CN102021144A - Method for separating funicle mesenchyme stem cell - Google Patents
Method for separating funicle mesenchyme stem cell Download PDFInfo
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- CN102021144A CN102021144A CN2009101959221A CN200910195922A CN102021144A CN 102021144 A CN102021144 A CN 102021144A CN 2009101959221 A CN2009101959221 A CN 2009101959221A CN 200910195922 A CN200910195922 A CN 200910195922A CN 102021144 A CN102021144 A CN 102021144A
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Abstract
The invention belongs to the field of cell biology and relates to a method for rapidly and fully obtaining funicle mesenchyme stem cells. The method comprises the following steps of: removing artery and vein of the funicle obtained under the sterile condition, preparing into tissue fragments, adding a phosphate buffer solution containing penicillin and streptomycin to clean, centrifuging to remove supernate, digesting the funicle fragments with collagenase NB4 and hyaluron, placing in a shaker in an ethylenediamine tetraacetate phosphate solution for oscillation, filtering with a filter screen of 100-200 meshes, collecting the filtrate, finally, centrifuging for 5-10 minutes with 600g, removing supernate, and cleaning for two to three times with DMEM (dulbecco's modified eagle medium) culture medium to obtain the funicle mesenchyme stem cells. In the invention, a large amount of funicle mesenchyme stem cells can be obtained in short time. Proved by experiments, the obtained funicle stem cells express the specific markers of the stem cells and ensure induction differentiation to fat, bone, cartilage, and the like.
Description
Technical field
The invention belongs to the cytobiology field, relate to a kind of method that fully obtains umbilical cord mesenchymal stem cells fast.
Background technology
Along with the research of tissue engineering tissue, organ deepens continuously, the research of artificial absorbable material is gradually ripe, but restricts main bottleneck---the seed cell shortage of organizational project development, and is more and more outstanding.Making up tissue engineering product in the past mainly is to utilize into somatocyte, but becoming somatocyte is terminally differentiated cells, division growth is limited in one's ability, or just can't increase (as neurone) at all, is difficult to obtain at short notice the seed cell structure tissue or the organ of q.s.In addition, become somatocyte to draw materials and also limit to very much, there is the immunity rejection in the allosome source, and draw materials from body, can bring new wound, more some just can't carry out drawing materials from body at all, so very urgent, necessary of the source of seeking a kind of new cell therapy or tissue engineering seed cell.Because the stem cell cell has how powerful self duplication, the amplification ability, and can multinomially be divided into different histocytes, so become the new source of cell therapy and tissue engineering seed cell.
Stem cell mainly contains embryonic stem cell and adult stem cell; Embryonic stem cell has very powerful self duplication, amplification ability, reduced immunogenicity and verifiedly almost can be divided into all histocytes.But embryonic stem cell research is subjected to the restriction of ethics, law etc., and self also exists differentiation of stem cells residual, forms teratomatous danger, so study at present and use significant limitation is arranged all.
(mesenchymal stem cells MSCs) is a type of adult stem cell to mescenchymal stem cell, possesses the key character of stem cell: powerful self duplication ability, multinomial differentiation potential and reduced immunogenicity.MSCs originates from mesoderm, and existing a large amount of Chinese and foreign documents reports can be induced to differentiate into the tissue in mesoderm sources such as bone, cartilage, fat.Bibliographical information is arranged again in the recent period, and MSCs not only can be divided into mesoblastic stroma under suitable vivo and vitro environment, can also be induced to differentiate into the histocyte in entoderms such as liver cell, neurocyte, ectoderm source.Midbrain or striatal cell co-cultivation with MSCs and tire mouse, it can be divided into neurone and spongiocyte, and MSCs is transplanted to around the myocardial ischemia necrotic area, after 3 weeks gradually with myocardial cell's plesiomorphism on every side, and can see a large amount of new vesseles in original necrotic zone.At present Most scholars is thought the differentiation of MSCs by special transcription factor decision, and different inductive conditions can induce it to different direction differentiation, and these inductive conditions have started that decision differentiation direction gene must be transcribed, the rise of expression or expression amount.
Adult stem cell mainly refers to MSCs.The research of MSCs the earliest is mainly derived from marrow, and existing a large amount of at present Chinese and foreign documents report, and mesenchymal stem cells MSCs can be induced to differentiate into various histocytes such as fat, bone, cartilage, neurone and neuroglia.But marrow stem mescenchymal stem cell (BMSCs) gatherer process causes suffering to donor, and the isolated cell amount is limited, and its stem cell population and also significantly decline of multiplication capacity with age, and difficulty meets clinical needs.Studies show that recently bleeding of the umbilicus and umbilical cord contain a large amount of MSCs, but bleeding of the umbilicus MSC content rareness, separation difficulty is not suitable for the mass-producing operation, and it is gathered and also is faced with ethical restriction.
It is to adopt the logical glue of the collagenase digesting fetal cord China method of spending the night that tradition obtains human umbilical cord mesenchymal stem cells.But the suspension thickness that this method digestion back obtains be difficult to a large amount of umbilical cord mesenchymal stem cells of centrifugal acquisition, and digestion time is long, and cytoactive is poor, and being difficult to obtain can be for the MSCs of amplification usefulness.
Summary of the invention
The purpose of this invention is to provide a kind of method that fully obtains umbilical cord mesenchymal stem cells fast.
The present invention adopts the novel method of composite gum protoenzyme and the digestion of hyaluronic acid enzyme sequence to obtain a large amount of umbilical cord mesenchymal stem cells at short notice.
Particularly, a kind of method of separating umbilical cord mesenchymal stem cells of the present invention is characterized in that it comprises the steps:
The umbilical cord that (1) will obtain under aseptic situation is removed artery and vein, makes and organizes fragment, adds the phosphate buffered saline buffer that contains penicillin and Streptomycin sulphate again and cleans the centrifugal supernatant of abandoning;
(2) with the umbilical cord fragment that obtains in the step (1), add 2-4 and doubly be 0.1-0.2% collagenase NB4 digestion to the mass/volume ratio of fragment amount volume, the digestion temperature is 37 ℃, vibration digested 2-3 hour in shaking table; Get mixture 1;
(3) in the mixture 1 that step (2) is obtained, the mass volume ratio that adds its volume 1/4-1/2 amount is the Unidasa of 0.1-0.2%, and 37 ℃ digested 10-30 minute; Get mixture 2.
(4) in the mixture 2 that step (3) is obtained, adding isopyknic mass volume ratio is 0.01-0.02% ethylenediamine tetraacetic acid (EDTA) phosphate solution, and 37 ℃ of shaking tables vibrated 10-20 minute; Get mixture 3.
(5) mixture 3 that step (4) is obtained filters with the 100-200 mesh filter screen, collects filtered solution.
(6) filtered solution that step (5) is collected was with the centrifugal 5-10 of 600g minute, and abandoning supernatant is cleaned 2-3 time with the DMEM substratum, promptly obtains umbilical cord mesenchymal stem cells.
In the aforesaid method, penicillin and Streptomycin sulphate mainly are to remain under the gnotobasis to operate.
In the aforesaid method, phosphate buffered saline buffer contains the penicillin of 60-120u/ml or the Streptomycin sulphate of 60-120u/ml usually in the step (1).
In the aforesaid method, remove the umbilical cord artery and vein and need in field of microscope, carry out, so that complete place to go vascular tissue.
In the aforesaid method, organize the volume of fragment the smaller the better in the step (1), but shear time is unsuitable long, fragment is 0.5-1.5mm usually
3More suitable.
In the aforesaid method, the collagenase NB4 of 0.1-0.2% can be solution with DMEM in the step (2).The DMEM substratum is a kind ofly to develop on the basis of MEM substratum, contains the cell culture medium of each seed amino acid and glucose.Compare with MEM, increased various composition consumptions, the content according to its D (+)-glucose is divided into high glycoform (being lower than 4.5g/L) and low-sugar type (being lower than 1.0g/L) again simultaneously.High glycoform helps cell and berths in a position growth, is suitable for growing comparatively fast, adheres to more difficult tumour cell etc.The clone cultivates better with high sugared effect, is usually used in the myeloma cell of hybridoma and the transformant of DNA transfection and cultivates.The DMEM substratum is widely used in production of vaccine and various first cell cultures and single cell for the virus host cell cultivated; Cultivation as clones such as A9,3T6, BALB/3T3, COS-1, COS-3, COS-7, L6, WEHI-3b.As BIBCO, companies such as SIGMA are all on sale.
In the aforesaid method, collagenase NB4 digestion time is better 2.5-3 hour in the step (3).
In the aforesaid method, the Unidasa digestion time preferably is controlled in half an hour in the step (3).For example, 15,18,20,23,25 minutes etc.Be preferably 20-25 minute.
In the aforesaid method, filter screen is preferably the 100-120 order in the step (5).
The present invention studies show that, people's umbilical cord is rich in mescenchymal stem cell (HUCMSCs), it is a seed cell source that possible be better than marrow and other tissue, can overcome above all defectives, possess following advantage: (1) source is sufficient, draw materials conveniently, as a kind of childbirth waste, it is gathered and is not had any ethics problem; (2) length of every umbilical cord is at 40～60cm, and cell quantity is abundant, and multiplication capacity is strong; (3) isolated MSC immunophenotype is immature, has more weak immunocyte antigenicity.(4) HUCMSCs has the stem cell characteristic similar to BMSCs, has good self and multidirectional differentiation and proliferation ability, therefore can obtain more cell quantity in the short period of time, to satisfy clinical demand.Bright studies show that: the HUCMSCs that sets out is fat, cartilage, bone, neuron or neuroglia like cell external can inducing, and transplantation treatment Spinal Cord Injury in Rats or Parkinson's disease are broken up to neurone and astroglia cell in the microenvironment in vivo.
The present invention has used the novel method of composite gum protoenzyme and the digestion of hyaluronic acid enzyme sequence, can obtain a large amount of umbilical cord mesenchymal stem cells at short notice.And through experiment confirm, the umbilical cord stem cell of acquisition is expressed the specific marker of stem cell, can induce differentiation to fat, bone, cartilage etc., can be used as the new source of cell therapy and tissue engineering seed cell.
Embodiment
Embodiment 1
The umbilical cord that (1) will obtain under aseptic situation is removed the umbilical cord arteriovenous with the phosphate buffered saline buffer wash and remove residual blood that contains 100u/ml penicillin and 100u/ml Streptomycin sulphate with microinstrument, shreds to 1mm
3The size tissue block.The phosphate buffered saline buffer that adds 3 times cleans the centrifugal supernatant of abandoning.
(2) with the umbilical cord fragment that obtains in the step (1), adding 3 times of mass/volume ratios to the fragment amount is the digestion of 0.1% collagenase NB4DMEM solution, and the digestion temperature is 37 ℃, and vibration digestion is 3 hours in shaking table; Get mixture 1.
(3) in the mixture 1 that step (2) is obtained, the mass volume ratio that adds its 1/3 amount is 0.1% Unidasa, and 37 ℃ digested 25 minutes; Get mixture 2.
(4) in the mixture 2 that step (3) is obtained, adding isopyknic mass volume ratio is 0.015% ethylenediamine tetraacetic acid (EDTA) (EDTA) phosphate solution, and 37 ℃ of shaking tables vibrated 15 minutes; Get mixture 3.
(5) mixture 3 that step (4) is obtained filters with 100 mesh filter screens, collects filtered solution.
(6) filtered solution that step (5) is collected is with 600g centrifugal 10 minutes, and abandoning supernatant is cleaned 3 times with the DMEM substratum, promptly obtains umbilical cord mesenchymal stem cells.
Embodiment 2
The umbilical cord that (1) will obtain under aseptic situation is removed the umbilical cord arteriovenous with the phosphate buffered saline buffer wash and remove residual blood that contains 100u/ml penicillin and 100u/ml Streptomycin sulphate with microinstrument, shreds to 1mm
3The size tissue block.The phosphate buffered saline buffer that adds 3 times cleans the centrifugal supernatant of abandoning.
(2) with the umbilical cord fragment that obtains in the step (1), adding 3 times of mass/volume ratios to the fragment amount is the digestion of 0.2% collagenase NB4DMEM solution, and the digestion temperature is 37 ℃, and vibration digestion is 3 hours in shaking table; Get mixture 1.
(3) in the mixture 1 that step (2) is obtained, the mass volume ratio that adds its 1/3 amount is 0.1% Unidasa, and 37 ℃ digested 25 minutes; Get mixture 2.
(4) in the mixture 2 that step (3) is obtained, adding isopyknic mass volume ratio is 0.015% ethylenediamine tetraacetic acid (EDTA) (EDTA) phosphate solution, and 37 ℃ of shaking tables vibrated 15 minutes; Get mixture 3.
(5) mixture 3 that step (4) is obtained filters with 100 mesh filter screens, collects filtered solution.
(6) filtered solution that step (5) is collected is with 600g centrifugal 10 minutes, and abandoning supernatant is cleaned 3 times with the DMEM substratum, promptly obtains umbilical cord mesenchymal stem cells.
Embodiment 3
The umbilical cord that (1) will obtain under aseptic situation is removed the umbilical cord arteriovenous with the phosphate buffered saline buffer wash and remove residual blood that contains 100u/ml penicillin and 100u/ml Streptomycin sulphate with microinstrument, shreds to 1mmw size tissue block.The phosphate buffered saline buffer that adds 3 times cleans the centrifugal supernatant of abandoning.
(2) with the umbilical cord fragment that obtains in the step (1), adding 3 times of mass/volume ratios to the fragment amount is the digestion of 0.1% collagenase NB4DMEM solution, and the digestion temperature is 37 ℃, and vibration digestion is 3 hours in shaking table; Get mixture 1.
(3) in the mixture 1 that step (2) is obtained, the mass volume ratio that adds its 1/3 amount is 0.1% Unidasa, and 37 ℃ digested 25 minutes; Get mixture 2.
(4) in the mixture 2 that step (3) is obtained, adding isopyknic mass volume ratio is 0.015% ethylenediamine tetraacetic acid (EDTA) (EDTA) phosphate solution, and 37 ℃ of shaking tables vibrated 15 minutes; Get mixture 3.
(5) mixture 3 that step (4) is obtained filters with 120 mesh filter screens, collects filtered solution.
(6) filtered solution that step (5) is collected is with 600g centrifugal 10 minutes, and abandoning supernatant is cleaned 3 times with the DMEM substratum, promptly obtains umbilical cord mesenchymal stem cells.
Embodiment 4
The umbilical cord that (1) will obtain under aseptic situation is removed the umbilical cord arteriovenous with the phosphate buffered saline buffer wash and remove residual blood that contains 100u/ml penicillin and 100u/ml Streptomycin sulphate with microinstrument, shreds to 1mm3 size tissue block.The phosphate buffered saline buffer that adds 3 times cleans the centrifugal supernatant of abandoning.
(2) with the umbilical cord fragment that obtains in the step (1), adding 3 times of mass/volume ratios to the fragment amount is the digestion of 0.1% collagenase NB4DMEM solution, and the digestion temperature is 37 ℃, and vibration digestion is 2 hours in shaking table; Get mixture 1.
(3) in the mixture 1 that step (2) is obtained, the mass volume ratio that adds its 1/3 amount is 0.1% Unidasa, and 37 ℃ digested 10 minutes; Get mixture 2.
(4) in the mixture 2 that step (3) is obtained, adding isopyknic mass volume ratio is 0.015% ethylenediamine tetraacetic acid (EDTA) (EDTA) phosphate solution, and 37 ℃ of shaking tables vibrated 15 minutes; Get mixture 3.
(5) mixture 3 that step (4) is obtained filters with 100 mesh filter screens, collects filtered solution.
(6) filtered solution that step (5) is collected is with 600g centrifugal 10 minutes, and abandoning supernatant is cleaned 3 times with the DMEM substratum, promptly obtains umbilical cord mesenchymal stem cells.
The result shows that embodiment 1-4 all can obtain umbilical cord mesenchymal stem cells.And through experiment confirm, the umbilical cord stem cell of acquisition is expressed the specific marker of stem cell, can induce differentiation to fat, bone, cartilage etc.Wherein embodiment 2 and 4 effect are better.
Claims (8)
1. a method of separating umbilical cord mesenchymal stem cells is characterized in that, this method comprises the steps:
The umbilical cord that (1) will obtain under aseptic situation is removed artery and vein, makes and organizes fragment, adds the phosphate buffered saline buffer that contains penicillin and Streptomycin sulphate again and cleans the centrifugal supernatant of abandoning;
(2) with the umbilical cord fragment that obtains in the step (1), add 2-4 and doubly be 0.1-0.2% collagenase NB4 digestion to the mass/volume ratio of fragment amount volume, the digestion temperature is 37 ℃, vibration digested 2-3 hour in shaking table; Get mixture 1;
(3) in the mixture 1 that step (2) is obtained, the mass volume ratio that adds its volume 1/4-1/2 amount is the Unidasa of 0.1-0.2%, and 37 ℃ digested 10-30 minute; Get mixture 2.
(4) in the mixture 2 that step (3) is obtained, adding isopyknic mass volume ratio is 0.01-0.02% ethylenediamine tetraacetic acid (EDTA) phosphate solution, and 37 ℃ of shaking tables vibrated 10-20 minute; Get mixture 3.
(5) mixture 3 that step (4) is obtained filters with the 100-200 mesh filter screen, collects filtered solution.
(6) filtered solution that step (5) is collected was with the centrifugal 5-10 of 600g minute, and abandoning supernatant is cleaned 2-3 time with the DMEM substratum, promptly obtains umbilical cord mesenchymal stem cells.
2. method according to claim 1 is characterized in that the middle phosphate buffered saline buffer of step (1) contains the penicillin of 60-120u/ml.
3. method according to claim 1 is characterized in that the middle phosphate buffered saline buffer of step (1) contains the Streptomycin sulphate of 60-120u/ml.
4. method according to claim 1 is characterized in that organizing the volume of fragment in the step (1) is 0.5-1.5mm
3
5. method according to claim 1 is characterized in that the collagenase NB4 of 0.1-0.2% in the step (2) is solution with DMEM.
6. method according to claim 1 is characterized in that collagenase NB4 digestion time is 2.5-3 hour in the step (3).
7. method according to claim 1 is characterized in that the Unidasa digestion time is 20-25 minute in the step (3).
8. method according to claim 1 is characterized in that filter screen is the 100-120 order in the step (5).
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| CN2009101959221A CN102021144A (en) | 2009-09-18 | 2009-09-18 | Method for separating funicle mesenchyme stem cell |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266081A (en) * | 2012-01-21 | 2013-08-28 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| CN104232572A (en) * | 2014-08-20 | 2014-12-24 | 北京瑞思德生物科技有限公司 | Kit for preparing umbilical cord mesenchymal stem cells |
| CN106399235A (en) * | 2016-10-19 | 2017-02-15 | 浙江译美生物科技有限公司 | Method for isolating human umbilical cord mesenchymal stem cells |
| CN106867981A (en) * | 2017-04-14 | 2017-06-20 | 青岛青春派生物科技有限公司 | A kind of human umbilical tissue digestive ferment and preparation method thereof |
| CN110656087A (en) * | 2018-06-29 | 2020-01-07 | 李陶 | MANF gene modified umbilical cord mesenchymal stem cell and preparation method and application thereof |
-
2009
- 2009-09-18 CN CN2009101959221A patent/CN102021144A/en active Pending
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| SESHAREDDY K. ET AL.: "Method to isolate mesenchymal-like cells from Wharton’s Jelly of umbilical cord", 《METHODS CELL BIOL》 * |
| 杨卿 等: "人脐带间充质干细胞分离扩增方法的优化", 《中山大学学报（医学科学版）》 * |
| 赵磊 等: "人脐带间充质干细胞的分离培养及成脂成骨分化", 《中国老年学杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103266081A (en) * | 2012-01-21 | 2013-08-28 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| CN103266081B (en) * | 2012-01-21 | 2015-05-13 | 中国人民解放军军事医学科学院附属医院 | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord |
| CN104232572A (en) * | 2014-08-20 | 2014-12-24 | 北京瑞思德生物科技有限公司 | Kit for preparing umbilical cord mesenchymal stem cells |
| CN106399235A (en) * | 2016-10-19 | 2017-02-15 | 浙江译美生物科技有限公司 | Method for isolating human umbilical cord mesenchymal stem cells |
| CN106867981A (en) * | 2017-04-14 | 2017-06-20 | 青岛青春派生物科技有限公司 | A kind of human umbilical tissue digestive ferment and preparation method thereof |
| CN110656087A (en) * | 2018-06-29 | 2020-01-07 | 李陶 | MANF gene modified umbilical cord mesenchymal stem cell and preparation method and application thereof |
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Application publication date: 20110420 |