CN104560872A - In-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells - Google Patents
In-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells Download PDFInfo
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Abstract
The invention discloses an in-vitro separation and cultivation method for tooth-sourced mesenchymal stem cells. The in-vitro separation and cultivation method comprises the following steps: (A), gingiva on a tooth surface and surrounding tissue are scraped off, and the tooth is stored in tooth preserving fluid; (B), a culture solution is added to a centrifuge tube, dental pulp is collected to the centrifuge tube and cut into pieces by scissors; (C), collagenase type I and dispase are added to digest dental pulp tissue; (D), single-cell suspension is prepared; (E), the obtained single cell is subjected to primary culture, and the planting density of primary cells is (1-5)*10<4>/cm<2>; and (F), the cells are subjected to subculturing. The tooth is crushed with a physical method to obtain the dental pulp tissue, the collagenase type I and the dispase are used for digestion, the single-cell suspension is obtained, primary planting and culturing are performed for 10-15 days, and then passage is performed; and the problems that the primary cells are mixed with other components of the tooth and aging occurs probably in a cell mass culture process are solved effectively.
Description
Technical field
The invention belongs to stem cell culture technique, particularly relate to tooth derived mesenchymal stem cell in-vitro separation and cultural method.
Background technology
Within 2000, first Gronthos proposes the concept of dental pulp stem cell (dental pulp stem cells, DPSCs), and a group is extracted in teeth cavity, has a group stem cell called after DPSCs of clonality and positive controls for high proliferation rates in vitro.2003, Miura isolated the stem cell with many differentiation capabilities from the hat of the deciduous teeth that come off and the dental pulp of root, by its called after deciduous teeth pulp matrix stem cell (stem cells from human exfoliated deciduous teeth, SHED).There is during SHED vitro culture very high multiplication capacity and clonality, and neurocyte, scleroblast and odontoblast can be divided into, the Transplanted cells of vitro culture is returned after in Mice Body and can form bone and dentine.
Show the research of dental pulp stem cell, dental pulp stem cell can be applicable to dental pulp regeneration, dentine regeneration, bore regenerating, nervous cell regenerating, cardiac muscle and revascularization etc.Dental pulp stem cell be applied to dental diagnostic, can promote that dentine regenerates, be separated from single tooth and obtain a large amount of dental pulp stem cells and can apply dentistry and carry out a large amount of Dental Erosion.Data show, the dental pulp stem cell of vitro culture, is the mescenchymal stem cell that ability of cell proliferation or cloning efficiency are all better than derived from bone marrow.
Consider, dental pulp stem cell has the following advantages: dental pulp stem cell is the very special stem cell of a class; The source ratio of dental pulp be easier to and also right and wrong invasive, the tooth that 7-8 year child comes off can be derived from, or the wisdom tooth of 19-35 year grownup; Dental pulp stem cell can differentiate polytype cell; Application prospect is very wide, and the new osseous tissue as dental pulp stem cell formation is transplanted to after in body and can be formed new blood vessel, graft can be made fully to incorporate in surrounding tissue or dentine can be made to regenerate etc.
Stem cell stores business stores the multiple source such as umbilical cord, placenta, tooth of developing into now stem cell storage from simple Cord blood, and its importance and necessity is by increasing people is approved.But still have considerable child, because its father and mother are in the past not enough to the understanding of stem cell, thus lose for the first time for child stores the opportunity of cord blood stem cell or umbilical cord stem cells, and deciduous teeth dental pulp stem cell stores carrying out of business and can make up this and regret, the health for child buys a insurance.
The cultural method of the present invention to dental pulp stem cell is optimized, and gets single cell suspension and carries out original cuiture, reduces Pollution risk, more easily obtains a large amount of primary cells that are more single, purifying, and in culturing process, maintain good dryness.
Summary of the invention
The present invention uses the broken tooth of physical method to obtain pulp tissue, NTx enzyme (Collagenase Type I) and neutral protease (Dispase) is utilized to digest, obtain single cell suspension, carry out primary plantation and cultivate 10-15 days, and then carry out passage; Effectively solve primary cell and mix other component of tooth, and easily aging problem occurs in cell mass culturing process.
The technical solution used in the present invention is:
1, tooth derived mesenchymal stem cell in-vitro separation and a cultural method, comprises step:
A) gum of dental surface and the tissue of surrounding are scraped off, use the tincture of iodine and medical alcohol teeth surfaces successively; Use brine 3-5 time again, remove the tincture of iodine and alcohol; Be kept in tooth conserving liquid;
B) add nutrient solution at centrifuge tube, dental pulp is collected in centrifuge tube, then shred with scissors;
The neutral protease (Dispase) of the NTx enzyme (Collagenase Type I) and 2-4 mg/ml that C) add 2-3 mg/ml digests pulp tissue;
D) preparation of single cell suspension: the complete culture solution adding 3-5 times of volume stops digestion, filters and obtains single cell suspension;
E) original cuiture is carried out, primary cell planting density 1-5 × 10 to the unicellular of acquisition
4/ cm
2;
F) passage is cultivated: often within 3-4 days, change liquid and cultivate, treat that cell grows to 70-90% degree of collecting, can carry out gathering in the crops and going down to posterity.
Further: the NTx enzyme concn added in described step C is 3 mg/ml, the neutral protein enzyme concn added is 4 mg/ml.
Further: the substratum used in described primary and Secondary Culture comprises α-MEM basic culture solution and new fetal calf serum.
Further: described new fetal calf serum concentration is 10%.
Adopt the separation and Culture scheme of technical solutions according to the invention, the single cell suspension that composition is comparatively single can be obtained, the mescenchymal stem cell that composition is single can be obtained, and in culturing process, maintain good dryness.
The present invention can from 7-8 year the healthy tooth that comes off of child or 19-35 year grownup wisdom tooth sharp separation go out mescenchymal stem cell, be easy to cultivate and preserve.
Because tooth is comparatively hard, open relative to the instrument such as curet, tweezers external force, or sucking-off, cell and surface contact, increase the possibility polluted, and easily make a fault.Sterile gauze wraps around tooth, squeezes broken tooth gently with bench vice and exposes dental pulp, simple to operate, reduces dental pulp and suffers contaminated chance.
For conventional trypsin digestion, often injure cell, and unstable when cultivating.The present invention uses gentle enzyme mixation digestion, while reaching same digestion object, can not bring injury, advantageously in cultivation and the results of cell to cytolemma.
Direct tissue mass cell culture cell is more assorted, organizes agglomerating cultivation, easily differentiation, aging, may bring the fragments such as tooth into simultaneously, increase the chance polluted.The present invention after digestion, obtains single cell suspension with the strainer filterings of 70 μm, and relatively directly tissue mass cell culture has superiority more.
α-MEM(Minimum Essential Medium) in a short time (7-11 days) cell clone of certain number can be obtained.And dental pulp stem cell rate of propagation quickly.Experimentally, in dental pulp stem cell vitro culture P2 generation, on average can gather in the crops 4.35 × 10
7cell, in P4 generation, on average can gather in the crops 2.03 × 10
9cell, harvest yield is considerable.Cellular form, in comparatively inmature short fusiformis or circle, has good homogeneity.
Digest and decompose method provided by the invention, can well obtain from tooth and unicellularly carry out original cuiture, in succeeding generations, can well keep cell uniformity and cell dryness to maintain.The pulp tissue that this cultural method adopts gathers convenient, uses the gentle injury of enzyme mixation digestion reduction to cell, reduces cell contamination by sterilization and cell filtration, simple to operate.
Accompanying drawing explanation
The primary cell Clone formation of Fig. 1 embodiment of the present invention two Tooth derived mesenchymal stem cell.
The cellular form of Fig. 2 embodiment of the present invention two Tooth derived mesenchymal stem cell.
The cell harvesting number of Fig. 3 embodiment of the present invention two Tooth derived mesenchymal stem cell.
The vitro culture accumulation doublings of Fig. 4 embodiment of the present invention two Tooth derived mesenchymal stem cell
Fig. 5 embodiment of the present invention three Tooth derived mesenchymal stem cell CFU-F assay result.
The result schematic diagram of Fig. 6 embodiment of the present invention four tooth derived mesenchymal stem cell surface antigen flow cytometer showed.
The Osteoinductive differentiation of Fig. 7 embodiment of the present invention two Tooth derived mesenchymal stem cell.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in further details.
The preparation of embodiment one tooth derived mesenchymal stem cell nutrient solution.
Monocyte culture solution provided by the invention comprises basic culture solution, new fetal calf serum, is specially: α-MEM cultivates+10% new fetal calf serum+L-AA-2-sodium phosphate+L-glutaminate+penicillin/streptomycin in basis.
The separation and ientification of embodiment two tooth derived mesenchymal stem cell.
The separation and ientification concrete steps of tooth derived mesenchymal stem cell:
1]. tooth conserving liquid: stroke-physiological saline solution, 4 degree of preservations are no more than 48 hours.
2]. the transport of sample and pre-treatment: the gum of dental surface and the tissue of surrounding are scraped off, use the tincture of iodine and 75% alcohol teeth surfaces successively, prevent the pollution of oral cavity bacterium.Use brine 5 times afterwards again, remove the tincture of iodine and alcohol.
3]. the separation and Culture of cell: wrapped by the bundle of tooth sterilizing, pulp cavity is placed towards centre, is placed in bench vice, squeeze broken tooth gently and expose dental pulp.Centrifuge tube adds nutrient solution, is collected in centrifuge tube by dental pulp with aseptic nipper, then shreds with scissors.The neutral protease (Dispase) 37 DEG C of the NTx enzyme (Collagenase Type I) and 4 mg/mL that add 3 mg/mL digests pulp tissue 30 minutes.
4]. the preparation of single cell suspension: the complete culture solution adding 5 times of volumes stops digestion, obtains single cell suspension with the strainer filterings of 70 μm.Centrifugal 10 minutes of 1000rpm, removes supernatant, then with complete culture solution, cell is resuspended.
5]. cell kind bottle: primary cell planting density 1 × 10
4/ cm
2, or according to dental implant T25 culturing bottle.
6]. Clone formation: remove after 2 days and do not have adherent cell, nutrient solution changes liquid in every 3 days, to 12.28 d ± 2.26 d (range 9-19 day, N=14) cultivated, form cell clone (Clone formation of cell number about about 100), carry out primary cell results and go down to posterity.
7]. passage: according to 4000/ cm
2repopulating cell, often can change liquid in 3-4 days, treat that cell grows to 70-90% degree of collecting, and can carry out gathering in the crops and going down to posterity.
Embodiment three tooth derived mesenchymal stem cell CFU-F assay.
Primary CFU-F analyzes: with 2 × 10
5/ cm
2density, pulp cells is planted in 3 holes of six orifice plates, add substratum, be placed in type culture case and cultivate, within 48 hours, change liquid; After this often within 3-4 days, liquid is changed.When attached cell formation is greater than the clone of 50 cells (about 7-11 days), in counted under microscope CFU-F, or by Toluidine blue staining, count CFU-F number.Averaging in 3 holes, is its CFU-F number.Remarks: because pulp cells amount to obtain is very low, do not make primary CFU-F and analyze.
Passage cell (dental pulp DPSCs) CFU-F analyzes: get 100 cell seedings in 3 holes of six orifice plates, and add substratum, be placed in type culture case and cultivate, every 3-4 days changes liquid.Cultivate after 10 days, in counted under microscope CFU-F, or by Toluidine blue staining, count CFU-F number.Averaging in 3 holes, is its CFU-F number.
Embodiment four tooth derived mesenchymal stem cell cell-surface antigens flow cytometer showed.
According to previous literature report and actual result, the human umbilical cord blood mononuclear cell heterogeneity obtained is very strong, in testing process, needs multiple index jointly to evaluate and test, therefore we to mainly contain some indexs of detection of levying property as follows:
Mesenchyme associated antibodies: CD73, CD90, CD105, CD29, CD44, mesenchymal cell;
Blood system associated antibodies: CD34, hemopoietic stem cell; CD45, white corpuscle wide spectrum identifies; CD14, monocyte and precursor thereof; CD19, B cell and precursor thereof;
Major histocompatibility complex: HLA-DR, histocompatibility is correlated with.
Flow cytometer detection operation steps:
A) sequentially arranged in batches by sample, every increment originally gets cell 6 × 10 to be measured
6individual, according to detect antibody type need get streaming dedicated pipe some, marked Isotype control pipe and antibody pipe respectively;
B) sample is moved into special streaming pipe, centrifugal 5 min of 500 g; Abandon supernatant, then add 1 mLPBS, fully mix; Centrifugal 5 min of 500 g, abandon supernatant, then add the PBS suspension cell precipitation of proper volume;
C) in pipe, add sample 100 μ L cell suspension respectively;
D) in corresponding pipe, add antibody and the corresponding each 10 μ L of Isotype control respectively, fully mix, put 4 DEG C of refrigerator lucifuges and hatch 30 minutes;
E) add 1 mL PBS, fully mix, centrifugal 5 min of 500 g;
F) abandon supernatant liquor, then add 1 mL PBS and clean, fully mix, centrifugal 5 min of 500 g;
G) abandon supernatant liquor, add 200 μ L PBS, fully mix, apply U.S. company BD FACS Aria flow cytomery subsequently.
Claims (4)
1. tooth derived mesenchymal stem cell in-vitro separation and a cultural method, comprises step:
A) gum of dental surface and the tissue of surrounding are scraped off, use the tincture of iodine and medical alcohol teeth surfaces successively; Use brine 3-5 time again, remove the tincture of iodine and alcohol; Be kept in tooth conserving liquid;
B) add nutrient solution at centrifuge tube, dental pulp is collected in centrifuge tube, then shred with scissors;
C) the NTx enzyme of 2-3 mg/ml and the neutral protein enzymic digestion pulp tissue of 2-4 mg/ml is added;
D) preparation of single cell suspension: the complete culture solution adding 3-5 times of volume stops digestion, filters and obtains single cell suspension;
E) original cuiture is carried out, primary cell planting density 1-5 × 10 to the unicellular of acquisition
4/ cm
2;
F) passage is cultivated: often within 3-4 days, change liquid and cultivate, treat that cell grows to 70-90% degree of collecting, can carry out gathering in the crops and going down to posterity.
2. tooth derived mesenchymal stem cell in-vitro separation as claimed in claim 1 and cultural method, it is characterized in that: the NTx enzyme concn added in described step C is 3 mg/ml, the neutral protein enzyme concn added is 4 mg/ml.
3. tooth derived mesenchymal stem cell in-vitro separation as claimed in claim 1 and cultural method, is characterized in that: the substratum used in described primary and Secondary Culture comprises α-MEM basic culture solution and new fetal calf serum.
4. tooth derived mesenchymal stem cell in-vitro separation as claimed in claim 3 and cultural method, is characterized in that: described new fetal calf serum concentration is 10%.
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106620763A (en) * | 2016-09-30 | 2017-05-10 | 广州赛莱拉干细胞科技股份有限公司 | Method for sterilizing tooth sample |
| CN106801033A (en) * | 2017-02-06 | 2017-06-06 | 贵州泛特尔细胞生物技术有限公司 | A kind of dental pulp stem cell isolated culture method |
| CN107227295A (en) * | 2016-03-23 | 2017-10-03 | 北京泰盛生物科技有限公司 | Come off separation and the in-vitro multiplication method of deciduous teeth stem cell |
| CN107287157A (en) * | 2017-05-18 | 2017-10-24 | 舟山医院 | The method of free serum culture people's gum mescenchymal stem cell |
| 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 |
| CN108888633A (en) * | 2018-09-21 | 2018-11-27 | 北京泰盛生物科技有限公司 | A kind of gum mescenchymal stem cell preparation and its application and preparation method |
| 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 |
| CN109777769A (en) * | 2017-11-14 | 2019-05-21 | 北京泰盛生物科技有限公司 | The source tooth screening technique extracted for dental pulp stem cell |
| CN110747164A (en) * | 2019-11-18 | 2020-02-04 | 北京泓信干细胞生物技术有限公司 | Preparation method of dental pulp stem cells |
| CN110951683A (en) * | 2020-01-06 | 2020-04-03 | 深圳华云生物科技发展有限公司 | Preparation method of dental pulp stem cells |
| 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 |
| CN115521906A (en) * | 2021-06-24 | 2022-12-27 | 东莞宣冠干细胞再生医学有限公司 | Method for preparing high-quality human-derived dental pulp stem cells |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107227295A (en) * | 2016-03-23 | 2017-10-03 | 北京泰盛生物科技有限公司 | Come off separation and the in-vitro multiplication method of deciduous teeth stem cell |
| CN106620763A (en) * | 2016-09-30 | 2017-05-10 | 广州赛莱拉干细胞科技股份有限公司 | Method for sterilizing tooth sample |
| CN106801033A (en) * | 2017-02-06 | 2017-06-06 | 贵州泛特尔细胞生物技术有限公司 | A kind of dental pulp stem cell isolated culture method |
| CN107287157A (en) * | 2017-05-18 | 2017-10-24 | 舟山医院 | The method of free serum culture people's gum mescenchymal stem cell |
| CN109777769A (en) * | 2017-11-14 | 2019-05-21 | 北京泰盛生物科技有限公司 | The source tooth screening technique extracted for dental pulp stem cell |
| 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 |
| CN108888633A (en) * | 2018-09-21 | 2018-11-27 | 北京泰盛生物科技有限公司 | A kind of gum mescenchymal stem cell preparation and its application and preparation method |
| CN110747164A (en) * | 2019-11-18 | 2020-02-04 | 北京泓信干细胞生物技术有限公司 | Preparation method of dental pulp stem cells |
| 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 |
| CN110951683A (en) * | 2020-01-06 | 2020-04-03 | 深圳华云生物科技发展有限公司 | Preparation method of dental pulp stem cells |
| CN115521906A (en) * | 2021-06-24 | 2022-12-27 | 东莞宣冠干细胞再生医学有限公司 | Method for preparing high-quality human-derived dental pulp stem cells |
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Application publication date: 20150429 |