CN107058219A - A kind of method that application stem cell self-characteristic prepares dental pulp stem cell - Google Patents
A kind of method that application stem cell self-characteristic prepares dental pulp stem cell Download PDFInfo
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Abstract
The invention discloses a kind of method that application stem cell self-characteristic prepares dental pulp stem cell, this method is included：Step 1：Extract dental pulp and be divided into some fritters, be put into centrifuge tube；Step 2：Prepare digestive juice and be added in centrifuge tube and digested；Step 3：Terminate digestion；Step 4：Centrifugation, removes supernatant, and the precipitation in bottom adds MSC primary culture mediums, is moved to after resuspension in blake bottle, carries out original cuiture；Step 5：When cell fusion degree reaches 80% 90%, supernatant is collected, the stem cell extract solution is added in MSC primary culture mediums, Secondary Culture base is obtained；Step 6：Pancreatin digestion is carried out, digestion time is no more than 1min, terminates digestion；Step 7：Bottle is planted, Secondary Culture is carried out；Step 8：Repeat the above steps 6 and 7 progress Secondary Culture several times；Step 9：Cell cryopreservation.The method of the present invention shortens the time of original cuiture, and multiplication capacity is strong, and the dental pulp stem cell of preparation has good cells and characteristic of stem, and differentiation potential is strong.
Description
Technical field
The present invention relates to a kind of preparation method of dental pulp stem cell, and in particular to prepared by one kind application stem cell self-characteristic
The method of dental pulp stem cell.
Background technology
Stem cell is the cell not broken up fully, with self-renewing and the ability for being divided into particular organization's organ.Dental pulp
Stem cell (dental pulp stem cells, DPSCs) is also known as dental pulp mescenchymal stem cell, refers to quickly increase in dental pulp
Grow and the pulp cells with certain clonality, proposed that it has and bone first equal to 2000 by Gronthos
The similar immunophenotype of bone marrow-drived mesenchymal stem.
Dental pulp stem cell has the advantages that abundance, collection are convenient, immunogenicity is low, without dispute of ethic.Therefore, dental pulp
Stem cell has a wide range of applications in regenerative medicine and organizational project reparation.Dental pulp stem cell periodontal tissue defect,
Stronger advantage is shown in periodontitis, pulp tissue's regeneration.The reliable and stable DPSCs of large-scale culture becomes as one kind
Gesture.
Conventional DPSCs multiplication capacities are more general, document " the Scaling-up of that Nelson F.Lizier etc. are delivered
Dental Pulp Stem Cells Isolated from Multiple Niches”（Literature reference：PLoS One.
2017; 7(6)）, which describe P3 and turn out 1.8*10 for highest8It is individual.
Confirm it being the conventional means of separation identification stem cell to the molecular labeling of stem cell surface, have now been found that
DPSCs expression mesodermal markers Stro-1 and CD146.Stro-1 is mescenchymal stem cell（mesenchymal stem
cells, MSCs）One of early stage cell marker molecules, DPSCs undifferentiated state can be identified.Farzaneh
Document that Aghajani etc. is delivered " Comparative Immunophenotypic Characteristics,
Proliferative Features, and Osteogenic Differentiation of Stem Cells Isolated
from Human Permanent and Deciduous Teeth with Bone Marrow”（Literature reference：Mol
Biotechnol (2016) 58:415–427）, which describe the DPSCs general ＜ 5% of surface marker Stro-1 surface markers.
The content of the invention
It is an object of the invention to provide the method for preparing dental pulp stem cell, this method shortens the time of original cuiture, and
And multiplication capacity is strong, the dental pulp stem cell of preparation has good cells and characteristic of stem, and differentiation potential is strong.
In order to achieve the above object, the invention provides the side that a kind of application stem cell self-characteristic prepares dental pulp stem cell
Method, this method is included：
Step 1：Tooth samples are chosen, corona is broken, dental pulp is extracted from the pulp cavity of tooth samples, tooth is washed using cushioning liquid
Marrow, is divided into some fritters by dental pulp, is put into centrifuge tube；
Step 2：Digestive juice is prepared, the digestive juice is included：Sodium chloride injection, Collagenase I, dispase II（DispaseⅡ）, it is white
Albumen, magnesium salts and calcium salt, the digestive juice is added in the centrifuge tube in step 1, the seal of tube will be centrifuged, at 37 DEG C after being well mixed
Lower digestion；
Step 3：After the fritter in centrifuge tube is loose, to the processing of centrifuge tube surface sterilizing, digestion terminate liquid is added, termination disappears
Change；
Step 4：The solution that step 3 is obtained is centrifuged, and removes supernatant, and the precipitation in bottom adds MSC primary culture mediums,
Precipitation is set to be transferred to after being resuspended in blake bottle, in 5%CO2, at 37 DEG C, carry out original cuiture；
Step 5：When cell fusion degree reaches 80%-90%, supernatant is collected, and carries out ultrafiltration, collects dry in upper screen
Cell extract, the stem cell extract solution is added in MSC primary culture mediums, Secondary Culture base is obtained；
Step 6：The cell of lower sediment in step 5 is inoculated into blake bottle, will when cell fusion degree reaches 80%-90%
Culture medium removal in blake bottle is standby, rinses blake bottle with phosphate buffer, the pancreatin of dilution is added in blake bottle, will
Pancreatin is laid in blake bottle bottom, carries out pancreatin digestion, and digestion time is no more than 1min, adds above-mentioned standby culture medium and terminates
Digestion；The pancreatin of dilution is the pancreatin that concentration is 0.025%；
Step 7：Step 6 is digested to obtained liquid centrifugation, the cell of bottom precipitation is added to the Secondary Culture base that step 5 is obtained
In, it is well mixed, by 1 × 102/cm2Cell density carry out kind of a bottle, in 5%CO2, Secondary Culture is carried out under conditions of 37 DEG C；
Step 8：Repeat the above steps 6 and 7, carry out Secondary Culture several times；
Step 9：Cells frozen storing liquid is placed in cryopreservation tube, carries out adding step 8 cell expansion in precooling, cryopreservation tube at 4 DEG C
Obtained stem cell Secondary Culture base is cultivated, cell density is 1.5 × 106~2×106/ 1.5mL/ is managed, and is gradually cooled to -80 DEG C,
The cell frozen is put into -196 DEG C of liquid nitrogen container after 24h and preserved.
Wherein, included in described MSC primary culture mediums：Growth factor.
Preferably, in step 1, described cushioning liquid is included：0.9% sodium chloride injection, 10,000Units/mL are blue or green
Mycin and 10,000 μ g/mL streptomysins.
Wherein, described sodium chloride injection：Penicillin：The volume ratio of streptomysin is 198:1:1.
Preferably, in step 2, described magnesium salts is included：Magnesium chloride；Described calcium salt is included：Calcium chloride.
Wherein, the concentration of each component is in described digestive juice：0.9% sodium chloride injection, 3% Collagenase I, 4% dispase
IIth, 1mol/L magnesium chlorides, 200mg/mL calcium chloride and 20% albumin.
Wherein, 0.9% described sodium chloride injection：1mol/L magnesium chlorides：200mg/mL calcium chloride：The body of 20% albumin
Product is than being 1mL：5μL：5μL ：100μL；
Wherein, the bioactivity of 3% described Collagenase I is 270U/mg；
Wherein, the bioactivity of 4% described dispase II is 0.85U/mg.
Preferably, in step 3, described terminate liquid is included：0.9% sodium chloride injection and 20% albumin, it is described
0.9% sodium chloride injection：The volume ratio of 20% albumin is 9：1.
Preferably, in step 4, described MSC primary culture mediums are included：The training of human mesenchymal stem cells' serum-free basis
Support base, stroma cell derivative factor and Glu.
Wherein, described growth factor is included：Fibroblast growth factor, HGF, epidermal growth factor
Son and VEGF.
Wherein, the volume of described human mesenchymal stem cells' serum-free basal medium：The matter of HGF
Amount：The quality of EGF：The quality of VEGF：The quality of stroma cell derivative factor：L- glutamy
Volume=100mL of amine：1μg：2μg：2μg：2μg：100μL.
Wherein, the content of described fibroblast growth factor is 100 IU/mL.
Preferably, in step 4, described blake bottle is treated blake bottle, and processing method is：By adherent reagent
The factor and phosphate buffer are with the μ L of volume ratio 10：1mL mixing be resuspended after, add blake bottle in, and rock blake bottle make it is adherent
The reagent factor covers all blake bottle bottom.
Preferably, in steps of 5, described stem cell extract solution：The volume ratio of MSC primary culture mediums is 1：50.
Preferably, in step 6,0.025% described pancreatin be 0.25% pancreatin and phosphate buffer using volume as
1：9 preparations are obtained.
Preferably, in step 9, cooling rate is 1 DEG C/min；Described cells frozen storing liquid is the jelly of non-animal derived serum
Liquid storage.
Preferably, in step 1, before tooth samples are chosen, tooth samples are preserved with liquid is preserved, and to tooth sample
This progress is screened.
Wherein, described preservation liquid is included：0.9% sodium chloride injection, mycillin and albumin, 0.9% described chlorine
Change sodium injection：Mycillin：The volume ratio of albumin is 100mL：1 mL：2 mL.
Wherein, described mycillin is included：10,000U/mL penicillin, 10,000 μ g/mL streptomysins.
Wherein, when carrying out tooth samples screening, it is provided simultaneously with following condition：
Comprising any one in hepatitis B, hepatitis, syphilis, AIDS, CMV, EBV and HTLV virus or two or more it is detected as the moon
Property；The non-inflammation of tooth；The non-natural tooth come off.
The method that the application stem cell self-characteristic of the present invention prepares dental pulp stem cell, with advantages below：
（1）The method of the present invention can be observed have attached cell to grow in the 2-3 days in original cuiture, and first time attached cell goes out
It is short between current；
（2）The method of the present invention was in original cuiture the 7-10 days, and cell fusion degree can reach more than 80%, can carry out passage behaviour
Make, be significantly reduced the original cuiture time；
（3）The method of the present invention promotes stem cell adherent growth using cell attachment reagent, is existed using the characteristic of stem cell in itself
Low-density species bottle is lower to carry out dental pulp stem cell of the screening with extremely strong multiplication capacity, utilizes cell of cytokine profiles joint itself
The extract of secretion is cultivated so that cell number has expanded more than ten times, the present invention than cell number prepared by other method
Cell number can be made to reach 2*10 in P3 generations9It is individual；
（4）Dental pulp stem cell prepared by the method for the present invention has good stem cell properties, the positive of its mescenchymal stem cell
The equal ＞ 95% of index CD73, CD90, CD105, the equal ＜ of negative indication CD34, CD31, CD45, HLA-DR of its mescenchymal stem cell
2%；
（5）Dental pulp stem cell prepared by the method for the present invention has good differentiation potential, and its P3 is for cell surface marker Stro-
1 ＞ 20%；
（6）Dental pulp stem cell prepared by the method for the present invention has good immunosuppressive activity, the characteristic with stem cell.
Brief description of the drawings
Fig. 1 is the cell state figure of dental pulp stem cell culture the 3rd day.
Fig. 2 is the cell state figure of dental pulp stem cell culture the 9th day.
Fig. 3 is the cell state figure after dental pulp stem cell is passed on（Cell fusion degree has reached 80%）.
Fig. 4 is the cell growth curve figure of dental pulp stem cell.
Fig. 5 is the dental pulp stem cell surface antigen CD31 of present invention detection figure.
Fig. 6 is the dental pulp stem cell surface antigen CD34 of present invention detection figure.
Fig. 7 is the dental pulp stem cell surface antigen CD45 of present invention detection figure.
Fig. 8 is the dental pulp stem cell surface antigen HLA-DR of present invention detection figure.
Fig. 9 is the dental pulp stem cell surface antigen CD73 of present invention detection figure.
Figure 10 is the dental pulp stem cell surface antigen CD90 of present invention detection figure.
Figure 11 is the dental pulp stem cell surface antigen CD105 of present invention detection figure.
Figure 12 is the dental pulp stem cell surface antigen S tro-1 of present invention detection figure.
Figure 13 is the result figure that dental pulp mescenchymal stem cell suppresses immunocyte ability（A is microscopic cells figure, and B is immune
Competent cell statistical chart）.
Embodiment
Technical scheme is described further below in conjunction with drawings and examples.
Embodiment 1
First, the preservation of tooth samples
Configure tooth samples and preserve liquid, the component of the preservation liquid is included： 0.9%（Mass fraction）Sodium chloride injection 100mL,
1mL mycillins and 2mL albumin, 1mL mycillins（Gibco, article No. 15140-122）In contain 10,000 unit
Penicillin（Alkali）（Penicillin）With 10,000 μ g streptomysin（Alkali）（Streptomycin）, tooth samples are stored in
State in preservation liquid, storage temperature is 2 DEG C -20 DEG C.Above-mentioned albumin is 20%（Mass fraction）Human serum albumins（It is purchased from：Mountain
Xikang treasured biological products limited company, 10g/50mL/20%/bottle）
2nd, the screening of tooth samples
Requirement to tooth samples, it is specific as follows：
（1）Hepatitis B, hepatitis, syphilis, AIDS, CMV, EBV, HTLV Viral diagnosis are feminine gender, and its detection method is normal for this area
Detection method is advised, such as hepatitis B HBV-DNA detection methods, antibody of HCV are detected；
（2）The non-inflammation of tooth；
（3）Exclude the tooth that adult and children come off naturally, the complete atrophy of tooth pulp tissue come off naturally, it is impossible to trained
Support；Deciduous teeth that children pull out, correction tooth, the wisdom tooth pulled out of being grown up can be used, can also be using not attacking the slight of pulp tissue
Decayed tooth.
3rd, the transport of tooth samples
Traffic condition is：At 2-8 DEG C, laboratory is transported in 12h.
4th, the preparation method of dental pulp stem cell
1st, blake bottle
Human mesenchymal stem cells promote adherent reagent（Bioind companies, product specification：05-752-1F）50 μ L+5mL phosphate delay
Fliud flushing（Phosphate buffer saline, PBS）After resuspension, it is added in T75 blake bottles, rocking blake bottle makes adherent examination
Agent covers all blake bottle bottom, is placed in stand-by in 37 DEG C of incubators.The pH value of above-mentioned phosphate buffer is 7.4.
2nd, the preparation of MSC primary culture mediums
Human mesenchymal stem cells' serum-free basal medium（Bioind companies, product specification：05-200-1A）500mL+bFGF
（Basic fibroblast growth factor）50000IU（International Unit, the international unit of biological value）+HGF（Liver
Porcine HGF）5μg+EGF（EGF）10μg+VEGF（VEGF）10μg +SDF-1（Matrix
Cell derived factor）The μ L of 10 μ g+L- glutamine 500.
The introduction of above-mentioned each factor, it is specific as follows：
（1）bFGF（Basic fibroblast growth factor）：bFGF（basic fibroblast growth factor）It is into
One member of fibroblast growth factor family.It has been demonstrated that：BFGF can support that undifferentiated human embryonic stem is thin
The stem cell properties of born of the same parents, can also stimulate the cell of mesoderma origin, and neuroderm, ectoderm and endoderm origin are thin
The propagation of born of the same parents.In vitro, bFGF Human Umbilical Vein Endothelial Cells have chemotactic and promote mitotic effect, and can promote nerve
Differentiation, survival and regeneration.It has been demonstrated regulation of embryonic development and differentiation in it is most important, it may be in angiogenesis, group
Played a role in the internal modulation for knitting reparation, embryonic development, and neuronal function.
（2）HGF（HGF）：HGF（Hepatocyte growth factor）A kind of bypolypeptide growth because
Son, includes the growth, migration and shape of the polytype cell such as liver cell, epithelial cell, endothelial cell, hematopoietic cell with promotion
The effect that state occurs.
（3）EGF（EGF）：EGF（Epidermal growth factor）A kind of multi-functional growth because
Son, all has strong rush splitting action to Various Tissues cell in vitro in vivo.Specific receptors knots of the EGF with responsive cell surface
Close, promote Receptor dimerization and make cytoplasm site phosphorylation.The acceptor being activated at least can have unlike signal sequence with 5 kinds
Protein binding, carry out signal transduction, the synthesis to protein in translation skill plays regulatory role.In addition EGF can be improved carefully
Intracellular DNA topoisomerase actives, can also promote some gene expressions relevant with propagation, such asmycGene（One group of oncogene）、fosGene（Proto-oncogene）Deng.
（4）VEGF（VEGF）：VEGF（vascular endothelial growth factor）It is early
Phase is also referred to as vascular permeability factor (vascular permeability factor, VPF), is vascular endothelial cell specificity
HBGF (heparin-binding growth factor, HBGF), can in vivo induction of vascular it is newborn.
（5）SDF-1（Stroma cell derivative factor）：SDF-1（stromal cell-derived factor 1）It is also known as
Change factor CXCL12, have strong chemotaxis to lymphocyte and played an important role in development, guided in embryonic development
Candidate stem cell migrating from fetus liver to marrow.
（6）Glu：Glu is particularly significant in cell culture.Take off after amino, Glu can
It is used as the energy source of culture cell, the synthesis and nucleic acid metabolism of participation protein.Glu passes through one section in the solution
It can be degraded after time, but definite degradation rate never has final determination, its degraded can cause the formation of ammonia, and ammonia is for one
A little cells have toxicity.
3rd, the method that dental pulp stem cell is prepared using stem cell self-characteristic, is specifically included：
Step 1：Tooth samples are chosen, corona is broken, dental pulp is extracted from the pulp cavity of tooth samples, tooth is washed using cushioning liquid
Marrow, is divided into some fritters by dental pulp, is put into 15mL centrifuge tubes；The component of above-mentioned buffer solution is included：0.9%（Mass fraction）Chlorine
Change sodium injection 495mL+10,000U/mL（Units/mL）Penicillin 2.5mL+10,000 μ g/mL streptomysins 2.5mL；
Step 2：Digestive juice is prepared, the digestive juice is included：0.9%（Mass fraction）Sodium chloride injection 2mL, 3%（Mass fraction）
Collagenase I 270U/mg, 4%（Mass fraction）Dispase II（DispaseⅡ）0.85U/mg（Units/mg）、20%（Quality point
Number）The μ L of 200 μ L, 1mol/L magnesium chloride of albumin, 10 μ L, 200mg/mL calcium chloride 10,2mL digestive juices are added in step 1
In 15mL centrifuge tubes, rolling makes tissue block be uniformly dispersed, and tightens lid, is obturaged with sealed membrane, posts label, rocks mixing
After be put into 37 DEG C of constant temperature water baths, it is per minute taking-up centrifuge tube rock once, tissue is fully mixed with digestive juice, digestion
15min；Above-mentioned albumin is 20%（Mass fraction）Human serum albumins（It is purchased from：Health precious biological products share in Shanxi is limited
Company, 10g/50mL/20%/bottle）；
Step 3：After the fritter in centrifuge tube is digested loosely, to the processing of centrifuge tube surface sterilizing, digestion terminate liquid is added extremely
14mL terminates digestion, and suspension is blown and beaten with pipette tips, cell is departed from as far as possible with tissue；Above-mentioned digestion terminate liquid includes 0.9%
（Mass fraction）Sodium chloride injection and 20%（Mass fraction）Albumin, 0.9% sodium chloride injection：20% albumin volume ratio
For 9：1；
Step 4：The solution that step 3 is obtained centrifuges 10min under 1200rpm rotating speeds, supernatant is removed, in the precipitation of bottom
Add in MSC primary culture mediums 10mL, blown and beaten with pipette tips, precipitation is transferred to 75cm after being resuspended2In blake bottle, pasted using cell
Wall reagent carries out coating and promotes stem cell adherent growth, and original cuiture condition is 5%CO2（Air：CO2Volume ratio be 95：5）,
37℃；At the 7th day of culture to 75cm25mLMSC primary culture mediums are added in blake bottle；
Step 5：When cell fusion degree reaches 80%-90%, supernatant is collected, ultrafiltration is carried out using 50mL super filter tubes,
10min is centrifuged under 12000rpm rotating speeds, the stem cell extract solution in upper screen is collected, the stem cell extract solution is added to
In MSC primary culture mediums, above-mentioned stem cell extract solution：The volume ratio of MSC primary culture mediums is 1：50, Secondary Culture base is obtained,
Unnecessary extract needs to place in -20 DEG C of refrigerators；
Step 6：The cell of lower sediment in step 5 is inoculated into blake bottle, when cell fusion degree reaches 80%-90%（Carefully
The area of born of the same parents' confluent cultures bottom of bottle portion 80%~90%）, the culture medium in blake bottle is removed to standby, pipette, extract PBS flushing trainings
Bottom of bottle portion is supported, PBS is suctioned out moved into waste liquid cylinder after rinsing, the pancreatin of dilution is added in blake bottle, pancreatin is laid in
Blake bottle bottom, carries out pancreatin digestion, and digestion time is no more than 1min, adds above-mentioned standby culture medium and terminates digestion；Dilution
Pancreatin be 0.25%（Mass fraction）Pancreatin：PBS volume ratios are 1:9 pancreatin；
Step 7：Step 6 is digested to obtained liquid centrifugation, the cell of bottom precipitation is added to the Secondary Culture base that step 5 is obtained
In, it is well mixed, dental pulp of the screening with extremely strong multiplication capacity is carried out under low-density species bottle using the characteristic of stem cell in itself
Stem cell, by 1 × 102/cm2Cell density carry out kind of a bottle, generation time, generation, sample number are indicated on blake bottle,
5%CO2, Secondary Culture is carried out under conditions of 37 DEG C；
Step 8：Repeat the above steps 6 and 7, carry out Secondary Culture several times；
Step 9：Cells frozen storing liquid is placed in cryopreservation tube, carries out adding step 8 cell expansion in precooling, cryopreservation tube at 4 DEG C
Obtained stem cell Secondary Culture base is cultivated, cell density is 1.5 × 106~2×106/ 1.5mL/ is managed, cell survival rate ＞ 95%,
It is cooled to that the cell frozen is put into -196 DEG C of liquid nitrogen container after -80 DEG C, 24h with 1 DEG C/min speed and preserves.It is above-mentioned thin
Born of the same parents' frozen stock solution is the frozen stock solution of non-animal derived serum（Bioind companies, product specification：05-712-1E）.
In above-mentioned steps 9, using this area conventional method, such as Trypan Blue and Microscopical Method For Detection determine cell survival rate,
Cell survival rate ＞ 95% is to freeze standard.
In the above-mentioned methods, the extract solution that dental pulp stem cell is added in MSC primary culture mediums is used as the training of Secondary Culture
Support base（Step 5）, typically cultivate cell and be required for adding cell factor, conventional way is artificially to add some known people
The cell factor of work synthesis, and the method for the present invention is then using the powerful secretion capacity of stem cell itself, in incubation
Stem cell can secret out of various cell factors and some elements into culture medium, be used as nutritional ingredient during passage.
In the above-mentioned methods, it is inoculated with a low density（Step 7）, with 100 times lower than prior art culture density of situation
Under, filter out multiplication capacity it is superpower and with stronger independent survival capacity stem cell.Stem cell compares other adults
Ability of cell proliferation is vigorous, and stem cell often can also grow under extremely low density.Pulp tissue is obtained by adherent digestion method
The cell obtained still has different cell subsets, some differences of their function and growth characteristic, some of which cell subset pair
Culture environment has larger adaptability and with stronger independent survival capacity, and cell colony rate is high, therefore is connect by low-density
Plant and filter out these propagation and the strong cell of survival ability.
5th, the detection of dental pulp stem cell
1st, the detection of the growth conditions of dental pulp stem cell
The upgrowth situation of cell is observed under the microscope using this area conventional method, it was observed that original cuiture the 2-3 days
It was observed that there is attached cell to grow, first time attached cell time of occurrence is short.Moreover, the 7-10 days cell fusion degree of original cuiture
Reach 80%, you can carry out passage operation, generation time first time is short, is significantly reduced the original cuiture time.
As shown in figure 1, being the cell state figure of dental pulp stem cell culture the 3rd day, as shown in Fig. 2 being dental pulp stem cell training
The cell state figure of the 9th day is supported, as shown in figure 3, being the cell state figure after dental pulp stem cell passage（Cell fusion degree has reached
To 80%）, cellular morphology is in largely fusiformis when can be seen that the 3rd day from Fig. 1,2 and 3, and density is small, cell colony at the 9th day
Significantly increase, cell density increase, orientation is inconsistent, as incubation time is longer, face of the cell on culture medium
Product is increasing, has been paved with whole culture medium when reaching 80% substantially, in whirllike arrangement, cell uniformity is good, cell state
It is good.
The growth curve of cell, such as mtt assay are determined using this area conventional method（Detect the side of cell survival and growth
Method）.As shown in figure 4, being the cell growth curve figure of dental pulp stem cell（P0 is primary）, by the 2nd passage（P2）Culture
Afterwards, the number of cell is sharply increased, from the 1st passage（P1）About 8*107Cell number increase to after P2 close to 8*108
Cell number, to the 3rd time passage（P3）When, cell number is already close to 2*109, amplification times close to 50 times, and
“Scaling-up of Dental Pulp Stem Cells Isolated from Multiple Niches”（PLoS
One. 2012; 7(6)）Middle P3 is 1.8*10 for cell number8It is individual.It is therefore shown that the preparation method of the present invention can make cell
Growth conditions are good, and multiplication capacity is strong, can largely expand.
2nd, the surface antigen detection of dental pulp stem cell
Using this area common detection methods（Such as flow cytomery）To P3 for dental pulp stem cell surface antigen CD31,
CD34, CD45, HLA-DR, CD73, CD90, CD105 and Stro-1 are detected.
CD31 is also known as platelet-endothelial cell adhesion molecule, contactin member；CD34 be I types across
Membrane glycoprotein, CD34 expresses decline after initial cell is divided into mature cell；CD45 is also known as LCA, can table
Up in mature erythrocyte, platelet surface；HLA-DR is HLA DR；CD73 is also known as extracellular 5 nucleotidase,
It is a kind of glycoprotein that glycosyl-phosphatidyl inositol (GPI) is anchored to membrane surface；CD 90（Also known as Thy-1）With cell
Stick, break up, cell-cell interaction it is relevant, be human microvascular endothelial cell activation mark；CD105 is also known as Endothelin,
It is the member of transforming growth factor β superfamily；Stro-1 be bone marrow stroma stem cell and normoblast precursor expression it is thin
Cellular surface albumen, for the positive cells of Stro-1, its fibroblast cloning formation unit is dramatically increased, and can be differentiated to form
Multidirectional interstitial cell system.
CD31, CD34, CD45 and HLA-DR are not expressed on MSCs surfaces, CD73, CD90, CD105 and Stro-1 is expressed.
As shown in figure 5, for dental pulp stem cell surface antigen CD31 of the invention detection figure, CD31 expression ratio is
0.60%；As shown in fig. 6, for dental pulp stem cell surface antigen CD34 of the invention detection figure, CD34 expression ratio is
0.94%；As shown in fig. 7, for dental pulp stem cell surface antigen CD45 of the invention detection figure, CD45 expression ratio is
0.47%；As shown in figure 8, for dental pulp stem cell surface antigen HLA-DR of the invention detection figure, HLA-DR expression ratio is
1.10%；As shown in figure 9, for dental pulp stem cell surface antigen CD73 of the invention detection figure, CD73 expression ratio is
99.2%；As shown in Figure 10, it is dental pulp stem cell surface antigen CD90 of the invention detection figure, CD90 expression ratio is
97.2%；As shown in figure 11, it is dental pulp stem cell surface antigen CD105 of the invention detection figure, CD105 expression ratio is
99.6%；As shown in figure 12, it is dental pulp stem cell surface antigen S tro-1 of the invention detection figure, Stro-1 expression ratio
For 27.0%.
The equal ＞ of positive indication CD73, CD90, CD105 of mescenchymal stem cell is can be seen that by above-mentioned testing result
95%, the equal ＜ 2% of negative indication CD34, CD31, CD45, HLA-DR of its mescenchymal stem cell.Therefore, the cell that the present invention is cultivated
Meet the characteristic of the surface molecular of human mesenchymal stem cell.
The P3 that the present invention is cultivated is for cell surface marker Stro-1 ＞ 20%, " Proliferative Features, and
Osteogenic Differentiation of Stem Cells Isolated from Human Permanent and
Deciduous Teeth with Bone Marrow”（Mol Biotechnol, 2016, 58:415-427）Describe DPSC
Middle P2, P3 are for cell surface marker Stro-1 ＜ 5%, " comparison of people's Periodontal Ligament Cells and pulp cells phenotypic characteristic "（West China
Dentistry impurity, 2 months 2009 phase of volume 27 the 1st）P3 is described in DPSC for cell surface marker Stro-1 ＜ 20%.Cause
This, the differentiation capability for the cell that the present invention is cultivated is high, the characteristic with dryness cell.
3rd, the detection of the immunosuppressive activity of dental pulp stem cell
MSCs not only has a variety of differentiation potentials, but also with immunoregulation capability and low immunogenicity, it is thin that it can suppress T
Other immunocytes are also had extensive adjustment effect by the propagation of born of the same parents, as it can suppress the lethal effect of NK mediations.
The present invention passes through flow cytometer（Flow Cytometry）Analyze the single core of human peripheral not being activated thin
Born of the same parents（Not Activated-human PMBC）, the human peripheral blood single nucleus cell that is activated（Activated-human PMBC）
Human peripheral blood single nucleus cell+P3 with activation is for dental pulp mescenchymal stem cell（Activated-human PMBC+ DPSC-
P3）Immunosuppressive activity（Immune Suppression Activity）, it is dental pulp mescenchymal stem cell as shown in figure 13
Suppress the result figure of immunocyte ability, A is microscopic cells figure, and B is outside immunocompetent cell statistical chart, the people that discovery is activated
The percentage with immunocompetent cell is close to zero in all blood mononuclear cells, in the human peripheral blood single nucleus cell being activated
Almost 90% cell has immunocompetence, but the human peripheral blood single nucleus cell+P3 of activation is in dental pulp mescenchymal stem cell
Drastically decline with immunocompetent cell number, only the cell less than 40% has immunocompetence.It is therefore shown that DPSC has
Significantly suppress immunocompetent effect, it was demonstrated that DPSC cells of the invention prepared have the characteristic of fine stem cell.
4th, freeze-stored cell recovery motility rate
Using common detection methods, freeze rear cell recovery motility rate and be slightly different according to the time length of freezing, detection freezes 1 year
The recovery motility rate of cell, its recovery motility rate is more than 90%.
The method of the present invention can be used in building dental pulp stem cell work storehouse.A large amount of same batches can be produced using this method
Cell sample, according to 1*106/ kg stem cell consumption, only one tooth can be treated more than 200 times for 50kg people, be
Large-scale production can the dental pulp stem cell that uses of clinicization there is provided new method.
In summary, the method that application stem cell self-characteristic of the invention prepares dental pulp stem cell, this method is shortened
The time of original cuiture, and multiplication capacity is strong, and the dental pulp stem cell of preparation has good cells and characteristic of stem, differentiation potential
By force.
Although present disclosure is discussed in detail by above preferred embodiment, but it should be appreciated that above-mentioned
Description is not considered as limitation of the present invention.After those skilled in the art have read the above, for the present invention's
A variety of modifications and substitutions all will be apparent.Therefore, protection scope of the present invention should be limited to the appended claims.
Claims (10)
1. a kind of method that application stem cell self-characteristic prepares dental pulp stem cell, it is characterised in that this method is included：
Step 1：Tooth samples are chosen, corona is broken, dental pulp is extracted from the pulp cavity of tooth samples, tooth is washed using cushioning liquid
Marrow, is divided into some fritters by dental pulp, is put into centrifuge tube；
Step 2：Digestive juice is prepared, the digestive juice is included：Sodium chloride injection, Collagenase I, dispase II, albumin, magnesium salts and
Calcium salt, the digestive juice is added in the centrifuge tube in step 1, the seal of tube will be centrifuged, and is digested after being well mixed at 37 DEG C；
Step 3：After the fritter in centrifuge tube is digested loosely, to the processing of centrifuge tube surface sterilizing, digestion terminate liquid is added, eventually
Only digest；
Step 4：The solution that step 3 is obtained is centrifuged, and removes supernatant, and the precipitation in bottom adds MSC primary culture mediums,
Precipitation is set to be transferred to after being resuspended in blake bottle, in 5%CO2, at 37 DEG C, carry out original cuiture；
Step 5：When cell fusion degree reaches 80%-90%, supernatant is collected, and carries out ultrafiltration, collects dry in upper screen
Cell extract, the stem cell extract solution is added in MSC primary culture mediums, Secondary Culture base is obtained；
Step 6：The cell of lower sediment in step 5 is inoculated into blake bottle, will when cell fusion degree reaches 80%-90%
Culture medium removal in blake bottle is standby, rinses blake bottle with phosphate buffer, the pancreatin of dilution is added in blake bottle, will
Pancreatin is laid in blake bottle bottom, carries out pancreatin digestion, and digestion time is no more than 1min, adds above-mentioned standby culture medium and terminates
Digestion；The pancreatin of dilution is the pancreatin that concentration is 0.025%；
Step 7：Step 6 is digested to obtained liquid centrifugation, the cell of bottom precipitation is added to the Secondary Culture base that step 5 is obtained
In, it is well mixed, by 1 × 102/cm2Cell density carry out kind of a bottle, in 5%CO2, Secondary Culture is carried out under conditions of 37 DEG C；
Step 8：Repeat the above steps 6 and 7, carry out Secondary Culture several times；
Step 9：Cells frozen storing liquid is placed in cryopreservation tube, carries out adding step 8 cell expansion in precooling, cryopreservation tube at 4 DEG C
Obtained stem cell Secondary Culture base is cultivated, cell density is 1.5 × 106~2×106/ 1.5mL/ is managed, and is gradually cooled to -80 DEG C,
The cell frozen is put into -196 DEG C of liquid nitrogen container after 24h and preserved；
Wherein, included in described MSC primary culture mediums：Growth factor.
2. the method that application stem cell self-characteristic according to claim 1 prepares dental pulp stem cell, it is characterised in that
In step 1, described cushioning liquid is included：0.9% sodium chloride injection, 10,000Units/mL penicillin and 10,000 μ g/mL
Streptomysin；
Described sodium chloride injection：Penicillin：The volume ratio of streptomysin is 198:1:1.
3. the method that application stem cell self-characteristic according to claim 1 prepares dental pulp stem cell, it is characterised in that
In step 2, described magnesium salts is included：Magnesium chloride；Described calcium salt is included：Calcium chloride；
The concentration of each component is in described digestive juice：0.9% sodium chloride injection, 3% Collagenase I, 4% dispase II, 1mol/L
Magnesium chloride, 200mg/mL calcium chloride and 20% albumin；
0.9% described sodium chloride injection：1mol/L magnesium chlorides：200mg/mL calcium chloride：The volume ratio of 20% albumin is
1mL：5μL：5μL ：100μL；
The bioactivity of 3% described Collagenase I is 270U/mg；
The bioactivity of 4% described dispase II is 0.85U/mg.
4. the method that application stem cell self-characteristic according to claim 1 prepares dental pulp stem cell, it is characterised in that
In step 3, described terminate liquid is included：0.9% sodium chloride injection and 20% albumin, 0.9% described sodium chloride injection：
The volume ratio of 20% albumin is 9：1.
5. the method that application stem cell self-characteristic according to claim 1 prepares dental pulp stem cell, it is characterised in that
In step 4, described MSC primary culture mediums are included：Human mesenchymal stem cells' serum-free basal medium, stroma cell derivative
The factor and Glu；
Described growth factor is included：Fibroblast growth factor, HGF, EGF and intravascular
Skin growth factor；
The volume of described human mesenchymal stem cells' serum-free basal medium：The quality of HGF：Epidermis is given birth to
The quality of the long factor：The quality of VEGF：The quality of stroma cell derivative factor：The volume of Glu=
100mL：1μg：2μg：2μg：2μg：100μL；
The bioactivity of described fibroblast growth factor is 100 IU/mL.
6. the method that application stem cell self-characteristic according to claim 1 prepares dental pulp stem cell, it is characterised in that
In step 4, described blake bottle is treated blake bottle, and processing method is：By the adherent reagent factor and phosphate-buffered
Liquid is with the μ L of volume ratio 10：After 1mL mixing is resuspended, add in blake bottle, and rocking blake bottle is completely covered the adherent reagent factor
Firmly blake bottle bottom.
7. the method that application stem cell self-characteristic according to claim 1 prepares dental pulp stem cell, it is characterised in that
In step 5, described stem cell extract solution：The volume ratio of MSC primary culture mediums is 1：50.
8. the method that application stem cell self-characteristic according to claim 1 prepares dental pulp stem cell, it is characterised in that
In step 6,0.025% described pancreatin is 0.25% pancreatin and phosphate buffer using volume as 1：9 preparations are obtained.
9. the method that application stem cell self-characteristic according to claim 1 prepares dental pulp stem cell, it is characterised in that
In step 9, cooling rate is 1 DEG C/min；Described cells frozen storing liquid is the frozen stock solution of non-animal derived serum.
10. the method that application stem cell self-characteristic according to claim 1 prepares dental pulp stem cell, it is characterised in that
In step 1, before tooth samples are chosen, tooth samples are preserved with liquid is preserved, and tooth samples are screened；
Described preservation liquid is included：0.9% sodium chloride injection, mycillin and albumin, 0.9% described chloride injection
Liquid：Mycillin：The volume ratio of albumin is 100mL：1 mL：2 mL；
Described mycillin is included：10,000U/mL penicillin, 10,000 μ g/mL streptomysins；
When carrying out tooth samples screening, following condition is provided simultaneously with：
Comprising any one in hepatitis B, hepatitis, syphilis, AIDS, CMV, EBV and HTLV virus or two or more it is detected as the moon
Property；
The non-inflammation of tooth；
The non-natural tooth come off.
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| CN108949682A (en) * | 2018-08-22 | 2018-12-07 | 广东唯泰生物科技有限公司 | A kind of preparation, culture and the purification process of dental pulp mescenchymal stem cell |
| CN109628389A (en) * | 2018-12-24 | 2019-04-16 | 北京博奥晶典启衡生物科技有限公司 | Culture for amplification in vitro dental pulp stem cell freezes system, method and kit |
| CN111172107A (en) * | 2020-03-02 | 2020-05-19 | 上海市口腔病防治院 | Dental pulp stem cell conditioned medium and preparation method and application thereof |
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| CN112251401A (en) * | 2020-10-16 | 2021-01-22 | 浙江优牙生物科技有限公司 | Large-scale normal-temperature digestion method for dental pulp stem cells |
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| CN113577107A (en) * | 2021-05-21 | 2021-11-02 | 施松涛 | Preparation method and application of stem cells |
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| CN108277202A (en) * | 2018-01-19 | 2018-07-13 | 深圳中生健康管理有限公司 | A kind of cultural method of dental pulp mescenchymal stem cell |
| CN108220230B (en) * | 2018-02-01 | 2022-01-18 | 上海莱馥医疗科技有限公司 | Method for separating and culturing human adipose-derived stem cells |
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| CN108949682A (en) * | 2018-08-22 | 2018-12-07 | 广东唯泰生物科技有限公司 | A kind of preparation, culture and the purification process of dental pulp mescenchymal stem cell |
| CN109628389A (en) * | 2018-12-24 | 2019-04-16 | 北京博奥晶典启衡生物科技有限公司 | Culture for amplification in vitro dental pulp stem cell freezes system, method and kit |
| CN111172107A (en) * | 2020-03-02 | 2020-05-19 | 上海市口腔病防治院 | Dental pulp stem cell conditioned medium and preparation method and application thereof |
| CN111172107B (en) * | 2020-03-02 | 2023-09-19 | 上海市口腔病防治院 | Dental pulp stem cell conditioned medium and preparation method and application thereof |
| 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 |
| 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 |
| CN112251401A (en) * | 2020-10-16 | 2021-01-22 | 浙江优牙生物科技有限公司 | Large-scale normal-temperature digestion method for dental pulp stem cells |
| CN112680412A (en) * | 2021-01-14 | 2021-04-20 | 辽宁省肿瘤医院 | Animal source-free passage method for passage stem cells |
| CN112680412B (en) * | 2021-01-14 | 2024-02-23 | 辽宁省肿瘤医院 | Animal source-free passage method for passage of stem cells |
| CN113577107A (en) * | 2021-05-21 | 2021-11-02 | 施松涛 | Preparation method and application of stem cells |
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