CN105936888A - Isolation culture and identification method of human tonsil epithelial cells - Google Patents

Isolation culture and identification method of human tonsil epithelial cells Download PDF

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CN105936888A
CN105936888A CN201510583200.9A CN201510583200A CN105936888A CN 105936888 A CN105936888 A CN 105936888A CN 201510583200 A CN201510583200 A CN 201510583200A CN 105936888 A CN105936888 A CN 105936888A
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tonsil
cell
culture
isolation
epithelial cell
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CN105936888B (en
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金玉
李树珍
李琦
孙真真
郑必霞
黄群
杨光
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Nanjing Children's Hospital Affiliated To Nanjing Medical University
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Nanjing Children's Hospital Affiliated To Nanjing Medical University
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Abstract

The invention relates to an isolation culture and identification method of human tonsil epithelial cells. The method mainly includes culture solution preparation, culture bottle treatment, tonsil tissue sampling, tonsil tissue grouping digestion comparison, isolation of tonsil epithelial cells, morphological observation and immunofluorescence assay. The invention improves the separation technique of oral epithelial cells, and explores an in vitro isolation and cultivation technology of tonsil epithelial cells. The technology has high success rate, simple steps, and high purification rate, and almost causes no pollution, can be applied to the in vitro cultivation and purification process of human tonsil epithelial cells, and plays an important role in the research on the mechanism of action of the follow-up EB and other viruses in the tonsil.

Description

The Isolation and identification method of human tonsil's epithelial cell
Technical field
The present invention relates to field of biomedicine technology, be specifically related to the extracorporeal culturing method of human tonsil's epithelial cell.
Background technology
Tonsil is peripheral lymphoid organ, belongs to again mucosa associated lymphoid tissue, has complicated and special crypts tubing, It is positioned at digestive tract and respiratory tract co-portal, is the important place producing immunne response, during wherein crypts epithelium is this organ First contacting antigen, retention antigen, and produce the position of immunne response, therefore crypts epithelium is the weight that tonsil exercises immunologic function Want tissue barrier.But under the repetitious stimulation of external antigen, usually cause the generation of chronic tonsillitis and adenoid vegetation, Its main cause is exactly antibacterial and the infection of virus, and increasing research finds the infection with Epstein-Barr virus, and relation is the closest. But concrete infection mechanism is still not clear, therefore in-vitro separation human tonsil Crypt Cells, it is possible to preferably inquire into EB further The infection mechanism of virus.
About the primary separation of tonsil epithelial cell, domestic still do not have pertinent literature to report, the tonsil cell of external report divides Be concentrated mainly on two kinds of separation methods from method, a kind of method the earliest be derived from oral cavity biology department of nineteen ninety University of Washington and The research of department of pharmacy, is the serum-free culture technology about mouth epithelial cells, belongs to enzyme digestion, becomes follow-up tonsil body Outer cultivation well use for reference foundation, but technically there are some differences and be not easy successfully multiple in the research method of each laboratory System [1].Another kind of method is the research of Bostonian TuftsUniversity medical college in 2004, utilizes fresh amygdaline outer implant, Carry out cell in vitro migration, and cultivate, but this kind of method, and process is relatively complicated, and separation cycle is longer, by individual diversity Different impact is relatively big, and easily pollutes [2].
1.Dolphine Oda,E.W.,Human oral epithelial cell culture I.Improved conditions for reproducible culture in serum-free medium.IN VITRO CELL DEV-AN,1990.26(6):p.589-595.
2.Pegtel,D.M.,J.Middeldorp,and D.A.Thorley-Lawson,Epstein-Barr virus infection in ex vivo tonsil epithelial cell cultures of asymptomatic carriers.J Virol,2004.78(22):p.12613-24..
Summary of the invention
It is an object of the invention to the isolation technics of mouth epithelial cells is improved, inquired into out that a kind of success rate is higher, step Simply, purification rate high, the in-vitro separation of tonsil epithelial cell and the culture technique polluted occurs hardly, to follow-up EB And study on mechanism that other virus is in tonsil is significant..
For reaching above-mentioned purpose, present invention provide the technical scheme that the Isolation and identification method of human tonsil's epithelial cell, Its step is as follows:
One, Preparatory work of experiment:
(1) culture fluid preparation: use K-SFM culture medium, adds epidermal growth factor, cattle pituitary extract, CaCl2, blue or green Mycin-streptomycin mixed liquor and amphotericin, mixing, filter, preserve;
(2) culture bottle processes;In culture bottle, add Type I/III collagen, be advisable, in bio-safety not have bottom culture bottle In cabinet, lucifuge is ventilated overnight, and next day irradiates under uviol lamp, saves backup;
Two, separation and Culture
(1) tonsil is drawn materials: tonsil is placed under Biohazard Safety Equipment the ice bath training filling PBS balanced salt solution Support in ware, remove periphery connective tissue, with the PBS after sterilizing for several times, be cut into piece of tissue, and transfer them to dispase In grade II solution;
(2) tonsil packet digestion: overnight incubation;
(3) separation of tonsil epithelial cell: the piece of tissue that will have digested, is sieved through filter in secondary Nikkei cell, cell suspension is centrifugal, Abandoning supernatant, cell precipitation trypsinization liquid is resuspended, digestion;It is subsequently adding the K-SFM culture medium containing serum, stops Digestion, then be centrifuged, abandon supernatant, finally resuspended by K-SFM culture medium, primary cell kind is entered in culture dish, stands training Support;
(4) morphological observation: the tonsil epithelial cell of different incubation times is carried out morphologic observation under inverted phase contrast microscope, Observe the change of its form and structure, and compare;
(5) identified by immunofluorescence: identified by immunofluorescence includes CK13 (CK13) and CK8 (CK8) Qualification: the cell density in culture dish to be trained reaches 70%-90%, discards culture fluid, adds fixative and fixes, discards With the permeabilized process of Triton X-100 after fixative, then close with BSA;Add one to resist, overnight;Add fluorescence mark The two of note resist, and subsequent step is both needed to lucifuge operation, incubated at room, then dyes, at fluorescence microscopy Microscopic observation with DAPI.
In Preparatory work of experiment, described culture fluid is formulated as: every 200 milliliters of K-SFM culture medium, adds 20-30 microlitre epidermis raw The long factor (EGF) and 4000-5000 microlitre cattle pituitary extract (BPE), the CaCl of 150-250 microlitre 60mM2, 2 micro- Rise Pen .-Strep mixed liquor and the amphotericin of 200 microlitre 25mg/ml, mixing, then filter through 0.22 micron membrane filter After, place 4 DEG C of preservations.
In Preparatory work of experiment, described culture bottle is processed as: add Type I/III collagen in 6 orifice plates or 10cm culture dish (Advanced BioMatrix, #5005-B), was advisable, in Biohazard Safety Equipment not have bottom 6 orifice plates or 10cm culture dish Lucifuge is ventilated overnight, and next day irradiates 1-2 hour under uviol lamp, can pack up 6 orifice plates or 10cm culture dish in 4 DEG C of preservations Standby.
Wherein, described tonsil packet digestion is: hatches 30min-1h, period shaken several times at 37 DEG C, is then placed in 4 DEG C of overnight incubation, referred to as gradient digestion method.
Another kind of scheme is: described tonsil packet digestion is: 4 DEG C of direct overnight incubation, referred to as 4 DEG C digestion are overnight Method.
Wherein, described tonsil draw materials for: under Biohazard Safety Equipment, tonsil is placed in and fills PBS balanced salt solution 4 DEG C of ice baths culture dish in, utilize clipper for surgical use carefully to remove periphery connective tissue, with sterilized PBS for several times, cut Become the piece of tissue of 3mm × 3mm size, and transfer them in the dispase grade II solution that concentration is 1-3mg/ml.
Wherein, being separated into of described tonsil epithelial cell: the piece of tissue digested, the cell in secondary Nikkei 220 mesh is sieved through filter, Cell suspension 1000rmp, centrifugal 5-10 minute;Abandoning supernatant, cell precipitation trypsinization liquid is resuspended, digests 5 in 37 DEG C Minute;It is subsequently adding the K-SFM culture medium containing 10-15% serum, stops digestion, then 1000rmp is centrifuged 5 minutes, abandons Supernatant, finally resuspended with keratin serum-free medium (K-SFM culture medium), primary cell kind is entered in culture dish, 37 DEG C, Quiescent culture under the conditions of 5%CO2, changes liquid for 2-3 days once.
Wherein, described trypsinization liquid contains pancreatin and the EDTA of 0.53mM of 0.05%-0.25%;Described keratin depletion of blood Clear culture medium contains 5-10ng/ml EGF, 20-30 μ g/ml BPE, 0.06mM CaCl2,1% Pen .-Strep, 25 μ g/ml Amphotericin.
Wherein, described identified by immunofluorescence includes CK13 (CK13) and the qualification of CK8 (CK8): Cell density in culture dish to be trained reaches 70%-90%, discards culture fluid, washes twice with PBS;Add 4% paraformaldehyde solid Determine liquid, fixing 20min-30min, then discard fixative, PBS washes three times, 3min/ time;Under room temperature condition, with 0.2% Triton X-100 permeabilized process 1h, PBS wash three times, 3min/ time;Under room temperature condition, 5%BSA closes 30min-1h, PBS washes three times, 3min/ time;Adding the one of 1:200-1:300 dilution to resist, 4 DEG C overnight, and PBS washes three times, 3min/ time;Add Fluorescently-labeled two resist, and subsequent step is both needed to lucifuge operation, incubated at room 1h, and PBS washes three times, 3min/ time;DAPI dyes 3-5min, PBS wash three times, 3min/ time;Fluorescence microscopy Microscopic observation.
The present invention inquires into the Isolation and identification method of human tonsil's epithelial cell, and it is applied to the body of human tonsil's epithelial cell Outer cultivation and purge process, lay the foundation for research Respirovirus mechanism of action in clinic.
Accompanying drawing explanation
Fig. 1: gradient digestion method separation tonsil epithelial cell, shows d0 days cell states (10 ×).
Fig. 2: 4 DEG C of digestion overnight separate tonsil epithelial cell, show d0 days cell states (10 ×).
Fig. 3: gradient digestion method separation morphological observation result (10 ×), shows the result cultivated the 5th, 8,14 days respectively.
Fig. 4: 4 DEG C digest overnight method separation morphological observation result (10 ×), show the result cultivated the 5th, 8,14 days respectively.
Fig. 5: identified by immunofluorescence is tonsil epithelial cell (10 ×), and blue-fluorescence display nucleus in figure, green fluorescence shows CK13。
Fig. 6: identified by immunofluorescence is Crypt Cells (10 ×), blue-fluorescence display nucleus in figure, green fluorescence display CK8.
Fig. 7: the flow chart of steps of human tonsil's epithelial cell Isolation and identification.
Detailed description of the invention
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1
One, Preparatory work of experiment:
(1) culture fluid preparation: cell culture fluid: every 200 milliliters of K-SFM culture medium, adds 20-30 microlitre epidermal growth factor And 4000-5000 microlitre cattle pituitary extract (BPE), 150-250 microlitre CaCl2 (60mM), 2 microlitre penicillium sp (EGF) Element-streptomycin mixed liquor and 200 microlitre amphotericins (25mg/ml), mixing, then after 0.22 micron membrane filter filters, put Put 4 DEG C of preservations;
(2) culture bottle processes: 6 orifice plates or 10cm culture dish are added Type I/III collagen (Advanced BioMatrix, #5005-B), being advisable not have bottom 6 orifice plates or 10cm culture dish, in Biohazard Safety Equipment, lucifuge is ventilated overnight, and next day is purple Irradiate 1-2 hour under outer lamp, 6 orifice plates or 10cm culture dish can be packed up and save backup in 4 DEG C;
Two, separation and Culture
(1) tonsil is drawn materials: tonsil is placed under Biohazard Safety Equipment the training of the 4 DEG C of ice baths filling PBS balanced salt solution Support in ware, utilize clipper for surgical use carefully to remove periphery connective tissue, with sterilized PBS for several times, be cut into 3mm × 3mm Left and right tissue piece, and transfer them in the dispase grade II solution that concentration is 1-3mg/ml;
(2) tonsil packet digestion: hatch 30min-1h for 37 DEG C, period manual shaken several times, it is then placed in 4 DEG C of overnight incubation;
(3) separation of tonsil epithelial cell: the piece of tissue digested, the cell in secondary Nikkei 220 mesh is sieved through filter, cell suspension 1000rmp, centrifugal 5-10 minute;Abandon supernatant, cell precipitation trypsinization liquid (pancreatin containing 0.05%-0.25% and 0.53mM EDTA) resuspended, in 37 DEG C digest 5 minutes;It is subsequently adding the K-SFM culture medium containing 10-15% serum, stops to disappear Change, then 1000rmp be centrifuged 5 minutes, abandon supernatant, finally with K-SFM culture medium (keratin serum-free medium, wherein Containing 5-10ng/ml EGF, 20-30 μ g/ml BPE, 0.06mM CaCl2, 1% Pen .-Strep, 25 μ g/ml both sexes are mould Element) resuspended, primary cell kind is entered in culture dish, 37 DEG C, quiescent culture under the conditions of 5%CO2, changes liquid for 2-3 days once.
(4) morphological observation: by the tonsil epithelial cell of different incubation times in inverted phase contrast microscope (Olympus CKX 41 Type) under carry out morphologic observation, observe the change of its form and structure, and compare.
(5) identified by immunofluorescence: identified by immunofluorescence includes CK13 (CK13) and CK8 (CK8) Identify: the cell density in culture dish to be trained reaches 70%-90%, discards culture fluid, washes twice with PBS;Add 4% poly Formaldehyde fixative, fixing 20min-30min, then discard fixative, PBS washes three times, 3min/ time;Under room temperature condition, use Triton X-100 permeabilized the process 1h, PBS of 0.2% washes three times, 3min/ time;Under room temperature condition, 5%BSA closes 30min-1h, PBS wash three times, 3min/ time;Adding one anti-(1:200-1:300 dilution), 4 DEG C overnight, and PBS washes three times, 3min/ Secondary;Adding fluorescently-labeled two anti-(subsequent step is both needed to lucifuge operation), incubated at room 1h, PBS washes three times, 3min/ time; Dye 3-5min, PBS of DAPI washes three times, 3min/ time;Fluorescence microscopy Microscopic observation.
Embodiment 2
One, Preparatory work of experiment:
(1) culture fluid preparation: cell culture fluid: every 200 milliliters of K-SFM culture medium, adds 20-30 microlitre epidermal growth factor And 4000-5000 microlitre cattle pituitary extract (BPE), 150-250 microlitre CaCl2 (60mM), 2 microlitre penicillium sp (EGF) Element-streptomycin mixed liquor and 200 microlitre amphotericins (25mg/ml), mixing, then after 0.22 micron membrane filter filters, put Put 4 DEG C of preservations;
(2) culture bottle processes: 6 orifice plates or 10cm culture dish are added Type I/III collagen (Advanced BioMatrix, #5005-B), being advisable not have bottom 6 orifice plates or 10cm culture dish, in Biohazard Safety Equipment, lucifuge is ventilated overnight, and next day is purple Irradiate 1-2 hour under outer lamp, 6 orifice plates or 10cm culture dish can be packed up and save backup in 4 DEG C;
Two, separation and Culture
(1) tonsil is drawn materials: tonsil is placed under Biohazard Safety Equipment the training of the 4 DEG C of ice baths filling PBS balanced salt solution Support in ware, utilize clipper for surgical use carefully to remove periphery connective tissue, with sterilized PBS for several times, be cut into 3mm × 3mm Left and right tissue piece, and transfer them in the dispase grade II solution that concentration is 1-3mg/ml;
(2) tonsil packet digestion: 4 DEG C of direct overnight incubation;
(3) separation of tonsil epithelial cell: the piece of tissue digested, the cell in secondary Nikkei 220 mesh is sieved through filter, cell suspension 1000rmp, centrifugal 5-10 minute;Abandon supernatant, cell precipitation trypsinization liquid (pancreatin containing 0.05%-0.25% and 0.53mM EDTA) resuspended, in 37 DEG C digest 5 minutes;It is subsequently adding the K-SFM culture medium containing 10-15% serum, stops to disappear Change, then 1000rmp be centrifuged 5 minutes, abandon supernatant, finally with K-SFM culture medium (keratin serum-free medium, wherein Containing 5-10ng/ml EGF, 20-30 μ g/ml BPE, 0.06mM CaCl2, 1% Pen .-Strep, 25 μ g/ml both sexes are mould Element) resuspended, primary cell kind is entered in culture dish, 37 DEG C, quiescent culture under the conditions of 5%CO2, changes liquid for 2-3 days once.
(4) morphological observation: by the tonsil epithelial cell of different incubation times in inverted phase contrast microscope (Olympus CKX 41 Type) under carry out morphologic observation, observe the change of its form and structure, and compare.
(5) identified by immunofluorescence: identified by immunofluorescence includes CK13 (CK13) and CK8 (CK8) Identify: the cell density in culture dish to be trained reaches 70%-90%, discards culture fluid, washes twice with PBS;Add 4% poly Formaldehyde fixative, fixing 20min-30min, then discard fixative, PBS washes three times, 3min/ time;Under room temperature condition, use Triton X-100 permeabilized the process 1h, PBS of 0.2% washes three times, 3min/ time;Under room temperature condition, 5%BSA closes 30min-1h, PBS wash three times, 3min/ time;Adding one anti-(1:200-1:300 dilution), 4 DEG C overnight, and PBS washes three times, 3min/ Secondary;Adding fluorescently-labeled two anti-(subsequent step is both needed to lucifuge operation), incubated at room 1h, PBS washes three times, 3min/ time; Dye 3-5min, PBS of DAPI washes three times, 3min/ time;Fluorescence microscopy Microscopic observation.
Embodiment 1, embodiment 2 comparison of test results analysis:
In embodiment 1, digestion method is the method using gradient digestion.Bacillus polymyxa Neutral proteinase activity enzyme at 37 DEG C that this institute utilizes Activity is the highest, therefore uses 37 DEG C of digestion 30min-1h then 4 DEG C of gradient digestion methods digested overnight, damages reducing cell membrane While wound, the yield of cell can be increased again, as shown in Figure 1.
In embodiment 2, digestion method is to use 4 DEG C to digest overnight method.It is low intensive that 4 DEG C of digested overnight can make cell be constantly in Under Bacillus polymyxa Neutral proteinase activity, without reducing cell viability, but this kind of method is more weak to the dispersion dynamics of piece of tissue, causes and is separated Cell is agglomerate distribution, and is unfavorable for that tonsil epithelial cell separates out from lymphocyte middle reaches.As shown in Figure 2.
By embodiment 1, the different incubation times of embodiment 2 tonsil epithelial cell in inverted phase contrast microscope (Olympus CKX 41 type) under carry out morphologic observation, observe the change of its form and structure, and compare.Result display human tonsil Epithelium primary cultured cell becomes the form of typical paving stone columnar epithelium cell, adherent growth, embodiment 1, embodiment 2 two kinds It is all in the majority with lymphocyte number that method separates the cell of first day, after cell attachment, through PBS with change liquid, and 5 days Time epithelial cell number the most substantially increase, lymphocyte all significantly reduces, it is seen that the tonsil of dozens of to tens thousand of cells composition Epithelial cell ball, cellular morphology rule, clear border, refractivity is strong.
As time goes on, forming cell monolayer layer after cultivating 14 days, cell shape also tends to typical case.Two kinds of digestion methods Relatively finding, cell that gradient digestion method separates and 4 DEG C digest the cell that overnight method separates and compare, and the speed of fusion is substantially very fast, And cell viability higher (Fig. 3,4).The cell of two kinds of method separation still may occur in which identical with original cuiture after passing on clone A large amount of second generation clones, single cell clone display individual cells cell clone when cultivating 3 days increases to dozens of, hereafter with density Increasing growth to accelerate, cellular morphology is constant, passes on latter 7 days cell numbers and i.e. can reach million cells.
The primary of two kinds of methods and the cell that passes on are carried out Keratin 13 (CK13) identified by immunofluorescence, result display major part Cell is CK13 antigen positive, and wherein the positive rate of gradient digestion method detection is higher than 4 DEG C of digested overnight methods (as shown in Figure 5,6). The positives rate of passage cell is higher, and result confirms that the primary cell separated is tonsil epithelial cell, further across CK8 (CK8) identified by immunofluorescence shows that the cell of more than 90% all has positive expression, illustrates that the tonsil epithelium being separated is thin In born of the same parents, major part is the one of tonsil epithelial cell for Crypt Cells, is also the Main Function target cell of the viruses such as EB, The mechanism infected for follow-up study virus is laid a good foundation by this,.
The above, be only presently preferred embodiments of the present invention, and the present invention not makees any pro forma restriction, and any to be familiar with basis special The technical staff of industry, in the range of without departing from technical solution of the present invention, according to the technical spirit of the present invention, to above example institute Any simple amendment, equivalent and the improvement etc. made, within all still falling within the protection domain of technical solution of the present invention.

Claims (9)

1. the Isolation and identification method of human tonsil's epithelial cell, it is characterised in that: step is as follows:
One, Preparatory work of experiment:
(1) culture fluid preparation: use K-SFM culture medium, adds epidermal growth factor, cattle pituitary extract, CaCl2, blue or green Mycin-streptomycin mixed liquor and amphotericin, mixing, filter, preserve;
(2) culture bottle processes;In culture bottle, add Type I/III collagen, be advisable, in bio-safety not have bottom culture bottle In cabinet, lucifuge is ventilated overnight, and next day irradiates under uviol lamp, saves backup;
Two, separation and Culture
(1) tonsil is drawn materials: tonsil is placed under Biohazard Safety Equipment the ice bath training filling PBS balanced salt solution Support in ware, remove periphery connective tissue, clean, be cut into piece of tissue, and transfer them in dispase grade II solution;
(2) tonsil packet digestion: overnight incubation;
(3) separation of tonsil epithelial cell: the piece of tissue that digested is sieved through filter in secondary Nikkei cell, cell suspension is centrifugal, Abandoning supernatant, cell precipitation trypsinization liquid is resuspended, digestion;It is subsequently adding the K-SFM culture medium containing serum, stops Digestion, then be centrifuged, abandon supernatant, finally resuspended by K-SFM culture medium, primary cell kind is entered in culture dish, stands training Support;
(4) morphological observation: the tonsil epithelial cell of different incubation times is carried out morphologic observation under inverted phase contrast microscope, Observe the change of its form and structure, and compare;
(5) identified by immunofluorescence: include CK13 (CK13) and the qualification of CK8 (CK8): wait to train Cell density in culture dish reaches 70%-90%, discards culture fluid, adds fixative and fixes, uses Triton after discarding fixative The permeabilized process of X-100, then closes with BSA;Add one to resist, overnight;Add fluorescently-labeled two to resist, follow-up step Suddenly it is both needed to lucifuge operation, incubated at room, then dyes, at fluorescence microscopy Microscopic observation with DAPI.
2. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 1, it is characterised in that: described is flat The packet digestion of Fructus Persicae soma is: hatches 30min-1h, period shaken several times at 37 DEG C, is then placed in 4 DEG C of overnight incubation.
3. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 1, it is characterised in that: described almond Soma's packet digestion is: 4 DEG C of direct overnight incubation.
4. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 1, it is characterised in that: in Preparatory work of experiment, Described culture fluid is formulated as: every 200 milliliters of K SFM culture medium, adds 20 30 microlitre epidermal growth factors (EGF) With 4,000 5000 microlitres cattle pituitary extract (BPE), the CaCl of 150 250 microlitre 60mM2, 2 microlitre penicillin chains Mycin mixed liquor and the amphotericin of 200 microlitre 25mg/ml, mixing, then after 0.22 micron membrane filter filters, place 4 DEG C of preservations.
5. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 1, it is characterised in that: in Preparatory work of experiment, Described culture bottle is processed as: add Type I/III collagen (Advanced in 6 orifice plates or 10cm culture dish BioMatrix, #5005 B), it to be advisable not have bottom 6 orifice plates or 10cm culture dish, in Biohazard Safety Equipment, lucifuge is ventilated Overnight, next day irradiates 12 hours under uviol lamp, 6 orifice plates or 10cm culture dish can be packed up and save backup in 4 DEG C.
6. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 1, it is characterised in that: described almond Soma draw materials for: under Biohazard Safety Equipment, tonsil is placed in the culture dish of the 4 DEG C of ice baths filling PBS balanced salt solution In, utilize clipper for surgical use carefully to remove periphery connective tissue, with sterilized PBS for several times, be cut into 3mm × 3mm big Little piece of tissue, and transfer them in the dispase grade II solution that concentration is 1 3mg/ml.
7. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 1, it is characterised in that: described almond Being separated into of body epithelial cell: the piece of tissue digested, the cell in secondary Nikkei 220 mesh is sieved through to be filtered, cell suspension 1000rmp, Centrifugal 5-10 minute;Abandoning supernatant, cell precipitation trypsinization liquid is resuspended, digests 5 minutes in 37 DEG C;It is subsequently adding and contains There is the K-SFM culture medium of 10-15% serum, stop digestion, then 1000rmp is centrifuged 5 minutes, abandons supernatant, finally uses Keratin serum-free medium (K-SFM culture medium) is resuspended, enters in culture dish by primary cell kind, 37 DEG C, 5%CO2 Under the conditions of quiescent culture, within 2-3 days, change liquid once.
8. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 7, it is characterised in that: described pancreatin Digestive system containing 0.05% 0.25% pancreatin and the EDTA of 0.53mM;Described keratin serum-free medium contains 5 10ng/ml EGF, 20 30 μ g/ml BPE, 0.06mM CaCl2,1% penicillin streptomycin, 25 μ g/ml amphotericins.
9. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 1, it is characterised in that: described immunity Fluorescence Identification includes CK13 (CK13) and the qualification of CK8 (CK8): in culture dish to be trained Cell density reaches 70%-90%, discards culture fluid, washes twice with PBS;Add 4% paraformaldehyde fixative, fixing 20min-30min, then discards fixative, and PBS washes three times, 3min/ time;Under room temperature condition, with the Triton X-100 of 0.2% Permeabilized process 1h, PBS wash three times, 3min/ time;Under room temperature condition, 5%BSA closes 30min-1h, PBS and washes three Time, 3min/ time;Adding the one of 1:200-1:300 dilution to resist, 4 DEG C overnight, and PBS washes three times, 3min/ time;Add fluorescence The two of labelling resist, and subsequent step is both needed to lucifuge operation, incubated at room 1h, and PBS washes three times, 3min/ time;DAPI dyes 3-5min, PBS wash three times, 3min/ time;Fluorescence microscopy Microscopic observation.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107236700A (en) * 2017-06-12 2017-10-10 新乡医学院 The culture of bronchus primary epithelial cells and authentication method
CN114829581A (en) * 2019-10-11 2022-07-29 类器官科学有限公司 Preparation method and application of tonsil organoid

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