CN101629165B - Preparation method of original mesenchymal stem cell - Google Patents
Preparation method of original mesenchymal stem cell Download PDFInfo
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Abstract
The invention discloses a preparation method of original mesenchymal stem cells. In the invention, the mesenchymal stem cells are prepared by adopting fresh human umbilical cord, extracting Huatong glue, digesting by collagenase, and separating and cultivating, and the mesenchymal stem cells are suitable for cryopreservation. The invention also relates to the original mesenchymal stem cells prepared by the method, and the original mesenchymal stem cells are verified as mesenchymal stem cells by the identification of biological phenotype according to standard formulated by the International Society of Cytotherapy. The multiplication capability and the purity of the mesenchymal stem cell are both higher than those of bone mesenchymal stem cells; and the amount of the released growth factors and cell factors is higher than that of the bone mesenchymal stem cells.
Description
Technical field
The present invention relates to the preparation method of primary mesenchymal stem cells, relating in particular to people's umbilical cord Wharton jelly (Wharton ' jelly) is method of material prepn mescenchymal stem cell and products thereof.
Background technology
The present age, RESEARCH ON CELL-BIOLOGY showed that the mescenchymal stem cell (MSCs) with self, multidirectional differentiation potential can be applied to multiple treatment of diseases and rehabilitation.But, find that in research with in using there is variety of problems in seed cell, for example, there are problems such as ethics, law, immunological rejection and tumorigenicity in the application of embryonic stem cell; Problems such as there is complicated component in the ABM mononuclearcell, and the stem cell content with multidirectional differentiation potential is low, and differentiation potential is limited; ABM MSCs originates limited, and cell proliferation and differentiation potential obviously descends with the growth at age, and external long-term amplification reduces breaks up the potential of going back to the nest in the body.
With compare from body MSCs; Bleeding of the umbilicus, umbilical cord, placenta MSCs source is sufficient, the virus pollution probability is low, immunogenicity weak, cell is more original; There are not society, ethics and legal dispute; But bleeding of the umbilicus source MSCs separation efficiency is low, has limited its widespread use in cell therapy and organizational project; Umbilical cord, placenta isolated cell purity are low, often are mixed with that vascular endothelial cell is clinical can not be used.
Show that more than current still do not have a kind of ideal seed cell and satisfy clinical demand, need the source of expansion multipotential stem cell.
Summary of the invention
The technical problem that the present invention will solve is; Provide a kind of material source to be easy to get; The mescenchymal stem cell preparation method that incubation time is short, the mescenchymal stem cell for preparing by the inventive method has stronger self and multidirectional differentiation potential, and is easy to freezing preservation.
The technical scheme that realizes the object of the invention does; Adopting a kind of particular tissues of fetal cord---the logical glue (Wharton glue) of China is the raw material preparing mescenchymal stem cell; Be called the umbilical cord Wharton jelly mescenchymal stem cell (Umbilical Cord Wharton ' s Jelly-Derived MSCs, UW-MSCs).The UW-MSCs preparation method is: get the fresh umbilical cord of people, ooze balanced salt solution or serum-free culture based sols flush away umbilical cord remained blood to wait, remove Umbilical artery, umbilical vein and umbilical cord adventitia; Extract the logical glue of China and also cut into piece, again with wait ooze balanced salt solution or serum-free DMEM culture medium solution (Dulbecco ' s modification of Eagle ' s mediumDulbecco) flushing after, be soaked in 0.05～0.3% collagenase solution; Put in 35～37.5 ℃ of incubators 10～30 hours; Tissue block digestion, it is thick that liquid is, and adds the foetal calf serum substratum that contains that is equivalent to 1～10 times of amount of stoste then and stop digestion; Be sub-packed in culturing bottle or petridish, put CO
2Incubator is treated to change the culture medium solution that contains foetal calf serum behind the cell attachment, whenever changes liquid once at a distance from 2～4 days later, the cultivation of going down to posterity after cell attachment merges; Go down to posterity and cultivated for 2～4 generations, use or freezing preservation are subsequent use immediately.
In order to clean the residual blood of umbilical cord and in order to wash the logical glue tissue block of China used etc. ooze balanced salt solution and comprise D-Hank balance liquid, 0.9% sodium chloride solution, phosphate buffered saline buffer (PBS).
Clean the preferred D-Hank balance liquid of the used liquid of the residual blood of umbilical cord; The logical preferred serum-free DMEM of the used liquid of the glue tissue block culture medium solution of flushing China.
As optimization, the umbilical cord scissors after cleaning is cut into the sections that length is 2～10cm, get into subsequent processing.
As optimization, the umbilical cord Wharton jelly that strips out cut into be not more than 2mm
3Tissue block, get into subsequent processing.
As optimization, the concentration of foetal calf serum is 5～30% in the substratum.
For cultivating the stem cell of preparation by aforesaid method of the present invention, carry out the biology phenotypic evaluation according to the standard that " international cell therapy association " (ISCT) works out, show following technical characterictic:
1. the plastics system that under the type culture condition, adheres to is cultivated vessel and is become the fiber-like growth;
2. express CD90, CD105, CD44, CD73, HLA-ABC; Do not express CD45, CD34, CD80, CD86, HLA-DR;
3. external scleroblast and the adipocyte of being induced to differentiate into.
Umbilical cord Wharton jelly is the gluey stroma between umbilical cord artery, vein and epidermis, and the people of not seing before uses it for the cultivation of mescenchymal stem cell.Above-mentioned qualification result shows that the present invention isolating stem cell from the logical glue of umbilical cord mesenchymal China belongs to mescenchymal stem cell, and does not contain hemopoietic stem cell.Above-mentioned technical characterictic can be used as the prepared mescenchymal stem cell product acceptance criteria of the inventive method.
Mescenchymal stem cell (UW-MSCs) by the inventive method makes is compared with the mescenchymal stem cell (BMSCs) of derived from bone marrow, and the primary cell culture time shortened to 5-6 days from 10-12 days; The MSCs multiplication capacity, the purity that make by the inventive method all are higher than BMSCs; The growth factor that discharges, cytokine amount comprise STEMCELLFACTOR, granulocyte-colony factor, pHGF, transforming growth factor-beta, interleukin-8 etc. apparently higher than BMSCs, have shown more medicinal use.
What adopt in view of the present invention is that the allogeneic stem cell is originated as seed cell, i.e. the recessive allele seed cell.Than from body BMSCs, the key issue that the recessive allele seed cell is transplanted is the transplantation immunology problem.The present invention has carried out the UW-MSCs inside and outside immunogenicity evaluation of system for this reason.The result is:
Observation in vitro: carry out the UW-MSCs immunophenotype with flow cytometer and identify that UW-MSCs only expresses HLA-ABC, do not express HLA-II class antigen HLA-DR and collaborative stimulator antigen CD80, CD86 shows the external no immunogenicity of UW-MSCs.
UW-MSCs transplants the whole immunology in back and observes: 1. dynamically mixed leukocyte culture test finds that UW-MSCs has obvious inhibition proliferative response function to the allosome lymphocyte; 2. t lymphocyte subsets of peripheral blood detects, and the flow cytometer detection of dynamic is transplanted the back rat, and blood T lymphocyte CD 3, CD4, CD8 change all non-stimulated increase of UW-MSCs on every side; 3. the local organization pathology detect: light microscopic detects UW-MSCs graft area tract tissue and does not see histogenic immunity changes of reactivity such as lymphocyte, macrophage proliferation infiltration.
Press the performance of the primordial stem cell of the inventive method preparation because of its material therefor source and finished product cell; Can solve a multinomial difficult problem that perplexs medical circle in the stem cell Application Areas at present, for example: the 1. problems such as ethics, law, immunological rejection and tumorigenicity that exist of embryonic stem cell; 2. the differentiation and proliferation of the adult stem cell existence ability of going back to the nest is hanged down many disadvantages such as reaching preparation cycle length.
Description of drawings
Fig. 1 is to 80% fusion finding cellular form figure behind the sticking wall of primary cell.Wherein, A is the 1st day cellular form; B is the 3rd day cellular form; C is the 6th day cellular form.
Fig. 2 is that III detects different phenotype cell content figure for the UW-MSCs flow cytometer.
Fig. 3 is that III is for external evoked scleroblast and the adipocyte aspect graph of being divided into of UW-MSCs.Wherein, A is alkaline phosphatase; B is Su Su red staining; C as an oil red "0" stain.
Embodiment
Below through embodiment the present invention is described further.
Embodiment 1～6
Below enumerate 6 embodiment, implementation method of the present invention is not limited to these 6 kinds.For the practical application of the selecting for use of the selectable experiment material of part in the implementation process, the adjustable technical data of part, and each embodiment gained finished product partly detects the index tabulation as follows.
The comprehensive listed content of table that goes up, by the inventive method, it is following to adopt people's umbilical cord Wharton jelly to prepare the process step of primary mesenchymal stem cells:
(1) material selection: select to comprise hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody, 4 feminine genders of aids antibody immunity, prepare the caesarean healthy pregnant women of row.Intercepting umbilical cord when cesarean section, speed is put in the D-Hank balance liquid and is delivered to cell culture chamber, in super clean bench, umbilical cord is handled.
(2) material pre-treatment: with residual blood on D-Hank balance liquid or 0.9% sodium chloride solution, phosphate buffered saline buffer (PBS), the abundant flush away umbilical cord of any liquid of serum free medium.
Umbilical cord scissors after cleaning is cut into the sections that length is 2～10cm, each sections is removed Umbilical artery, umbilical vein and umbilical cord adventitia, extract the logical glue of China and cut into and be not more than 2mm
3Tissue block, wash with serum-free DMEM nutrient solution (Dulbecco ' s modified Eagle mediia) or D-Hank balance liquid, 0.9% sodium chloride solution, the logical to China glue tissue block of any liquid of phosphate buffered saline buffer.
Wherein, clean the preferred D-Hank balance liquid of the used liquid of the residual blood of umbilical cord; The logical preferred serum-free DMEM of the used liquid of the glue tissue block culture medium solution of flushing China.
(3) tissue block digestion: the logical glue tissue block of the China that will rinse well places 0.05～0.3% collagenase solution to digest.I～IV Collagen Type VI enzyme all can use, preferred II Collagen Type VI enzyme.The consumption of collagenase at least can the logical glue tissue block of submergence China.Placed 35～37.5 ℃ of incubators then 10～30 hours, the logical glue tissue block of China is digested, and it is thick that liquid is, and adds then to be equivalent to the substratum termination digestion that 1～10 times of amount of stoste contains foetal calf serum.The concentration of foetal calf serum is 5～30% in the substratum.Be sub-packed in culturing bottle or petridish.
(4) cell cultures: culturing bottle or petridish that the logical glue digest of China will be housed are put CO
2Incubator is cultivated.Treat to change the culture medium solution that contains foetal calf serum behind the cell attachment, whenever changed liquid once later, the cultivation of going down to posterity after cell attachment merges at a distance from 2～4 days.Go down to posterity and cultivated for 2～4 generations, be finished product umbilical cord Wharton jelly primary mesenchymal stem cells, use or freezing preservation are subsequent use immediately.
2～4 generation UW-MSCs to each embodiment obtained carry out identification and detection respectively, and the identification and detection project comprises
1. morphological observation: cell attachment becomes the fiber-like growth.
2. biology phenotypic evaluation: identify by international cell therapy association (ISCT) required standard in 2007.
1. the plastics system that under the type culture condition, adheres to is cultivated vessel and is become the fiber-like growth; (as shown in Figure 1)
2. express CD90, CD105, CD44, CD73, HLA-ABC.Their content is all above 90%; (as shown in Figure 2) do not expressed CD45, CD34, CD80, CD86, HLA-DR.Their content is all below 3%.
③ vitro differentiation into osteoblasts and adipocytes, alkaline phosphatase staining, Su Su red stain, oil red "0" staining were positive.(as shown in Figure 3)
Above qualification result shows by the inventive method isolating stem cell from umbilical cord mesenchymal Wharton glue and belongs to MSCs, and do not contain hemopoietic stem cell.
3. safety index detects: 2～4 generation UW-MSCs that each embodiment obtained are carried out safety index respectively detect, comprise 1. microbial culture; 2. virus detects; 3. detection of mycoplasma; 4. the immunology detection that comprises hepatitis B surface antigen, c-hepatitis antibody, syphilis antibody, aids antibody.These four detections of all batches are all negative.
Claims (4)
1. the preparation method of a primary mesenchymal stem cells is characterized in that, its process method is: get the fresh umbilical cord of people; With isoosmotic balanced salt solution or serum-free culture based sols flush away umbilical cord remained blood, remove Umbilical artery, umbilical vein and umbilical cord adventitia, extract the logical glue of China and cut into piece; With after the flushing of isoosmotic balanced salt solution or serum-free culture based sols, be soaked in 0.05～0.3% collagenase solution again, put in 35～37.5 ℃ of incubators 10～30 hours; Tissue block digestion, it is thick that liquid is, and adds the foetal calf serum substratum that contains that is equivalent to 1～10 times of amount of stoste then and stop digestion; Be sub-packed in culturing bottle or petridish, put CO
2Incubator is treated to change the culture medium solution that contains foetal calf serum behind the cell attachment, whenever changes liquid once at a distance from 2～4 days later, the cultivation of going down to posterity after cell attachment merges; Go down to posterity and cultivated for 2～4 generations, use or freezing preservation are subsequent use immediately.
2. the preparation method of primary mesenchymal stem cells according to claim 1; It is characterized in that it is that D-Hank balance liquid, 0.9% sodium chloride solution, phosphate buffered saline buffer are any that the grade that is used for cleaning residual blood and flushing China logical glue tissue block is oozed balanced salt solution.
3. according to the preparation method of the said primary mesenchymal stem cells of claim 1, it is characterized in that being used for digesting the magnificent collagenase that leads to the glue tissue block is that I～IV Collagen Type VI enzyme is any.
4. according to the preparation method of the said primary mesenchymal stem cells of claim 1, it is characterized in that the concentration of foetal calf serum is 5～30% in the cell culture medium.
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Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102198292B (en) * | 2010-03-26 | 2013-06-26 | 卢世璧 | Scaffolds of umbilical cord decellularized Wharton jelly for tissue engineering and preparation method thereof |
| PL2576768T3 (en) | 2010-06-01 | 2017-10-31 | Auxocell Laboratories Inc | Native wharton's jelly stem cells and their purification |
| CN102258809A (en) * | 2011-07-15 | 2011-11-30 | 中国人民解放军海军总医院 | Application of mesenchymal stem cells from human umbilical cord Wharton's jelly for preparation of cell transplantation materials |
| CN102517251A (en) * | 2012-01-12 | 2012-06-27 | 苏州大学附属第一医院 | Mesenchymal stem cells, as well as preparation method and application thereof |
| CN104164451A (en) * | 2014-08-09 | 2014-11-26 | 高连如 | Gene engineering stem cell for treating Type 2 diabetes |
| TWI525193B (en) * | 2014-10-20 | 2016-03-11 | 國璽幹細胞應用技術股份有限公司 | Uses of trans-cinnamaldehyde |
| CN105420183A (en) * | 2015-12-11 | 2016-03-23 | 郭镭 | Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord Wharton jelly tissue |
| CN105462919A (en) * | 2015-12-11 | 2016-04-06 | 郭镭 | Method for separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells) from wharton jelly tissue of umbilical cord |
| CN108456655A (en) * | 2018-02-23 | 2018-08-28 | 深圳至博生物科技有限公司 | Mescenchymal stem cell suspension and the preparation method and application thereof |
| CN108315296A (en) * | 2018-02-23 | 2018-07-24 | 深圳至博生物科技有限公司 | It the isolated culture method of mescenchymal stem cell and freezes, method for resuscitation |
| CN108496957A (en) * | 2018-06-04 | 2018-09-07 | 北京中广天生物科技有限公司 | The deepfreeze store method of umbilical cord mesenchymal stem cells culture supernatant |
| CN109762783A (en) * | 2019-03-20 | 2019-05-17 | 江苏瑞思坦生物科技有限公司 | A kind of preparation method of people's umbilical cord Wharton jelly mescenchymal stem cell |
| CN111544453B (en) * | 2020-04-27 | 2021-01-22 | 高连如 | Mixed stem cell injection containing three angiogenesis attributes and preparation method thereof |
| CN114231484A (en) * | 2022-01-06 | 2022-03-25 | 高连如 | Preparation method of ApoB antigen specific mesenchymal stem cells, and product and application thereof |
| CN115323498A (en) * | 2022-08-18 | 2022-11-11 | 宁波希诺赛生物科技有限公司 | Construction method and application of ready-to-use clinical-grade umbilical cord mesenchymal stem cell working library |
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