culture medium of 150 μ l, and BEGM is BEBM (Bronchial Epithelial Basal
Medium) 500ml adds epidermal growth factor (hEGF, 0.5ml), bovine insulin (Insulin, 0.5ml), transferrins
(Transferrin, 0.5ml), Lithyronine (Triiodothyronine, 0.5ml), hydrocortisone (Hydrocortisone,
0.5ml), retionic acid (Retinoic Acid, 0.5ml), epinephrine (Epinephrine, 0.5ml), calf pituitary extract (BPE,
2.0ml)、GA,0.5ml.Culture plate inserts incubator, and condition of culture is 37 DEG C, 5%CO2。
(5) treat that 3-5 days epithelial cells climb out of from piece of tissue edge, move to piece of tissue when density reaches 70% new overlay IV Placenta Hominis
Continue in the hole of collagen to cultivate, piece of tissue repeatable paste block 5-8 time.
Embodiment 3: the separation and Culture of primary human airway epithelial cells
Concrete steps:
(1) Carcinoma side normal tissue of excision is collected.Lung tissue is stored in 4 DEG C and contains in dual anti-aseptic PBS solution,
Dual anti-formula is 100mg/ml penicillin and 100mg/ml streptomycin.
(2) with sterile deionized water 5ml+ glacial acetic acid 200ul dissolving 5mg IV type placental collagen, final concentration of 0.1mg/ml, often
Individual 24 orifice plates add 250 μ l, overnight air-dry standby in being placed in super-clean bench.
(3) tissue sample is put in the sterile petri dish containing pre-cooling PBS, go out diameter about 1cm with the fracture of sterilization
With the air flue of cartilaginous ring, the most carefully remove fat and the fibrous tissue of air flue outer layer.With pre-cooling containing dual anti-nothing
Bacterium PBS solution rinses air flue endocrine.Tracheal tissue is cut into the piece of tissue of size about 3 × 3mm, and airway epithelia face is close to
On the culture plate being covered with IV placental collagen in advance.
(4) each culture hole adds the BEGM culture medium of 150 μ l, and BEGM is BEBM (Bronchial Epithelial Basal
Medium) 500ml adds epidermal growth factor (hEGF, 0.5ml), bovine insulin (Insulin, 0.5ml), transferrins
(Transferrin, 0.5ml), Lithyronine (Triiodothyronine, 0.5ml), hydrocortisone (Hydrocortisone,
0.5ml), retionic acid (Retinoic Acid, 0.5ml), epinephrine (Epinephrine, 0.5ml), calf pituitary extract (BPE,
2.0ml)、GA,0.5ml.Culture plate inserts incubator, and condition of culture is 37 DEG C, 5%CO2。
(5) treat that 3-5 days epithelial cells climb out of from piece of tissue edge, move to piece of tissue when density reaches 70% new overlay IV Placenta Hominis
Continue in the hole of collagen to cultivate, piece of tissue repeatable paste block 5-8 time.
Embodiment 4: the Secondary Culture of the primary human airway epithelial cells of people
Concrete steps:
(1) human airway epithelial cells is expanded to during 80-90% (Fig. 4), and after cleaning by room-temperature sterile PBS solution, addition contains
0.1%EDTA trypsin solution, every hole dosage 0.2-0.3ml, when cell occurs that retraction, intercellular substance increase, addition contains
In 10% hyclone DMEM and after digestion reaction, it is allowed to form cell suspending liquid.
(2) 1000rpm removes supernatant after being centrifuged 1 minute, by 37 DEG C of water-bath preheating DMEM culture medium re-suspended cell precipitations, connects
Plant in Nostoc commune Vanch ware.
(3) at 37 DEG C, 5%CO2Supernatant, 1000rpm recentrifuge 1 minute is carefully drawn after incubator stands 10min
After remove supernatant, be inoculated in after precipitation BEGM culture medium is resuspended in the pre-culture dish being covered with IV placental collagen, the ratio of passing on is
1:2 (Fig. 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。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112501110A (en) * | 2020-11-26 | 2021-03-16 | 海西纺织新材料工业技术晋江研究院 | Standardized culture medium and method for three-dimensional culture of lung and lung cancer tissue organoid |
| CN114891725A (en) * | 2022-03-29 | 2022-08-12 | 南京医科大学 | Mouse airway culture method |
| CN115089713A (en) * | 2022-06-29 | 2022-09-23 | 浙江大学 | Use of lysosomal inhibitors for the preparation of a medicament for the prevention, treatment and/or amelioration of acute lung injury/acute respiratory distress syndrome |
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| CN102433296A (en) * | 2011-12-13 | 2012-05-02 | 广州医学院第一附属医院 | Method for culturing human airway epithelial cells |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501110A (en) * | 2020-11-26 | 2021-03-16 | 海西纺织新材料工业技术晋江研究院 | Standardized culture medium and method for three-dimensional culture of lung and lung cancer tissue organoid |
| CN112501110B (en) * | 2020-11-26 | 2023-08-25 | 海西纺织新材料工业技术晋江研究院 | Standardized culture medium for three-dimensional culture of lung and lung cancer tissue organoids and culture method |
| CN114891725A (en) * | 2022-03-29 | 2022-08-12 | 南京医科大学 | Mouse airway culture method |
| CN114891725B (en) * | 2022-03-29 | 2024-01-16 | 南京医科大学 | Mouse airway culture method |
| CN115089713A (en) * | 2022-06-29 | 2022-09-23 | 浙江大学 | Use of lysosomal inhibitors for the preparation of a medicament for the prevention, treatment and/or amelioration of acute lung injury/acute respiratory distress syndrome |
| CN115089713B (en) * | 2022-06-29 | 2023-11-28 | 浙江大学 | Application of lysosomal inhibitor in preparation of medicine for preventing, treating and/or relieving acute lung injury/acute respiratory distress syndrome |
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