CN102807967A - Application of deciduous tooth pulp to preparation of mesenchymal stem cell and culturing method of deciduous tooth pulp mesenchymal stem cell - Google Patents
Application of deciduous tooth pulp to preparation of mesenchymal stem cell and culturing method of deciduous tooth pulp mesenchymal stem cell Download PDFInfo
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- CN102807967A CN102807967A CN201210318439XA CN201210318439A CN102807967A CN 102807967 A CN102807967 A CN 102807967A CN 201210318439X A CN201210318439X A CN 201210318439XA CN 201210318439 A CN201210318439 A CN 201210318439A CN 102807967 A CN102807967 A CN 102807967A
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Abstract
The invention relates to an application of deciduous tooth pulp to preparation of a mesenchymal stem cell and a culturing method of the deciduous tooth pulp mesenchymal stem cell. The method comprises the following steps of: performing primary culture on a deciduous tooth to obtain a deciduous tooth pulp mesenchymal stem cell primary cell mixed solution; and performing subculturing on the deciduous tooth pulp mesenchymal stem cell primary cell mixed solution to obtain a deciduous tooth pulp mesenchymal stem cell. In the method, deciduous tooth pulp is taken as a tissue material for preparing the mesenchymal stem cell, which is different from marrow, cord blood and placenta, and the collection way is natural fall or surgical removal of complete deciduous teeth of children. A child has 20 deciduous teeth, deciduous tooth stem cells cultured with six deciduous teeth of upper and lower jaws are best, sampling is not required at the birth of the child, and 20 collection chances are available, so that the deciduous tooth pulp mesenchymal stem cell can be obtained possibly by using any one of the 20 deciduous teeth even for a person misses the collection change of placenta and umbilical cord tissues and a child whose teeth fall off and are not collected in time.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of deciduous teeth pulp tissue mescenchymal stem cell, application and the cultural method of deciduous teeth dental pulp mescenchymal stem cell of especially a kind of deciduous teeth dental pulp in the preparation mescenchymal stem cell.
Background technology
Stem cell is a kind of undifferentiated cell; Two fundamental characteristics are arranged, the one, have the of self-replication capacity, the 2nd, can be divided into more than one functioning cell; Size according to differentiation potential; Stem cell generally is divided into three types, and the first kind is that myeloid-lymphoid stem cell (totipotent stem cell) promptly can be categorized as the consistent totipotent cell of function, can grow to be fetus; Second type is pluripotent stem cell (pturipotent stem cell), and it can be divided into every kind of cell type in the health, but can not form placenta or the necessary sustentacular tissue of fetation.Because the differentiation potential of pluripotent stem cell is not " all ", therefore this cell is not called " all-round ", and they is not the embryo.The further specialization of pluripotent stem cell is multipotency (multiPotent) stem cell, and it is exclusively used in the cell of the specific kind system that is divided into the specific function specialization.Multipotential stem cell can be divided into the cell type that contains in the tissue that they are derived from; For example blood stem cell is merely able to be divided into red blood cell, white cell and thrombocyte.Embryonic stem cell (Embryonic stem cell) has potential widely, can generate to remove all histocytes of extraplacental body; The 3rd type is that adult stem cell (aduit stem cell) is a kind of undifferentiated cell that will have lifelong self-replacation (identical copies) and self (self-renewal) ability; It is distributed in different tissues, and can develop and become various types of qualification cells.The classification of stem cell mainly is divided into hemopoietic stem cell and non-hematopoietic stem cell.Hemopoietic stem cell is dominated the disease of blood, immunology, the for example Cord blood of originating; Non-hematopoietic stem cell is main with cytodifferentiation and reparation then, can be applicable to regenerative medicines such as apoplexy, mellitus, senile dementia, and there are umbilical cord, deciduous teeth etc. in the source.
Mescenchymal stem cell (mesenchymal stem cell MSC) is the multipotential stem cell that can form the various kinds of cell type.MSC finds in marrow, also finds subsequently to be present in many kind tissues of human body generation, growth course.In view of mescenchymal stem cell have multidirectional differentiation potential, can hematopoiesis support and promote hemopoietic stem cell to implant, regulate characteristics such as immunity and separation and Culture be easy and simple to handle, receive people's attention just day by day.Increasingly mature along with mescenchymal stem cell and correlation technique thereof, clinical study is carried out in many countries.As seed cell, be mainly used in the treatment histocyte that can't repair naturally of body and the multiple refractory disease of organ damage clinically; As immunity regulatory cell, treatment immunological rejection and autoimmune disorder.
At present, can from tissue such as marrow, fat, synovial membrane, bone, muscle, lung, liver, pancreas and amniotic fluid, Cord blood, separate and the preparation mescenchymal stem cell, with the most use is the mescenchymal stem cell of derived from bone marrow.But there is following problem in the mescenchymal stem cell of derived from bone marrow: along with wearing out of age, the stem cell number significantly reduces, the proliferation and differentiation ability fails significantly; The preparation process is not easy Quality Control; Transplant allosome and possibly cause immunoreation; Healthy donor when drawing materials the patient had damage, can not gather when the patient has bone marrow disease, even also can not extract too many marrow.This has all limited the mesenchymal stem cells MSCs clinical application, makes that seeking marrow becomes an important problem in other alternative mescenchymal stem cells sources in addition.
Isolated mescenchymal stem cell in placenta and the umbilical cord tissue has not only kept the biological characteristics of mescenchymal stem cell, and it is more original to have a cell, and stronger proliferation and differentiation ability is arranged; Immunocyte is comparatively inmature, and functionally active is low, can not trigger immunoreation and cause graft versus host disease; Stem cell is easy to separate, and purity is high, is convenient to Quality Control; During collection puerpera and newborn infant there are not any harm and damage, advantage such as ethics dispute is few.Though placenta or umbilical cord contain profuse stem cell, its problem is: the collection of organization material can only once be accomplished, and does collection in a flash when being confined to produce, and can not obtain once more the opportunity of missing.
Cord blood, marrow, dental pulp stem cell function are had nothing in common with each other, and Cord blood is primarily aimed at blood disease, marrow then in the repairing of organ.
Deciduous teeth dental pulp stem cell (stem cells from human exfoliated deciduousteeth; SHED) belong to multi-functional adult stem cell; After utilizing the biochip technical Analysis, show that deciduous teeth and gum stem cell can show various growth factors in a large number.Its effect comprises: the regenerative medicine that 1. can apply to tissue repair; These cells via special processing after; Can be divided into sclerocyte, make the chondrocyte, neurocyte, vascular cell, adipocyte, make ivory's cell etc., can apply to the regenerative medicine that various stromas are repaired future.2. be used for medical cosmetology, Clostridium botulinum and Hyaluronic Acid that medical cosmetology at present is commonly used can only be kept 6 to 12 months effect, if with gum autologous stem cells treatment wrinkle of skin and decree line etc., then can reach 3 years, and not have repellency.3. to the treatment neurodegenerative disorders such as and even Alzheimer's disease is anti-aging, skin and inostosis, organ and vascular repair, ivory, pulp tissue, serious periodontopathy reparation, even the improvement and the regenerating bone or cartilage aspect of Parkinsonism, mellitus all have very big application potential.4. can in time carry out the reproducibility treatment to diseased region and the impaired place of wound.Stem cell storage at present is the most popular with Cord blood and marrow, but in comparison, SHED has sizable advantage and irreplaceable effect on following cell therapy is used.The proliferation regeneration power of deciduous teeth stem cell is strong, histocompatibility is high, convenient for storing, provides the 2nd chance of modern to store stem cell (the 1st time is Cord blood, umbilical cord and placenta).If can stem cell be stored, be expected future to benefit oneself, father and mother even grand parents.
The method of existing culturing human stem cell need use feeder layer, raise conditioned medium (feeder conditioned media) or in substratum supply human or animal cell extract with expanding stem cells with prevent spontaneous differentiation.In addition, in existing method, if the human stem cell that is produced is used for clinical treatment, culture condition possibly be derived from the product of animal, and such product has the risk of transmission disease.Therefore, if the stem cell of cultivating is used for the human treatment, this method is unaccommodated.The clinical application of human stem cell need be at the environment (culture environment that is called " no xenobiotics ") of standardized, as to receive excellent control no animal product, and it reduces the method for culturing cell in the risk of transmitting disease.In addition, need be under the situation that does not have feeder layer or sustenticular cell the method for culturing human stem cell to reduce feeder cell or to be derived from the risk of the contaminants stem-cell therapy product of feeder cell.
The World Health Organization is to rehabilitation, antidotal being newly defined as: " cure diseases, the most basic anti-ageing approach be repair cell, improve cellular metabolism, activate the function of senile cell ".Have only in time damaged cell, senile cell are effectively treated, make damaged cell obtain rehabilitation, senile cell obtains activating, and organ-tissue and physiological function could be recovered normally fully, and human body could be from truly keeping fit, keep young attitude.Stem-cell therapy promptly is that a healthy stem cell transplantation is in the patient body, to reach the purpose of repairing sick cell or rebuilding normally functioning cell and tissue.Stem cell therapy is just as injecting new vitality to body, and " self-healing function " of human activin self replenished and regulated and control the cell of pathology; Activate cell functions increases Normocellular quantity, improves cell activity; Improve the quality of cell; Prevent and delay the pathology of cell, recover the normal physiological function of cell, thereby reach rehabilitation, antidotal purpose.
Application of stem cells treatment disease and traditional therapy relatively have lot of advantages: hypotoxicity or nontoxicity, and one time medicine is effective; Do not need to understand fully disease incidence cutter reason really; There is not pathophorous risk; Also possibly use self stem cell transplantation, avoid producing immunological rejection.American National food and medicine Surveillance Authority (FDA) has ratified treatment technologies such as cell therapy cardiomyopathy, cell therapy nervous system disorders in succession in the period of 2004～2009; Promote carrying out of whole world cell therapy, have in the whole America that tame hospital has carried out with treatment technologies such as cell therapy cardiomyopathy and cell therapy nervous system disorderss in succession more than 40.Simultaneously, developed countries such as the U.S., Britain, Japan make laws in succession, and cell therapy is legalized with it.Also have how tame hospital to carry out cell therapy at present in China, annual ten hundreds of patients therefore are benefited.
Through retrieval, find the following patent publication us relevant with patented claim of the present invention:
Utilize epithelial cells stem cell and dental pulp stem cell to carry out the technology (CN200410071721.8) of regeneration of tooth, the invention belongs to organic regeneration technology, specifically, the present invention relates to utilize epithelial cells stem cell and dental pulp stem cell to carry out the technology of regeneration of tooth.Technical scheme of the present invention: from the mortar tooth, take out pulp tissue, after digestive ferment digestion, change in the nutrient solution and cultivate; After cultivating 24h,, remove not attached cell with PBS washing three times, when the cell of adherent growth continues to be cultured to 14～16 days, the cultivation of going down to posterity; Dental pulp stem cell is recombinated the epithelial cells stem cell and the dental pulp stem cell of vitro culture after growth factor B MP4 and FGF10 induce 24h, forms the reorganization agglomerate; The agglomerate of will recombinating places 37 ℃, 5% CO
2Cultivate 24h in the incubator, be transplanted to again in the oral cavity, after cultivating for 10～12 weeks, in the oral cavity, form tooth.The invention has the advantages that and utilize epithelial cells stem cell and dental pulp stem cell to carry out regeneration of tooth; Epithelial cells stem cell and dental pulp stem cell can obtain through external a large amount of cultivations according to disclosed technique means; Enlarged the source of material greatly, conveniently promoted the use of.
Through contrast, there are the different of essence in patented claim of the present invention with above-mentioned patent publication us.
Summary of the invention
The objective of the invention is to overcome the weak point of prior art; Provide a kind of deciduous teeth dental pulp at application and the cultural method of deciduous teeth dental pulp mescenchymal stem cell of preparation in the mescenchymal stem cell; Preparation convenient, that in culturing process, do not need special trophocyte that the deciduous teeth pulp tissue that is adopted in this cultural method gathers; Particularly need not add any animal serum and other any animal source composition; Adopt the cell culture technology of no external source animal serum, and use highly purified animal derived trypsinase surrogate to carry out the digestion of cell, method is simple.
The present invention realizes that the technical scheme of purpose is:
The application of deciduous teeth dental pulp in the preparation mescenchymal stem cell.
A kind of deciduous teeth dental pulp mescenchymal stem cell cultural method; Step is following: deciduous teeth are supported acquisition deciduous teeth dental pulp mescenchymal stem cell primary cell mixed solution through former being commissioned to train, deciduous teeth dental pulp mescenchymal stem cell primary cell mixed solution is cultivated through going down to posterity promptly obtained deciduous teeth dental pulp mescenchymal stem cell again.
And the said former breeding method of being commissioned to train is:
⑴ cut into deciduous teeth to organize fragment through aseptically process, physics, adds the mescenchymal stem cell complete culture solution and promptly obtain pulp tissue's mixed solution;
⑵ place 36～38 ℃, volume(tric)fraction with the pulp tissue's mixed solution among the step ⑴ is 4～6% CO
2Cultivate in the incubator;
⑶ cultivate and promptly obtained deciduous teeth dental pulp mescenchymal stem cell primary cell mixed solution in 10～15 days.
And the described cultured method that goes down to posterity is:
⑴ supernatant gets attached cell in the sucking-off deciduous teeth dental pulp mescenchymal stem cell primary cell mixed solution, in attached cell, adds pancreatin surrogate Digestive system to the submergence attached cell, is placed on CO again
2In the incubator, 36～38 ℃ of insulations digested in 5 minutes;
⑵ after finishing, digestion, adds the flushing of mescenchymal stem cell complete culture solution with the Digestive system sucking-off, and centrifugal; After the centrifugal end, the sucking-off supernatant must precipitate;
⑶ add the mescenchymal stem cell complete culture solution to immersion precipitation in the deposition of step ⑵, placing 36～38 ℃, volume(tric)fraction is 4～6% CO
2Cultivate in the incubator, cultivated 5～7 days, promptly obtain deciduous teeth dental pulp mescenchymal stem cell.
And the composition of described pancreatin surrogate Digestive system is: mass percent is 0.25% the tryptic phosphate buffered saline buffer of recombinant animal.
A kind of store method of deciduous teeth dental pulp mescenchymal stem cell, step is following:
⑴ the collection of freeze-stored cell
Collect the prepared deciduous teeth dental pulp mescenchymal stem cell of cultural method as claimed in claim 2; Add pancreatin surrogate Digestive system; Add-on is a submergence deciduous teeth dental pulp mescenchymal stem cell, digest enchylema, with enchylema centrifugal cell precipitation; Again cell precipitation is got cell suspension with the mescenchymal stem cell complete culture solution is resuspended, and carry out cell counting;
⑵ the preparation of cell cryopreservation suspension
According to cell count in the cell suspension is 1-5 * 10
6The time, the dose volume ratio of cell suspension and DMSO is that 3-5:1 adds DMSO in cell suspension, mixing, and packing gets the cell cryopreservation suspension then;
⑶ cell cryopreservation
With the cell cryopreservation suspension among the step ⑵ with 1 ℃ of .min
-1Cooling rate be cooled to-85 ℃, place-196 ℃ of liquid nitrogen to preserve then to deciduous teeth dental pulp mescenchymal stem cell.
The method for resuscitation of a kind of deciduous teeth pulp tissue mescenchymal stem cell, step is following:
⑴ take out the deciduous teeth dental pulp mescenchymal stem cell like the said preservation of claim 7, put into immediately 36-38 ℃ of water-bath melt cell suspension;
⑵ wash the centrifugal cell precipitation that gets with cell suspension with the mescenchymal stem cell complete culture solution;
⑶ resuspended with the mescenchymal stem cell complete culture solution with cell precipitation, and placing 37 ℃, volume(tric)fraction then is that 4～6% CO2 incubator is cultivated i.e. recovery in 5～7 days and obtained deciduous teeth pulp tissue mescenchymal stem cell.
Advantage of the present invention and beneficial effect are:
1, the present invention has adopted the organization material of deciduous teeth dental pulp as the preparation mescenchymal stem cell, and these are different with marrow, Cord blood, placenta, and the mode of collection is the complete deciduous teeth of children that nature comes off or performs the operation and pull out.It has with the identical advantage of isolated mescenchymal stem cell in placenta and the umbilical cord tissue, simultaneously, because children have 20 deciduous teeth; Deciduous teeth stem cell so that each 6 deciduous teeth of the upper jaw and the lower jaw are turned out is best, because needn't be once the sampling of being born, and the chance of 20 collections in addition; Even so miss the people of placenta and the meeting of umbilical cord tissue harvester; With the children that at present once dropped and have little time to collect, any deciduous teeth in 20 all have an opportunity to obtain the deciduous teeth mescenchymal stem cell.
2, the present invention's conditioned medium name of preparing the deciduous teeth mescenchymal stem cell is called
MSC SFM XenoFree; Article No.: A10675-01; Commercially available culture medium for the production of U.S. GIBCO company; Its composition comprises:
MSC SFMBasal Medium (500mL), article No.: A13829-01;
MSC SFM XenoFree Supplement (5mL)
Article No.: A11577-01.It does not contain any animal component; With no heat source water preparation; Packing after Sterile Filtration, antibiotic-free adds, and this reagent need not add animal serum and other any animal source composition when using; Can reduce the influence of inhuman source factor to greatest extent, make the mescenchymal stem cell of production be applicable to clinical treatment.
3, the deciduous teeth mescenchymal stem cell of the present invention pancreatin surrogate name of using in the cultural method that goes down to posterity is called TrypLE
TMSelect, article number 12563-029, the commercialization cell dissociation buffer for U.S. GIBCO company produces uses it that attached cell is carried out digestion process, so that pair cell carries out amplification cultivation.This pancreatin surrogate is a recombinase, is highly purified animal derived trypsinase surrogate, acellular lytic enzyme; Use it to carry out the digestion of anchorage-dependent cell; Cell growth amplification is in good condition, and can repeatedly go down to posterity, and does not have significant difference with the level that obtains with traditional method.Be applicable to any culture condition that contains serum and serum-free, do not need trypsin inhibitor during use, this reagent keeps proterties stable under all temps condition, stores easily and uses.
4, deciduous teeth mescenchymal stem cell store method of the present invention, this method can make cell temporarily break away from growth conditions and make its cell characteristics be able to preserve, when needing again the recovery cell increase.During cell cryopreservation, in substratum, add protective material DMSO 99.8MIN. (DMSO), DMSO is a kind of important osmosis type cytoprotective.Preserve in the process of cell in profound hypothermia (subzero 200 degree), can prevent the damage that ice crystal formation in the cell, osmotic pressure change, cellularstructure disorder etc. cause.DMSO can get in the cell by the quick penetration cytolemma, reduces freezing point, delays frozen process, improves intracellular ion concentration simultaneously, reduces the formation of ice crystal in the cell, thereby reduces cell injury.The difference not statistically significant of stem cells hyperplasia rate after frozen, apoptosis rate and multidirectional differentiation capability and fresh culture SHED.
5, the present invention is devoted to develop and is suitable for keeping with the method for expanding stem cells of scale operation and thinks that clinical application provides stem cell or its differentiation verivate of sufficient amount.The method of the application of the invention is cultivated the condition of SHED and has been eliminated the demand that feeder cell are supported, can keep not differentiation in 150 days time at as many as under the situation that does not have feeder cell.Method of the present invention then is to develop the cultural method of improvement mesenchymal cell to be used for amplification and further when needed differentiated stem cells, makes it be suitable for the production of treating standardization of application, regulation and control and expansion SHED.
Description of drawings
Fig. 1 is deciduous teeth dental pulp mescenchymal stem cell primary cultured cell figure of the present invention, i.e. grown cell figure around pulp tissue's piece;
Fig. 2 is dispersed in individual cells figure for deciduous teeth dental pulp mescenchymal stem cell cultured cell line of the present invention;
Fig. 3 is the cultured cell line figure of cell stand density more than 80% of deciduous teeth dental pulp mescenchymal stem cell of the present invention;
Fig. 4 is deciduous teeth dental pulp mescenchymal stem cell cultured cell line of the present invention growth conditions figure day by day;
Wherein, Fig. 4-1 is 1 day growth conditions figure of deciduous teeth dental pulp mescenchymal stem cell cultured cell line of the present invention; Fig. 4-2 is 3 days growth conditions figure of deciduous teeth dental pulp mescenchymal stem cell cultured cell line of the present invention; Fig. 4-3 is 5 days growth conditions figure of deciduous teeth dental pulp mescenchymal stem cell cultured cell line of the present invention; Fig. 4-4 is 7 days growth conditions figure of deciduous teeth dental pulp mescenchymal stem cell cultured cell line of the present invention.
Embodiment
Through specific embodiment the present invention is made further detailed description below, following examples are descriptive, are not determinate, can not limit protection scope of the present invention with this.
Employed method among the present invention like no specified otherwise, is ordinary method; Employed reagent among the present invention like no specified otherwise, is conventional reagent.
Employed pancreatin surrogate Digestive system is TrypLE among the present invention
TMSelect, article number 12563-029, U.S. GIBCO company produces; Its composition is: the tryptic phosphate buffered saline buffer of recombinant animal, the tryptic mass percent of recombinant animal is 0.25%.
Mescenchymal stem cell complete culture solution and compound method thereof are following in the patented claim of the present invention:
1, the mescenchymal stem cell nutrient solution is formed: totally 2 bottles of commercialization mescenchymal stem cell substratum
the MSC SFM XenoFree that U.S. GIBCO company produces; Comprise: basic medium
MSC SFM Basal Medium (500mL), additive
MSC SFM XenoFree Supplement (5mL);
Its composition comprises: people's derived collagen albumen (being produced by the cell autocrine), Transferrins,iron complexes, RWJ-60235, recombinant human epidermal growth factor, ɑ-MEM.
2, compound method: get 1 bottle of 5ml of additive; Aseptic technique joins in the 500ml basic medium; Make into mescenchymal stem cell complete culture solution
MSC SFM XenoFree, 37 ℃ of insulations are subsequent use.
One, a kind of deciduous teeth dental pulp mescenchymal stem cell cultural method, the concrete steps that each deciduous teeth is handled respectively are following:
(1) deciduous teeth dental pulp mescenchymal stem cell former is commissioned to train foster
1, the condition and the standard of the collection of deciduous teeth organization material
⑴ the pacing items that collect deciduous teeth is: the child age of gathering deciduous teeth is 5～10 years old children who is in the stage of growing permanent teeth, totally 20, and preferred each 6 deciduous teeth of the upper jaw and the lower jaw.
⑵ the mode of deciduous teeth collection is: the complete deciduous teeth of children that come off naturally or perform the operation and pull out.
⑶ the standard that collect deciduous teeth is: the fresh deciduous teeth of pulling out or coming off naturally, the cleaning of selection tooth body and symptoms such as healthy pulp, no carious tooth, no dental pulp inflammation and pulp necrosis does not have the deciduous teeth of dirt settling.This is the prerequisite that SHED is obtained in success.
⑷ gather the preservation condition of deciduous teeth: come off naturally or fresh each that pull out deciduous teeth should be put into the transfer pipeline that contains 10ml deciduous teeth preservation liquid in 4 hours; 2～8 ℃ of cryopreservation are transported to the laboratory, and should be above 40～120 hours from the collection deciduous teeth to the time of handling dental pulp.
2, deciduous teeth are preserved the preparation of liquid
⑴ deciduous teeth are preserved the moity of liquid:
The DMEM/F12 liquid medium
Penicillin (penicillium mould) 100IU/ml
Streptomycin (Streptomycin sulphate) 100IU/ml
⑵ compound method: add mycillin mixed solution (containing Penicillin 10000IU/ml, Streptomycin 10000IU/ml) 10ml in the 500ml DMEM/F12 liquid medium.
⑶ deciduous teeth are preserved the liquid preservation condition: 2～8 ℃.
3, the treatment process of deciduous teeth pulp material
⑴ prepare antimicrobial
1. composition: penicillium mould 800,000 units/Streptomycin sulphate 1,000,000 unit/;
2. compound method: add penicillium mould 100IU/ml, Streptomycin sulphate 100IU/ml in the normal saline solution, mix.
⑵ the aseptically process of deciduous teeth pulp material
1. under gnotobasis, from deciduous teeth preservation liquid, take out tooth, put into antiseptic-germicide and wash repeatedly, and embathed 10 minutes with tweezers;
2. rive corona or split bore with dentistry and open corona takes out crown root portion dental pulp, puts into new antiseptic-germicide 15ml and embathes 10 minutes, with the ophthalmology curved scissors pulp tissue is cut into fragment;
The antiseptic-germicide that 3. will contain pulp tissue is put into the 50ml centrifuge tube, centrifugal 10 minutes of 200g;
4. after centrifugal, with pasteur pipet sucking-off supernatant, the deposition that will contain pulp tissue's fragment is with the resuspended one-tenth of 3ml mescenchymal stem cell complete culture solution pulp tissue suspension.
4, the cultivation of deciduous teeth dental pulp mescenchymal stem cell primary cell
⑴ preparation mescenchymal stem cell complete culture solution:
1. the mescenchymal stem cell nutrient solution is formed: totally 2 bottles of commercialization mescenchymal stem cell substratum
the MSC SFM XenoFree that U.S. GIBCO company produces; Comprise: basic medium
MSC SFM Basal Medium (500mL), additive
MSC SFM XenoFree Supplement (25mL).
2. compound method: get 1 bottle of 25ml of additive; Aseptic technique joins in the 500ml basic medium; Make into mescenchymal stem cell complete culture solution
MSC SFM XenoFree, 37 ℃ of insulations are subsequent use.
⑵ be transferred to 25cm with the aforesaid suspension 3ml of pulp tissue (2,3 macroscopic dental pulps of cutting off are arranged, and the weight of dental pulp is 10ug) that is suspended with pulp tissue's fragment in nutrient solution
2Tissue Culture Flask in, other gets 2ml mescenchymal stem cell complete culture solution flushing centrifuge tube, and all adds in the above-mentioned Tissue Culture Flask, abundant mixing, the bottle cap of screwing, it is 4～6% CO that culturing bottle is placed 36～38 ℃, volume(tric)fraction
2Cultivate in the incubator.
⑶ after cultivate about 10～15 days; Grow adherent fibroblast-like cells from pulp tissue's block edge; This moment, pair cell changed liquid; Promptly draw cultured cells suspension 3ml with aseptic pipettor and abandon it, add fresh mescenchymal stem cell complete culture solution 3ml to culturing bottle, it is 4～6% CO that culturing bottle is placed 36～38 ℃, volume(tric)fraction
2Continue in the incubator to cultivate.
⑷ continue to cultivate about about 10 days; Attached cell is proliferated into bigger cell colony gradually; The Tissue Culture Flask wall forms a plurality of not of uniform size, cell islands that cell quantity is intensive; Promptly obtain deciduous teeth dental pulp mescenchymal stem cell primary cell mixed solution, can obtain deciduous teeth dental pulp mescenchymal stem cell primary cell through centrifugal again.Should carry out passage this moment.
(2) cultivation of going down to posterity of deciduous teeth dental pulp mescenchymal stem cell
1, gets TrypLE
TM1 bottle of Select, article number 12563-029, this uses the pancreatin surrogate for cell dissociation, and U.S. GIBCO company produces, and room temperature is placed and is preserved;
2, with aseptic pipettor with 25cm
2The whole sucking-offs of tissue block in the cell bottle and nutrient solution are drawn the 3ml Digestive system and are added in the culturing bottle, cover tight bottle cap, wave and culture bottle gently, cause Digestive system to be paved with bottle at the bottom of;
The culturing bottle that 3, will add Digestive system is put into CO
2In the incubator, 37 ℃ are incubated 5 minutes;
4, after insulation finishes, take out culturing bottle, pat bottle gently; Mirror is observed down, and the attached cell circle contracts, at the bottom of the part cell has broken away from bottle after; Be that available pipettor is with the Digestive system sucking-off; Add mescenchymal stem cell complete culture solution washing bottle floor cells, and whole cell suspensions are moved in the 50ml centrifuge tube centrifugal 5 minutes of 100g～200g room temperature;
5, centrifugal end, the sucking-off supernatant is drawn a small amount of mescenchymal stem cell complete culture solution, and with 3～5 speed p.s., the limit edged is the rotating centrifugal pipe gently, and nutrient solution is joined in the cell precipitation, blows and beats mixing gently;
6, draw above-mentioned cell suspension, join 1 175cm
2In the Tissue Culture Flask, supply nutrient solution to 35～40ml;
7, culturing bottle being placed 36～38 ℃, volume(tric)fraction is 4～6% CO
2Cultivate in the incubator;
8, after passage is cultured to 5～7 days, after attached cell has 70～90% to cover with individual layer, promptly get deciduous teeth dental pulp mescenchymal stem cell passage cell, this cell can carry out subculture and go down to posterity.
Two, the store method of deciduous teeth dental pulp mescenchymal stem cell passage cell
1, cell cryopreservation is with protection liquid
Composition: DMSO 99.8MIN. (DMSO)
2, the compound method of freeze-stored cell suspension
⑴ the collection of freeze-stored cell
When the cell attachment density in the back culturing bottle that goes down to posterity reaches 60～90%,, add 10mlGIBCO TrypLE earlier with the whole sucking-offs of enchylema
TMThe Select Digestive system; 37 ℃ of insulations 5 minutes with the Digestive system sucking-off, are washed the culturing bottle inner cell with the mescenchymal stem cell complete culture solution when treating that the observation of cell circle contracts under the mirror repeatedly; And all be collected into cell suspension in the centrifuge tube; Centrifugal 5 minutes of 100～200g abandons supernatant, and cell precipitation is resuspended with the mescenchymal stem cell complete culture solution.
⑵ cell counting
Get cell suspension resuspended and that mix; Suitably dilute with sterilized water; Get the 10ul cell diluent and join in the cell counting count board deckglass lower rim, this moment, cell suspension was that the surface tension of liquid capable of using is full of the count block, left standstill 30 seconds; Tally is placed on the microscope stage; After finding the count block, four jiaos of big grids (each big grid divides 16 lattices again) are added up to counting, the line ball cell is taked to remember that upper limit cell is disregarded the lower limit cell, meter left side limit cell is disregarded right limit cell.Calculate the contained cell quantity of every ml cell suspension according to formula then.
Cell calculation formula: cell count/ml=(4 big grid total number/4) * 10000 * extension rate
⑶ the preparation of cell cryopreservation suspension
Cell suspension is 4:1 with the dose volume of frozen protection liquid DMSO than ml:ml ratio, is that 2ml is a standard with loading amount in the frozen pipe promptly, and the amount that adds cell suspension is 1.6ml, and the amount that adds DMSO is 0.4ml.According to the freeze-stored cell quantity in every frozen pipe, it is some to get the cell suspension of having counted, and in proportion itself and DMSO solution is mixed, and joins in the cell cryopreservation pipe.The go down to posterity freeze-stored cell number of usefulness of being used to recover should be 2 * 10
6/ pipe.
3, cell cryopreservation method
With the cell cryopreservation pipe with 1 ℃ of .min
-1Cooling rate be cooled to-85 ℃.The cell cryopreservation pipe that will be cooled to-85 ℃ is then put into liquid nitrogen (196 ℃) preservation, can preserve deciduous teeth dental pulp mescenchymal stem cell.
Three, the method for resuscitation of deciduous teeth dental pulp mesenchymal stem cell cryopreserving cell
1, the thawing of freeze-stored cell
From liquid nitrogen, take out the cell cryopreservation pipe of preparing recovery, put into 37 ℃ of water-baths immediately and melt rapidly;
2, the cultivation of freeze-stored cell
⑴ put into the centrifuge tube that contains the mescenchymal stem cell complete culture solution with the enchylema in the frozen pipe of aseptic pipettor sucking-off, the piping and druming mixing;
⑵ carried out centrifuge washing in centrifugal 8 minutes with centrifuge tube with 200g, abandons supernatant, and cell precipitation is resuspended with the mescenchymal stem cell complete culture solution;
⑶ join 2 175cm respectively with resuspended enchylema
2In the Tissue Culture Flask, supply the mescenchymal stem cell complete culture solution according to every bottle of 35～40ml amount of liquid then;
⑷ place 37 ℃, volume(tric)fraction with culturing bottle is 4～6% CO
2Cultivate in the incubator.
⑸ after passage is cultured to 5～7 days, and after attached cell has 70～90% to cover with individual layer, i.e. recovery obtains deciduous teeth pulp tissue mescenchymal stem cell.
Four, the deciduous teeth dental pulp can be applicable in the preparation of mescenchymal stem cell
Deciduous teeth dental pulp stem cell (stem cells from human exfoliated deciduousteeth; SHED) belong to multi-functional adult stem cell; After utilizing the biochip technical Analysis, show that deciduous teeth and gum stem cell can show various growth factors in a large number.Stem cell storage at present is the most popular with Cord blood and marrow, but in comparison, SHED has sizable advantage and irreplaceable effect on following cell therapy is used.The proliferation regeneration power of deciduous teeth stem cell is strong, histocompatibility is high, convenient for storing, provides the 2nd chance of modern to store stem cell (the 1st time is Cord blood, umbilical cord and placenta).If can stem cell be stored, be expected future to benefit oneself, father and mother even grand parents.
With the organization material of deciduous teeth dental pulp as the preparation mescenchymal stem cell, these are different with marrow, Cord blood, placenta, and the mode of collection is the complete deciduous teeth of children that nature comes off or performs the operation and pull out.It has with the identical advantage of isolated mescenchymal stem cell in placenta and the umbilical cord tissue, simultaneously, because children have 20 deciduous teeth; Deciduous teeth stem cell so that each 6 deciduous teeth of the upper jaw and the lower jaw are turned out is best, because needn't be once the sampling of being born, and the chance of 20 collections in addition; Even so miss the people of placenta and the meeting of umbilical cord tissue harvester; With the children that at present once dropped and have little time to collect, any deciduous teeth in 20 all have an opportunity to obtain the deciduous teeth mescenchymal stem cell.Therefore, the deciduous teeth dental pulp can be applicable in the preparation of mescenchymal stem cell.
The employed conditioned medium name of preparation deciduous teeth mescenchymal stem cell is called
MSCSFM XenoFree in the patented claim of the present invention; Article No.: A10675-01; Commercially available culture medium for the production of U.S. GIBCO company; Its composition comprises:
MSC SFM Basal Medium (500mL), article No.: A13829-01;
MSC SFM XenoFreeSupplement (5mL), article No.: A11577-01.It does not contain any animal source composition, with no heat source water preparation, and packing after Sterile Filtration, antibiotic-free adds.Physical behavior is a liquid colourless, clear, pH7.2～7.6.This reagent need not add animal serum and other any animal source composition when using, can reduce the influence of inhuman source factor to greatest extent, makes the mescenchymal stem cell of production be applicable to clinical treatment.
The deciduous teeth mescenchymal stem cell goes down to posterity and uses the pancreatin surrogate to be TrypLE in the cultural method in the patented claim of the present invention
TMSelect, article number 12563-029, the commercialization cell dissociation buffer for U.S. GIBCO company produces is used for attached cell is carried out digestion process, so that pair cell carries out amplification cultivation.This pancreatin surrogate is a recombinase, is highly purified animal derived trypsinase surrogate, acellular lytic enzyme; Use it to carry out the digestion of anchorage-dependent cell; Cell growth amplification is in good condition, and can repeatedly go down to posterity, and does not have significant difference with the level that obtains with traditional method.Be applicable to any culture condition that contains serum and serum-free, do not need trypsin inhibitor during use, this reagent keeps proterties stable under all temps condition, stores easily and uses.
Deciduous teeth mescenchymal stem cell store method in the patented claim of the present invention, this method can make cell temporarily break away from growth conditions and make its cell characteristics be able to preserve, when needing again the recovery cell increase.During cell cryopreservation, in substratum, add protective material DMSO 99.8MIN. (DMSO), DMSO is a kind of important osmosis type cytoprotective.Preserve in the process of cell in profound hypothermia (subzero 200 degree), can prevent the damage that the formation of intracellular fluid ice crystal, osmotic pressure change, cellularstructure disorder etc. cause.DMSO can get in the cell by the quick penetration cytolemma, reduces freezing point, delays frozen process, improves intracellular ion concentration simultaneously, reduces the formation of ice crystal in the cell, thereby reduces cell injury.The difference not statistically significant of stem cells hyperplasia rate after frozen, apoptosis rate and multidirectional differentiation capability and fresh culture SHED.Cell cryopreservation quantity contains (1 ~ 2) * 10 for the cells frozen storing liquid with every 2mL
6Individual cell is advisable, the too high or too low decline that all can cause the cell recovery rate of density; The temperature of stem cell cryopreserving is-196 ℃, is stored in the liquid nitrogen, to preserve the activity of stem cell to greatest extent.Frozen method is: with the cell cryopreservation pipe with 1 ℃ of .min
-1Cooling rate be cooled to-85 ℃, be put in subsequently and carry out standing storage in the liquid nitrogen.
The method for resuscitation of deciduous teeth mesenchymal stem cell cryopreserving cell in the patented claim of the present invention; The cell cryopreservation pipe of the preparation recovery that is about to take out in the liquid nitrogen is put into 37 ℃ of water-baths immediately and is melted rapidly, wash with nutrient solution immediately then centrifugal, to remove frozen protective material DMSO; The toxicity of the DMSO pair cell that adds during because of freeze-stored cell is very big; Therefore action wants fast during recovery, wash DMSO 99.8MIN. as early as possible off, in order to avoid cause the serious toxicity of pair cell.
The result:
1, the dental pulp mescenchymal stem cell of deciduous teeth mescenchymal stem cell cultural method of the present invention institute separation and Culture still can keep multidirectional differentiation potential through repeatedly going down to posterity, and purity can reach more than 90%.The deciduous teeth mescenchymal stem cell that is obtained is an anchorage-dependent cell, and its growth traits is long shuttle type or irregular type, and the cultivation back of going down to posterity can adherently cover with individual layer in 5～7 days.
The form of the deciduous teeth mescenchymal stem cell that 2, the present invention cultivated is an anchorage-dependent cell, has fibroblast-like shape, and long shuttle type or irregular type have projection different in size, the karyon oval.Its growth characteristics do, the primary cell growth is slower, and cell is the growth of clone's appearance after about 10～15 days, forms colony not of uniform size, the intensive growth of 2 all left and right sides colonies; The back cell growth that is evenly distributed of going down to posterity, two to three days doubling times.
Claims (7)
1. the application of deciduous teeth dental pulp in the preparation mescenchymal stem cell.
2. deciduous teeth dental pulp mescenchymal stem cell cultural method; It is characterized in that: step is following: deciduous teeth are supported acquisition deciduous teeth dental pulp mescenchymal stem cell primary cell mixed solution through former being commissioned to train, deciduous teeth dental pulp mescenchymal stem cell primary cell mixed solution is cultivated through going down to posterity promptly obtained deciduous teeth dental pulp mescenchymal stem cell again.
3. deciduous teeth dental pulp mescenchymal stem cell cultural method according to claim 2 is characterized in that: the said former breeding method of being commissioned to train is:
⑴ cut into deciduous teeth to organize fragment through aseptically process, physics, adds the mescenchymal stem cell complete culture solution and promptly obtain pulp tissue's mixed solution;
⑵ place 36～38 ℃, volume(tric)fraction with the pulp tissue's mixed solution among the step ⑴ is 4～6% CO
2Cultivate in the incubator;
⑶ cultivate and promptly obtained deciduous teeth dental pulp mescenchymal stem cell primary cell mixed solution in 10～15 days.
4. deciduous teeth dental pulp mescenchymal stem cell cultural method according to claim 2 is characterized in that: the described cultured method that goes down to posterity is:
⑴ supernatant gets attached cell in the sucking-off deciduous teeth dental pulp mescenchymal stem cell primary cell mixed solution, in attached cell, adds pancreatin surrogate Digestive system to the submergence attached cell, is placed on CO again
2In the incubator, 36～38 ℃ of insulations digested in 5 minutes;
⑵ after finishing, digestion, adds the flushing of mescenchymal stem cell complete culture solution with the Digestive system sucking-off, and centrifugal; After the centrifugal end, the sucking-off supernatant must precipitate;
⑶ add the mescenchymal stem cell complete culture solution to immersion precipitation in the deposition of step ⑵, placing 36～38 ℃, volume(tric)fraction is 4～6% CO
2Cultivate in the incubator, cultivated 5～7 days, promptly obtain deciduous teeth dental pulp mescenchymal stem cell.
5. deciduous teeth dental pulp mescenchymal stem cell cultural method according to claim 4, it is characterized in that: the composition of described pancreatin surrogate Digestive system is: mass percent is 0.25% the tryptic phosphate buffered saline buffer of recombinant animal.
6. the store method of a deciduous teeth dental pulp mescenchymal stem cell, it is characterized in that: step is following:
⑴ the collection of freeze-stored cell
Collect the prepared deciduous teeth dental pulp mescenchymal stem cell of cultural method as claimed in claim 2; Add pancreatin surrogate Digestive system; Add-on is a submergence deciduous teeth dental pulp mescenchymal stem cell, digest enchylema, with enchylema centrifugal cell precipitation; Again cell precipitation is got cell suspension with the mescenchymal stem cell complete culture solution is resuspended, and carry out cell counting;
⑵ the preparation of cell cryopreservation suspension
According to cell count in the cell suspension is 1-5 * 10
6The time, the dose volume ratio of cell suspension and DMSO is that 3-5:1 adds DMSO in cell suspension, mixing, and packing gets the cell cryopreservation suspension then;
⑶ cell cryopreservation
With the cell cryopreservation suspension among the step ⑵ with 1 ℃ of min
-1Cooling rate be cooled to-85 ℃, place-196 ℃ of liquid nitrogen to preserve then to deciduous teeth dental pulp mescenchymal stem cell.
7. the method for resuscitation of a deciduous teeth pulp tissue mescenchymal stem cell, it is characterized in that: step is following:
⑴ take out the deciduous teeth dental pulp mescenchymal stem cell like the said preservation of claim 7, put into immediately 36-38 ℃ of water-bath melt cell suspension;
⑵ wash the centrifugal cell precipitation that gets with cell suspension with the mescenchymal stem cell complete culture solution;
⑶ resuspended with the mescenchymal stem cell complete culture solution with cell precipitation, and placing 37 ℃, volume(tric)fraction then is 4～6% CO
2Cultivate i.e. recovery in 5～7 days in the incubator and obtain deciduous teeth pulp tissue mescenchymal stem cell.
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| CN104560872A (en) * | 2014-12-29 | 2015-04-29 | 深圳市北科生物科技有限公司 | In-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells |
| CN104630141A (en) * | 2015-02-03 | 2015-05-20 | 黑龙江天晴干细胞股份有限公司 | Method for preparing dental pulp mesenchymal stem cells and establishing dental pulp mesenchymal stem cell bank and cell resuscitation method |
| CN105602895A (en) * | 2016-02-02 | 2016-05-25 | 江苏赫柏慧康再生医疗技术研究院有限公司 | Preparation method of deciduous tooth pulp mesenchymal 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 |
| 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 |
| CN110229787A (en) * | 2019-06-17 | 2019-09-13 | 华夏源（上海）细胞基因工程股份有限公司 | A kind of primary extracting method of dental pulp stem cell |
| CN110408592A (en) * | 2019-07-25 | 2019-11-05 | 四川大学 | For the regenerated multipotential stem cell MDPSCs of dental pulp and its isolated culture method and application |
| CN110468098A (en) * | 2019-07-05 | 2019-11-19 | 北京中瑞联合生物科技有限公司 | A kind of dental pulp stem cell preparation method |
| CN110616255A (en) * | 2019-10-14 | 2019-12-27 | 康妍葆(北京)干细胞科技有限公司 | Primer group, kit and identification method for identifying dental pulp stem cells and gingival stem cells by RNA level |
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| CN104560872A (en) * | 2014-12-29 | 2015-04-29 | 深圳市北科生物科技有限公司 | In-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells |
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| CN105602895A (en) * | 2016-02-02 | 2016-05-25 | 江苏赫柏慧康再生医疗技术研究院有限公司 | Preparation method of deciduous tooth pulp mesenchymal 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 |
| 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 |
| CN110229787A (en) * | 2019-06-17 | 2019-09-13 | 华夏源（上海）细胞基因工程股份有限公司 | A kind of primary extracting method of dental pulp stem cell |
| CN110229787B (en) * | 2019-06-17 | 2022-10-14 | 华夏源细胞工程集团股份有限公司 | Primary extraction method of dental pulp stem cells |
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| CN110408592A (en) * | 2019-07-25 | 2019-11-05 | 四川大学 | For the regenerated multipotential stem cell MDPSCs of dental pulp and its isolated culture method and application |
| CN110616255A (en) * | 2019-10-14 | 2019-12-27 | 康妍葆(北京)干细胞科技有限公司 | Primer group, kit and identification method for identifying dental pulp stem cells and gingival stem cells by RNA level |
| CN110616255B (en) * | 2019-10-14 | 2023-01-24 | 康妍葆(北京)干细胞科技有限公司 | Primer group, kit and identification method for identifying dental pulp stem cells and gingival stem cells by RNA level |
| CN111334465A (en) * | 2019-12-30 | 2020-06-26 | 上海循益生物技术有限公司 | Preparation method of dental pulp mesenchymal stem cells |
| CN111548990A (en) * | 2020-05-22 | 2020-08-18 | 深圳至博生物科技有限公司 | Dental pulp stem cell polymer for exfoliating deciduous teeth and preparation method and application thereof |
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