CN101914496B - Simplified method for preparing umbilical mesenchymal stem cells in large scale - Google Patents
Simplified method for preparing umbilical mesenchymal stem cells in large scale Download PDFInfo
- Publication number
- CN101914496B CN101914496B CN 201010246359 CN201010246359A CN101914496B CN 101914496 B CN101914496 B CN 101914496B CN 201010246359 CN201010246359 CN 201010246359 CN 201010246359 A CN201010246359 A CN 201010246359A CN 101914496 B CN101914496 B CN 101914496B
- Authority
- CN
- China
- Prior art keywords
- substratum
- umbilical cord
- days
- tissue
- stem cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a method for preparing umbilical mesenchymal stem cells in a large scale. In the method, although umbilical cord cutouts are larger, the primary culture time is greatly shortened. A medium contains bFGF and FBS, promotes adherence growth of mesenchymal stem cells and does not influence differentiation capacity of the stem cells. After 8 to 9 days, about 80 percent of cells are fused, the cells grow fast and the generation time is short.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of large-scale preparation method of mescenchymal stem cell.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSC) is the important member of stem cell family, derives to grow early stage mesoderm and ectoderm.MSC finds in marrow at first, because it has the concern that multi-lineage potential, hematopoiesis support and the characteristics such as the implantation of promotion stem cell, immunoregulation and self-replacation are subject to people day by day.As mescenchymal stem cell in vivo or under external specific inductive condition, can be divided into the Various Tissues cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, still have multi-lineage potential after continuous passage cultivation and freezing preservation, can be used as desirable seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes.
The mescenchymal stem cell clinical application is in solving multiple disease in the blood system, diabetes and diabetic foot, cardiovascular disorder, liver cirrhosis, Spinal injury, the nervous system disorderss such as Parkinson's disease, meniscus of knee joint Partial Resection injury repairing, the aspects such as autoimmune disorder have obtained important breakthrough, have saved the life of many sufferers.
Mescenchymal stem cell is distributed widely in the marrow, periosteum, spongy bone, fat, synovial membrane, skeletal muscle, tire liver, deciduous teeth, umbilical cord, Cord blood of fetus and adult, and wherein the mescenchymal stem cell quality in umbilical cord source is high, pure, quantity is many.The mescenchymal stem cell in umbilical cord source not only can become the desirable surrogate of mesenchymal stem cells MSCs, and has larger application potential.
Umbilical cord mesenchymal stem cells is expressed the peculiar molecular marker of multiple embryonic stem cell, have that differentiation potential is large, multiplication capacity is strong, immunogenicity is low, draw materials conveniently, the features such as restriction of amoral ethics problem, therefore might become the multipotential stem cell of tool potential applicability in clinical practice.But the separation and Extraction process of umbilical cord mesenchymal stem cells is loaded down with trivial details and extraction efficiency is low, and the mass-producing of mescenchymal stem cell has been prepared into the bottleneck of its widespread use.
Mescenchymal stem cell preparation at present to be adopted enzyme digestion and organizes adherent method to prepare.It is after umbilical cord tissue is cut into fragment that enzyme digestion extracts umbilical cord mesenchymal stem cells, with trypsinase and collagenase digesting 1-2 hour, stop enzymic digestion with the substratum that contains foetal calf serum, then washed cell discards digestive ferment, and the cell after washing is seeded to and cultivates amplification in culture vessel.Washed cell takes approximately 1 hour, and heavier to cell injury in washing process, and complex operation step is easily polluted, and digestive ferment is also heavier to cell injury, causes finally that the cell viability that obtains is low, propagation is slow.Organizing adherent method is more a kind of method of using at present, and traditional adherent method of organizing is that umbilical cord tissue is shredded, and requires tissue to shred degree very high, cut to the meat gruel shape, shear time more than 30min, time and effort consuming; Centrifugally wash away inoculation culture after tissue juice, the mescenchymal stem cell adherent growth is slower, needs 10-14 days more.
Enzyme digestion prepares mescenchymal stem cell length consuming time, and is larger to cell injury in operating process, and the mescenchymal stem cell vigor that makes separation obtain is low, propagation is slow.Organize adherent method requirement umbilical cord tissue to shred degree very high, the operation effort, and cell attachment is slow, and growth is slow.
Summary of the invention
The inventor is for above-mentioned shortcoming, extensively consult a large amount of domestic and foreign literatures, on the basis of existing mescenchymal stem cell technology of preparing, the adherent method technology of preparing is organized in improvement, the tissue block shear history is no longer required great effort, and add bFGF (Prostatropin) in nutrient solution, can accelerate the adherent of cell and growth, just make whole primary incubation time greatly shorten by original 10-14 days.
The present invention adopts following technical scheme:
The invention provides a kind of preparation method of umbilical cord mesenchymal stem cells, comprise the following steps:
1) fresh umbilical cord rinses with the PBS damping fluid; Described PBS damping fluid contains 100kU/L penicillin and 100mg/L Streptomycin sulphate;
2) the long umbilical cord of clip 6-8cm is cut into the long segment of 2cm with umbilical cord, repeatedly rinses with the PBS damping fluid; Described PBS damping fluid contains 100kU/L penicillin and 100mg/L Streptomycin sulphate;
3) the umbilical cord tissue piece is shredded into 2-3mm
3Fritter;
4) will shred tissue and add L-DMEM substratum washing, under the 650g condition centrifugal 5 minutes, abandon supernatant;
5) with tissue block and substratum 2.5-3 by volume: 1 ratio adds substratum, and the mixing tissue block is seeded in Tissue Culture Dish, puts incubator and cultivates; Described substratum is for containing the L-DMEM substratum of 1-10ng/ml Prostatropin (bFGF) and volume percent 10%-15% foetal calf serum (FBS);
6) add the described substratum of step 5 after 24 hours.
7) changed a subculture in every 3 days, change the L-DMEM substratum that contains volume ratio 10% foetal calf serum after the 6th day, reach to 8-9 days cells and go down to posterity when merge 80% left and right; The substratum that goes down to posterity is the L-DMEM substratum that contains volume ratio 10% foetal calf serum;
8) went down to posterity once in every 3 days after.
Description of drawings
Fig. 1: the cellular form of the mescenchymal stem cell that people's umbilical cord is originated: (A-C) be the mescenchymal stem cell cellular form of primary the 4th, 5,7 day that adopts method of the present invention to obtain; (D-F) be the mescenchymal stem cell first-generation the 1st, the cellular form after 2,3 days; (G) be the mescenchymal stem cell s-generation cellular form of the 3rd day; (H-J) be the mescenchymal stem cell third generation the 1st, the cellular form of 2,3 days.
Fig. 2: the cell growth curve of human umbilical cord mesenchymal stem cells
Fig. 3: the flow cytometry cell phenotype is analyzed
Technique effect
1, umbilical cord rinses thoroughly, contains microbiotic in washing fluid, can maximum wash away dirt and remaining blood, avoids polluting.
2, the umbilical cord scissors stripping and slicing is larger, and it is time saving and energy saving to shear.The primary incubation time of shortening that in the case still can be a large amount of.
Add the optimum proportion of substratum when 3, having determined the mixing tissue block, avoided substratum deficiency or unnecessary impact on cell cultures.
4, substratum is for containing the fresh perfect medium L-DMEM of bFGF (concentration 1-10ng/ml) and 10%-15%FBS.The lower concentration bFGF that adds had both promoted the mescenchymal stem cell adherent growth, did not affect again the differentiation capability of stem cell.
5, change substratum after the 6th day and be (L-DMEM+FBS), reach to 8-9 days cells and go down to posterity when merge 80% left and right, Growth of Cells is fast, and the generation time is short.
The tool present embodiment
1. fresh umbilical cord, through virus detect qualified after, ((pH7.2～7.4) contain NaCl 137mmol/L, KCl 2.7mmol/L, Na with the PBS damping fluid that contains 100kU/L penicillin and 100mg/L Streptomycin sulphate
2HPO
44.3mmol/L, KH
2PO
41.4mmol/) wash away dirt and blood.
2. the about 6-8cm umbilical cord of clip is cut into umbilical cord the segment of 2cm left and right, washes away umbilical vein and endarterial remaining blood with the PBS damping fluid that contains 100kU/L penicillin and 100mg/L Streptomycin sulphate as far as possible.
3. with umbilical cord scissors, the umbilical cord tissue piece is shredded into 2-3mm
3Fritter.
4. will shred tissue adds the L-DMEM substratum (Gibco company, USA) washing under the 650g condition centrifugal 5 minutes, is abandoned supernatant.
5. according to 2.5-3: 1 ratio (tissue block: substratum, volume ratio) add substratum (L-DMEM+FBS+bFGF) (namely, the L-DMEM substratum that contains 1-10ng/ml bFGF and volume percent 10%-15%FBS) mixing tissue block, be seeded in Tissue Culture Dish, put 37 ℃, 5%CO
2The saturated humidity incubator cultivate.Described FBS is foetal calf serum, and bFGF is Prostatropin.
6.24 add the described substratum of step 5 after hour.
7. changed a subculture in every 3 days, see that spindle cell climbs out of around 5-6 days tissue block.Change substratum after the 6th day and be (L-DMEM+ volume ratio 10%FBS), reach to 8-9 days cells and go down to posterity when merge 80% left and right; The substratum that goes down to posterity is L-DMEM+ volume ratio 10%FBS.
8. went down to posterity once in every 3 days later on, identify to the third generation.
Fig. 1, Fig. 2, Fig. 3 are seen in the cellular form of the mescenchymal stem cell in each stage, upgrowth situation, cell phenotype analysis.Control experiment (conventional organization piece adherent method)
1. get umbilical cord, stroke-physiological saline solution is rinsed crude removal and blood;
2. aseptic hammer-shears umbilical cord is to 1-2mm
3Size.
3. tissue block is transferred in the centrifuge tube of 50ml, adds the PBS damping fluid 40ml that contains 1% mycillin, room temperature under the 250g condition centrifugal 5 minutes is abandoned supernatant.
The tissue block that obtains with containing the DMEM culture medium culturing of volume ratio 10%, changed once in every 3 days and to cultivate.
5.7 have fusiformis and polygon cell to climb out of around it left and right tissue block, 10-14 days left and right cells just reach 80% left and right and merge, and go down to posterity with 0.25% trysinization.
Experimental result: method of the present invention, contain microbiotic in the umbilical cord washing fluid, can maximum wash away dirt and remaining blood, avoided pollution; Though the umbilical cord scissors stripping and slicing is larger, but still the primary incubation time of shortening that can be a large amount of; Add the optimum proportion of substratum when having determined the mixing tissue block, substratum is for containing the fresh perfect medium L-DMEM of bFGF (concentration 1-10ng/ml) and 10%-15%FBS, both promoted the mescenchymal stem cell adherent growth, do not affect again the differentiation capability of stem cell, namely reaching 80% left and right to 8-9 days cells merges, Growth of Cells is fast, and the generation time is short.
Claims (1)
1. the preparation method of a umbilical cord mesenchymal stem cells is characterized in that comprising the following steps:
1) fresh umbilical cord is with containing NaCl 137mmol/L, KCl 2.7mmol/L, Na
2HPO
44.3mmol/L, KH
2PO
41.4mmol/ and the PBS damping fluid that contains 100kU/L penicillin and 100mg/L Streptomycin sulphate pH7.2～7.4 washes away dirt and blood;
2) the long umbilical cord of clip 6-8cm is cut into the long segment of 2cm with umbilical cord, washes away umbilical vein and endarterial remaining blood with the PBS damping fluid that contains 100kU/L penicillin and 100mg/L Streptomycin sulphate as far as possible;
3) the umbilical cord tissue piece is shredded into 2-3mm
3Fritter;
4) will shred tissue and add L-DMEM substratum washing, under the 650g condition centrifugal 5 minutes, abandon supernatant;
5) with tissue block and substratum 2.5-3 by volume: 1 ratio adds substratum, and the mixing tissue block is seeded in Tissue Culture Dish, puts incubator and cultivates; Described substratum is the L-DMEM substratum that contains 1-10ng/ml Prostatropin and volume percent 10%-15% foetal calf serum;
6) add the described substratum of step 5 after 24 hours;
7) changed a subculture in every 3 days, change the L-DMEM substratum that contains volume ratio 10% foetal calf serum after the 6th day, reach to 8-9 days cells and go down to posterity when merge 80% left and right; The substratum that goes down to posterity is the L-DMEM substratum that contains volume ratio 10% foetal calf serum;
8) went down to posterity once in every 3 days after.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010246359 CN101914496B (en) | 2010-08-06 | 2010-08-06 | Simplified method for preparing umbilical mesenchymal stem cells in large scale |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201010246359 CN101914496B (en) | 2010-08-06 | 2010-08-06 | Simplified method for preparing umbilical mesenchymal stem cells in large scale |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101914496A CN101914496A (en) | 2010-12-15 |
| CN101914496B true CN101914496B (en) | 2013-06-12 |
Family
ID=43322142
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201010246359 Active CN101914496B (en) | 2010-08-06 | 2010-08-06 | Simplified method for preparing umbilical mesenchymal stem cells in large scale |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101914496B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104805058A (en) * | 2014-01-23 | 2015-07-29 | 上海欧易生物医学科技有限公司 | Human umbilical cord-derived mesenchymal stem cell strain stably expressing exogenous GLP-1 gene |
| CN108165527A (en) * | 2018-02-09 | 2018-06-15 | 王巍然 | A kind of enrichment method of beauty and skin care stem cell factor and its application |
| CN110540959A (en) * | 2019-10-08 | 2019-12-06 | 孟明耀 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
-
2010
- 2010-08-06 CN CN 201010246359 patent/CN101914496B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Rao MS.Stem cells and aging:expanding the possibilities.《Mech Ageing Rev》.2001,第122卷(第7期),713-734. * |
| Wagner W.Comparative characteristics of mesenchymal stem cells from human bone marrow,adipose tissue,and umbillical cord blood.《Exp Hematol》.2005,第33卷(第11期),1402-1416. * |
| 巴云涛.人脐带Wharton’s jeIly源间充质干细胞培养中碱性成纤维细胞生长因子的作用.《中国组织工程研究与临床康复》.2010,第14卷(第19期),3513-3517. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101914496A (en) | 2010-12-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN102127522B (en) | Human umbilical mesenchymal stem cell and preparation method thereof | |
| CN105238751B (en) | Isolated culture method of umbilical cord tissue mesenchymal stem cells | |
| CN103266081B (en) | Efficient method for isolating and culturing mesenchymal stem cells from umbilical cord | |
| CN110540959A (en) | Umbilical cord mesenchymal stem cell isolation culture amplification method | |
| CN102676452B (en) | Culture medium containing human umbilical cord mesenchymal stem cell exudates and preparation method and applications thereof | |
| CN101922048B (en) | Method for constructing public library of umbilical mesenchymal stem cells | |
| CN105062959A (en) | Isolated culture method of human amnia mesenchymal stem cells | |
| CN109234229B (en) | Method for separating mesenchymal stem cells from placental blood vessels and digestive enzyme composition used in same | |
| CN113249317A (en) | Isolated culture and amplification method and system for human umbilical cord mesenchymal stem cells | |
| CN106635968A (en) | Method of inducing and differentiating human umbilical cord derived mesenchymal stem cells into cartilage cells | |
| CN106282104A (en) | The method of effective acquisition mescenchymal stem cell from umbilical cord tissue | |
| CN101591644A (en) | The preparation of umbilical cord-mesenchymal stem cells (UC-MSCs) used in clinical therapy and storage | |
| CN102154200B (en) | Preparation and storage of mesenchymal stem cells for clinical treatment | |
| CN105970300A (en) | Method for establishing human umbilical cord mesenchymal stem cell bank by adopting blood serum substituent | |
| CN110628708A (en) | Separation and purification method of high-purity pig muscle stem cells | |
| CN104673748A (en) | Method for extracting mesenchymal stem cells from human umbilical cord | |
| CN104651305A (en) | Method for acquiring bioactive proteins by utilizing umbilical cord mesenchymal stem cells | |
| CN105434468A (en) | Preparation method of skin cell damage repairing reagent | |
| CN111346051A (en) | Preparation method of umbilical cord mesenchymal stem cell injection for treating cerebral infarction | |
| CN103223194A (en) | Cartilage graft for cartilage injury repair and preparation method thereof | |
| CN101914496B (en) | Simplified method for preparing umbilical mesenchymal stem cells in large scale | |
| CN105505865A (en) | Separation method for umbilical cord mesenchymal stem cells | |
| CN102146359A (en) | Method for extracting original mesenchymal stem cells from placenta and serum-free amplification | |
| CN109628388A (en) | With digestive enzyme compositions from placenta blood vessel separating mesenchymal stem cell |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Huang Yuxiang Inventor after: Gao Hong Inventor after: Wang Li Inventor after: Hu Jianxia Inventor after: Zhang Xuefeng Inventor after: Zhang Meirong Inventor before: Wei Sili Inventor before: Gao Hong Inventor before: Wang Li Inventor before: Hu Jianxia Inventor before: Zhang Xuefeng Inventor before: Zhang Meirong |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: WEI SILI GAO HONG WANG LI HU JIANXIA ZHANG XUEFENG ZHANG MEIRONG TO: HUANGYUXIANG GAO HONG WANG LI HU JIANXIA ZHANG XUEFENG ZHANG MEIRONG |