CN105936888B - The Isolation and identification method of human tonsil's epithelial cell - Google Patents
The Isolation and identification method of human tonsil's epithelial cell Download PDFInfo
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Abstract
The present invention relates to a kind of Isolation and identification method of human tonsil's epithelial cell, mainly include the steps such as nutrient solution is prepared, blake bottle is handled, tonsil is drawn materials, tonsil packet digestion is compared, the separation of tonsillotome epithelial cell, morphological observation, identified by immunofluorescence.The present invention is improved to the isolation technics of mouth epithelial cells, inquire into out that a kind of success rate is higher, step simple, purifying rate is high, hardly there is the in-vitro separation and culture technique of the tonsillotome epithelial cell polluted, it is applied to the in vitro culture and purge process of human tonsil's epithelial cell, significant to the study on mechanism of follow-up EB and other viruses in tonsillotome.
Description
Technical field
The present invention relates to field of biomedicine technology, and in particular to the extracorporeal culturing method of human tonsil's epithelial cell.
Background technology
Tonsillotome is both peripheral lymphoid organ, and mucosa associated lymphoid tissue is belonged to again, the special crypts pipe with complicated
Road system, is the important place for producing immune response, wherein crypts epithelium is this at alimentary canal and respiratory tract co-portal
Antigen, retention antigen are contacted in organ at first, and produces the position of immune response, therefore crypts epithelium is that tonsillotome enforcement is immune
The vital tissue barrier of function.But under the repetitious stimulation of external antigen, usually cause chronic tonsillitis and proventriculus type fertilizer
Big generation, its main cause is exactly the infection of bacterium and virus, and increasing research finds the infection with Epstein-Barr virus, relation
It is the closest.But specific infection mechanism is still not clear, therefore in-vitro separation human tonsil's Crypt Cells, can preferably be entered
One step inquires into the infection mechanism of Epstein-Barr virus.
It is domestic still to be reported without pertinent literature on the primary separation of tonsillotome epithelial cell, the tonsillotome of foreign countries' report
Cell isolation method is concentrated mainly on two kinds of separation methods, and a kind of earliest method is derived from nineteen ninety University of Washington oral cavity
Biology department and the research of department of pharmacy, are the free serum culture technologies on mouth epithelial cells, belong to enzyme digestion, as follow-up
The good reference foundation of tonsillotome in vitro culture, but technically there are some differences and not in the research method in each laboratory
Easily it is successfully, reproduced[1].Another method is the research of Bostonian medical college of TuftsUniversity in 2004, utilizes fresh almond
The explant of body, carries out cell in vitro migration, and is cultivated, but such a method, and process is relatively complicated, separation cycle compared with
It is long, influenceed larger by individual difference, and easily pollution[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..
The content of the invention
The purpose of the present invention is that the isolation technics of mouth epithelial cells is improved, inquired into out a kind of success rate compared with
High, step is simple, purifying rate is high, the in-vitro separation and culture technique of the tonsillotome epithelial cell polluted hardly occurs,
It is significant to the study on mechanism of follow-up EB and other viruses in tonsillotome.
To reach above-mentioned purpose, the technical scheme that the present invention is provided is：Human tonsil's epithelial cell being separately cultured and reflects
Determine method, its step is as follows：
First, Preparatory work of experiment：
(1) nutrient solution is prepared：Using K-SFM culture mediums, EGF, ox pituitary extract, CaCl are added2, it is blue or green
Mycin-streptomysin mixed liquor and anphotericin, are mixed, and are filtered, and are preserved；
(2) blake bottle is handled；Type I/III collagens are added into blake bottle, not have blake bottle bottom to be advisable, in life
Lucifuge ventilation is stayed overnight in thing safety cabinet, is irradiated, is saved backup under next day uviol lamp；
2nd, it is separately cultured
(1) tonsil is drawn materials：Tonsil is placed in the ice for filling PBS balanced salt solutions under Biohazard Safety Equipment
Bathe in culture dish, remove periphery connective tissue, be cut into tissue block, and transfer them to the PBS after sterilizing for several times
In dispase grade II solution；
(2) tonsil packet digestion：It is incubated overnight；
(3) separation of tonsillotome epithelial cell：By the tissue block digested, in the filtering of secondary Nikkei cell sieve, cell suspension
Centrifuge, abandon supernatant, cell precipitation is resuspended with pancreatin digestive juice, digestion；Then the K-SFM culture mediums containing serum are added, are stopped
Digestion, then centrifuge, abandon supernatant, finally it is resuspended, primary cell kind is entered in culture dish, quiescent culture with K-SFM culture mediums；
(4) morphological observation：By the tonsillotome epithelial cell of different incubation times in carrying out shape under inverted phase contrast microscope
State is observed, and observes the change of its form and structure, and be compared；
(5) identified by immunofluorescence：Identified by immunofluorescence includes CK13 (CK13) and CK8 (CK8)
Identification：Treat that the cell density in culture dish reaches 70%-90%, discard nutrient solution, add fixer and fix, discard fixer
The permeabilized processing of Triton X-100 is used afterwards, is then closed with BSA；Primary antibody is added, overnight；The secondary antibody of fluorescence labeling is added, after
Continuous step is both needed to lucifuge operation, and then incubation at room temperature is dyed with DAPI, in fluorescence microscopy Microscopic observation.
In Preparatory work of experiment, described nutrient solution is formulated as：Every 200 milliliters of K-SFM culture mediums, add 20-30 microlitres of epidermis
Growth factor (EGF) and 4000-5000 microlitres of ox pituitary extract (BPE), 150-250 microlitres of 60mM CaCl2, 2 microlitres of green grass or young crops
The anphotericin of mycin-streptomysin mixed liquor and 200 microlitres of 25mg/ml, is mixed, and after then being filtered through 0.22 micron membrane filter, is put
Put 4 DEG C of preservations.
In Preparatory work of experiment, described blake bottle is processed as：Type I/III glue is added into 6 orifice plates or 10cm culture dishes
Former (Advanced BioMatrix, #5005-B), not have 6 orifice plates or 10cm culture dishes bottom to be advisable, in Biohazard Safety Equipment
Lucifuge ventilation is stayed overnight, and is irradiated 1-2 hours under next day uviol lamp, you can pack up 6 orifice plates or 10cm culture dishes standby in 4 DEG C of preservations
With.
Wherein, described tonsil packet digestion is：30min-1h is incubated at 37 DEG C, during which shaken several times, then
It is put into 4 DEG C of overnight incubations, referred to as gradient digestion method.
Another scheme is：Described tonsil is grouped digestion：Directly it is incubated overnight at 4 DEG C, referred to as 4 DEG C digestion
Method overnight.
Wherein, described tonsil materials are：Tonsil is placed under Biohazard Safety Equipment and fills PBS balance salt
In the culture dish of 4 DEG C of ice baths of solution, periphery connective tissue is carefully removed using clipper for surgical use, with sterilized PBS for several times,
The tissue block of 3mm × 3mm sizes is cut into, and is transferred them in the dispase grade II solution that concentration is 1-3mg/ml.
Wherein, described tonsillotome epithelial cell is separated into：The tissue block digested, in the secondary aim cell of Nikkei 220
Filter is sieved through, cell suspension 1000rpm is centrifuged 5-10 minutes；Supernatant is abandoned, cell precipitation is resuspended with pancreatin digestive juice, disappeared in 37 DEG C
Change 5 minutes；Then the K-SFM culture mediums containing 10-15% serum are added, stop digestion, then 1000rpm is centrifuged 5 minutes, is abandoned
Clearly, finally it is resuspended, primary cell kind is entered in culture dish, 37 DEG C, 5% with keratin serum free medium (K-SFM culture mediums)
CO2Under the conditions of quiescent culture, change within 2-3 days liquid once.
Wherein, the EDTA of pancreatin and 0.53mM of the described pancreatin digestive juice containing 0.05%-0.25%；Described keratin
Serum free medium contains 5-10ng/ml EGF, 20-30 μ g/ml BPE, 0.06mM CaCl2,1% Pen .- Strep, 25
μ g/ml anphotericins.
Wherein, described identified by immunofluorescence includes the mirror of CK13 (CK13) and CK8 (CK8)
It is fixed：Treat that the cell density in culture dish reaches 70%-90%, discard nutrient solution, washed with PBS twice；4% paraformaldehyde is added to consolidate
Determine liquid, then fixed 20min-30min discards fixer, PBS washes three times, 3min/ times；Under room temperature condition, with 0.2%
Triton X-100 permeabilized processing 1h, PBS wash three times, 3min/ times；Under room temperature condition, 5%BSA closings 30min-1h, PBS
Wash three times, 3min/ times；Add 1:200-1:The primary antibody of 300 dilutions, 4 DEG C overnight, and PBS washes three times, 3min/ times；Add fluorescence mark
The secondary antibody of note, subsequent step is both needed to lucifuge operation, is incubated at room temperature 1h, and PBS washes three times, 3min/ times；DAPI dyes 3-5min, PBS
Wash three times, 3min/ times；Fluorescence microscopy Microscopic observation.
The present invention inquires into the Isolation and identification method of human tonsil's epithelial cell, and it is thin that it is applied to human tonsil's epithelium
The in vitro culture and purge process of born of the same parents, lays the foundation for mechanism of action of the research Respirovirus in clinic.
Brief description of the drawings
Fig. 1：Gradient digestion method separates tonsillotome epithelial cell, shows d0 days cell states (10 ×).
Fig. 2：4 DEG C of digestion separate tonsillotome epithelial cell overnight, show d0 days cell states (10 ×).
Fig. 3：Gradient digestion method separation morphological observation result (10 ×), shows culture the 5th, the knot of 8,14 days respectively
Really.
Fig. 4：Method separation morphological observation result (10 ×) is stayed overnight in 4 DEG C of digestion, show respectively cultivate the 5th, 8,14 days
Result.
Fig. 5：Identified by immunofluorescence is blue-fluorescence display nucleus in tonsillotome epithelial cell (10 ×), figure, and green is glimmering
Light shows CK13.
Fig. 6：Identified by immunofluorescence is blue-fluorescence display nucleus, green fluorescence in Crypt Cells (10 ×), figure
Show CK8.
Fig. 7：The step flow chart of human tonsil's epithelial cell Isolation and identification.
Embodiment
With reference to specific embodiment, the invention will be further described.
Embodiment 1
First, Preparatory work of experiment：
(1) nutrient solution is prepared：Cell culture fluid：Every 200 milliliters of K-SFM culture mediums, add 20-30 microlitres of epidermal growth factor
Sub (EGF) and 4000-5000 microlitres of ox pituitary extract (BPE), 150-250 microlitres of CaCl2 (60mM), 2 microlitres of penicillin-chains
Mycin mixed liquor and 200 microlitres of anphotericins (25mg/ml), mix, after then being filtered through 0.22 micron membrane filter, place 4 DEG C of guarantors
Deposit；
(2) blake bottle is handled：6 orifice plates or 10cm culture dishes are added into Type I/III collagens (Advanced
BioMatrix, #5005-B), not have 6 orifice plates or 10cm culture dishes bottom to be advisable, lucifuge was divulged information in Biohazard Safety Equipment
Irradiated 1-2 hours under night, next day uviol lamp, you can 6 orifice plates or 10cm culture dishes are packed up and saved backup in 4 DEG C；
2nd, it is separately cultured
(1) tonsil is drawn materials：Tonsil is placed in fill PBS balanced salt solutions 4 DEG C under Biohazard Safety Equipment
In the culture dish of ice bath, periphery connective tissue is carefully removed using clipper for surgical use, with sterilized PBS for several times, be cut into 3mm ×
3mm or so tissues piece, and transfer them in the dispase grade II solution that concentration is 1-3mg/ml；
(2) tonsil packet digestion：37 DEG C of incubation 30min-1h, during which manual shaken several times, are then placed in 4 DEG C and incubate
Educate overnight；
(3) separation of tonsillotome epithelial cell：The tissue block digested, filter, cell are sieved through in the secondary aim cell of Nikkei 220
Suspension 1000rpm, is centrifuged 5-10 minutes；Abandon supernatant, cell precipitation with pancreatin digestive juice (pancreatin containing 0.05%-0.25% and
0.53mM EDTA) it is resuspended, digested 5 minutes in 37 DEG C；Then the K-SFM culture mediums containing 10-15% serum are added, stop to disappear
Change, then 1000rpm is centrifuged 5 minutes, supernatant is abandoned, finally with K-SFM culture mediums (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 anphotericins) and weight
It is outstanding, primary cell kind is entered in culture dish, 37 DEG C, 5%CO2Under the conditions of quiescent culture, change within 2-3 days liquid once.
(4) morphological observation：By the tonsillotome epithelial cell of different incubation times in inverted phase contrast microscope (Olympus
The types of CKX 41) under carry out morphologic observation, observe the change of its form and structure, and be compared.
(5) identified by immunofluorescence：Identified by immunofluorescence includes CK13 (CK13) and CK8 (CK8)
Identification：Treat that the cell density in culture dish reaches 70%-90%, discard nutrient solution, washed with PBS twice；Add 4% poly first
Aldehyde fixer, fixed 20min-30min, then discards fixer, PBS washes three times, 3min/ times；Under room temperature condition, with 0.2%
Triton X-100 permeabilized processing 1h, PBS wash three times, 3min/ times；Under room temperature condition, 5%BSA closing 30min-1h,
PBS washes three times, 3min/ times；Add primary antibody (1:200-1:300 dilutions), 4 DEG C are overnight, and PBS washes three times, 3min/ times；Add glimmering
The secondary antibody (subsequent step is both needed to lucifuge operation) of signal, is incubated at room temperature 1h, PBS washes three times, 3min/ times；DAPI dyes 3-
5min, PBS wash three times, 3min/ times；Fluorescence microscopy Microscopic observation.
Embodiment 2
First, Preparatory work of experiment：
(1) nutrient solution is prepared：Cell culture fluid：Every 200 milliliters of K-SFM culture mediums, add 20-30 microlitres of epidermal growth factor
Sub (EGF) and 4000-5000 microlitres of ox pituitary extract (BPE), 150-250 microlitres of CaCl2 (60mM), 2 microlitres of penicillin-chains
Mycin mixed liquor and 200 microlitres of anphotericins (25mg/ml), mix, after then being filtered through 0.22 micron membrane filter, place 4 DEG C of guarantors
Deposit；
(2) blake bottle is handled：6 orifice plates or 10cm culture dishes are added into Type I/III collagens (Advanced
BioMatrix, #5005-B), not have 6 orifice plates or 10cm culture dishes bottom to be advisable, lucifuge was divulged information in Biohazard Safety Equipment
Irradiated 1-2 hours under night, next day uviol lamp, you can 6 orifice plates or 10cm culture dishes are packed up and saved backup in 4 DEG C；
2nd, it is separately cultured
(1) tonsil is drawn materials：Tonsil is placed in fill PBS balanced salt solutions 4 DEG C under Biohazard Safety Equipment
In the culture dish of ice bath, periphery connective tissue is carefully removed using clipper for surgical use, with sterilized PBS for several times, be cut into 3mm ×
3mm or so tissues piece, and transfer them in the dispase grade II solution that concentration is 1-3mg/ml；
(2) tonsil packet digestion：Directly it is incubated overnight at 4 DEG C；
(3) separation of tonsillotome epithelial cell：The tissue block digested, filter, cell are sieved through in the secondary aim cell of Nikkei 220
Suspension 1000rpm, is centrifuged 5-10 minutes；Abandon supernatant, cell precipitation with pancreatin digestive juice (pancreatin containing 0.05%-0.25% and
0.53mM EDTA) it is resuspended, digested 5 minutes in 37 DEG C；Then the K-SFM culture mediums containing 10-15% serum are added, stop to disappear
Change, then 1000rpm is centrifuged 5 minutes, supernatant is abandoned, finally with K-SFM culture mediums (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 anphotericins) and weight
It is outstanding, primary cell kind is entered in culture dish, 37 DEG C, 5%CO2Under the conditions of quiescent culture, change within 2-3 days liquid once.
(4) morphological observation：By the tonsillotome epithelial cell of different incubation times in inverted phase contrast microscope (Olympus
The types of CKX 41) under carry out morphologic observation, observe the change of its form and structure, and be compared.
(5) identified by immunofluorescence：Identified by immunofluorescence includes CK13 (CK13) and CK8 (CK8)
Identification：Treat that the cell density in culture dish reaches 70%-90%, discard nutrient solution, washed with PBS twice；Add 4% poly first
Aldehyde fixer, fixed 20min-30min, then discards fixer, PBS washes three times, 3min/ times；Under room temperature condition, with 0.2%
Triton X-100 permeabilized processing 1h, PBS wash three times, 3min/ times；Under room temperature condition, 5%BSA closing 30min-1h,
PBS washes three times, 3min/ times；Add primary antibody (1:200-1:300 dilutions), 4 DEG C are overnight, and PBS washes three times, 3min/ times；Add glimmering
The secondary antibody (subsequent step is both needed to lucifuge operation) of signal, is incubated at room temperature 1h, PBS washes three times, 3min/ times；DAPI dyes 3-
5min, PBS wash three times, 3min/ times；Fluorescence microscopy Microscopic observation.
Embodiment 1, the analysis of the comparison of test results of embodiment 2：
In embodiment 1, digestion method is the method digested using gradient.The scattered enzymatic activity that this research institute utilizes is at 37 DEG C
Lower enzymatic activity highest, therefore the gradient digestion method using 37 DEG C of digestion 30min-1h and then 4 DEG C of digestion overnight, are reducing cell
While membrane damage, the yield of cell can be increased again, as shown in Figure 1.
In embodiment 2, digestion method is to stay overnight method using 4 DEG C of digestion.It is low strong that 4 DEG C of digested overnights can be constantly in cell
Under the scattered enzymatic activity of degree, without reducing cell viability, but such a method is weaker to the scattered dynamics of tissue block, causes point
From cell be distributed in agglomerate, and be unfavorable for tonsillotome epithelial cell separate out from lymphocyte.As shown in Figure 2.
By embodiment 1, the different incubation times of embodiment 2 tonsillotome epithelial cell in inverted phase contrast microscope
Morphologic observation is carried out under (Olympus CKX41 types), the change of its form and structure is observed, and be compared.As a result people is shown
Tonsillotome epithelium primary cultured cell is into the form of typical paving stone columnar epithelium cell, adherent growth, embodiment 1, embodiment 2
The cell that two methods separate first day is in the majority with lymphocyte number, after after cell attachment, by PBS with change liquid, 5
It when epithelial cell number substantially increase, lymphocyte is significantly reduced, it is seen that it is flat that dozens of to tens thousand of cells is constituted
Peach body epithelial cell ball, cellular morphology rule, clear border, refractivity is strong.
Over time, cell monolayer layer is formed after cultivating 14 days, cell shape also tends to typical case.Two kinds of digestion sides
Method it was found that, the cell of gradient digestion method separation is compared with the cell that separates of method is stayed overnight in 4 DEG C of digestion, and the speed of fusion is obvious
Comparatively fast, and cell viability is higher (Fig. 3,4).The cell of two methods separation still may occur in which and original cuiture phase after passage clone
With a large amount of second generations clone, single cell clone shows that individual cells cell clone when cultivating 3 days increases to dozens of, hereafter with
Density increase growth is accelerated, and cellular morphology is constant, and 7 days cell numbers are that can reach million cells after passage.
The cell of the primary of two methods and passage is subjected to Keratin 13 (CK13) identified by immunofluorescence, as a result shows big
Part cell is in CK13 antigen positives, and the positive rate of wherein gradient digestion method detection is higher than 4 DEG C of digested overnight methods (such as Fig. 5,6 institutes
Show).The positives rate of passage cell is higher, and the primary cell for as a result confirming separation is tonsillotome epithelial cell, further across angle
The identified by immunofluorescence of albumen 8 (CK8) shows that more than 90% cell has positive expression, illustrates on separated tonsillotome
In chrotoplast largely for Crypt Cells be tonsillotome epithelial cell one kind, be also EB etc. virus main function target
Cell, this lays a good foundation the mechanism for follow-up study virus infection.
It is described above, only it is presently preferred embodiments of the present invention, any formal limitation not is made to the present invention, it is any ripe
Professional and technical personnel is known, it is without departing from the scope of the present invention, real to more than according to the technical spirit of the present invention
Apply any simple modification, equivalent substitution that example made and improve etc., still fall within technical solution of the present invention protection domain it
It is interior.
Claims (3)
1. the Isolation and identification method of human tonsil's epithelial cell, it is characterised in that：Step is as follows：
First, Preparatory work of experiment：
(1) nutrient solution is prepared:Using K-SFM culture mediums, EGF, ox pituitary extract, CaCl are added2, penicillin-
Streptomysin mixed liquor and anphotericin, are mixed, and are filtered, and are preserved；
(2) blake bottle is handled；Type I/III collagens are added into blake bottle, not have blake bottle bottom to be advisable, in biology peace
Lucifuge ventilation is stayed overnight in full cabinet, and next day irradiates under uviol lamp, saves backup；
2nd, it is separately cultured
(1) tonsil is drawn materials：Tonsil is placed in the ice bath training for filling PBS balanced salt solutions under Biohazard Safety Equipment
Support in ware, remove periphery connective tissue, cleaning is cut into tissue block, and transfer them in dispase grade II solution；
(2) tonsil packet digestion：It is incubated overnight：30min-1h is incubated at 37 DEG C, during which shaken several times, are then placed in 4
DEG C be incubated overnight；
(3) separation of tonsillotome epithelial cell：The tissue block digested is filtered in secondary Nikkei cell sieve, cell suspension centrifugation,
Supernatant is abandoned, cell precipitation is resuspended with pancreatin digestive juice, digestion；Then the K-SFM culture mediums containing serum are added, stop digestion,
Centrifuge again, abandon supernatant, be finally resuspended, primary cell kind entered in culture dish, quiescent culture with K-SFM culture mediums；
(4) morphological observation：By the tonsillotome epithelial cell of different incubation times in carrying out Appearance View under inverted phase contrast microscope
Examine, observe the change of its form and structure, and be compared；
(5) identified by immunofluorescence：Identification including CK13 and CK8：Treat the cell density in culture dish
70%-90% is reached, nutrient solution is discarded, fixer is added and fixes, discard and the permeabilized processing of Triton X-100 is used after fixer,
Then closed with BSA；Primary antibody is added, overnight；The secondary antibody of fluorescence labeling is added, subsequent step is both needed to lucifuge operation, is incubated at room temperature,
Then dyed with DAPI, in fluorescence microscopy Microscopic observation；
In Preparatory work of experiment, described nutrient solution is formulated as：Every 200 milliliters of K-SFM culture mediums, add 20-30 microlitres of epidermal growth
The CaCl of the factor and 4000-5000 microlitres of ox pituitary extract, 150-250 microlitres of 60mM2, 2 microlitres of Pen .- Strep mixing
The anphotericin of liquid and 200 microlitres of 25mg/ml, is mixed, after then being filtered through 0.22 micron membrane filter, places 4 DEG C of preservations；
In Preparatory work of experiment, described blake bottle is processed as：Type I/III collagens are added into 6 orifice plates or 10cm culture dishes,
Not have 6 orifice plates or 10cm culture dishes bottom to be advisable, lucifuge ventilation is stayed overnight in Biohazard Safety Equipment, and 1- is irradiated under next day uviol lamp
2 hours, you can 6 orifice plates or 10cm culture dishes are packed up and saved backup in 4 DEG C；
Described tonsil is drawn materials：Tonsil is placed in fill PBS balanced salt solutions 4 DEG C under Biohazard Safety Equipment
In the culture dish of ice bath, periphery connective tissue is carefully removed using clipper for surgical use, with sterilized PBS for several times, be cut into 3mm ×
The tissue block of 3mm sizes, and transfer them in the dispase grade II solution that concentration is 1-3mg/ml；
Described tonsillotome epithelial cell is separated into：The tissue block digested, is sieved through filter, carefully in the secondary aim cell of Nikkei 220
Born of the same parents suspension 1000rpm, is centrifuged 5-10 minutes；Supernatant is abandoned, cell precipitation is resuspended with pancreatin digestive juice, is digested 5 minutes in 37 DEG C；So
The K-SFM culture mediums containing 10-15% serum are added afterwards, stop digestion, then 1000rpm is centrifuged 5 minutes, is abandoned supernatant, is finally used
Keratin serum free medium is resuspended, and primary cell kind is entered in culture dish, 37 DEG C, 5%CO2Under the conditions of quiescent culture, 2-3 days
Change liquid once.
2. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 1, it is characterised in that：Described
The EDTA of pancreatin and 0.53mM of the pancreatin digestive juice containing 0.05%-0.25%；Described keratin serum free medium contains 5-
10ng/ml EGF, 20-30 μ g/ml BPE, 0.06mM CaCl2, 1% Pen .- Strep, 25 μ g/ml anphotericins.
3. the Isolation and identification method of human tonsil's epithelial cell as claimed in claim 1, it is characterised in that：Described
Identified by immunofluorescence includes the identification of CK13 and CK8：Treat that the cell density in culture dish reaches 70%-
90%, nutrient solution is discarded, is washed with PBS twice；4% paraformaldehyde fixer is added, then fixed 20min-30min discards solid
Determine liquid, PBS washes three times, 3min/ times；Under room temperature condition, three are washed with the permeabilized processing 1h of 0.2% Triton X-100, PBS
Time, 3min/ times；Under room temperature condition, 5%BSA closings 30min-1h, PBS wash three times, 3min/ times；Add 1:200-1:300 is dilute
The primary antibody released, 4 DEG C overnight, and PBS washes three times, 3min/ times；The secondary antibody of fluorescence labeling is added, subsequent step is both needed to lucifuge operation, room
Temperature is incubated 1h, and PBS washes three times, 3min/ times；DAPI dyes 3-5min, and PBS washes three times, 3min/ times；Fluorescence microscopy Microscopic observation.
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