CN105695394A - In vitro culture method of mouse salivary gland organ - Google Patents

In vitro culture method of mouse salivary gland organ Download PDF

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CN105695394A
CN105695394A CN201610213597.7A CN201610213597A CN105695394A CN 105695394 A CN105695394 A CN 105695394A CN 201610213597 A CN201610213597 A CN 201610213597A CN 105695394 A CN105695394 A CN 105695394A
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mouse
gland
mouse salivary
organoid body
cell
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张雁
吴愁
肖洒
郑骏恒
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Sun Yat Sen University
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Abstract

The invention discloses an in vitro culture method of a mouse salivary gland organ. The in vitro culture method of the mouse salivary gland organ is characterized in that a primary or passage salivary gland single cell is mixed with matrigel, and then the mixture is added into a stem cell culture solution to be cultured. The invention establishes a method for culturing the mouse salivary gland organ in vitro, enrichment of saliva stem cells is realized by adopting the cell line, and material basis is provided for study of development of salivary glands and stem cell treatment of xerostomia.

Description

A kind of extracorporeal culturing method of mouse salivary glands organoid body
Technical field
The invention belongs to cell engineering field, relate generally to the extracorporeal culturing method of a kind of mouse salivary glands organoid body。
Background technology
1 salivary gland and salivary gland stem cell
The salivary gland of people and Mus is mainly by three pairs of common bodies of gland, the parotid gland, submaxillary gland and sublingual gland, and many small glandula compositions。In mouse salivary glands, body of gland maximum, topmost is submaxillary gland, this body of gland multiple-limb leaflet, is positioned at the centre of cervical region veutro, two, left and right body of gland relative。Submaxillary gland, near submandibular lymph nodes, connects sublingual gland, and extends to the parotid gland of dorsal part。Submaxillary gland is less, is made up of single leaflet。The parotid gland is the multiple leaflet of diffusion-type, is separated by connective tissue and fatty tissue。And in human body, maximum body of gland is the parotid gland, this body of gland is inverted taper, is positioned at leading edge and the lower edge of external ear。The parotid gland has the barrier film of prosperity, body of gland is divided into many lobules, keeps apart with its hetero-organization。Submaxillary gland is positioned at the leading edge of digastric, mylohyoid be divided into shallow table and deeply feel two parts。And sublingual gland is positioned at submaxillary gland front, below tongue。
In mice and people, three main bodies of gland have some similar anatomical structures: acinous cell is connected to profit pipe, imports lobule conduit, then interlobar duct, is finally combined into secretory duct, flows into oral cavity。Submaxillary gland and sublingual secretory duct opening are near front tooth, and the conduit of the parotid gland is near molar。And in the submaxillary gland of mice, there is the vessel arrangment of uniqueness: the conduit of large grained is connected between profit pipe and lobule conduit。This catheter segment of mouse submandibular gland has obvious asyngamy, and compared with female, the vessel cell of male Mus linear arrays is bigger, in obvious cylindrical particle shape。The dimorphism of this sex is determined by androgen, and is absent from this difference in human body。
Body of gland stem cell or CFU-GM, have the function repairing damage and tissue regeneration。Cell labeling experiment and duct ligation are it is experimentally confirmed that body of gland stem cell or CFU-GM are present in the conduit of body of gland。Utilize nucleotide analog, such as bromine deoxyguanosine (BdU) and3H-thymidine, the proliferative cell of labelling is primarily targeted in secretory duct and the profit pipe of salivary gland。The Major Secretory conduit of ligation salivary gland so that acinar cell apoptosis, causes profit pipe and secretory duct cell proliferation。After going ligation, the afunction that this duct ligation causes can be restored, and by the propagation of vessel cell and differentiation, salivation amount can quickly recover to the level before ligation。The studies above illustrates, and the multiplication capacity of acinous cell is limited, it is impossible to recover Intervention；And inhabit salivary gland conduit place, there is this group of cells of propagation and differentiation capability, it may be possible to effective body of gland stem cell or CFU-GM monoid。
2 incidence cancer and xerostomias
Up to now, the main flow that radiation and chemotherapy is still that in world wide treatment of cancer。Due to higher survival rate, the whole world has the chemotherapy close to 500,000 people's accepting header cervical region cancers every year。Research confirm, the improvement of medicine mode can be in harmonious proportion roentgenization produce side effect。But the uncertainty of the physiological structure complicated due to incidence and disease locus so that the treatment of incidence cancer and difficulty thereof, pharmaceutical intervention is extremely limited to alleviating the side effect that radiotherapy causes。Therefore, the patient of about 40% can suffer the Intervention imbalance caused by radiation and the side effect xerostomia caused thereof。
Xerostomia is complication the most prominent after incidence cancer radiotherapy, and radiotherapy causes the damage of salivary gland, changes the secretory volume of saliva, viscosity and pH value, allows originally thin, neutral saliva become dense increase thick, acid。Patient oral cavity is uncomfortable or pain, speaks, chews, dysphagia, too increases the risk of decayed tooth and oral cavity infection, ultimately results in appetite and decline and lose weight。
Although incidence cancer patient is advantageous for by radiotherapy and Drug therapy, but the xerostomia that radiation causes, not only substantially reduce the quality of life of patient, also bring new health problem to them。It would therefore be desirable to take new Clinics means, improve the quality of life of incidence cancer patient。
3 stem-cell therapies
After accepting radiotherapy, the recovery of salivary gland function depends primarily on the dosage of radiation and the quantity of body of gland autologous stem cells。Except after radiotherapy decades ago for the bone marrow transplantation of hematopoietic reconstitution, the complication that stem-cell therapy reduces its hetero-organization is utilized to be still that a brand-new research field fast-developing again。Nearly ten years, stem-cell therapy is as potential scheme a kind of in regenerative medicine, it is possible to the disease that healing Various Tissues or dysfunction of organ are correlated with。
Radiation-induced salivary gland damages, and mainly the scarce capacity of the functional cell of the abundant maturation of stem cell generation causes。It is interesting that still complete after most of non-functional conduit radiotherapies, this is stem cell transplantation to suitable environment, regeneration salivary gland provides chance。Prophylactic agent pilocarpine and keratinocyte growth factor (KGF) precisely due to the propagation of stimulating catheter stem cell or CFU-GM and play the effect of protection body of gland。The factor of the emiocytosis of derived from bone marrow also can stimulating catheter stem cell or CFU-GM, some researchs also state that, the salivary gland of radiation damage can also be replaced by mescenchymal stem cell, these cells can transdifferentiation formed acinus sample phenotype。
Current research is concentrated mainly on the qualification of tissue autologous stem cells, separation and transplanting aspect。The salivary gland stem cell of mice and people can separate from the saliva ball cultivated and obtain, but real saliva stem cell identifies still unpredictable。C-Kit+Cell has the ability significantly improving RADI, and the cell that CD24/CD29, CD49f and CD133 express can be divided into all of gland cell monoid equally, and the salivary gland after radiating can be allowed to regenerate。In the stem-cell therapy of xerostomia, research worker has made some progress, but before clinical practice, it would be desirable to solve the safety problem of cell itself。
4 Stem cells cultured in vitro technology
Traditional plane cultural method, under suitable cytokine conditions, it is possible to allow pluripotent stem cell and tumor cell form stable cell line in vitro。But, the adult stem cell of many tissues or organ origin, but cannot obtain stable amplification in vitro。Having benefited from the understanding of signal of interest function that is stem cell microenvironment and control stem cell is stable and that break up, external dimensional culture technology obtains quick development。This driven by stem cell, form the micro-assembly robot of approximate physiological structure, in vitro self renewal, be referred to as " organoid body " (organoid)。
Having been reported title, efficient dimensional culture system can obtain an unbounded quantity of stem cell or CFU-GM in vitro, from esophagus, stomach, small intestinal, rectum, pancreas to prostate。It is different from traditional In vitro culture, this dimensional culture system can expand the primary epithelium " organoid body " of mice or people for a long time, and can maintain the stability of organoid body genotype and phenotype in vitro, and organoid body is similar to original structure on the Nomenclature Composition and Structure of Complexes。
The appearance of organoid body is that conventional planar is cultivated and the important bridge of in vivo model, this technology compared with plane cultivate can the physiological environment in analogue body better, microenvironment component, signal path and gene editing are easier to manipulation than in vivo model。In the epoch of current Personalized medicine, organoid body technique is regenerative medicine, tumor occurs and drug detection has opened up the brand-new visual field。
Summary of the invention
It is an object of the invention to provide the extracorporeal culturing method of a kind of mouse salivary glands organoid body
The technical solution adopted in the present invention is:
A kind of extracorporeal culturing method of mouse salivary glands organoid body, be specially by primary or the salivary gland that goes down to posterity is unicellular mix with matrigel after, add stem cell medium cultivate。
Further, the cytokine in described stem cell medium includes EGF, IGF, FGF10, Noggin, Rspondin1 and Y-27632。
Further, described stem cell medium consists of the following composition: DF+10%FBS, 100 × N2,50 × B27,100 × Glutamax, 100 × NEAA, 50ng/mlEGF, 50ng/mlIGF, 100ng/mlFGF10,100ng/mlNoggin, 200ng/mlRspondin1 and 10mMY-27632。
Further, described matrigel is Matrigel substrate。
Further, described primary salivary gland is unicellular for adopting mechanical digestion and enzyme digestion to process mouse salivary glandular tissue, and separates acquisition with cold stimulation。
Further, cultivate described primary salivary gland unicellular time, the density of inoculating cell is 1～2x104Cells/ hole/every 50uL substrate。
Further, the salivary gland that goes down to posterity described in is unicellular for adopting the cell of centrifugation and enzyme digestion process original cuiture and culture medium mixture to obtain。
Further, when the salivary gland that goes down to posterity described in cultivation is unicellular, the density of inoculating cell is 0.5～1x104Cells/ hole/every 50uL substrate。
Further, the extracorporeal culturing method of described mouse salivary glands organoid body, comprise the following steps: a) adopt mechanical digestion and enzyme digestion to process mouse salivary glandular tissue, and primary unicellular with cold stimulation separation salivary gland；B) enter to have by 1-2 × 104cells/ hole kind in 24 orifice plates of Matrigel substrate, add stem cell and cultivate；C) go down to posterity after cultivating 14 days, enter to have by 0.5-1 × 104cells/ hole kind in 24 orifice plates of Matrigel substrate, stably go down to posterity and namely obtain described mouse salivary glands organoid body。
Present disclosure also includes a kind of stem cell medium for mouse salivary glands organoid body In vitro culture, consist of the following composition: DF+10%FBS, 100 × N2,50 × B27,100 × Glutamax, 100 × NEAA, 50ng/mlEGF, 50ng/mlIGF, 100ng/mlFGF10,100ng/mlNoggin, 200ng/mlRspondin1 and 10mMY-27632。
First, present invention demonstrates three kinds of cell population of existence in mouse salivary glands growth course: basal cell, chamber cell and salivary acinous cell。Secondly, the invention provides the stem cell medium of the cultural method of a kind of mouse salivary glands organoid body and use, in this culture fluid, the propagation of salivary gland organoid body is had indispensable effect by each cytokine。The mouse salivary glands organoid that the method for the present invention obtains, it is possible to the low cell concentration ratio of 1:50, stably go down to posterity more than 1 year。To the organoid body utilizing the method to cultivate, detect the situation of mRNA and protein expression through RT-PCR and IF, it was demonstrated that this organoid body contains all three salivary gland cell monoid, simulates the growth of body of gland in vitro。
The invention have the benefit that the method that the present invention establishes Cultured Mouse salivary gland organoid body, and achieve the enrichment of saliva stem cell by this cell line, the stem-cell therapy of growth and xerostomia for studying salivary gland provides material base。
In order to be more fully understood that and implement, describe the present invention in detail below in conjunction with accompanying drawing。
Accompanying drawing explanation
Fig. 1, embryonic stage (E15), mouse salivary glandular tissue HE dyeing in the 1st day, the 2nd week, the 10th week, scale: 50 μm after birth。
Fig. 2, embryonic stage (E15), the IF dyeing of the 1st day, the 2nd week, the 10th week mouse salivary glandular tissue CK14-CK8, scale: 50 μm after birth。
Fig. 3, embryonic stage (E15), the IF dyeing of the 1st day, the 2nd week, the 10th week mouse salivary glandular tissue CDH1-AQP5, scale: 50 μm after birth。
Fig. 4, " 6-1 " cytokine remove the light microscopic figure to salivary gland organoid bulk-growth, scale: 50 μm。
Fig. 5, the cytokine cartogram to organoid body Clone formation number。
The light microscopic figure that Fig. 6, organoid body grow in stem cell medium, scale: 50 μm。
Fig. 7, the organoid body HE of the 14th day dye, scale: 50 μm。
The IF dyeing of CK14-CK8, scale: 50 μm in Fig. 8, organoid body growth course in vitro。
Fig. 9, the 14th day CDH1-AQP5 of organoid body IF dyeing, scale: 50 μm。
Figure 10, the 14th day CDH1-Ki67 of organoid body IF dyeing, scale: 50 μm。
Figure 11, organoid body ideograph。
The expression of Figure 12, nucleic acid electrophoresis detection organoid body CK5, CK14, CK8, CDH1, AQP5, AMY α。
Detailed description of the invention
Material illustrates:
1 mice: the mice used by this experiment is purchased from Guangdong Medical Lab Animal Center。
2 main solution and formula
2.1 cells are cultivated
1) 10 × PBSBuffer:NaCl80g, KCl2g, Na2HPO411.55g, KH2PO42g, adds the ultra-pure water of about 800ml, and magnetic stirring apparatus is sufficiently stirred for dissolving, regulates pH value to 7.4, is settled to 1L, after autoclave sterilization, and room temperature preservation。
2) hyclone (FBS): be made into proper proportion purchased from Hyclone company, 4 DEG C of preservations, and culture medium。
3) 10 × Trypsin-EDTA (TE): weigh 1.25gtrypsin, 1.00gEDTA and be dissolved in the 1 × PBS of 250ml, filtration sterilization ,-20 DEG C of preservations。It is diluted to debita spissitudo peptic cell with 1 × PBSbuffer before using。
4) F12 (DF) culture medium: Sigma company, is dissolved in the ultra-pure water of 1L, regulates pH value to 7.2, utilizes peristaltic pump and 0.22 μm of membrane filtration degerming。4 DEG C of preservations。
5) Dispase:BD company, uses during original cuiture。
6) stem cell medium: DF+10%FBS, 100 × N2 (Gibco), 50 × B27 (Gibco), 100 × Glutamax (Gibco), 100 × NEAA (Gibco), 50ng/mlEGF (PeproTech), 50ng/mlIGF (PeproTech), 100ng/mlFGF10 (PeproTech), 100ng/mlNoggin (PeproTech), 200ng/mlRspondin1 (PeproTech) and 10mMY-27632 (Sigma)。
2.2 nucleic acid electrophoresis
1) 50 × TAE electrophoresis Buffer (pH8.5): add 242gTris, 18.6gNa2EDTA 2H2O in the vial of 1L, be subsequently adding two grades of water of about 800ml, it is sufficiently stirred for dissolving, adds the acetic acid of 57.1ml, be sufficiently stirred for, add two grades of water and be settled to 1L, room temperature preservation。Dilute with 1:50 during use。
2) ethidium bromide (EB): storing liquid concentration is 10mg/ml, and working concentration is 0.5 μ g/ml。
3) 1% Normal Agarose Gel: weigh 1g agarose (agarose), adds 100ml1 × TAEbuffer, and heated and boiled is completely dissolved to agarose, it is cooled to about 50 DEG C, add 5 μ lEB and store liquid, make final concentration of 0.5 μ g/ml, shake up rear Casting of gels flat board。
2.3 immunofluorescences
1) 4%PFA:20g paraformaldehyde is dissolved in 400ml1 × PBS, and 70 DEG C of heating for dissolving are about 3-4h。When bottom residue precipitates on a small quantity, add 40 μ l1NNaOH dissolutions, and be settled to 500ml, 4 DEG C of preservations。
2) 30% sucrose solution: 3g sucrose powder is dissolved in 4%PFA, is settled to 10ml, 4 DEG C of preservations。
3) confining liquid: 10% nonimmune lowlenthal serum, stock solution is diluted to this concentration by 1 × PBS, 4 DEG C of preservations。
4) antibody diluent: for primary antibodie, has confining liquid to be diluted to respective concentration and uses；Resist for two, use after diluting by 1:500 confining liquid。
5) primary antibodie: CK14 (Abcam, ab7800), CK8 (Abcam, ab53280), AQP5 (Abcam, ab104751), CDH1 (Abcam, ab11512)
Method and result
The growth of 3.1 mouse salivary glandular tissues
3.1.1 mouse salivary glandular tissue HE dyeing
1) salivary organization 10% formalin solution is fixing overnight；
2) automatic dehydration instrument carries out dehydration: 70% ethanol 2 hours, 80% ethanol 2 hours, 95% ethanol 30 minutes, 95% ethanol 30 minutes, dehydrated alcohol 30 minutes, dehydrated alcohol 2 hours, dehydrated alcohol+TO (1:1) 30 minutes, TO30 minute, TO2 hour, TO+ paraffin (1:1) 30 minutes, paraffin 3 hours, paraffin 2 hours。Totally 12 barrels, 16 hours；
3) embedding machine embeds with paraffin, microtome is cut into slices；
4) 60 DEG C of dewaxings of tissue, dimethylbenzene 5 minutes, dehydrated alcohol 1 minute, 90% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, distillation washing 4 minutes；
5) haematoxylin liquid dyes 3-4 minute, tap water 10 minutes, water solublity eosin stains 1-2 minute；
6) dehydration, transparent and mounting: section is added 70% ethanol 30 seconds, 80% ethanol 30 seconds, 90% ethanol 1 minute, dehydrated alcohol 1 minute, dimethylbenzene 5 minutes, finally use resinene mounting。
Interpretation of result: as it is shown in figure 1, along with the growth of mice, salivary gland is reached maturity gradually。The body of gland of embryonic stage is in the crudity of conduit, and after birth, body of gland, by catheter tip proliferation and differentiation, forms acinous cell, buds into functional salivary gland gradually。And after mice 4-5 week sexual maturity, profit pipe and the lobule conduit of Mus submaxillary gland have obvious asyngamy, compared with female, the vessel cell of male Mus linear arrays is bigger, in obvious cylindrical particle shape (the 10th week)。
3.1.2 mouse salivary glandular tissue IF dyeing
1) sucrose solution of mouse salivary glandular tissue 30% is fixing overnight；
2) 1 × PBS shaking table rinses 3 times, each 10min；
3) confining liquid room temperature closes 1h；
4) removing confining liquid, dropping is diluted to the primary antibodie of respective concentration, 4 DEG C of overnight incubation；
5) remove primary antibodie, 1 × PBS shaking table rinses 3 times, each 10min；
6) dropping be diluted to respective concentration two resist, incubated at room 1h；
7) remove two to resist, 1 × PBS shaking table rinses 3 times, each 10min；
8) dropping is diluted to the DAPI, incubated at room 5-10min of respective concentration；
9) rinsing 3 times, each 10min on DAPI, 1 × PBS shaking table is removed；
10) fluorescence mountant mounting, 4 DEG C keep in Dark Place, take pictures。
Interpretation of result: as shown in Figure 2,3, comprises the cell of three kinds of monoids, respectively CK14 in mouse salivary glands+Basal cell, CK8+Chamber cell and AQP5+Acinous cell。Basal cell and chamber cell are two conservative monoid cells, form maturation from salivary gland and all exist always, are positioned at gland conduit place；After being born 2 weeks, the cell proliferation and differentiation of catheter tip, form ripe acinous cell。And, three monoid cells of body of gland are in female male middle indifference (the 10th week)。
The foundation of 3.2 mouse salivary glands organoid body cultural methods
3.2.1 organoid body original cuiture
1) taking salivary organization, after 75% soak with ethanol 15s, pre-cooling 1 × PBS rinses 2 times, is shredded by tissue as meat pulp sample；
2) tissue shredded is transferred in 15ml centrifuge tube, adds 50 units/mlDispase37 DEG C and hatches 1h；
3), after 1 × PBS of pre-cooling rinses 2 times, in 15ml centrifuge tube, again add 10ml1 × PBS, stir 10min on ice；
4) cell after 40 μm of membrane filtrations is collected, after counting, according to 1-2 × 104Cells/ hole/every 50uL substrate is planted in 24 orifice plates after mixing with Matriel, adds stem cell medium and cultivates。
3.2.2 organoid body Secondary Culture
1) in 24 orifice plates, every hole adds 1ml1 × PBS, is smashed to pieces with cell mixture by Matrigel, joins in centrifuge tube, 1,200rpm centrifugal 5min, collects precipitation；
2) add 10 × TE peptic cell, hatch 30min for 37 DEG C；
3) after 1 × PBS rinses 2 times, according to 0.5-1 × 104Cells/ hole/every 50uL substrate is planted in 24 orifice plates after mixing with Matriel, adds stem cell medium and cultivates。
Adopt the mouse salivary glands organoid body that the method is cultivated, when stem cell medium, it is possible to the low cell concentration ratio of 1:50, stably go down to posterity, so far to go down to posterity more than 1 year。
Interpretation of result: as Figure 4-Figure 6, mouse salivary glands, only in complete culture solution, could be stablized and breed, goes down to posterity, 6 important factors, wherein some remove the amplification in vitro that all can affect organoid body。In complete culture solution, cell can steady in a long-term grow, and maintains good form。
The analysis of the structure and function of ecosystem of 3.3 mouse salivary glands organoid bodies
3.3.1 organoid fluid-structure analysis
3.3.1.1 organoid body HE dyeing
Taking the organoid body cultivated the 14th day, 10% formalin solution is fixed after overnight, dehydration, paraffin embedding, section。Paraffin section is carried out HE dyeing。
3.3.1.1 organoid body whole-mountIF dyeing
Taking the organoid body cultivated the 3rd, 5,7,10,14 days, 4%PFA fixes after overnight, and organoid body is carried out whole-mount immunofluorescence dyeing:
1) 1 × PBS shaking table rinses 3 times, each 10min；
2) confining liquid room temperature closes 2-3h；
3) removing confining liquid, dropping is diluted to the primary antibodie of respective concentration, hatches 48h for 4 DEG C；
4) remove primary antibodie, 1 × PBS shaking table rinses 3 times, each 10min；
5) dropping is diluted to two anti-and DAPI, 4 DEG C of overnight incubation of respective concentration；
6) remove two to resist and rinsing 3 times on DAPI, 1 × PBS shaking table, each 10min；
7) 4 DEG C keep in Dark Place, and Lightsheet takes pictures。
Interpretation of result: as is seen in figs 7-10, mouse salivary glands organoid body forms a structure hollow, multiple-limb from a solid ball propagation。And growing as salivary gland, organoid body is originally present within CK14 from formation+Basal cell and CK8+Chamber cell, the 14th day in vitro, there is AQP5 in the outer layer of organoid body branch+Acinous cell。And, Ki67+Proliferating-cell population be present near basal cell。As shown in figure 11,3 cell population that whole organoid body exists are similar with body of gland。
3.3.2 organoid body gene expression
3.3.2.1 the extraction of cell RNA
1) take the cell cultivated the 14th day, centrifugal collecting precipitation, centrifuge tube adds 1mlTrizol, complete to lysis with pipettor piping and druming, liquid is drawn to 1.5ml without in enzyme centrifuge tube；
2) often using 1mlTrizol to add chloroform 0.2ml, violent jolting 15s, room temperature stands 5min so as to be layered, 4 DEG C, 12,000rpm centrifugal 15min；
3) upper strata aqueous phase being carefully transferred to another 1.5ml without, in the clean centrifuge tube of enzyme, adding isopropanol isopyknic with aqueous phase, mix gently, room temperature stands 10 minutes, 4 DEG C, 12,000rpm centrifugal 10min；
4) abandon supernatant, add without enzyme 75% ethanol 1ml along wall, mix gently, 4 DEG C of centrifugal 5min of 7500xg, abandon supernatant；
5) being inverted by 1.5ml centrifuge tube, with the micropipettor of the 100 μ l remaining ethanol of exhaustion, dry, precipitation is dissolved in the water without RNase of 20～40 μ l；
8) taking 1 μ l, measure RNA concentration with ultraviolet spectrophotometer (Nanodrop) ,-80 DEG C save backup。
3.3.2.2RT-PCR reverse transcription reaction (TOYOBOReverTraAce test kit)
1) it is sequentially added in PCR reaction tube:
Total serum IgE y μ l (1000ng)
65 DEG C of effect 5min；
2) RNA after degeneration is placed on ice, adds:
3) it is placed on ice, adds 5 × RTMasterMix II；
4) PCR instrument is set, 37 DEG C of 15min, 50 DEG C of 5min, 98 DEG C of 5min, 4 DEG C of hold, put-20 DEG C and save backup。
3.3.2.3PCR program
1) it is sequentially added in PCR pipe:
2) different primers was arranged according to various annealing temperatures, period, extension time etc.；
3) nucleic acid electrophoresis testing result。
Interpretation of result: as shown in figure 12, organoid body is enriched salivary gland epithelia cell (CDH1), wherein there is the mrna expression of basal cell (CK5, CK14), chamber cell (CK8) and acinous cell (AQP5), and exocrine amylase (AMY α) is expressed inconspicuous。
The invention is not limited in above-mentioned embodiment, if to the various changes of the present invention or deformation without departing from the spirit and scope of the present invention, if these are changed and deform within the claim and the equivalent technologies scope that belong to the present invention, then the present invention is also intended to comprise these changes and deformation。

Claims (10)

1. the extracorporeal culturing method of a mouse salivary glands organoid body, it is characterised in that by primary or the salivary gland that goes down to posterity is unicellular mix with matrigel after, add stem cell medium cultivate。
2. the extracorporeal culturing method of mouse salivary glands organoid body according to claim 1, it is characterised in that the cytokine in described stem cell medium includes EGF, IGF, FGF10, Noggin, Rspondin1 and Y-27632。
3. the extracorporeal culturing method of mouse salivary glands organoid body according to claim 2, it is characterized in that, described stem cell medium is grouped into by the one-tenth of following concentration: DF+10%FBS, 100 × N2,50 × B27,100 × Glutamax, 100 × NEAA, 50ng/mlEGF, 50ng/mlIGF, 100ng/mlFGF10,100ng/mlNoggin, 200ng/mlRspondin1 and 10mMY-27632。
4. the extracorporeal culturing method of mouse salivary glands organoid body according to claim 1, it is characterised in that described matrigel is Matrigel substrate。
5. the extracorporeal culturing method of mouse salivary glands organoid body according to claim 1, it is characterised in that described primary salivary gland is unicellular for adopting mechanical digestion and enzyme digestion to process mouse salivary glandular tissue, and separates acquisition with cold stimulation。
6. the extracorporeal culturing method of mouse salivary glands organoid body according to claim 5, it is characterised in that cultivate described primary salivary gland unicellular time, the density of inoculating cell is 1～2x104Cells/ hole/every 50uL substrate。
7. the extracorporeal culturing method of mouse salivary glands organoid body according to claim 5, it is characterised in that described in go down to posterity the unicellular cell for adopting centrifugation and enzyme digestion to process original cuiture of salivary gland and culture medium mixture obtains。
8. the extracorporeal culturing method of mouse salivary glands organoid body according to claim 7, it is characterised in that when the salivary gland that goes down to posterity described in cultivation is unicellular, the density of inoculating cell is 0.5～1x104Cells/ hole/every 50uL substrate。
9. the extracorporeal culturing method of the mouse salivary glands organoid body according to any bar in claim 1-8, it is characterized in that, comprise the following steps: a) adopt mechanical digestion and enzyme digestion to process mouse salivary glandular tissue, and primary unicellular with cold stimulation separation salivary gland；B) by 1～2x104Cells/ hole/every 50uL substrate kind enters to have in 24 orifice plates of Matrigel substrate, adds stem cell and cultivates；C) go down to posterity after cultivating 14 days, by 0.5～1x104Cells/ hole/every 50uL substrate kind enters to have in 24 orifice plates of Matrigel substrate, stably goes down to posterity and namely obtains described mouse salivary glands organoid body。
10. the stem cell medium for mouse salivary glands organoid body In vitro culture, consist of the following composition: DF+10%FBS, 100 × N2,50 × B27,100 × Glutamax, 100 × NEAA, 50ng/mlEGF, 50ng/mlIGF, 100ng/mlFGF10,100ng/mlNoggin, 200ng/mlRspondin1 and 10mMY-27632。
CN201610213597.7A 2016-04-07 2016-04-07 In vitro culture method of mouse salivary gland organ Pending CN105695394A (en)

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Cited By (3)

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CN106190980A (en) * 2016-07-12 2016-12-07 张云霞 A kind of special culture media and cultural method being used for In vitro culture esophageal carcinoma tumor organoid based on Human Esophageal Carcinoma
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CN109321528A (en) * 2018-09-18 2019-02-12 梁廷波 Chinese B7-H5 expresses positive pancreatic cancerous cell line sipanc-1076 method for building up

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