CN103966158B - A kind of preparation method and applications of periodontal tissue specific cell epimatrix ECM - Google Patents
A kind of preparation method and applications of periodontal tissue specific cell epimatrix ECM Download PDFInfo
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Abstract
The preparation method of a kind of periodontal tissue specific cell epimatrix, comprises the following steps: 1) separation of people's periodontal ligament cell and cultivation；2) preparation of periodontal tissue's specificity ECM；The application of periodontal tissue's specific cell epimatrix, periodontal tissue specificity ECM is as the application of amplification in vitro Periodontal ligament stem cell；And promote its directed differentiation；Have that preparation method is simple, can remarkably promote the in-vitro multiplication of periodontal ligament stem cell and the feature of directed differentiation.
Description
Technical field
The invention belongs to biological technical field, relate to the preparation method of the extracellular matrix in a kind of periodontal tissue source,
And it is in the application of the aspects such as periodontal ligament stem cell cultivation, amplification.
Background technology
Periodontitis is one of mankind's disease the most ancient, most common.It is to be caused by the Institute of Micro-biology in dental plaque
The chronic infectious disease of Periodontal Supporting Tissue, can cause the formation of the inflammation of Periodontal Supporting Tissue, periodontal pocket, Progressive symmetric erythrokeratodermia attachment
Lose and frontal resorption, ultimately cause odontoseisis and pulled out.According to statistics, periodontitis prevalence and seriousness are with the age
Increasing and increase, after 35 years old, prevalence substantially increases, and peaks when 50 years old to 60 years old.At present, periodontitis is the trouble of China
Sick rate, more than 80%, is China adult the first reason of losing tooth.Meanwhile, periodontitis or whole body system disease example
Such as diabetes, the important risk factor of cardiovascular disease.
Include the following four stage currently for the treatment procedure of periodontitis: first stage, Primary Care, i.e. use clean
Control the conventional methods such as art, Occlusal adjustment, Drug therapy and eliminate paathogenic factor, control inflammation；Second stage, periodontal surgery treatment,
I.e. remove infected tissue by flap operation, bone grafting operation, guided tissue regeneration etc., promote periodontal regenerative；Phase III, repair
And/or orthodontic treatment, i.e. while repairing agomphosis, fix agomphiasis, set up stable occlusion；Finally, periodontal support is treated,
So that curative effect is kept for a long time.But, all there is respective limitation in existing Therapeutic Method, still can not realize preferable tooth
Zhou Zaisheng and completely recovery periodontal tissue structure.
In recent years, the fast development of stem-cell research and regenerative medicine brings dawn for treatment mankind's difficult diseases.Seo
Deng isolating a kind of adult stem cell, named periodontal ligament stem cell (periodontal first from people's periodontal membrane tissue
Ligament stem cells, PDLSCs) (Seo BM, Miura M, Gronthos S, et al.
Investigation of multipotent postnatal stem cells from human periodontal
ligament. Lancet, 2004,364:149-55.).Periodontal ligament stem cell is considered to possess to cementoblast, fibroblast
Dimension cell and the potential of osteoblast differentiation, can form cementum, periodontal ligament and alveolar bone respectively, recovers complicated and fine
" sandwich sample " periodontal tissue's structure, bring glad tidings for patients with periodontitis.But, adult stem cell includes periodontal ligament stem cell
In vitro after repeatedly passing on, can gradually lose the characteristic of its stem cell, Spontaneous Differentiation or aging occur, and group cannot be met
Knit regeneration and the demand rebuild.Therefore, high-quality periodontal ligament stem cell is amplified the most in a large number？The most accurately control
The directed differentiation of periodontal ligament stem cell？These are all to realize key urgently to be resolved hurrily from laboratory to clinical practice successful conversion
Problem.
Extracellular matrix (extracellular matrix, ECM) refers to be positioned in the organism inner cell external space, by
The network structure general name that the protein of emiocytosis and polysaccharide are constituted.It or be filled in cell with the form of extracellular matrix
And constitute iuntercellular cement substance between cell, or be positioned at connective tissue its important component part of composition, or with basement membrane shape
Formula is positioned at epithelium and connective tissue junctional area, for cell attachment, breed and break up and can produce material impact.Meanwhile, base
The physical property of matter also is able to affect the state of cell.The research such as Engler finds that the elastic modelling quantity of substrate can instruct mesenchyme
Stem cell generation directed differentiation, the soft substrate being such as similar to cerebral tissue can be induced mesenchymal stem cells into neurons to break up, is similar to
The substrate of muscular tissue can induce stem cell to break up to myocyte, and the hard substrate being similar to osseous tissue then induces stem cell to one-tenth
Bone cell differentiation (Engler AJ, Sen S, Sweeney HL, et al. Matrix elasticity directs
stem cell lineage specification. Cell, 2006,126:677-89).Chen etc. take the lead in rate in the world
First propose that " ECM that medullary cell (Bone marrow cells, BMCs) is originated can be with microenvironment in analogue body, between holding
The stem cell properties of mesenchymal stem cells " hypothesis, and carried out effective checking.BMCs is cultivated on Nostoc commune Vanch plate by they,
BMCs is made to secrete a large amount of ECM, after effective de-cell processes, the remaining membranaceous ECM with complete structure and protein component.He
First have studied mice BMCs source ECM, with common Plastic cultivate compared with, this ECM can promote mice BMMSCs
Propagation, keeps the bone of cell to, fat to differentiation capability simultaneously, and internal transplantation experiments result show the mice of cultivation on ECM
Bone amount and bone marrow amount that BMMSCs generates add 5 times and 8 times (Chen XD, Dusevich V, Feng than Plastic respectively
JQ, et al. Extracellular matrix made by bone marrow cells facilitates
expansion of marrow-derived mesenchymal progenitor cells and prevents their
Differentiation into osteoblasts. J Bone Miner Res, 2007,22:1943-56).They enter one
Step have studied the people BMCs source ECM effect for hBMMSCs, finds that ECM can promote cell proliferation, keeps embryonal,
Reduce the generation producing oxygen-derived free radicals simultaneously, improve telomerase activation, strengthen the osteogenic response to BMP2；Experiment in vivo result table
Bright, ECM expands the bone formation performance that the hBMMSCs passed on can keep good, and the osteogenic ability of the cell of Nostoc commune Vanch
Generally fell sharply after 6 ~ 7 generations (Lai Y, Sun Y, Skinner CM, et al. Reconstitution of marrow-
derived extracellular matrix ex vivo: a robust culture system for expanding
large-scale highly functional human mesenchymal stem cells. Stem Cells Dev,
2010,19:1095-107).Why ECM can play its mechanism of such effect may 2 points: first, ECM can be temporary
Time " binding " emiocytosis Endogenous Growth Factors such as BMP etc., play slow release effect, so that cell obtains sufficient propagation
Time, and the sensitivity of exogenous somatomedin holding height；Second, the structure of scanning electron microscope display ECM is as arranged in order
" track " of row, cell can migrate along " track " thereon, the fewest generation " collision ", decreases contact inhibition, thus promotes
Cell proliferation, reduces the probability of differentiation.
According to above research, it is presumed that " extracellular matrix in periodontal membrane source can provide suitable for periodontal ligament stem cell
Suitable growing environment, promotes its propagation and directed differentiation ".We utilize periodontal ligament cell successfully to construct periodontal tissue's specificity
ECM, and find this ECM can efficient amplification periodontal ligament stem cell in vitro, and maintain the dryness that it is higher.Up to now,
Have not seen the preparation about periodontal tissue specificity ECM and the report on periodontal ligament stem cell impact thereof.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of periodontal tissue specific cell
The preparation method and its usage of epimatrix, and utilize this tissue specificity ECM high-quality in vitro to expand periodontal ligament stem cell
And promote its directed differentiation, there is preparation method is simple, can remarkably promote periodontal ligament stem cell in-vitro multiplication and directed differentiation
Feature.
To achieve these goals, the technical solution used in the present invention is: a kind of periodontal tissue specific cell epimatrix
Preparation method, comprise the following steps:
1) separation of people's periodontal ligament cell and cultivation, specific practice is:
Collect clinically 12～need fresh permanent bicuspid and third molar, periodontal health, the nothing pulled out because of correction in 29 years old
Dental caries are bad, without tip of a root inflammation, in superclean bench with 75% alcohol disinfecting corona, PBS liquid rinses 3～5 times, in scraping root 1/3
Periodontal membrane, moves into centrifuge tube, and the type i collagen enzyme and the 2.4units/mlDispase that add 625units/ml shake gently, 37 DEG C
The digested 40min of incubator, vibrates 1 time every 5min, centrifugal rotational speed 1200r/min, centrifugal 5min, abandoning supernatant；Add
0.5～the 1mL α MEM culture fluid containing 15% hyclone, moves into 25mL culture bottle diapire, uniformly inoculates, culture bottle is inserted
37℃、5%CO2The incubator of saturated humidity is hatched 24h, adds culture fluid after cell attachment gently to 3mL；Change after 3d and cultivate
Liquid discards non-attached cell, changes liquid 1 time every 2～3d later, and the culture supernatant of the periodontal ligament cell collecting exponential phase is standby
With, cell growth reaches 8O%～90% and uses 0.25% trypsinization when converging, and passes on 1:2 ratio；
2) preparation of periodontal tissue's specificity ECM, specific practice is:
By P3 for people periodontal ligament cell hPDLCs with 2x104The density of cells/ml is inoculated in 6 orifice plates, cultivates 15 days,
Often within 3-4 days, changing liquid once, within last 8 days, change film induction liquid into, PBS liquid rinses 2 times, adds de-cell treatment fluid, reacts 5 under room temperature
Minute, PBS liquid rinses 3 times, it is thus achieved that hPDLCs-ECM, is attached to 6 orifice plates, 4 ° of C cryopreservation.
Described de-cell treatment fluid contains 0.5%TritonX-100 and 20mM NH4The PBS liquid of OH.
Described PBS liquid penicillin Han 100U/ml, 100 g/ml streptomycins and amphotericin 0.25 g/ml.
Described α MEM culture fluid contains 15% hyclone, 2mmol/L mono-glutamine, 100U/ml penicillin and 100
G/ml streptomycin.
Described film induction liquid contains ascorbic acid 50 M.
The application of a kind of periodontal tissue specific cell epimatrix, it is characterised in that periodontal tissue specificity ECM is as body
The application of outer amplification Periodontal ligament stem cell, and promote its directed differentiation.
Compared with prior art, the present invention has a following useful technique effect:
1) the periodontal tissue specificity ECM prepared by the present invention can remarkably promote with altitude simulation periodontal tissue microenvironment
The in-vitro multiplication of periodontal ligament stem cell and directed differentiation, and without adding somatomedin in culture fluid or cell being carried out gene
Modify.
2) the periodontal tissue specificity ECM prepared by the present invention establishes a periodontal ligament stem cell simple, effective
The external model of growth, contributes to studying the microenvironment impact on stem cell, is simultaneously also beneficial to from now on by periodontal ligament stem cell
Study the conversion to clinical practice.
Accompanying drawing explanation
Fig. 1 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) through taking off knot under the microscope before and after cell processes
Really；Wherein, before Figure 1A processes for de-cell, 4 ×；Before Figure 1B processes for de-cell, 10 ×；After Fig. 1 C processes for de-cell, 4
×；After Fig. 1 D processes for de-cell, 10 ×.
Fig. 2 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) through taking off under the scanning electron microscope before and after cell processes
Result；Wherein, Fig. 2 A is de-cell figure before treatment；Fig. 2 B is the figure after de-cell processes.
Fig. 3 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) through taking off under the transmission electron microscope before and after cell processes
Result；Wherein, before Fig. 3 A processes for de-cell；After Fig. 3 B processes for de-cell.
Fig. 4 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) through taking off the type i collagen immunity before and after cell processes
Fluorescence staining；Wherein, before Fig. 4 A processes for de-cell；After Fig. 4 B processes for de-cell.
Fig. 5 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) Biglycan before and after de-cell processes exempts from
Epidemic disease fluorescence staining；Wherein, before Fig. 5 A processes for de-cell；After Fig. 5 B processes for de-cell.
Fig. 6 is periodontal tissue specificity ECM(hPDLCs-ECM of the present invention) through taking off the Decorin immunity before and after cell processes
Fluorescence staining；Wherein, before Fig. 6 A processes for de-cell；After Fig. 6 B processes for de-cell.
Fig. 7 is periodontal tissue specificity ECM(hPDLCs-ECM) through taking off the Perlecan immunofluorescence before and after cell processes
Dyeing；Wherein, before Fig. 7 A processes for de-cell；After Fig. 7 B processes for de-cell.
Fig. 8 is that the periodontal tissue specificity ECMhPDLCs-ECM of the present invention Fibronectin before and after de-cell processes exempts from
Epidemic disease fluorescence staining；Wherein, Fig. 8 A is de-cell figure before treatment；Fig. 8 B is the figure after de-cell processes.
Fig. 9 is that the microscope of the present inventor's periodontal ligament stem cell hPDLSCs upgrowth situation on tissue specificity ECM is observed
Figure.
Figure 10 is the growth curve chart after the present inventor's periodontal ligament stem cell hPDLSCs is cultivated on tissue specificity ECM.
Figure 11 is the Clone formation detection on tissue specificity ECM of the present inventor's periodontal ligament stem cell hPDLSCs and becomes
Bone becomes fat to break up, gross examination of skeletal muscle figure.
Figure 12 is the Clone formation detection on tissue specificity ECM of the present inventor's periodontal ligament stem cell hPDLSCs and becomes
Bone becomes fat to break up, and microscope observes figure.
Detailed description of the invention
Below in conjunction with specific embodiments and the drawings, the present invention is described in further detail, described in be explanation of the invention
Rather than limit.
The invention provides a kind of periodontal tissue specificity ECM(hPDLCs-ECM simple, efficient) preparation method, profit
Periodontal ligament stem cell hPDLSCs can be expanded the most in a large number with prepared ECM and promote its directed differentiation.
As a example by the preparation method of people periodontal membrane tissue specificity ECM, concrete steps include the primary of people's periodontal ligament cell
Cultivation, Secondary Culture, the preparation of ECM and detection, and apply this ECM amplification in vitro Periodontal ligament stem cell hPDLSCs.
1) separation of people's periodontal ligament cell hPDLCs and cultivation, specific practice is:
Collect clinically 12～need fresh permanent bicuspid and third molar, periodontal health, the nothing pulled out because of correction in 29 years old
Dental caries are bad, without tip of a root inflammation, rinse 3～5 times with PBS liquid after 75% alcohol disinfecting corona in superclean bench, in scraping root 1/3
Periodontal membrane, moves into centrifuge tube, and the type i collagen enzyme and the 2.4 units/ml Dispase that add 625units/ml shake gently, and 37
DEG C digested 40 min of incubator, vibrate 1 time every 5min, centrifuge speed 1200r/min, centrifugal 5min, abandoning supernatant.
Add the 0.5～1mL α MEM culture fluid containing 15% hyclone, move into 25mL culture bottle diapire, uniformly inoculate. culture bottle
Put people 37 DEG C, 5%CO2The incubator of saturated humidity is hatched 24 h, adds culture fluid after cell attachment gently to 3mL.After 3d more
Change culture fluid and discard non-attached cell, change liquid 1 time every 2～3d later, collect the cultivation of the periodontal ligament cell of exponential phase
Supernatant is standby, and cell growth reaches 8O%～90% and uses 0.25% trypsinization when converging, and passes on 1:2 ratio.
2) preparation of periodontal tissue's specificity ECM
By P3 for hPDLCs with 2x 104The density of cells/ml is inoculated in 6 orifice plates, cultivates 15 days, every 3-4
It changes liquid, within last 8 days, changes film induction liquid into, and PBS liquid rinses 2 times, adds de-cell treatment fluid, reacts 5 minutes, PBS under room temperature
Liquid rinses 3 times, it is thus achieved that hPDLCs-ECM, it is attached to 6 orifice plates, 4 ° of C cryopreservation.
Described de-cell treatment fluid contains 0.5%TritonX-100 and 20mM NH4The PBS liquid of OH.
Described PBS liquid penicillin Han 100U/ml, 100 g/ml streptomycins and amphotericin 0.25 g/ml.
Described α MEM culture fluid contains 15% hyclone, 2mmol/L mono-glutamine, 100U/ml penicillin and 100
G/ml streptomycin.
Described film induction liquid contains ascorbic acid 50 M.
The constructed observation of periodontal tissue specificity ECM
Ordinary optical microscope is observed: directly observe hPDLCs-ECM surface before and after Cell extraction under inverted microscope
Form, as shown in Figure 1.
Scanning electron microscope (SEM) morphological observation hPDLCs-ECM: washed three times by hPDLCs-ECM PBS, with containing 2% penta
After the 0.1M sodium cacodylate buffer liquid (pH value 7.2) of dialdehyde fixes 1 hour, the 0.1M cacodylate buffer transferred to
In.Specimen is after gradient concentration dehydration (from 70% to 100% ethanol), and metal spraying processes, and specimen uses Flied emission scanning electron to show
Micro mirror (S-4800, Hitachi, Japan) observes hBMCs-ECM ultrastructure.As shown in Figure 2.
Transmission electron microscope ultrastructure observation of experimental: fix hPDLCs-ECM and hPDLCs 2 with 2% glutaraldehyde PBS fixative
H, collects precipitate, through rinsing, fix, replace, be impregnated with, embed, cutting into slices, with lead citrate electron staining, JEM 1 transmission
Ultramicroscope (Philips, Holland) observation of cell ultrastructure.As shown in Figure 3.
The immunofluorescence dyeing of periodontal tissue specificity ECM
Being rinsed 2 times for hPDLCs and hPDLCs-ECM cell climbing sheet PBS by P3,4% paraformaldehyde fixes 20 points on ice
Clock, rinses 3 times with PBS, and hPDLCs cell climbing sheet adds 0.5%Triton-100,1% BSA37 DEG C of closing 20min, hPDLCs-
ECM cell climbing sheet 1% BSA 37 DEG C closes 20min, adds COL I, Biglycan, Decorin, Perlecan, Fibronectin
One anti-(1:500), 4 DEG C overnight, and after next day rewarming, PBS washes 3 times.The goat antirabbit of FITC labelling it is separately added under the conditions of lucifuge
Anti-(1:200) with goat anti-mouse two, to hatch 1 hour for 37 DEG C, dye 5min, PBS of DAPI washes 3 times, mounting, fluorescence microscope
Observing, experimentation lucifuge operates.Negative control group replaces one to resist with PBS.As shown in Fig. 4 to Fig. 8.
The separation and Culture of Periodontal ligament stem cell hPDLSCs
Limiting dilution assay clone's culture purified periodontal ligament stem cell: by the exponential phase periodontal ligament cell of above-mentioned collection
Culture supernatant is centrifuged 10 min by 1500 r/min, after the filter of 0.22 m diameter filters, mixes with 1:1 ratio with culture medium
Cooperation is adaptability culture medium. the 1st generation cell adaptability culture medium doubling dilution of trophophase of taking the logarithm, adjust cell density
To 1O～15/mL, piping and druming mixing, being inoculated in 96 well culture plates, lO0 l/ hole, after cultivating 12 h, labelling individual cells is adherent
Hole, and fluid infusion is to 200 l/ holes, changes liquid, light Microscopic observation Clone formation situation after 5 days.Cellar culture 7～14 d, to going out
Existing cell clone (cell number >=5O is criterion), trypsinization after clone is long at the bottom of hole 1/3～1/2, it is transferred to 24 holes
Plate amplification culture.
The effect that periodontal tissue specificity ECM breeds for hPDLSCs
By P3 for hPDLSCs with 2x 104The density of cells/ml is inoculated in Nostoc commune Vanch plate, hPDLCs-ECM respectively
Culture plate and hBMCs-ECM culture plate, 4 hours after inoculation, 3 days and 7 days observation of cell growing state respectively.As it is shown in figure 9,
The hPDLSCs adherent and growth vigor on hPDLCs-ECM culture plate is obvious.
Take growth logarithmic (log) phase P3 for hPDLSCs, be inoculated in Nostoc commune Vanch plate, hPDLCs-ECM culture plate and hBMCs-
ECM culture plate, trypsinization respectively after seven days.After cell counting, adjusting cell density is 2 × 103/ hole is inoculated in 96 orifice plates,
Every hole adds liquid 100 L, altogether 48 holes of inoculation, overnight incubation.Next day changes culture fluid, treats that gaging hole adds 100 L CCK8
Liquid, measures 12 holes every time, and incubator is cultivated 4 hours.After 4 hours, enzyme-linked immunosorbent assay instrument 450 nm goes out to measure 12 hole light absorption values
Go meansigma methods.Repeated measure, measured 7 days continuously, was repeated 3 times next day.As shown in Figure 10, hPDLSCs cultivates at hPDLCs-ECM
After cultivating on plate, propagation is substantially.
Periodontal tissue specificity ECM is for the effect of hPDLSCs directed differentiation
P3 is inoculated in three groups of common six orifice plates respectively for hPDLSCs low concentration (100 cells/well), hPDLCs-
ECM six orifice plate and hBMCs-ECM six orifice plate, conventional α MEM culture fluid is (containing 15% hyclone, 2 mmol/L mono-paddy amine acyls
Amine, 100 U/ml penicillins, 100 g/ml streptomycins) cultivate, within every two days, change liquid, after cultivating 14 days, use one group of six orifice plate inspection
Survey CFU-F clone, i.e. with PBS rinse, methanol fix, 0.1% violet staining 10min；Other two group of six orifice plate continues to cultivate,
(osteogenic induction liquid is the α MEM culture fluid containing 15%FBS, 10mmol/L β glycerol to carry out osteogenic induction and adipogenic induction respectively
Sodium phosphate, 0.05mmol/L vitamin C and 100mmol/L dexamethasone；Adipogenic induction liquid is that the α MEM containing 15%FBS cultivates
Liquid, 10 μ g/mL insulins, 1mmoL/L dexamethasone, 0. 5 mmoL/L isobutyl methylxanthine (IBMX), 200 μ
MoL/L indomethacin), after 20 days, carry out Von kossa dyeing respectively and oil red dyes, detect CFU-OB and CFU-AD.As
Figure 11, shown in 12, hPDLSCs clonality on hPDLCs-ECM, Osteoblast Differentiation with become fat differentiation to be remarkably reinforced, its
The differentiation of middle one-tenth fat is better than hBMCs-ECM group, Osteoblast Differentiation is weaker than this group, indicates the specific effect of periodontal tissue ECM.
The invention discloses the preparation method of a kind of periodontal tissue specificity ECM, and apply this ECM to periodontal ligament stem cell
Carry out amplification in vitro and directed differentiation.Periodontal tissue specificity ECM prepared by the present invention establishes one simply, effectively
The external model of periodontal ligament stem cell growth, contributes to studying the microenvironment impact on stem cell, from now on will be simultaneously also beneficial to
Periodontal ligament stem cell research converts to clinical practice.
Claims (3)
1. the preparation method of periodontal tissue's specific cell epimatrix, it is characterised in that comprise the following steps:
1) separation of people's periodontal ligament cell and cultivation, specific practice is:
Collect clinically 12～need fresh permanent bicuspid and the third molar pulled out because of correction in 29 years old, periodontal health, bad without dental caries,
Inflammation without the tip of a root, in superclean bench with 75% alcohol disinfecting corona, PBS liquid rinse 3～5 times, scraping root in 1/3 periodontal
Film, moves into centrifuge tube, and the type i collagen enzyme and the 2.4units/mlDispase that add 625units/ml shake gently, 37 DEG C of cultivations
The digested 40min of case, vibrates 1 time every 5min, centrifugal rotational speed 1200r/min, centrifugal 5min, abandoning supernatant；Add 0.5～
1mL, containing the α MEM culture fluid of 15% hyclone, moves into 25mL culture bottle diapire, uniformly inoculates, culture bottle insert 37 DEG C,
5%CO2The incubator of saturated humidity is hatched 24h, adds culture fluid after cell attachment gently to 3mL；Change culture fluid after 3d to discard
Non-attached cell, changes liquid 1 time every 2～3d later, and the culture supernatant of the periodontal ligament cell collecting exponential phase is standby, cell
Growth reaches 80%～90% and uses 0.25% trypsinization when converging, and passes on 1:2 ratio；
2) preparation of periodontal tissue's specificity ECM, specific practice is:
By P3 for people periodontal ligament cell hPDLCs with 2 × 104The density of cells/ml is inoculated in 6 orifice plates, cultivates 15 days, every 3-4
It changes liquid once, within last 8 days, changes film induction liquid into, and PBS liquid rinses 2 times, adds de-cell treatment fluid, reacts 5 minutes under room temperature,
PBS liquid rinses 3 times, it is thus achieved that hPDLCs-ECM, is attached to 6 orifice plates, 4 ° of C cryopreservation；
Described film forming induction liquid contains ascorbic acid 50 M；
Described de-cell treatment fluid is containing 0.5%TritonX-100 and 20mM NH4The PBS liquid of OH.
The preparation method of a kind of periodontal tissue the most according to claim 1 specific cell epimatrix, it is characterised in that institute
The PBS liquid penicillin Han 100U/ml stated, 100 g/ml streptomycins and amphotericin 0.25 g/ml.
The preparation method of a kind of periodontal tissue the most according to claim 1 specific cell epimatrix, it is characterised in that institute
The α MEM culture fluid stated contains 15% hyclone, 2mmol/L glutamine, 100U/ml penicillin and 100 g/ml streptomycins.
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