CN101717750A - Method for constructing banks of human dental pulp stem cells - Google Patents
Method for constructing banks of human dental pulp stem cells Download PDFInfo
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Abstract
A method for constructing banks of human dental pulp stem cells comprises the steps of acquiring basic information, obtaining dental pulp stem cells, cryopreserving and putting dental pulp stem cells into cell banks and recording and checking information. When the banks of dental pulp stem cells are used, recovery and multiplication culture are carried out in accordance with the actual demands. In the method, the dental pulp stem cells are obtained from dental pulp of waste healthy human teeth and a reserve bank for effective resources is established by using system engineering. A great quantity of dental pulp stem cells with functional activities can be obtained through short-term culture of the human dental pulp stem cells stored in the banks and can be stored for long term without losing the activities. Compared with the prior art, the invention is characterized in that the construction operation specification is simple, easy to grasp, safe and feasible; the bank construction cost is low and the standardization degree is high; and the cells from different age groups adopt different cryopreservation solution so that the activities of the recovered cells are enhanced, thus expanding the sources of the cells and enhancing the activities of the cells. The teeth used in the method are those which are extracted for various reasons and are waste, thus greatly saving the medical resources; therefore, the construction has wide application prospect.
Description
Technical field
The invention belongs to the biological organization material technical field, be specifically related to the preparation of people's dental pulp stem cell and the construction process of cell bank thereof.
Background technology
Because defect of teeth that a variety of causes causes and absence of tooth have become the principal disease of harm oral cavity and even HUMAN HEALTH, its sickness rate increases year by year.Mainly be to deal with the tooth defect disease at present,, can not solve root problem though can stop the continuation development of pathology to a certain extent by means of various dental material fillings.Although a lot of for the absence of tooth solution route, the most effective and the most basic way still is the biological regeneration of tooth.
Along with the fast development of tissue regeneration medical science, for effective treatment of defect of teeth and absence of tooth has brought hope.Dental tissue has been born at present existing many research and utilization teeth source property epithelium and the mesochymal interaction of property of tooth source again.Tooth source property mesenchyme commonly used comprises dental papilla cells and pulp tissue's cell.Because dental papilla cells is difficult to obtain clinically, therefore, the seed cell first-selection that is used for tooth body and regeneration of tooth at present is the cell in pulp tissue source, wherein the most important thing is dental pulp stem cell.
Found dental pulp stem cell (S.Gronthos, M.Mankani, J.Brahim in 2000 first, P, et al, Postnatal human dental pulp stem cells (DPSCs) in vitro andin vivo, Proc Natl Acad Sci U S is December 5 A.2000; 97 (25): 13625-13630.).This is that a class has the high proliferation ability, highly self ability, multidirectional differentiation capability equal the cell of cell characteristics.Existing this cell of research and utilization has successfully made up dental pulp, dentine and dental tissue.Therefore, dental pulp stem cell is the desirable seed cell that makes up tooth body and dental tissue.
Stem cell bank is the place that stores stem cell and collect storage stem cell resource related data in profound hypothermia.Perfect stem cell bank should possess the ability that the stem cell of health is provided in clinical use whenever and wherever possible.Successfully establish some stem cell banks (as the cord blood stem cell storehouse) at present, and still unmanned so far dental pulp stem cell storehouse.
Replace to impacted tooth, accessory tooth and orthodontic tooth from newborn permanent teeth, a large amount of teeth go out of use, and dental pulp stem cell resource wherein runs off in vain.Therefore, the preparation dental pulp stem cell is set up banks of human dental pulp stem cells, for providing the seed cell of treatment of tooth body and regeneration of tooth to have great importance from body and other people.
Summary of the invention
The purpose of this invention is to provide a kind of constructing banks of human dental, and propose a kind of preparation method who makes full use of deciduous teeth, impacted tooth, accessory tooth and just abnormal tooth for people's dental pulp stem cell in source; For the preparation of the structure of banks of human dental pulp stem cells and people's dental pulp stem cell provides easy grasp, safe and feasible, working specification with low cost.
Constructing banks of human dental proposed by the invention has that essential information collection, dental pulp stem cell obtain, frozen, the warehouse-in of dental pulp stem cell and an information record and the step of checking; Specifically comprise:
Step 1, collection essential information: as retrieve encoded, the relevant information of donor name, age, ID card No., date of birth, race, address, contact method, routine inspection result and subsequent step; Described routine inspection comprises: the detection of ABO/Rh blood group, the detection of HLA somatotype, syphilis antibody detection, special-shaped hepatitis antigen antibody test, CMV antibody test and the HIV immunodetection etc. of donor blood can also comprise the detection of donor three generations with interior above-mentioned project;
Step 2, obtain dental pulp stem cell: (1) adopts the fresh dental tissue of donor that to contain V/V be that the DMEM nutrient solution of 5%～20% new-born calf serum is finished transfer, under aseptic condition, remove periodontal tissue with the flushing of PBS liquid, use the crusher in crushing tooth, select pulp tissue and wash with PBS liquid; (2) centrifugal 3～10 minutes at 1000～2000 rev/mins, remove supernatant liquor, after adding the Digestive system of 5～10 times of volumes, dispel evenly with suction pipe, inserting in 37 ℃ of incubators ventilative hatching 10～60 minutes, described Digestive system is 3～9U/ml collagenase solution and the mixing of 0.2～5U/ml Dispase solution of equal volume; (3) hatch finish after, in suspension, add the described DMEM nutrient solution that contains new-born calf serum, dispel the histocyte suspension and stop continuation digestion, centrifugal 3～10 minutes at 800～2000 rev/mins, after abandoning supernatant, the perfect medium re-suspended cell that adds 5～10 times of volumes, with cell suspension change over to carry out in the culturing bottle former be commissioned to train foster; (4) cultivate after 7～9 days with the digestion of pancreatin solution and the piping and druming cell of 3～9U/ml concentration, make the cell back renewed vaccination cultivation that comes off fully; Treat cell cover with bottle at the bottom of 80～90% o'clock, adopt the magnetic bead partition method to isolate dental pulp stem cell; Use stem cell markers (as STRO-1 and CD146) that resulting cell is identified again, adopt cell factory to carry out amplification cultivation then;
Described perfect medium is to be added with 10～30% foetal calf serum in α-MEM nutrient solution by volume, and final concentration is that Noggin and the final concentration of 400～600ng/ml is the fibroblast growth factor of 20～60ng/ml; In addition, cell conditioned medium is dental pulp stem cell is cultivated 2～4 days in perfect medium after, collects the liquid behind its broth out impurity.
Frozen, the warehouse-in of step 3, dental pulp stem cell: with the dental pulp stem cell that obtained by 1 * 10
7～8 * 10
7The density of individual/ml is resuspended in frozen storing liquid, divides the frozen pipe of packing into, and frozen pipe under 0～4 ℃ of condition, is refrigerated to-80～-120 ℃ with 0.5～2 ℃/minute speed; Again frozen pipe is transferred to and stores warehouse-in in-196 ℃ of liquid nitrogen; Storing warehouse-in can be by the preservation of classifying of ABO/Rh and HLA somatotype;
Described frozen storing liquid comprises following four kinds: the glycerine that 1. contains 10% volume in the perfect medium; 2. the dimethyl sulfoxide (DMSO) (DMSO) that contains 10% volume in the perfect medium; 3. described frozen storing liquid 1. with mixing with the volume cell conditioned medium; 4. described frozen storing liquid 2. with mixing with the volume cell conditioned medium.Wherein, 1. and 2. be applicable to the structure in the permanent teeth dental pulp stem cell storehouse of deciduous teeth and≤40 years old donor; 3. and 4. be applicable to the structure in the dental pulp stem cell storehouse of 40-60 year donor.Dimethyl sulfoxide (DMSO) in the described frozen storing liquid and glycerine, can enter cell by the quick penetration cytolemma, reduce freezing point, delay refrigerating process, improve ionic concn in the born of the same parents simultaneously, the cell injury of avoiding the formation of liquid ice crystal, osmotic pressure change, cellularstructure disorder etc. to cause.
Step 4, information write down and check: establish relevant information on the container carrier of storing warehouse-in, with the aforementioned relevant information input computer information storage of gathering donor essential information and preparation of corresponding dental pulp stem cell and cell bank building process thereof; And access associated note and the donor data is examined by retrieve encoded, include the cell bank management in; Promptly finish the structure of banks of human dental pulp stem cells.
The application method of the constructed banks of human dental pulp stem cells of the present invention is, after in cell bank management, checking relevant information by retrieve encoded, with the storage cell cryopreservation pipe of correspondence by taking out in the liquid nitrogen, at once put into 37 ℃～45 ℃ water-bath, melt in 30 seconds to 1 minute, melt recovery rapidly and can prevent to damage cell; Institute's recovery cell is carried out can using after the amplification cultivation according to actual needs.
The construction process of people's dental pulp stem cell preparation of the present invention and cell bank thereof can obtain a large amount of dental pulp stem cells from the dental pulp of depleted people healthy tooth, utilize systems engineering, sets up the warehouse of efficient resource.The present invention is stored in the people's dental pulp stem cell in the storehouse, can obtain a large amount of (1 * 10 through Short-term Culture
10～7 * 10
10) be rich in the dental pulp stem cell of functionally active, and can prolonged preservation and do not lose its activity.Compare with existing stem cell bank, structure working specification of the present invention, simple grasp easily, safe and feasible, this is cheap to build Kucheng, the standardization level height adopts different frozen storing liquids to make recovery back cell activity strengthen at the cell in different ages section source, enlarged cell the source, strengthened cell activity.In addition, tooth used in the present invention is a variety of causes and is pulled out the depleted tooth, has saved medical resource greatly; Therefore, the structure of banks of human dental pulp stem cells is with a wide range of applications.
Embodiment
Below in conjunction with example technical solution of the present invention is described further, but is not limited to following example.Operation in the example is all carried out in strict gnotobasis, and is not contaminated for guaranteeing cell, all is added with 100U/ml penicillin and 100U/ml Streptomycin sulphate in the solution of PBS, the α-MEM of all uses and DMEM.
The preparation of embodiment 1, people's wisdom tooth dental pulp stem cell and the structure of stem cell bank thereof
Step 1, gather essential information: extract donor venous blood and determine record after the security through trace routine (detections of ABO/Rh blood group, the detection of HLA somatotype, syphilis antibody detection, special-shaped hepatitis antigen antibody test, CMV antibody test and HIV immunodetection etc.), collection donor essential information comprises: retrieve encoded, name, age, identification card number, date of birth, race, address and contact method etc.
Step 2, obtain dental pulp stem cell: obtain two of donor impacted wisdom tooths and insert in the centrifuge tube of DMEM nutrient solution that the ready V/V of containing is 15% calf serum, be transferred to aseptic super clean bench after sealing with film, wash dental tissue repeatedly with the PBS liquid of pH7.4 and remove remained on surface blood and periodontium; Be transferred to broken tooth in the crusher, expose complete pulp tissue, carefully choose out pulp tissue's fragment and wash with PBS liquid; Adopt 1200 rev/mins centrifugal 5 minutes, remove supernatant liquor, add 2ml Digestive system (for mixing of the 1U/ml Dispase solution of the collagenase solution of 6.25U/ml and equal volume) and then dispel pulp tissue's fragment with suction pipe, put into 37 ℃, 5%CO
2Ventilative hatching 50 minutes in the incubator of condition; Hatch finish after, in suspension, add the described DMEM nutrient solution 5ml that contains calf serum, dispelling the histocyte suspension stops it to continue digestion, 1000 rev/mins centrifugal 5 minutes, remove supernatant, (perfect medium is to be added with 20% foetal calf serum in α-MEM nutrient solution by volume to add the perfect medium re-suspended cell of 4ml, final concentration is that Noggin and the final concentration of 400ng/ml is the fibroblast growth factor of 60ng/ml), with cell suspension move into T type culturing bottle carry out former be commissioned to train foster, change liquid after 12 hours, after this changed liquid at interval in 2～3 days; With the digestion of pancreatin solution and the piping and druming cell of 3U/ml concentration, the cell back renewed vaccination that comes off is fully cultivated after 7 days; Treat cell cover with bottle at the bottom of 80～90% o'clock, adopt the magnetic bead partition method to isolate dental pulp stem cell; The dental pulp stem cell that is obtained is carried out STRO-1 and CD146 identification and detection; Then dental pulp stem cell is inoculated in amplification cultivation in the cell factory;
Step 3, frozen, the warehouse-in of dental pulp stem cell: the dental pulp stem cell of the gained that will increase is by 3 * 10
7The density of individual/ml is resuspended in frozen storing liquid (cell conditioned medium mixes with the perfect medium that contains 10% dimethyl sulfoxide (DMSO) with volume), divide and put into frozen pipe, frozen pipe is placed 4 ℃ of environment, speed with 1 ℃/minute is refrigerated to-120 ℃, frozen pipe is transferred to stored warehouse-in in-196 ℃ of liquid nitrogen containers then; By the preservation of classifying of ABO/Rh and HLA somatotype;
Step 4, information write down and check: set up relevant information (as retrieve encoded and donor name) on the container carrier of storing warehouse-in, with aforementioned relevant information (as cell culture fluid formation and concentration, pancreatin and collagenase concentration and digestion time, cell inoculation density and former generation and amplification cultivation time, frozen storing liquid formation and frozen time etc.) the input computer information storage of gathering donor essential information and dental pulp stem cell preparation and cell bank building process; And access associated note and the donor data is examined by retrieve encoded, and confirm all data and operate accurately, include the cell bank management in; Promptly finish the structure in people's wisdom tooth dental pulp stem cell storehouse.During application above-mentioned cell is recovered and amplification cultivation according to actual needs.
The just abnormal preparation of sound of baby talk marrow stem cell and the structure of stem cell bank thereof pulled out of embodiment 2, people
Step 1, gather essential information: extract donor venous blood and determine record after the security through trace routine (detections of ABO/Rh blood group, the detection of HLA somatotype, syphilis antibody detection, special-shaped hepatitis antigen antibody test, CMV antibody test and HIV immunodetection etc.), collection donor essential information comprises: retrieve encoded, name, age, identification card number, date of birth, race, address and contact method etc.;
Step 2, obtain dental pulp stem cell: obtain 4 of the canine teeth that donor pulls out because of just abnormal need and put into the centrifuge tube that the ready V/V of containing is the DMEM nutrient solution of 10% calf serum, be transferred to aseptic super clean bench after sealing with film, wash tooth repeatedly with the PBS liquid of pH7.4 and remove remained on surface blood and periodontium; Be transferred to crusher and carry out broken tooth, expose complete pulp tissue; Carefully choosing out pulp tissue's fragment washes with PBS liquid, adopt 1200 rev/mins centrifugal 5 minutes, abandon supernatant liquor, add 2ml Digestive system (for mixing of the 0.5U/ml Dispase solution of the collagenase solution of 6.25U/ml and equal volume) and then dispel pulp tissue's fragment, put into 37 ℃, 5%CO with suction pipe
2Ventilative hatching 50 minutes in the incubator of condition; Hatch finish after, in suspension, add the described DMEM nutrient solution 2ml that contains calf serum, dispelling the histocyte suspension stops it to continue digestion, 1000 rev/mins centrifugal 5 minutes, remove supernatant, (perfect medium is to be added with 20% foetal calf serum in α-MEM nutrient solution by volume to add the perfect medium re-suspended cell of 4ml, final concentration is that Noggin and the final concentration of 600ng/ml is the fibroblast growth factor of 40ng/ml), with cell suspension move into T type culturing bottle carry out former be commissioned to train foster, change liquid after 12 hours, liquid was changed at after this every interval in 2 days; With the digestion of pancreatin solution and the piping and druming cell of 6U/ml concentration, the cell back renewed vaccination that comes off is fully cultivated after 8 days; Treat cell cover with bottle at the bottom of 80～90% o'clock, adopt the magnetic bead partition method to isolate dental pulp stem cell; The dental pulp stem cell that is obtained is carried out STRO-1 and CD146 identification and detection; Then dental pulp stem cell is inoculated in amplification cultivation in the cell factory;
Step 3, frozen, the warehouse-in of dental pulp stem cell: the dental pulp stem cell of the gained that will increase is by 4 * 10
7The density of individual/ml is resuspended in frozen storing liquid (perfect medium that contains the dimethyl sulfoxide (DMSO) of 10% volume), divide and put into frozen pipe, frozen pipe is placed 2 ℃ of environment, be refrigerated to-100 ℃, frozen pipe is transferred to stored warehouse-in in-196 ℃ of liquid nitrogen containers then with 1 ℃/minute speed; By the preservation of classifying of ABO/Rh and HLA somatotype;
Step 4, information write down and check: set up relevant information (as retrieve encoded and donor name) on the container carrier of storing warehouse-in, with aforementioned relevant information (as cell culture fluid formation and concentration, pancreatin and collagenase concentration and digestion time, cell inoculation density and former generation and amplification cultivation time, frozen storing liquid formation and frozen time etc.) the input computer information storage of gathering donor essential information and dental pulp stem cell preparation and cell bank building process; And access associated note and the donor data is examined by retrieve encoded, and confirm all data and operate accurately, include the cell bank management in; Promptly finish the structure of banks of human dental pulp stem cells.During application above-mentioned cell is recovered and amplification cultivation according to actual needs.
The preparation of embodiment 3, human milk sound of baby talk marrow stem cell and the structure of stem cell bank thereof
Step 1, gather essential information: extract donor venous blood and determine record after the security through trace routine (detections of ABO/Rh blood group, the detection of HLA somatotype, syphilis antibody detection, special-shaped hepatitis antigen antibody test, CMV antibody test and HIV immunodetection etc.), collection donor essential information comprises: retrieve encoded, name, age, identification card number, date of birth, race, address and contact method etc.
Step 2, obtain dental pulp stem cell: obtain donor and replace 3 of deciduous teeth pulling out because of newborn permanent teeth and insert in the centrifuge tube of DMEM nutrient solution that the ready V/V of containing is 10% calf serum, be transferred to aseptic super clean bench after sealing with film, wash dental tissue repeatedly with the PBS liquid of pH7.4 and remove remained on surface blood and periodontium; Be transferred to broken tooth body in the crusher, expose complete pulp tissue, carefully choose out pulp tissue's fragment and wash with PBS liquid; Adopt 1200 rev/mins centrifugal 3 minutes, remove supernatant liquor, add 2ml Digestive system (for mixing of the 0.4U/ml Dispase solution of the collagenase solution of 4.25U/ml and equal volume) and then dispel pulp tissue's fragment with suction pipe, put into 37 ℃, 5%CO
2Ventilative hatching 45 minutes in the incubator of condition; Hatch finish after, in suspension, add the described DMEM nutrient solution 3ml that contains calf serum, dispelling the histocyte suspension stops it to continue digestion, 800 rev/mins centrifugal 5 minutes, remove supernatant, (perfect medium is to be added with 20% foetal calf serum in α-MEM nutrient solution by volume to add the perfect medium re-suspended cell of 3ml, final concentration is that Noggin and the final concentration of 500ng/ml is the fibroblast growth factor of 50ng/ml), with cell suspension move into T type culturing bottle carry out former be commissioned to train foster, change liquid after 12 hours, liquid was changed at after this every interval in 3 days; With the digestion of pancreatin solution and the piping and druming cell of 5U/ml concentration, the cell back renewed vaccination that comes off is fully cultivated after 9 days; Treat cell cover with bottle at the bottom of 80～90% o'clock, adopt the magnetic bead partition method to isolate dental pulp stem cell; The dental pulp stem cell that is obtained is carried out STRO-1 and CD146 identification and detection; Then dental pulp stem cell is inoculated in amplification cultivation in the cell factory;
Step 3, frozen, the warehouse-in of dental pulp stem cell: the dental pulp stem cell of the gained that will increase is by 4 * 10
7The density of individual/ml is resuspended in frozen storing liquid (perfect medium that contains 10% volume glycerine), divide and put into frozen pipe, frozen pipe is placed 4 ℃ of environment, be refrigerated to-100 ℃, frozen pipe is transferred to stored warehouse-in in-196 ℃ of liquid nitrogen containers then with 1 ℃/minute speed; By the preservation of classifying of ABO/Rh and HLA somatotype;
Step 4, information write down and check: set up relevant information (as retrieve encoded and donor name) on the container carrier of storing warehouse-in, with aforementioned relevant information (as cell culture fluid formation and concentration, pancreatin and collagenase concentration and digestion time, cell inoculation density and former generation and amplification cultivation time, frozen storing liquid formation and frozen time etc.) the input computer information storage of gathering donor essential information and dental pulp stem cell preparation and cell bank building process; And access associated note and the donor data is examined by retrieve encoded, and confirm all data and operate accurately, include the cell bank management in; Promptly finish the structure of human milk sound of baby talk marrow stem cell bank.During application above-mentioned cell is recovered and amplification cultivation according to actual needs.
Claims (4)
1. a constructing banks of human dental is characterized in that, has that essential information collection, dental pulp stem cell obtain, frozen, the warehouse-in of dental pulp stem cell and an information record and the step of checking; Specifically comprise:
Step 1, collection essential information: comprise retrieve encoded, the relevant information of donor name, age, ID card No., date of birth, race, address, contact method, routine inspection result and subsequent step;
Step 2, obtain dental pulp stem cell: 1. the fresh dental tissue of donor is adopted that to contain V/V be that the DMEM nutrient solution of 5%～20% new-born calf serum is finished transfer, remove periodontal tissue with the flushing of PBS liquid under aseptic condition, broken tooth is selected pulp tissue and is washed with PBS liquid; 2. 1000～2000 rev/mins centrifugal 3～10 minutes, remove supernatant liquor, add the Digestive system of 5～10 times of volumes after, dispel evenly with suction pipe, inserting breathes freely in 37 ℃ of incubators hatched 10～60 minutes; Described Digestive system is 3～9U/ml collagenase solution and the mixing of 0.2～5U/ml Dispase solution of equal volume; 3. hatch finish after, in suspension, add the described DMEM nutrient solution that contains new-born calf serum, dispel the histocyte suspension and stop continuation digestion, centrifugal 3～10 minutes at 800～2000 rev/mins, after abandoning supernatant, the perfect medium re-suspended cell that adds 5～10 times of volumes, change over to carry out in the culturing bottle former be commissioned to train foster; 4. cultivate after 7～9 days digestion of pancreatin solution and piping and druming cell, the cell back renewed vaccination that comes off is fully cultivated with 3～9U/ml concentration; Treat cell cover with bottle at the bottom of 80～90% o'clock, adopt the magnetic bead partition method to isolate dental pulp stem cell; With stem cell markers resulting cell is identified again, adopted cell factory to carry out amplification cultivation then;
Frozen, the warehouse-in of step 3, dental pulp stem cell: with the dental pulp stem cell that obtained by 1 * 10
7～8 * 10
7The density of individual/ml is resuspended in frozen storing liquid, divides the frozen pipe of packing into, under 0～4 ℃ of condition, is refrigerated to-80～-120 ℃ with 0.5～2 ℃/minute speed; Again frozen pipe is transferred to and stores warehouse-in in-196 ℃ of liquid nitrogen;
Step 4, information write down and check: set up relevant information on the container carrier of storing warehouse-in, with the aforementioned relevant information input computer information storage of gathering donor essential information and preparation of corresponding dental pulp stem cell and cell bank building process thereof; And access associated note and the donor data is examined by retrieve encoded, include the cell bank management in; Promptly finish the structure of banks of human dental pulp stem cells.
2. cell bank construction process according to claim 1, it is characterized in that, described perfect medium is to be added with 10～30% foetal calf serum in α-MEM nutrient solution by volume, and final concentration is that Noggin and the final concentration of 400～600ng/ml is the fibroblast growth factor of 20～60ng/ml; Described cell conditioned medium is dental pulp stem cell is cultivated 2～4 days in perfect medium after, collects the liquid behind its broth out impurity.
3. cell bank construction process according to claim 1 is characterized in that, described frozen storing liquid comprises following four kinds: the glycerine that 1. contains 10% volume in the perfect medium; 2. the dimethyl sulfoxide (DMSO) that contains 10% volume in the perfect medium; 3. described frozen storing liquid 1. with mixing with the volume cell conditioned medium; 4. described frozen storing liquid 2. with mixing with the volume cell conditioned medium.
4. cell bank construction process according to claim 1 and 2 is characterized in that, all is added with 100U/ml penicillin and 100U/ml Streptomycin sulphate in the solution of PBS, the α-MEM of all uses and DMEM.
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| CN102807967A (en) * | 2012-08-31 | 2012-12-05 | 沙船（天津）生物科技发展有限公司 | Application of deciduous tooth pulp to preparation of mesenchymal stem cell and culturing method of deciduous tooth pulp mesenchymal stem cell |
| CN103387959A (en) * | 2012-10-19 | 2013-11-13 | 夏佑红 | Stem cell from deciduous teeth, and preparation method and application thereof |
| CN103849595A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Large scale production technology for dental pulp stem cell |
| CN104694464A (en) * | 2015-02-09 | 2015-06-10 | 安徽新生命干细胞科技有限公司 | Clinical dental pulp stem cell and preparation method thereof |
| CN104705289A (en) * | 2015-03-01 | 2015-06-17 | 安徽新生命干细胞科技有限公司 | In-vitro tooth preservation liquid |
| CN105145547A (en) * | 2015-10-28 | 2015-12-16 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells |
| CN105165804A (en) * | 2015-10-28 | 2015-12-23 | 广州赛莱拉干细胞科技股份有限公司 | Adipose tissue-derived stem cell frozen stock protection fluid and frozen stock method |
| CN105331578A (en) * | 2015-11-27 | 2016-02-17 | 华中科技大学同济医学院附属协和医院 | Stem cell culture method for promoting periapical periodontitis bone healing and stem cell application |
| 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 |
| CN105754934A (en) * | 2016-03-08 | 2016-07-13 | 大连大学 | Dental pulp stem cell, preparation method thereof and related bone tissue engineering material |
| CN106417250A (en) * | 2016-07-28 | 2017-02-22 | 广州赛莱拉干细胞科技股份有限公司 | Dental pulp mesenchymal stem cell cryopreservation solution and cryopreservation method thereof |
| CN107267451A (en) * | 2017-07-27 | 2017-10-20 | 温州优牙生物科技有限公司 | A kind of preparation method of primary dental pulp stem cell and the method for building dental pulp stem cell storehouse |
| WO2018064732A1 (en) * | 2016-10-03 | 2018-04-12 | SILY DE ASSIS BUMACHAR, Brunela | Process for cryopreserving stem cells obtained from the pulp of deciduous teeth |
| 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 |
| CN112167246A (en) * | 2020-10-31 | 2021-01-05 | 郑州贝贝生物科技有限公司 | Dental pulp mesenchymal stem cell cryopreservation liquid and cryopreservation method |
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| CN102807967A (en) * | 2012-08-31 | 2012-12-05 | 沙船（天津）生物科技发展有限公司 | Application of deciduous tooth pulp to preparation of mesenchymal stem cell and culturing method of deciduous tooth pulp mesenchymal stem cell |
| CN103387959A (en) * | 2012-10-19 | 2013-11-13 | 夏佑红 | Stem cell from deciduous teeth, and preparation method and application thereof |
| CN103849595A (en) * | 2012-12-05 | 2014-06-11 | 上海坤爱生物科技有限公司 | Large scale production technology for dental pulp stem cell |
| CN104694464A (en) * | 2015-02-09 | 2015-06-10 | 安徽新生命干细胞科技有限公司 | Clinical dental pulp stem cell and preparation method thereof |
| CN104705289A (en) * | 2015-03-01 | 2015-06-17 | 安徽新生命干细胞科技有限公司 | In-vitro tooth preservation liquid |
| CN105145547A (en) * | 2015-10-28 | 2015-12-16 | 广州赛莱拉干细胞科技股份有限公司 | Cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells |
| CN105165804A (en) * | 2015-10-28 | 2015-12-23 | 广州赛莱拉干细胞科技股份有限公司 | Adipose tissue-derived stem cell frozen stock protection fluid and frozen stock method |
| CN105331578A (en) * | 2015-11-27 | 2016-02-17 | 华中科技大学同济医学院附属协和医院 | Stem cell culture method for promoting periapical periodontitis bone healing and stem cell application |
| 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 |
| CN105754934A (en) * | 2016-03-08 | 2016-07-13 | 大连大学 | Dental pulp stem cell, preparation method thereof and related bone tissue engineering material |
| CN105754934B (en) * | 2016-03-08 | 2019-07-12 | 大连大学 | Dental pulp stem cell, preparation method and its related engineering material of bone tissue |
| CN106417250A (en) * | 2016-07-28 | 2017-02-22 | 广州赛莱拉干细胞科技股份有限公司 | Dental pulp mesenchymal stem cell cryopreservation solution and cryopreservation method thereof |
| WO2018064732A1 (en) * | 2016-10-03 | 2018-04-12 | SILY DE ASSIS BUMACHAR, Brunela | Process for cryopreserving stem cells obtained from the pulp of deciduous teeth |
| GB2569753A (en) * | 2016-10-03 | 2019-06-26 | Brunela Sily De Assis Bumachar | Process for cryopreserving stem cells obtained from the pulp of deciduous teeth |
| CN107267451A (en) * | 2017-07-27 | 2017-10-20 | 温州优牙生物科技有限公司 | A kind of preparation method of primary dental pulp stem cell and the method for building dental pulp stem cell storehouse |
| 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 |
| CN112167246A (en) * | 2020-10-31 | 2021-01-05 | 郑州贝贝生物科技有限公司 | Dental pulp mesenchymal stem cell cryopreservation liquid and cryopreservation method |
| CN112167246B (en) * | 2020-10-31 | 2021-07-27 | 北京泰盛生物科技有限公司 | Dental pulp mesenchymal stem cell cryopreservation liquid and cryopreservation method |
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