CN102517251A - Mesenchymal stem cells, as well as preparation method and application thereof - Google Patents
Mesenchymal stem cells, as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of cell engineering and discloses mesenchymal stem cells, as well as a preparation method and an application thereof. The method comprises the following steps of cutting an umbilical cord into small pieces after removing an umbilical artery and umbilical veins, digesting with an alpha MEM (minimum essential medium) culture medium containing 0.1% of type IV collagenase and a complete medium, then centrifugating cell suspension obtained after digestion, removing supernatant liquid, then washing, further performing resuspension with the complete medium, transferring suspension after resuspension into a culture bottle coated by a wall-adhered matrix for culture till the emergence of shuttle-shaped wall-adhered cells, washing, and replacing the new complete medium for continuing the culture so as to get the shuttle-shaped wall-adhered cells after the culture, namely the mesenchymal stem cells. According to the preparation method disclosed by the invention, the culture medium without serum or foreign protein is used for preparation, a recombinase with a single component is used for passage, and the prepared mesenchymal stem cells are smaller in diameter, have no sensibiligen and can improve the vitality of the cells and increase the safety in regeneration treatment of a patient.
Description
Technical field
The present invention relates to the cell engineering field, a kind of mescenchymal stem cell specifically is provided.
Background technology
Mescenchymal stem cell is found in marrow at first, because of it has multidirectional differentiation potential, hematopoiesis support and promotes characteristics such as stem cell implantation, immunoregulation and self-replacation to receive people's attention day by day.Mescenchymal stem cell is in vivo or under the external specific inductive condition; Can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium; Still have multidirectional differentiation potential after continuous passage cultivation and the freezing preservation, can be used as the ideal seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes.
Bibliographical information people umbilical cord Wharton ' s is arranged in recent years, and (transliteration: magnificent Dun Shi) colloid also is rich in mescenchymal stem cell.The mescenchymal stem cell of umbilical cord mesenchymal stem cells and derived from bone marrow is similar, expresses some total surface markers, has higher self-regeneration ability, can be divided into adipocyte, chondrocyte, scleroblast, neurocyte, myocardial cell and endotheliocyte.Experimental results such as Baksh D show that the umbilical cord mesenchymal stem cells multiplication capacity is higher than mesenchymal stem cells MSCs.Because bone marrow aspiration operation is that the operation of wound property is arranged for donor, and gathers bone marrow fluid to the mescenchymal stem cell of cultivating the clinical treatment order of magnitude certainly and need the 4-6 time-of-week.Health is cutd open the palace and is produced umbilical cord and belonged to clinical waste in the past, gather umbilical cord puerpera and newborn infant are had no effect, and the qualified umbilical cord mesenchymal stem cells of preparation can set up the mesenchyma stem cell liquid nitrogen and preserves, and can increase in a short time obtains clinical treatment order of magnitude cell.The stroma cell in people's umbilical cord Wharton ' s colloid source has broad application prospects aspect regenerative therapy.
The cultivation umbilical cord derived mesenchymal stem cell methods of domestic and foreign literature report is that low sugar DMEM or α MEM substratum add the 5-20% foetal calf serum at present; Simultaneously can add the human mesenchymal stem cell relative growth factor, like fibroblast growth factor FGF or platelet derived growth factor PDGF.Umbilical cord mesenchymal stem cells goes down to posterity and is cultured to P2 generation or P3 can reach the requirement that is applied to regenerative therapy for purity, existing preparation method when going down to posterity with the pancreatin and the EDTA.Na of pig pancreas extraction
2The cultivation of going down to posterity behind the digestion previous generation cell.But mix a small amount of bovine serum albumin, pig pancreatin in the mescenchymal stem cell goods of the existing method preparation of application; The two all is clear and definite high sensitinogen; The mesenchyma stem cell suspension that the patient accepts present method preparation can produce corresponding antibody; Reduce mescenchymal stem cell treatment curative effect and increase anaphylaxis and take place, and the potential risk that infects the bovine viral that comprises ox brain spongiform disease is arranged.
In addition; Researchs such as Furlani D show that the mescenchymal stem cell that utilizes foetal calf serum to cultivate has cell dia more than 10% greater than 45 μ m; The bigger blocking action for the microcirculation capillary bed of cell dia is obvious more; Can cause dead mouse when serious under heavy dose of venoclysis test conditions, this has brought the potential threat for the patient utilizes mescenchymal stem cell to carry out regenerative therapy.Simultaneously, when going down to posterity, pig trysinization target spot is more, and is big to the toxicity of mescenchymal stem cell, and then influences the vigor of cell.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of mescenchymal stem cell, make that the mescenchymal stem cell diameter of this preparing method's preparation is less, reduce the probability that causes anaphylaxis, improve the passage cell vigor.
For realizing above goal of the invention, the present invention provides following technical scheme:
A kind of preparation method of mescenchymal stem cell is cut into small pieces after umbilical cord removed Umbilical artery and umbilical vein, with α MEM substratum that contains 0.1%IV Collagen Type VI enzyme and perfect medium digestion; Then that the cell suspension that obtains after umbilical cord fritter and the digestion is centrifugal; Go the supernatant after scouring to remove collagenase and collagen, then resuspended with perfect medium, the suspension after resuspended is transferred in the culturing bottle that was encapsulated by adherent matrix to be cultivated; Not attached cell is removed in time washing to the fusiformis attached cell occurring; And the perfect medium that more renews continue to cultivate, and changes a perfect medium in later per 2 days, reaches 80-85% to the degree of converging of fusiformis attached cell and promptly gets;
Said perfect medium is the α MEM substratum that contains 15-25ng/ml FGF, 15-25ng/ml PDGF, 15-25ng/m1 human albumin, 15-25ng/ml Regular Insulin, 15-25ng/ml sodium selenate and 15-25ng/ml ferritin;
The umbilical cord that the present invention adopts is provided by University Of Suzhou's first affiliated hospital's Obstetric and Gynecologic Department, and the pluripara agrees and the signature Informed Consent Form before gathering.The existing method for preparing umbilical cord mesenchymal stem cells; Owing to adopt foetal calf serum, make mescenchymal stem cell have heterogenous animal albumen, increase it causes anaphylaxis in patient's body probability; The mescenchymal stem cell diameter of existing preparation is bigger simultaneously; When being used for regenerative therapy, can play blocking action to the microcirculation capillary bed, threaten to the patient.
For this reason; The present invention optimizes medium component; The perfect medium of selecting for use new serum-free not have the animal foreign protein prepares, and reduced the probability of mescenchymal stem cell initiation anaphylaxis greatly, and the mescenchymal stem cell diameter of final preparation detects through inverted microscope; Average out to 26 μ m, and the cell dia average out to 35 μ m that utilize existing method to prepare.Simultaneously, detect through the injected in mice test, mescenchymal stem cell injection of the present invention back mouse death rate is merely 10%, and the mescenchymal stem cell mouse death rate of existing method preparation is 100%.
Wherein, perfect medium according to the invention preferably adopts the MesenCult-ACF serum free medium, and available from Canadian Stemcell company, catalog number is 05420.Said adherent matrix can adopt the conventional adherent matrix in this area, and the present invention preferably adopts the adherent matrix that contains fibronectin, more preferably adopts the adherent matrix of MesenCult, available from Canadian Stemcell company.The α MEM substratum that contains 0.1%IV Collagen Type VI enzyme is available from Worthington company.α MEM substratum is the substratum that often use this area, is the Earle substratum of alpha improvement, available from U.S. Gibco company.
Digestion according to the invention was preferably under 37 ℃, 250rpm digestion 2 hours, and said cultivation is preferably at 37 ℃, 5%CO
2Cultivate under concentration, 95% relative humidity.
In the digestive process of reality, the umbilical cord fritter can not complete digestion become cell suspension, still has the less fritter of part residual, contains many mescenchymal stem cells on it, can get into next preparation link in the lump with cell suspension, and starting material avoid waste.The present invention is in order to remove collagenase and collagen in postdigestive washing; And the washing after the fusiformis attached cell occurs is in order to remove not attached cell; Twice washing can be adopted the conventional reagent that does not influence the cell growth in this area, and like PBS, the present invention preferably adopts α MEM substratum.
Continue in the culturing process in the present invention; Need regularly replace substratum according to general knowledge known in this field; The present invention is preferred whenever to change a perfect medium at a distance from 2 days, and in order to guarantee certain cell quantity, the present invention preferably collects when fusiformis attached cell degree of converging reaches 80-85%.
Prior art adopts the pig pancreatin to digest attached cell when going down to posterity; But the pig pancreatin is a kind of mixture that contains multiple protein; Mescenchymal stem cell can have some pig pancreatin, and the multiple heterogenous animal albumen in the pig pancreatin can increase the probability that causes anaphylaxis undoubtedly.And the pig pancreatin is bigger for the toxicity of mescenchymal stem cell, when going down to posterity, influences the vigor of cell.
For this reason, as preferred version, the present invention also comprises the culturing step that goes down to posterity; Fusiformis attached cell (being the previous generation mescenchymal stem cell) washing with above-mentioned cultivation; Digest to the fusiformis attached cell with TrypLE cell Dispase II then and separate, the cell suspension that obtains after the digestion is added the centrifugal supernatant that goes of perfect medium, mix the centrifugal supernatant that goes with solution after the adherent matrix of washing from adherent matrix; Washing is gone behind the supernatant resuspended with perfect medium once more; The gained suspension inoculation in the culturing bottle that was encapsulated by adherent matrix, is added perfect medium and cultivates, and the fusiformis attached cell after the cultivation is mescenchymal stem cell of future generation; Said perfect medium, adherent matrix, cultural method are with above-mentioned preparation method.
In cultivation is gone down to posterity in the present invention; Washing is in order to remove the albumen in the perfect medium residual in the previous generation mescenchymal stem cell for the first time; Washing for the second time is to be used to the mescenchymal stem cell that goes down to posterity and cultivate in order to maximize to collect; And for the third time the purpose of washing with wash for the first time identical, these three times washings all can adopt this area conventional do not influence the reagent that cell is grown, like PBS.
Suspension after the present invention is will be with perfect medium resuspended is inoculated in certain cell density in the culturing bottle that was encapsulated by adherent matrix, can obtain mescenchymal stem cell of future generation through cultivating.Wherein, as preferably, the cell density of inoculation is preferably 3 * 10
3Individual/cm
2In order to guarantee cell sufficient nutrition material and collect a certain amount of cell that the present invention is preferred in the cultivation of going down to posterity whenever to change a perfect medium at a distance from 2 days, and when fusiformis attached cell degree of converging of future generation reaches 80-85%, collected.
The digestion that the present invention adopts TrypLE Select recombinase to carry out cell separates; It is a kind of reconstitution cell digestive ferment that does not contain protein for animal; Do not contain ox and pig source foreign protein, can be used to digest attached cells such as attached cell such as CHO, HEK 293, A529, be a kind of enzyme of ability degradation of cell surface protein, a little less than the pancreatin that the proteic degraded damage of pair cell protrusion of surface is used always; Available from U.S. Gibco company, catalog number (Cat.No.) is 12563-001.Propagating method of the present invention is compared with existing propagating method, and the cell viability after it goes down to posterity has improved about 5% than the latter's cell viability.
Because the mescenchymal stem cell of preparing method's preparation according to the invention has above-mentioned advantage, so the present invention also provides a kind of mescenchymal stem cell by preparing method's preparation according to the invention, it can be applied in the preparation process of regenerative therapy goods.
Can know by above technical scheme; Preparing method according to the invention optimizes medium component; Adopt serum-free not have the medium preparation of animal foreign protein, prepared mescenchymal stem cell diameter is less and can reduce the occurrence probability of anaphylaxis, has increased the security of patient's regenerative therapy; Go down to posterity to cultivate and adopt little, the single recombinase of composition of cytotoxicity, improved the vigor of cell.
Description of drawings
Shown in Figure 1 is the fluidic cell that detects of mescenchymal stem cell CD29, CD73 immunophenotype figure as a result;
Shown in Figure 2 is the fluidic cell that detects of mescenchymal stem cell CD105, CD44 immunophenotype figure as a result;
Shown in Figure 3 is the fluidic cell that detects of mescenchymal stem cell HLA-ABC, CD45 immunophenotype figure as a result;
Shown in Figure 4 is the fluidic cell that detects of mescenchymal stem cell HLA-DR, CD31 immunophenotype figure as a result;
Shown in Figure 5 is the fluidic cell that detects of mescenchymal stem cell CD34 immunophenotype figure as a result.
Embodiment
The invention discloses a kind of mescenchymal stem cell, those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Method of the present invention is described through preferred embodiment, and the related personnel obviously can change or suitably change and combination methods and applications as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: utilize preparation method according to the invention to prepare mescenchymal stem cell
Umbilical cord transports: choose transmissible diseases such as hepatitis, syphilis, AIDS and detect the palace of cuing open negative and that do not have an obstetric complication and produce umbilical cord, before the art through puerpera's informed consent and sign Informed Consent Form.Prepare the aseptic PS liquid storage bottle of 250ml before the art and transport bottle as umbilical cord, interior dress 100ml α MEM substratum (Gibco Company products) adds 1000U heparin sodium and 100U penicillin/streptomycin.The long umbilical cord of 10-30cm is packed in the bottle, and 4 ℃ of conditions are delivered to the laboratory in following 2 hours.
Umbilical cord digestion: after umbilical cord is transported to the laboratory, use phosphate buffered saline buffer PBS (Gibco Company products) washing to remove blood and amniotic fluid 2 times.In 2 grades of Biohazard Safety Equipments, carrying out umbilical cord handles.Prepare an aseptic bag and include 1 medium size operation staight scissors, 1 medium size operation straight forceps, 1 glass culture dish, 1 100ml beaker that diameter is 10cm.With umbilical cord scissors is the umbilical cord section of 2-3cm, takes out Umbilical artery respectively and umbilical vein discards.The umbilical cord section is cut into 3mm * 3mm * 5mm fritter; Being transferred to the 50ml tapered bottom gathers in the third ethene centrifuge tube; Perfect medium (Stemcell company) to contain 0.1%IV Collagen Type VI enzyme α MEM substratum (Worthington company) and
(catalog number (Cat.No.) 05420) serum-free and do not have animal component is a Digestive system; Add 25ml and place the constant temperature shaking table with 37 ℃, 250rpm digestion 2 hours, the digestion back is with 3 times of α MEM substratum washing 3 times removals collagenase and collagens with the Digestive system volume.
Tissue Culture Flask wrapper sheet: carried out wrapper sheet in advance in 1 day.Dilute adherent matrix components (Stemcell company), T-75cm with 1: 28 ratio with PBS
2Culturing bottle adds the adherent matrix of 5ml, tightens bottle cap horizontal positioned ambient temperature overnight.Exhaust liquid component with the 10ml transfer pipet before using next day and avoid adherent of exposing cell.Aqua sterilisa is cleaned in aseptic aqua sterilisa washing 1 time, and room temperature leaves standstill 15min.
Umbilical cord mesenchymal stem cells is former be commissioned to train foster: will digest back umbilical cord fritter and cell suspension with 75-150ml
(Catalog#05420) serum-free not have the perfect medium of animal component resuspended.Suspension is transferred to the T-75cm that adherent matrix components encapsulated
2Culturing bottle places 37 ℃ and 5%CO
2, cultivated 48 hours in the 95% humidity incubator.Cultivate 48 as a child visible fusiformis attached cells; Exhaust nutrient solution also with α MEM substratum washing 1 time; Add fresh
serum free medium, changed liquid once in after this per 2 days.Reach 80%-85% until fusiformis attached cell degree of converging, the general time is 8-12 days, promptly gets P0 for mescenchymal stem cell.
Embodiment 2: the mescenchymal stem cell immunophenotype detects
P0 is for mesenchyma stem cell suspension in preparation, and the adjustment cell density is 4 * 10
5Individual/ml, every pipe adds the 0.5ml cell suspension, and every pipe adds the anti-CD29 of PE mark, CD105, CD73, CD44, HLA-DR, HLA-ABC, CD34, CD45, CD31 monoclonal antibody respectively, and the FC500 flow cytometer detects, and the result sees Fig. 1.Can know that by Fig. 1 the mescenchymal stem cell cellular immunization phenotype of the present invention's preparation meets generally acknowledged mescenchymal stem cell phenotype fully.
Embodiment 3: the mescenchymal stem cell differentiation potential detects
1, adipocyte differentiation
P0 presses 5 * 10 for mescenchymal stem cell
3Individual/cm
2Cell density is inoculated 6 orifice plates, induces the differentiation perfect medium for adding the low sugar DMEM substratum of 1 μ M DEXAMETHASONE BP98,500 μ M isobutyl methylxanthines, 60 μ M indomethacins, 5mg/L Regular Insulin and 10% foetal calf serum.Renewed in per 3 days and induce differentiation liquid once.Inducing culture 21 days exhausts nutrient solution, and fixing 1 hour of 10% neutral formalin is got 1ml0.16% oil red O stain 20min.Exhaust dye liquor, 70% ethanol washs 1 time fast, adds the 1mlPBS mirror and observes down.The microscopy result shows that prepared mescenchymal stem cell can be divided into adipocyte.
2, osteoblast differentiation
P0 presses 5 * 10 for mescenchymal stem cell
3Individual/cm2 born of the same parents' density is inoculated 6 orifice plates, induces the differentiation perfect medium for adding the low sugar DMEM substratum of 10nM DEXAMETHASONE BP98,5mM β-glycerophosphate, 10nM Vitamin D3 500,000 I.U/GM, 50mg/L xitix and 10% foetal calf serum.Renewed in per 3 days and induce differentiation liquid once.Inducing culture 21 days carries out von Kossa dyeing.Exhaust nutrient solution, fixing 1 hour of 10% neutral formalin is got 1ml 5% Silver Nitrate room temperature lucifuge and is left standstill 20min.Exhaust Silver Nitrate, distilled water wash 1 time, uv lamp is irradiation 20min down, 5% hypo solution washing 1 time, 1 time mirror of distilled water wash is observed down.The microscopy result shows that prepared mescenchymal stem cell can be divided into osteocyte.
3, chondrocyte's differentiation
P0 is adjusted into 1.6 * 10 for mescenchymal stem cell
7Individual/the ml cell density, connect aseptic yellow rifle head 10-20 μ l multiple spot with pipettor and inoculate 6 orifice plates, the following 37 ℃ of incubators of saturated humidity leave standstill hatches 20min.Leave standstill the back and slowly add that to induce the differentiation perfect medium be to contain the low sugar DMEM substratum that adds 100nM DEXAMETHASONE BP98,10 μ g/L TGF-β 3 and 10% foetal calf serum.Renewed in per 3 days and induce differentiation liquid once.Inducing culture 21 days carries out Alcian Blue dyeing.Differentiation liquid is induced in exhaustion, and fixing 1 hour of 10% neutral formalin is got the acid Alcian Blue of 1ml1% incubated at room 20min.Exhaust dye liquor, distilled water wash 1 time, mirror is observed down.The microscopy result shows that prepared mescenchymal stem cell can be divided into the chondrocyte.
Embodiment 4: the cell dia of mescenchymal stem cell detects
Adopt the umbilical cord of identical source, quality; Utilize the method for the invention and traditional method (adding foetal calf serum cultivates) preparation mescenchymal stem cell respectively; Other culture condition are consistent; The mescenchymal stem cell for preparing separately being processed cell suspension be statically placed in the culturing bottle, adopt the high-end inverted microscope of LEICA or NIKON band camera to measure down, is unit with the micron.
The result shows, the cell dia of the mescenchymal stem cell of preparing method's preparation according to the invention is between 18-39 μ m, average 26 μ m; The cell dia of mescenchymal stem cell that utilizes traditional method preparation is between 20-61 μ m, average 35 μ m.
Embodiment 5: the injected in mice test of mescenchymal stem cell
In two kinds of mescenchymal stem cells difference intravenous injection to 10 20-23 gram normal female BALB/c mouse with embodiment 4 preparations, injection volume is 1.5 * 10
6Individual cell.Only there is 1 respiratory and circulatory failure to occur and death in 10 mouse of the mescenchymal stem cell that injection the present invention is prepared, and respiratory and circulatory failure all occurs and death in 10 mouse of the mescenchymal stem cell of injection traditional method preparation.Show capillary blood vessel degree of congestion when the prepared mescenchymal stem cell of injection the present invention can alleviate intravenous injection, reduce the injection untoward reaction, the danger when reducing regenerative therapy.
Embodiment 6: utilize P0 to cultivate the preparation mescenchymal stem cell for going down to posterity
Exhaust substratum, the P0 that embodiment 1 is prepared discards with PBS washing 1 time and exhaustion for mescenchymal stem cell.Add 2ml TrypLE Select recombinase (Gibco company, catalog number (Cat.No.) 12563-011) in the culturing bottle and cover the cell aufwuchsplate, place in 37 ℃ of incubators digestion 7-10min until attached cell from surface isolation.The cell suspension sucking-off is placed 50ml tapered bottom centrifuge tube; Add equal-volume
serum free medium mixing; 300 * g is centrifugal, and 8min removes supernatant; And with mix with the solution behind the 3ml PBS washing culturing bottle 1 time; The centrifugal supernatant that goes; After removing supernatant with the PBS washing once more, use
serum free medium washed cell suspension 1 time.T-75cm
2Culturing bottle encapsulated adherent matrix in advance in 1 day, by 3 * 10
3Individual/cm
2Cell density inoculation mescenchymal stem cell adds temperature in advance to 37 ℃
Serum free medium changed liquid once in after this per 2 days, reached 80%-85% until fusiformis attached cell degree of converging, and promptly got P1 for mescenchymal stem cell.Reaching P2 generation from P1 generation can be with reference to this method.
Embodiment 7: the mescenchymal stem cell vigor detects
The P0 that adopts embodiment 1 is for mescenchymal stem cell; Adopt go down to posterity cultural method and the tradition according to the invention cultural method (adopting pig trysinization isolated cell) that goes down to posterity respectively, other culture condition are consistent, and the cell of future generation of preparation is adopted 0.4% trypan blue dyeing back microscopy; Be that cell suspension mixes counting after back 2 minutes with equal-volume 0.4% trypan blue; Viable cell has refractivity and does not dye, and dead cell dyes blueness, cell viability=viable count ÷ (viable count+dead cell number) * 100%.
The result shows, utilizes the present invention to go down to posterity the mescenchymal stem cell vigor of future generation of cultural method preparation between 94-96%, average 95%; Utilize tradition to go down to posterity the mescenchymal stem cell vigor of future generation of cultural method preparation between 88-94%, average 90%, the more traditional propagating method of cell viability of the present invention is high by about 5%.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (10)
1. the preparation method of a mescenchymal stem cell is characterized in that, is cut into small pieces after umbilical cord is removed Umbilical artery and umbilical vein; With α MEM substratum that contains 0.1%IV Collagen Type VI enzyme and perfect medium digestion; The cell suspension that will obtain after will digesting then is centrifugal, removes the supernatant after scouring, and is then resuspended with perfect medium; Suspension after resuspended is transferred in the culturing bottle that was encapsulated by adherent matrix and cultivates; Wash when the fusiformis attached cell occurring, and the perfect medium that more renews continues to cultivate, the fusiformis attached cell after the cultivation is mescenchymal stem cell;
Said perfect medium is the α MEM substratum that contains 15-25ng/ml FGF, 15-25ng/ml PDGF, 15-25ng/ml human albumin, 15-25ng/ml Regular Insulin, 15-25ng/ml sodium selenate and 15-25ng/ml ferritin; The α MEM substratum of the said 0.1%IV of containing Collagen Type VI enzyme and the volume ratio of perfect medium are 4: 1.
2. according to the said preparation method of claim 1, it is characterized in that said perfect medium is the MesenCult-ACF serum free medium.
3. according to the said preparation method of claim 1, it is characterized in that said adherent matrix is the adherent matrix that contains fibronectin.
4. according to the said preparation method of claim 3, it is characterized in that said adherent matrix is the adherent matrix of MesenCult.
5. according to the said preparation method of claim 1, it is characterized in that said digestion is digestion 2 hours under 37 ℃, 250rpm.
6. according to the said preparation method of claim 1, it is characterized in that said cultivation is at 37 ℃, 5%CO
2Cultivate under concentration, 95% relative humidity.
7. according to the said preparation method of claim 1, it is characterized in that, also comprise the culturing step that goes down to posterity; With the said fusiformis attached cell washing of claim 1; Digest to the fusiformis attached cell with TrypLE Select recombinase then and separate, the cell suspension that obtains after the digestion is added the centrifugal supernatant that goes of perfect medium, mix the centrifugal supernatant that goes with solution after the adherent matrix of washing from adherent matrix; Washing is gone behind the supernatant resuspended with perfect medium once more; The gained suspension inoculation in the culturing bottle that was encapsulated by adherent matrix, is added perfect medium and cultivates, and the fusiformis attached cell after the cultivation is mescenchymal stem cell of future generation;
Said perfect medium, adherent matrix, cultural method are with claim 1.
8. according to the said preparation method of claim 7, it is characterized in that the cell density of said inoculation is 3 * 10
3Individual/cm
2
9. the mescenchymal stem cell of any said preparation method preparation of claim 1 to 8.
10. the application of the said mescenchymal stem cell of claim 9 in preparation regenerative therapy goods.
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014201986A1 (en) * | 2013-06-21 | 2014-12-24 | Zhang Wenwei | Monoclonal mesenchymal stem cell generation method and use thereof |
| CN105602893A (en) * | 2015-12-28 | 2016-05-25 | 上海吉泉生物技术有限公司 | Umbilical cord mesenchymal stem cell serum-free cultivation method and application |
| CN105891512A (en) * | 2016-05-11 | 2016-08-24 | 天津普瑞赛尔生物科技有限公司 | Kit for identifying umbilical cord mesenchymal stem cells |
| CN109337867A (en) * | 2018-11-23 | 2019-02-15 | 北京太东生物科技有限公司 | A kind of umbilical cord mesenchymal stem cells separation method |
| CN110172440A (en) * | 2019-04-18 | 2019-08-27 | 青岩生物科技(湖州)有限公司 | It is a kind of for cultivating the serum free medium of mesodermal stroma cell |
| CN110964693A (en) * | 2019-12-27 | 2020-04-07 | 河北银丰鼎诚生物技术有限公司 | Separation method of umbilical cord mesenchymal stem cells |
| CN111019891A (en) * | 2019-12-31 | 2020-04-17 | 上海林望生物科技有限公司 | Method for promoting human umbilical cord mesenchymal stem cell proliferation and application |
| CN112116555A (en) * | 2020-08-12 | 2020-12-22 | 清华大学深圳国际研究生院 | Method for detecting physiological function of mesenchymal stem cells and application thereof |
| CN113073077A (en) * | 2021-04-07 | 2021-07-06 | 德泉生物医学技术(深圳)有限公司 | Method for culturing clinical-grade umbilical cord blood mesenchymal stem cells by using closed system |
| CN114181899A (en) * | 2021-12-22 | 2022-03-15 | 中山大学孙逸仙纪念医院 | Method for obtaining mesenchymal stem cells from gum tissue of mice |
| CN117210401A (en) * | 2023-11-09 | 2023-12-12 | 江苏睿源生物技术有限公司 | Preparation and culture medium for promoting mesenchymal stem cell adherent growth and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101451124A (en) * | 2008-12-10 | 2009-06-10 | 戴育成 | Preparation of human umbilical cord mesenchymal stem cells wound surface smearing agent and storage application method |
| CN101629165A (en) * | 2009-07-24 | 2010-01-20 | 中国人民解放军海军总医院 | Preparation method of umbilical cord Huatong glue mesenchymal stem cells and product thereof |
| CN102311940A (en) * | 2010-07-09 | 2012-01-11 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing differentiation of stem cells into islet-like cells |
-
2012
- 2012-01-12 CN CN2012100089054A patent/CN102517251A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101451124A (en) * | 2008-12-10 | 2009-06-10 | 戴育成 | Preparation of human umbilical cord mesenchymal stem cells wound surface smearing agent and storage application method |
| CN101629165A (en) * | 2009-07-24 | 2010-01-20 | 中国人民解放军海军总医院 | Preparation method of umbilical cord Huatong glue mesenchymal stem cells and product thereof |
| CN102311940A (en) * | 2010-07-09 | 2012-01-11 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing differentiation of stem cells into islet-like cells |
Non-Patent Citations (4)
| Title |
|---|
| 侯克东等: "人脐带Wharton 胶中间充质干细胞的分离、培养与鉴定", 《解放军医学杂志》 * |
| 孙国栋等: "人脐带间充质干细胞的分离培养及向成骨成脂分化的实验研究", 《西安交通大学学报（医学版）》 * |
| 尹富华等: "人脐带间充质干细胞3 种分离制备方法的比较", 《临床检验杂志》 * |
| 梁旭竞: "永生化人肝细胞和人端粒酶基因脐带间充质干细胞分化的研究", 《中国博士学位论文全文数据库》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014201986A1 (en) * | 2013-06-21 | 2014-12-24 | Zhang Wenwei | Monoclonal mesenchymal stem cell generation method and use thereof |
| CN105602893A (en) * | 2015-12-28 | 2016-05-25 | 上海吉泉生物技术有限公司 | Umbilical cord mesenchymal stem cell serum-free cultivation method and application |
| CN105891512A (en) * | 2016-05-11 | 2016-08-24 | 天津普瑞赛尔生物科技有限公司 | Kit for identifying umbilical cord mesenchymal stem cells |
| CN109337867A (en) * | 2018-11-23 | 2019-02-15 | 北京太东生物科技有限公司 | A kind of umbilical cord mesenchymal stem cells separation method |
| CN109337867B (en) * | 2018-11-23 | 2019-10-25 | 北京太东生物科技有限公司 | A kind of umbilical cord mesenchymal stem cells separation method |
| CN110172440A (en) * | 2019-04-18 | 2019-08-27 | 青岩生物科技(湖州)有限公司 | It is a kind of for cultivating the serum free medium of mesodermal stroma cell |
| CN110964693A (en) * | 2019-12-27 | 2020-04-07 | 河北银丰鼎诚生物技术有限公司 | Separation method of umbilical cord mesenchymal stem cells |
| CN111019891A (en) * | 2019-12-31 | 2020-04-17 | 上海林望生物科技有限公司 | Method for promoting human umbilical cord mesenchymal stem cell proliferation and application |
| CN112116555A (en) * | 2020-08-12 | 2020-12-22 | 清华大学深圳国际研究生院 | Method for detecting physiological function of mesenchymal stem cells and application thereof |
| CN112116555B (en) * | 2020-08-12 | 2024-03-26 | 清华大学深圳国际研究生院 | Method for detecting physiological functions of mesenchymal stem cells and application thereof |
| CN113073077A (en) * | 2021-04-07 | 2021-07-06 | 德泉生物医学技术(深圳)有限公司 | Method for culturing clinical-grade umbilical cord blood mesenchymal stem cells by using closed system |
| CN114181899A (en) * | 2021-12-22 | 2022-03-15 | 中山大学孙逸仙纪念医院 | Method for obtaining mesenchymal stem cells from gum tissue of mice |
| CN117210401A (en) * | 2023-11-09 | 2023-12-12 | 江苏睿源生物技术有限公司 | Preparation and culture medium for promoting mesenchymal stem cell adherent growth and preparation method thereof |
| CN117210401B (en) * | 2023-11-09 | 2024-02-20 | 江苏睿源生物技术有限公司 | Preparation and culture medium for promoting mesenchymal stem cell adherent growth and preparation method thereof |
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