CN1590537A - Separation and culturing method of ectomesenchyme stem cell - Google Patents
Separation and culturing method of ectomesenchyme stem cell Download PDFInfo
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- CN1590537A CN1590537A CNA031345360A CN03134536A CN1590537A CN 1590537 A CN1590537 A CN 1590537A CN A031345360 A CNA031345360 A CN A031345360A CN 03134536 A CN03134536 A CN 03134536A CN 1590537 A CN1590537 A CN 1590537A
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Abstract
A process for separating and culturing the ectomesenchyme stem cells which can be used for bone tissue engineering, muscle tissue engineering, tooth tissue engineering and repairing peripheral nerve nicludes such steps as preparing culture medium, separating, culturing and purifying.
Description
Technical field
The invention belongs to the biomass cells technical field, relate to the ecto-mesenchymal stem cell isolation cultivation method.
Background technology
In the growth course of facial each projection of jaw, the neural crest cell (neural crest stem cell is hereinafter to be referred as NCSC) that derives from neurocele and surperficial ectoderm junction is through directional migration, propagation, and the arrival face mesoderm of dashing forward is called outer embryo mesenchyme.In embryo's growth course, ecto-mesenchymal stem cell can further break up to a plurality of pedigrees such as odontoblast, pulp cells, scleroblast, sarcoplast, spongiocyte, neurones again.Compare with other stem cell (as embryonic stem cell, mesenchymal stem cells MSCs, neural crest cell and neural stem cell) that is applied to organizational project, it has the following advantages: 1. the source is abundant, draws materials easily; 2. rate of propagation is fast, the sustainable biography of people's gnathism mescenchymal stem cell tens of generations; 3. can be used for heteroplastic transplantation, clinical application range is wide; 4. under effect of cytokines, can keep undifferentiated state, can break up to other pedigree under the natural situation; 5. induce down at exogenous factor, can be divided into the cell of the multiple differentiation of end eventually,, in organizational project tooth and peripheral nerve reparation, have important theoretical research and use value comprising odontoblast, schwann cell etc.Ecto-mesenchymal stem cell is taken from the organism body early embryo, the characteristic that has kept stem cell, has stronger multiplication capacity, studies show that, under the effect of exogenous somatomedin, ecto-mesenchymal stem cell can be divided into odontoblast, also can be induced to differentiate into multiple functioning cell, as scleroblast, Skeletal Muscle Cell, schwann cell, odontoblast etc., be suitable as the seed cell of organizational project, combine, utilize the multidirectional differentiation potential of this cell with various biomaterials, be used to make up tissue engineered bone, muscle, neural, tooth, and be used for defect repair.
At present, both at home and abroad the research of NCSC mainly is confined in the non-oviparity system, Shang Weijian separates, turns out any reported in literature and the relevant patent of NCSC.
Summary of the invention
Have above-mentioned so many advantage in view of NCSC and do not studied, the present situation of development and utilization, the object of the present invention is to provide the isolation cultivation method of ecto-mesenchymal stem cell.And the new purposes of the NCSC of proposition the inventive method preparation.
Isolation cultivation method of the present invention is characterised in that: it comprises the purifying of the separation of ecto-mesenchymal stem cell culture medium preparation, ecto-mesenchymal stem cell and cultivation, ecto-mesenchymal stem cell, and concrete steps have:
(1) ecto-mesenchymal stem cell culture medium preparation:
A. get commercial minimum must nutrient solution DMEM and commercial nutrient solution F12 by the basic culture solution that is mixed and made at 1: 1;
B. in basic culture solution, add sodium bicarbonate, foetal calf serum, L-glutamy ammonium, sodium bicarbonate, penicillin/streptomycin, Prostatropin (bFGF), Niu Chuiti extract (BPE) and leukaemia inhibitory factor (LIF), fully stirring and evenly mixing; Accent pH is 7.4-7.5;
C. super clean bench suction filtration sterilization, 4 ℃ of preservations are standby;
(2) separation of ecto-mesenchymal stem cell and cultivation:
A. under aseptic condition, cut the 40-50 days fetuses of voluntary termination of pregnancy or the last mandibular process of pregnant 11-12 days mouse embryos, cut into 1mm
3Size, 37 ℃ of digestion of 2.5g/l pancreatin;
B. the nutrient solution that contains serum that adds equivalent stops digestion, and piping and druming makes tissue be dispersed into cell suspension, and is centrifugal, abandons supernatant, adds nutrient solution branch is packed in the culturing bottle, puts in 37 ℃, the incubator of 5%CO2;
(3) purifying of ecto-mesenchymal stem cell:
A. be commissioned to train when supporting former, single cell suspension is abandoned supernatant after being inoculated in and being cultured to most of cell attachment in the culturing bottle, adds new nutrient solution and continues cultivation;
B. in the time of at the bottom of cell is paved with bottle, had digestive transfer culture has the magnetic bead separation and purification of specific antibody HNK-1 (CD57) to obtain ecto-mesenchymal stem cell by crosslinked again.
In the cultural method of above-mentioned outer embryo mesenchymal cell, the amount that adds each component in basic culture solution is:
Composition 1000ml volume
Foetal calf serum (5-15%) 50-150ml
Sodium bicarbonate 4-6g
L-glutamy ammonium 0.1-0.3g
Prostatropin 5-15 μ g
Ox hypophysis extract 0.1-20ml
Leukaemia inhibitory factor 106U
Penicillin/streptomycin 50-150i.u
The purposes of ecto-mesenchymal stem cell is:
1. ecto-mesenchymal stem cell is used for bone tissue engineer
The ecto-mesenchymal stem cell that the jaw facial skeleton is then mainly originated from the cranial nerve ridge.Mouse, rat, the prominent ecto-mesenchymal stem cell of people embryo face have osteogenic ability and are proved.Experiment in vitro shows that in containing the mineralising induced liquid of sodium, vitamins C, dexamethasone, a series of variation has appearred in outer embryo mesenchymal cell.Cellular form becomes polygon, cube by the one-tenth fiber-like of fusiformis, and volume increases simultaneously, and cell proliferation rate is slack-off, expresses type i collagen.Alkaline phosphatase activities increases and the expression of Bone Gla protein shows that further this cell has been divided into scleroblast.With the prolongation of incubation time, the nodular formation confirmation of mineralising inducing cell has been secreted mineralising matrix.These a series of changes show that outer embryo mesenchymal cell successfully is induced to differentiate into scleroblast.Cell after external evoked is directly implanted in the animal body, perhaps compound with collagen, forging bone, coral, repairing bone defect, all visible defective region has sophisticated new bone forming, and begins to rebuild the bone defective region.Show that ecto-mesenchymal stem cell is used to make up tissue engineered bone and is used for defect repair is feasible after inducing.
2. the embryo mescenchymal stem cell is used for the muscle tissue engineering
The research of muscle tissue engineering mostly is conceived to the angle of Transplanted cells and sets out.Sarcoplast has stronger differentiation and proliferation ability, and it is seed cell that sarcoplast is adopted in the research of muscle tissue engineering aspect at present more.Another approach in seed cell source is to obtain corresponding seed cell by Stem Cell Engineering.Embryonic stem cell and mescenchymal stem cell have the ability that is divided into muscle cell.And studies show that of ecto-mesenchymal stem cell, it is that the another useful of stem cell muscle tissue engineering seed cell source replenished.Ecto-mesenchymal stem cell has the ability that is divided into muscle cell.Studies show that, very obvious when ecto-mesenchymal stem cell exists at no differentiation inhibitors to the trend of smooth muscle cell differentiation, and under TGF-β effect, the ratio of differentiation can reach 95%.Under the effect of DEMSO, ecto-mesenchymal stem cell can be divided into Skeletal Muscle Cell.If ecto-mesenchymal stem cell is at external evoked smooth muscle cell, the Skeletal Muscle Cell of being divided into, mutually compound with collagen or other biological material again, be transplanted in the body, it is damaged to repair muscle.
3. ecto-mesenchymal stem cell is used for the peripheral nerve reparation
Studies show that ecto-mesenchymal stem cell can be divided into schwann cell under the effect of Buddhist screw thread woods (forskolin), Niu Chuiti extract.By directional induction gnathism mescenchymal stem cell directed differentiation is schwann cell, might search out the new seed cell source of tissue engineering nerve.By compound with the chitosan/collagen nerve trachea of development, the bridge joint sciatic nerve broken ends of fractured bone is observed the regeneration of neural axon, and new thinking and valid approach more will be provided for the treatment of exploring neurotization, injury repairing.
4. ecto-mesenchymal stem cell is used for the dental tissue engineering
Except that enamel, comprise that dentine, dental cement, dental pulp, periodontal tissue are all from ecto-mesenchymal stem cell.Oral epithelium and mesenchyme interact, and regulate and control the growth of tooth jointly.Early stage in dental germ during development, the oral epithelium of partial thickening forms dental lamina and also stretches in the deep layer reticular tissue, is provided as tooth information in FX, instruct its below and around ecto-mesenchymal stem cell break up to tooth source property mesenchymal cell.The dental lamina epithelium of this moment claims tooth source property epithelium again, grows in the future for becoming enamal organ, and forms enamel.Under the inducing of tooth source property epithelium, the part of tooth source property mesenchyme next-door neighbour IEE will be divided into odontoblast, the justacrine dentin matrix, mineralising forms dentine, the dental papilla cells of below further is divided into pulp tissue's cell, and part Dental Follicle Cells on every side is differentiated to form dental cement, thereby causes the morphology of tooth to take place.Ecto-mesenchymal stem cell is the most suitable seed cell of dental tissue engineering.As the derived cell of the multiple structure of tooth, its multidirectional differentiation potential is the guarantee to the multiple structure differentiation of tooth.Studies show that under the effect of exogenous somatomedin, ecto-mesenchymal stem cell can be divided into odontoblast.
Embodiment
Present embodiment is the separation and the cultural method of the outer embryo mesenchymal cell of SD rat gnathism, may further comprise the steps:
1.SD the separation of the outer embryo mesenchymal cell of rat gnathism and former be commissioned to train foster
Disconnected neck is put to death pregnant 11-12 days SD rat, cut open the belly under the aseptic condition and take out the tire mouse, it is prominent to cut tire mouse top and bottom in Hank ' the s liquid, and the chopping back digested 8 minutes for 37 ℃ with the 2.5g/l pancreatin, and the nutrient solution that contains serum that adds equivalent stops digestion, piping and druming makes tissue be dispersed into cell suspension gently, 800 rev/mins centrifugal 6 minutes, abandon supernatant, add and to divide in the culturing bottle of packing into after nutrient solution makes the cell resuspending, add an amount of nutrient solution, put 37 ℃, 5%CO
2Incubator in 40 minutes, it is adherent that inverted microscope is observed down most of cell, abandons supernatant, add fresh medium and continue to cultivate, the next day observation of cell upgrowth situation, 2-3 day is changed nutrient solution.
2. go down to posterity and cultivate and preliminary purification
When cell covers with at the bottom of the culturing bottle, with 37 ℃ of digestion of 2.5g/l pancreatin 6 minutes, see that at inverted microscope most of cell has bounced back and floats in the nutrient solution, the nutrient solution that contains serum with equivalent stops digestion, and centrifugal outstanding again precipitation goes down to posterity at 1: 2.Abandon supernatant after 40 minutes for 37 ℃ and add that fresh medium is conventional to be cultivated.
3. immunomagnetic beads separation and purification
The single cell suspension of preparation is screened by magnetic bead, and the specific antibody that is adopted is CD57.Pretreated magnetic bead is put into the well-bound cell suspension with CD57, and uniform mixing is placed on hatched under the room temperature 30 minutes.The cell suspension that combines magnetic bead is put into high high-intensity magnetic field left standstill 5-10 minute, inhale the liquid that goes in the test tube gently; Test tube broken away from magnetic field and with D-Hank liquid flushing test tube wall the cell (being the CD57 positive cell) that sticks on the tube wall is washed make the purifying cells suspension.
4. before the 5th generation, adopting amplification culture medium, is 10% DMEM/F12, wherein adds the recombinant human LIF (production of Sigma company) of 1000U/ml, with recombinant human bfgf's (production of Sigma company) of 10ng/ml, 5% Niu Chuiti extract (production of Sigma company).
5. before the 5th generation, with cell be applied to make preparations for sowing research and produce.
The inventive method is simple, and is repeatable strong, easy to operation, for the scientific research of ecto-mesenchymal stem cell and application thereof, teaching, clinical application etc. can play inestimable and effect, also breeding huge social benefit and economic benefit simultaneously.
Claims (6)
1, the separation of ecto-mesenchymal stem cell and cultural method is characterized in that: comprise the purifying of the separation of ecto-mesenchymal stem cell culture medium preparation, ecto-mesenchymal stem cell and cultivation, ecto-mesenchymal stem cell, concrete steps have:
(1) ecto-mesenchymal stem cell culture medium preparation:
A. get minimum must nutrient solution DMEM and nutrient solution F12 by the basic culture solution that mixes at 1: 1;
B. in basic culture solution, add foetal calf serum, L-glutamy ammonium, sodium bicarbonate, penicillin/streptomycin, Prostatropin, Niu Chuiti extract and leukaemia inhibitory factor, stirring and evenly mixing;
C. transferring pH is 7.4-7.5, and the sterilization of super clean bench suction filtration is preserved standby;
(2) separation of ecto-mesenchymal stem cell and cultivation:
A. under aseptic condition, cut the 40-50 days fetuses of voluntary termination of pregnancy or the last mandibular process of pregnant 11-12 days mouse embryos, trysinization;
B. the nutrient solution that contains serum that adds equivalent stops digestion, and piping and druming makes tissue be dispersed into cell suspension, and is centrifugal, abandons supernatant, adds to divide in the culturing bottle of packing into after nutrient solution makes the cell resuspending, puts in the incubator and hatches;
(3) purifying of ecto-mesenchymal stem cell:
A. be commissioned to train when supporting former, single cell suspension is abandoned supernatant after being inoculated in and being cultured to most of cell attachment in the culturing bottle, adds new nutrient solution and continues cultivation;
B. in the time of at the bottom of cell is paved with bottle, had digestive transfer culture has the magnetic bead separation and purification of specific antibody HNK-1 (CD57) to obtain ecto-mesenchymal stem cell by crosslinked again.
2, the separation of ecto-mesenchymal stem cell according to claim 1 and cultural method is characterized in that: substratum used in the cultural method of ecto-mesenchymal stem cell the ratio of each component is (in cumulative volume 1L): foetal calf serum 50-150ml, sodium bicarbonate 4-6g, L-glutamy ammonium 0.1-0.3g, Prostatropin 5-15ug, Niu Chuiti extract 0.1-20ml, leukaemia inhibitory factor 10
6U, penicillin or Streptomycin sulphate 50-150i.u.
3, according to the new purposes of the prepared ecto-mesenchymal stem cell of claim 1, it is characterized in that: ecto-mesenchymal stem cell is used to make up tissue engineered bone and be used for defect repair after inducing.
4, according to the new purposes of the prepared ecto-mesenchymal stem cell of claim 1, it is characterized in that: with ecto-mesenchymal stem cell at external evoked smooth muscle cell, the Skeletal Muscle Cell of being divided into, mutually compound with collagen or other biological material again, be transplanted in the body, it is damaged to repair muscle.
5, according to the new purposes of the prepared ecto-mesenchymal stem cell of claim 1, it is characterized in that: ecto-mesenchymal stem cell is a schwann cell by directional induction, can be used as the new seed cell source of tissue engineering nerve, is used for the peripheral nerve reparation; By compound with chitosan/collagen nerve trachea, but the bridge joint sciatic nerve broken ends of fractured bone.
6, according to the new purposes of the prepared ecto-mesenchymal stem cell of claim 1, it is characterized in that: ecto-mesenchymal stem cell is the most suitable seed cell of dental tissue engineering and be used for the dental tissue engineering; Under the effect of exogenous somatomedin, ecto-mesenchymal stem cell can be divided into odontoblast.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008071074A1 (en) * | 2006-12-13 | 2008-06-19 | Songling Wang | The use of mesenchymal stem cells and the separating and preserving method of stem cells from human tissues |
| WO2010040302A1 (en) * | 2008-10-10 | 2010-04-15 | 深圳市嘉天源生物科技有限公司 | Methods for isolating mesenchymal stem cells from embryos of human or animals and extracting secretion substances thereof |
| CN101215547B (en) * | 2008-01-15 | 2011-06-08 | 刘岱良 | Method for separating, purifying and extracting fat mesenchymal stem cell |
| CN101808672B (en) * | 2007-04-25 | 2013-05-01 | 干细胞旋转股份公司 | New stem cell lines, their application and culture methods |
| CN101802174B (en) * | 2007-09-11 | 2013-06-05 | 北海道公立大学法人札幌医科大学 | Cell growth method and pharmaceutical preparation for tissue repair and regeneration |
| CN109847098A (en) * | 2019-01-22 | 2019-06-07 | 江南大学 | A kind of compound bio timbering material for repairing bone defect |
| WO2021035679A1 (en) * | 2019-08-30 | 2021-03-04 | 江南大学 | Tissue engineered nerve graft and preparation method therefor |
-
2003
- 2003-09-02 CN CNA031345360A patent/CN1590537A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008071074A1 (en) * | 2006-12-13 | 2008-06-19 | Songling Wang | The use of mesenchymal stem cells and the separating and preserving method of stem cells from human tissues |
| CN101808672B (en) * | 2007-04-25 | 2013-05-01 | 干细胞旋转股份公司 | New stem cell lines, their application and culture methods |
| CN101802174B (en) * | 2007-09-11 | 2013-06-05 | 北海道公立大学法人札幌医科大学 | Cell growth method and pharmaceutical preparation for tissue repair and regeneration |
| CN101215547B (en) * | 2008-01-15 | 2011-06-08 | 刘岱良 | Method for separating, purifying and extracting fat mesenchymal stem cell |
| WO2010040302A1 (en) * | 2008-10-10 | 2010-04-15 | 深圳市嘉天源生物科技有限公司 | Methods for isolating mesenchymal stem cells from embryos of human or animals and extracting secretion substances thereof |
| CN109847098A (en) * | 2019-01-22 | 2019-06-07 | 江南大学 | A kind of compound bio timbering material for repairing bone defect |
| WO2021035679A1 (en) * | 2019-08-30 | 2021-03-04 | 江南大学 | Tissue engineered nerve graft and preparation method therefor |
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