CN107267452A - A kind of method for resuscitation of dental pulp stem cell resuscitation fluid and dental pulp stem cell - Google Patents
A kind of method for resuscitation of dental pulp stem cell resuscitation fluid and dental pulp stem cell Download PDFInfo
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Abstract
A kind of method for resuscitation of dental pulp stem cell resuscitation fluid and dental pulp stem cell, new stem cell resuscitation fluid is built by adding a certain amount of basic fibroblast growth factor in convenient stem cells culture medium, and DPSCs resuscitation process is applied to this resuscitation fluid, DPSCs vigor fast quick-recovery in a short time can be made, and keep the dryness and multi-lineage potential of original stem cell, the stem cell recovery time can be shortened and stem cell Clinical practice survival rate is improved, growth rate exceedes conventional medium group, cell dryness and multi-lineage potential are maintained simultaneously, effectively performance and the preferable seed cell of state can be provided for clinical stem-cell therapy, save the substantial amounts of time, manpower and material resources cost.
Description
Technical field
Present invention relates particularly to technical field of stem cell culture, and in particular to a kind of dental pulp stem cell resuscitation fluid and dental pulp are done
The method for resuscitation of cell.
Background technology
Stem cell is the cell that a class has self-renewing, hyperproliferation and Multidirectional Differentiation ability, under given conditions can
Induction differentiation is carried out to human body mature tissue, organ, has been widely used in the research of regenerative medicine various aspects.Stem cell
Source is mainly embryonic stem cell and mescenchymal stem cell, and wherein embryonic stem cell limits it and faced due to being related to ethnics Problem
Bed Study on Transformation；Mescenchymal stem cell main source marrow, i.e. mesenchymal stem cells MSCs, but it is in clinical Study on Transformation process
It is middle to there is the shortcomings of causing invasive damage to donor.Since Gronthos in 2000 etc. [GRONTHOS S, MANKANI M,
BRAHIM J, et al.Postnatal human dental pulp stem cells (DPSCs) in vitro and in
Vivo.Proc Natl Acad Sci USA, 2000,97 (25)：13625-13630.] found from health adult's third molar
Since dental pulp stem cell (dental pulp stem cells, DPSCs), in the past as the tooth that comes off of oral medical discarded object
" turn waste into wealth " with extraction of wisdom tooth etc., the main source studied as DPSCs.Dental pulp stem cell is a kind of with height increasing
The mescenchymal stem cell of ability and multi-lineage potential is grown, with abundance, materials safe ready, is not related to ethics knowledge
Topic, the low advantage of immunogenicity, have made great progress in regenerative medicine field, have broad application prospects.Meanwhile,
DPSCs can be induced to differentiate into bone tissue, cartilaginous tissue, adipose tissue, nerve fiber, muscular tissue, cornea under proper condition
Deng Various Tissues cell, and it can be induced as the induced multi-potent stem cell (induced with embryonic stem cell-like characteristic
Pluripotent stem cells, iPSCs), can be as the seed cell in later a variety of Disease Clinical Study on Transformation.
DPSCs is applied to during clinical research as seed cell, is not to take immediately most of the time, but thing
First setting up has stem cell bank, stores DPSCs, cell recovery and amplification is carried out when waiting clinic to need in time, to reach clinical treatment
Demand.Therefore, a kind of effective DPSCs method for resuscitation is set up so that the fast quick-recovery of vigor and keep related after cell recovery
Dryness function is particularly important.
The content of the invention
In order to overcome the defect that above-mentioned prior art is present, there is provided a kind of dental pulp stem cell resuscitation fluid and dental pulp stem cell
Method for resuscitation, can make DPSCs vigor fast quick-recovery in a short time, and keep the dryness and Multidirectional Differentiation of original stem cell to dive
Can, the stem cell recovery time can be shortened and stem cell Clinical practice survival rate is improved.
The technical solution that the present invention is used is：A kind of dental pulp stem cell resuscitation fluid, it is characterised in that described dental pulp
Stem cell resuscitation fluid is made up of mescenchymal stem cell conventional medium and basic fibroblast growth factor (bFGF), described
Mescenchymal stem cell conventional medium is by the μ g/mL chains of α-MEM culture medium+10-20% hyclone+100U/mL penicillin+100
Mycin is constituted, the final concentration of 10-20ng/mL that described basic fibroblast growth factor (bFGF) is acted on.
The final concentration of 20ng/mL of described basic fibroblast growth factor (bFGF) effect
A kind of method for resuscitation of dental pulp stem cell, it is characterised in that comprise the following steps：
(1) separation, culture and the identification of dental pulp stem cell：Healthy patients tooth is collected, first with 75% alcohol wipe tooth body
Surface, then with the sterile PBS containing penicillin and streptomysin embathe 2 times it is standby, aseptically, using splitting jumping through rings incisor tooth,
Dental pulp is taken out, tissue block is cut into, 30min, digestion knot are digested in 37 DEG C of environment with type i collagen enzyme and neutral proteinase mixed enzyme
Dispelled after beam, cell is collected by centrifugation, be inoculated in sterile petri dish, complete α-MEM culture mediums are added dropwise, 37 DEG C, 5%CO is put into2
Cultivated in cell culture incubator, the first subculture is changed after 5d, subsequent routine 3d changes a subculture, treats cell fusion
After reaching 80% one 90%, pancreatin/EDTA conventional digestion cells are utilized, the good second generation of growth conditions is chosen in passage
DPSCs, adjustment cell concentration is 1 × 106Individual/mL, dispense into 1.5mL EP pipes, be separately added into the anti-human CD90 of appropriate mouse,
The mixed liquor of cell and antibody is incubated 1h, flow cytomery by CD73 and CD14, room temperature lucifuge；
(2) dental pulp stem cell freezes：Second generation dental pulp stem cell degrees of fusion reached after 80-90%, conventional digestion,
5min is centrifuged under the conditions of 1000rpm and collects cell, cells frozen storing liquid is added and dispels, then by cell using concentration as 3 × 106Individual/
ML is distributed into cell cryopreservation tube, is moved into the special freezing storing box of cell, under the conditions of being put into -80 DEG C overnight, is finally transferred to liquid nitrogen ring
Long-term Cryopreservation is carried out in border；
(3) DPSCs recovery：It is chosen at and the dental pulp stem cell of 3 months is frozen in liquid nitrogen, in 37 DEG C of water bath with thermostatic control environment
Quick-thawing, then using routine α-MEM culture dilutions, is collected by centrifugation cell, then adds the dental pulp stem cell containing bFGF
Resuscitation fluid, is put into 37 DEG C, 5%CO2Cultivated in incubator, a subculture is changed every other day.
Complete α-MEM culture mediums are by the μ g/mL streptomysins of 20%FBS+100U/mL penicillin+100 in described step (1)
Composition.
Cells frozen storing liquid is made up of 10%DMSO+90%FBS in described step (2).
Routine α-MEM cultures are by the μ g/mL streptomysin groups of 10%FBS+100U/mL penicillin+100 in described step (3)
Into.
The beneficial effects of the invention are as follows：The invention provides the recovery of a kind of dental pulp stem cell resuscitation fluid and dental pulp stem cell
Method, new do is built by adding a certain amount of basic fibroblast growth factor in convenient stem cells culture medium
Cell recovery liquid, and the resuscitation process with this resuscitation fluid applied to DPSCs, can make DPSCs vigor quick in a short time
Recover, and keep the dryness and multi-lineage potential of original stem cell, the stem cell recovery time can be shortened and improve dry thin
Born of the same parents' Clinical practice survival rate, growth rate exceedes conventional medium group, while cell dryness and multi-lineage potential are maintained, can
Performance and the preferable seed cell of state effectively are provided for clinical stem-cell therapy, substantial amounts of time, manpower and thing is saved
Power cost.
Figure of description
Fig. 1 is the 7d and 14d of DPSCs original cuitures light microscopic picture.
Fig. 2 is DPSCs surface markers CD90, CD73 and CD14 flow cytomery result.
Fig. 3 is the CCK-8 results containing various concentrations bFGF resuscitation fluid culture first generation DPSCs (P1-ac).
Fig. 4 is that first generation DPSCs is acted on by various concentrations bFGF, and second generation DPSCs (P2-ac) reverts to cellar culture
The CCK-8 results of base
Fig. 5 is that first generation DPSCs is acted on by 20ng/mL bFGF, and second generation DPSCs (P2-ac) reverts to cellar culture
The immunofluorescence results of base.
Fig. 6 is that first generation DPSCs is acted on by 20ng/mL bFGF, and second generation DPSCs (P2-ac) reverts to cellar culture
The western-blotting results of base.
Fig. 7 is that first generation DPSCs is acted on by 20ng/mLbFGF, and second generation DPSCs (P2-ac) reverts to cellar culture
Base into surface marker Nestin/GFAP immunofluorescence results after nerve-inducing.
Fig. 8 is that first generation DPSCs is acted on by 20ng/mL bFGF, and second generation DPSCs (P2-ac) reverts to cellar culture
The coloration result of fat drips oil red -0 after the adipogenic induction of base.
Fig. 9 is that first generation DPSCs is acted on by 20ng/mL bFGF, and second generation DPSCs (P2-ac) reverts to cellar culture
Calcium scoring alizarin red-S coloration results after the osteogenic induction of base.
Embodiment
With reference to embodiment, the present invention will be described in detail, and embodiment is only the preferred embodiment of the present invention,
It is not limitation of the invention.
The preparation of dental pulp stem cell resuscitation fluid：
Mainly collectively constituted by mescenchymal stem cell conventional medium and basic fibroblast growth factor (bFGF), its
In, conventional cell culture medium is：A-MEM culture mediums (Gibco, USA)+10% hyclone (FBS, Gibco, USA)+100U/
The final concentration of mL penicillin+100 μ g/mL streptomysins (Gibco, USA) .bFGF effects is respectively 5ng/mL, 10ng/mL, 20ng/
ML, 50ng/mL, 80ng/mL and 100ng/mL.
The resuscitation process of dental pulp stem cell：
DPSCs separation, culture and identification：Clinically 19-29 Sui healthy patients tooth is collected, 75% alcohol wipe is first used
Tooth surface, then with the sterile PBS containing penicillin and streptomysin embathe 2 times it is standby.Aseptically, using splitting jumping through rings incisor
Tooth, takes out dental pulp, is cut into size about 1mm2Tissue block, with type i collagen enzyme and neutral proteinase mixed enzyme in 37 DEG C of environment
30min is digested, digestion dispels after terminating, cell is collected by centrifugation, is inoculated in sterile petri dish, and complete α-MEM culture mediums are added dropwise
(the μ g/mL streptomysins of 20%FBS+100U/mL penicillin+100), is put into 37 DEG C, 5%CO2Cultivated in cell culture incubator, 5d
After change the first subculture, one subculture of subsequent routine 3d replacings.After cell fusion reaches 80%-90%, pancreas is utilized
The good second generation DPSCs of growth conditions is chosen in enzyme/EDTA conventional digestion cells, passage, and adjustment cell concentration is 1 × 106
Individual/mL, is dispensed into 1.5mL EP pipes, is separately added into appropriate mouse anti-human CD90, CD73 and CD14, and room temperature lucifuge will be thin
The mixed liquor of born of the same parents and antibody is incubated 1h, flow cytomery.
DPSCs's freezes：Second generation DPSCs degrees of fusion reaches after 80-90% that cell is collected by centrifugation in conventional digestion
(1000rpm, 5min), adds cells frozen storing liquid (10%DMSO+90%FBS) and dispels, then by cell using concentration as 3 × 106
Individual/mL is distributed into cell cryopreservation tube, is moved into the special freezing storing box of cell, is put into -80 DEG C of refrigerator overnights, is finally transferred to liquid nitrogen
Long-term Cryopreservation is carried out in environment.
DPSCs recovery：It is chosen at and the DPSCs of 3 months is frozen in liquid nitrogen, is quickly solved in 37 DEG C of water bath with thermostatic control environment
Freeze, then diluted, be collected by centrifugation using conventional α-MEM culture mediums (the μ g/mL streptomysins of 10%FBS+100U/mL penicillin+100)
Cell, then adds recovery medium (the μ g/mL strepto-s of α-MEM+10%FBS+100U/mL penicillin+100 containing bFGF
Element), it is put into 37 DEG C, 5%CO2Cultivated in incubator, a subculture is changed every other day.BFGF effect final concentration be respectively
5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 80ng/mL and 100ng/mL, and bFGF acts only on the recovery first generation
DPSCs, recovery second generation DPSCs culture mediums are replaced by convenient stem cells culture medium.The DPSCs of the recovery first generation and the second generation points
Biao Ji not be and P2-ac.
Dental pulp stem cell related biological performance detection after bFGF effects：
Cell-proliferation activity detects (CCK-8 detections)：Choose the preferable DPSCs of growth conditions(P1-ac), adjust cell concentration
For 2.0 × 103It is inoculated in 96- porocyte culture plates, adds recovery nutrient solution, be put into 37 DEG C, 5%CO2Trained in incubator
Support, a subculture is changed every other day.Meanwhile, (DPSCs after passage(P2-ac)) culture medium is all replaced by conventional medium
(the μ g/mL streptomysins of α-MEM+10%FBS+100U/mL penicillin+100), respectively at after culture 1,3,5 and 7d, 10 are added per hole
μ L CCK-8 solution, is put into cell culture incubator and continues to cultivate 1h, and the absorbance at 450nm is then observed on ELIASA
(OD values).Under equal conditions, conventional medium is set to control group.
Immunofluorescence dyeing：Mainly examined by mescenchymal stem cell surface marker CD146 and STRO-1 fluorescent staining
DPSCs dryness function is surveyed, is comprised the following steps that：Cell is inoculated on six orifice plate creep plates, after culture terminates, PBS rinses 3
Secondary, 4% paraformaldehyde fixes 30min, and PBS is rinsed 3 times, and BSA combines 0.1 TritonX-100 ruptures of membranes closing 30min；CD146,
4 DEG C of incubation 16h of STRO-1 primary antibodies；PBST is rinsed 3 times, and secondary antibody lucifuge is incubated 1h, and PBST is rinsed 3 times, and DAPI lucifuges are incubated 5min,
PBST is rinsed 3 times；Fluorescence microscope cell, is photographed to record.
Western-blotting is detected：Cell is inoculated in six orifice plates, after culture terminates, PBS is rinsed 3 times, RIPA and
PMSF cell lysis on ice, 4 DEG C of centrifuging and taking supernatants, western blotting detect CD146 and Nanog expression.Per hole
Protein content is consistent, and 10%SDS-PAGE glue carries out electrophoresis, and albumen is gone into pvdf membrane after terminating, and milk closing 2h, TBST rinse 3
It is secondary, incubation 16h, the TBST flushing 3 times of 4 DEG C of primary antibody, secondary antibody is incubated 1h, and TBST is rinsed 3 times.Finally, Bio-rad protein gels are passed through
The amount of relevant target protein is observed and analyzed to imaging system.
Into nerve-inducing：By certain density cells (4 × 103cells/cm2) be inoculated in after six orifice plates, 1d, change nerve and lure
Lead culture medium (DMEM high glucose mediums dexamethasone containing 10-7M, 50 μ g/mL vitamin Cs, 50 μM of Indomethacins, 10 μ g/mL pancreases
Island element, 45mM IBMX), a nutrient solution is changed per 3d.Induce after 6d, PBS is rinsed 3 times, 4% paraformaldehyde fixes 30min,
PBS is rinsed 3 times, BSA joint 0.1TritonX-100 rupture of membranes closings 30min；4 DEG C of incubation 16h of Nestin and GFAP primary antibodies；PBST
Rinse 3 times, secondary antibody lucifuge is incubated 1h, PBST is rinsed 3 times, DAPI lucifuges are incubated 5min, PBST is rinsed 3 times；Fluorescence microscope is seen
Cell is examined, is photographed to record.
Adipogenic induction：By certain density cells (2 × 104 cells/cm2) six orifice plates are inoculated in, 2mL is added per hole conventional
Culture medium, when cell fusion degree reaches 100%, culture medium is replaced by 2mLOriCellTMAdipogenic induction differential medium
After (Cyagen, USA) A liquid, induction 3d, 2mLOriCell is replaced byTMA is gained again after adipogenic induction differential medium B liquid, 1d
After liquid, A liquid and B liquid alternating action 4 times (16d), continue to maintain culture 6d with B liquid, changed per 3d and once induces liquid.Adipogenic induction
It is fixed after differentiation terminates, the dyeing of oil red O dyestuffs working solution, micro- Microscopic observation is into fat Color.
Osteogenic induction：By cell according to certain density (2 × 104cells/cm2) six orifice plates are inoculated in, it is conventional that hole adds 2mL
Culture medium, when cell fusion degree reaches 60%-70%, is replaced by 2mLOriCellTMStem cell Osteoinductive differentiation is trained completely
Support base (Cyagen, USA).Change fixed after once fresh induction liquid, 2w per 3d, Alizarin red staining, micro- Microscopic observation skeletonization
Color.
Experimental result
As shown in figure 1, when result shows original cuiture 7d, existing dental pulp stem cell is climbed out of around tissue block, thin during 14d
Born of the same parents cover with substantially, in typical fibroblast sample shape.
As shown in Fig. 2 result is shown, DPSCs positive expression mescenchymal stem cell surface marker CD90 (98.82%),
CD73 (99.72%) and radiolucent table reach CD14 (0.85%).
As shown in figure 3, result is shown, the trend of a sustainable growth is presented in DPSCs under various concentration bFGF effects, and
When bFGF acts on final concentration of 20ng/mL, cell proliferation rate is apparently higher than other groups, in 5d and 7d, and OD values are respectively
0.9970 and 2.0912, it is 1.37 times and 1.24 times of control group.
As shown in figure 4, result is shown, the DPSCs growth rates pre-processed by 20ng/mL bFGF apparently higher than other
Group, and reach in 7d a peak value (OD：2.0482), illustrate to only need to utilize the recovery training containing 20ng/mLbFGF
Nutrient solution acts on first generation DPSCs after recovery, after passage, and second generation DPSCs equally has preferably growth and proliferation function, because
This, subsequent experimental experimental group bFGF concentration only selects 20ng/mL.
As shown in figure 5, result is shown, DPSCs (P2-ac) positive expression mescenchymal stem cell surface marker CD146 and
STRO-1, DPSCs (P2-ac) still have the dryness function of mescenchymal stem cell.
As shown in fig. 6, result shows DPSCs (P2-ac) expression CD146 and NANOG, illustrate DPSCs (P2-ac) dryness
Function is uninfluenced.
As shown in fig. 7, as a result show, DPSCs (P2-ac) positive expression NSC surface marker Nestin and
GFAP, can succeed and carry out induction differentiation to neural-like cells.
As shown in figure 8, result is shown, DPSCs (P2-ac) has obvious fat drips to occur, and explanation can succeed into fat-like
Cell direction carries out induction differentiation.
As shown in figure 9, result is shown, DPSCs (P2-ac) has obvious calcium scoring to occur, and explanation can succeed to skeletonization
Cell direction carries out induction differentiation.
The various concentrations bFGF of table 1 acts on OD values at recovery first generation DPSCs (P1-ac) 450nm
Table 2 by various concentrations bFGF pretreatment after second generation DPSCs (P2-ac) 450nm at OD values
Described above is only the preferred embodiment of the present invention, and protection scope of the present invention is not limited merely to above-mentioned implementation
Example, all technical schemes belonged under thinking of the present invention belong to protection scope of the present invention.It should be pointed out that for the art
Those of ordinary skill for, some improvements and modifications without departing from the principles of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (6)
1. a kind of dental pulp stem cell resuscitation fluid, it is characterised in that described dental pulp stem cell resuscitation fluid is normal by mescenchymal stem cell
Advise culture medium and basic fibroblast growth factor (bFGF) composition, described mescenchymal stem cell conventional medium by α-
The μ g/mL streptomysins of MEM culture medium+10-20% hyclone+100U/mL penicillin+100 are constituted, and described basic fibroblast is thin
The final concentration of 10-20ng/mL of the intracellular growth factor (bFGF) effect.
2. a kind of dental pulp stem cell resuscitation fluid according to claim 1, it is characterised in that described basic fibroblast
The final concentration of 20ng/mL of growth factor (bFGF) effect.
3. a kind of method for resuscitation of the dental pulp stem cell of the dental pulp stem cell resuscitation fluid described in usage right requirement 1, its feature exists
In comprising the following steps：
(1) separation, culture and the identification of dental pulp stem cell：Healthy patients tooth is collected, first with 75% alcohol wipe Tooth surface,
Again with the sterile PBS containing penicillin and streptomysin embathe 2 times it is standby, aseptically, using jumping through rings incisor tooth is split, take out tooth
Marrow, is cut into tissue block, and 30min is digested in 37 DEG C of environment with type i collagen enzyme and neutral proteinase mixed enzyme, and digestion terminates after-blow
Dissipate, cell is collected by centrifugation, is inoculated in sterile petri dish, complete α-MEM culture mediums are added dropwise, 37 DEG C, 5%CO is put into2Cell is trained
Support and cultivated in case, the first subculture is changed after 5d, subsequent routine 3d changes a subculture, treats that cell fusion reaches
After 80%-90%, using pancreatin/EDTA conventional digestion cells, the good second generation DPSCs of growth conditions, adjustment are chosen in passage
Cell concentration is 1 × 106Individual/mL, dispense into 1.5mL EP pipes, be separately added into appropriate mouse anti-human CD90, CD73 and
The mixed liquor of cell and antibody is incubated 1h, flow cytomery by CD14, room temperature lucifuge；
(2) dental pulp stem cell freezes：Second generation dental pulp stem cell degrees of fusion reached after 80-90%, conventional digestion,
5min is centrifuged under the conditions of 1000rpm and collects cell, cells frozen storing liquid is added and dispels, then by cell using concentration as 3 × 106Individual/
ML is distributed into cell cryopreservation tube, is moved into the special freezing storing box of cell, under the conditions of being put into -80 DEG C overnight, is finally transferred to liquid nitrogen ring
Long-term Cryopreservation is carried out in border；
(3) DPSCs recovery：It is chosen at and the dental pulp stem cell of 3 months is frozen in liquid nitrogen, it is quick in 37 DEG C of water bath with thermostatic control environment
Thaw, then using routine α-MEM culture dilutions, cell is collected by centrifugation, then add the dental pulp stem cell recovery containing bFGF
Liquid, is put into 37 DEG C, 5%CO2Cultivated in incubator, a subculture is changed every other day.
4. the method for resuscitation of dental pulp stem cell according to claim 3, it is characterised in that in described step (1) completely
α-MEM culture mediums are made up of the μ g/mL streptomysins of 20%FBS+100U/mL penicillin+100.
5. the method for resuscitation of dental pulp stem cell according to claim 3, it is characterised in that cell in described step (2)
Frozen stock solution is made up of 10%DMSO+90%FBS.
6. the method for resuscitation of dental pulp stem cell according to claim 3, it is characterised in that conventional in described step (3)
α-MEM cultures are made up of the μ g/mL streptomysins of 10%FBS+100U/mL penicillin+100.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110338189A (en) * | 2019-07-09 | 2019-10-18 | 浙江优牙生物科技有限公司 | A kind of pulp tissue's serum-free freezes and method for resuscitation and frozen stock solution |
| CN111635860A (en) * | 2020-06-17 | 2020-09-08 | 山东大学 | System and method for stem cell culture |
| CN111727962A (en) * | 2020-08-10 | 2020-10-02 | 医微细胞生物技术(广州)有限公司 | Stem cell cryopreservation liquid |
| CN111727961A (en) * | 2020-08-10 | 2020-10-02 | 医微细胞生物技术(广州)有限公司 | Dental pulp stem cell cryopreservation liquid and cryopreservation method thereof |
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| CN110338189A (en) * | 2019-07-09 | 2019-10-18 | 浙江优牙生物科技有限公司 | A kind of pulp tissue's serum-free freezes and method for resuscitation and frozen stock solution |
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| CN111727962A (en) * | 2020-08-10 | 2020-10-02 | 医微细胞生物技术(广州)有限公司 | Stem cell cryopreservation liquid |
| CN111727961A (en) * | 2020-08-10 | 2020-10-02 | 医微细胞生物技术(广州)有限公司 | Dental pulp stem cell cryopreservation liquid and cryopreservation method thereof |
| CN111727962B (en) * | 2020-08-10 | 2020-12-01 | 医微细胞生物技术(广州)有限公司 | Stem cell cryopreservation liquid |
| CN111727961B (en) * | 2020-08-10 | 2020-12-01 | 医微细胞生物技术(广州)有限公司 | Dental pulp stem cell cryopreservation liquid and cryopreservation method thereof |
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