CN104845935A - Separation culture method for endothelial progenitor cells and kit of method - Google Patents
Separation culture method for endothelial progenitor cells and kit of method Download PDFInfo
- Publication number
- CN104845935A CN104845935A CN201510288551.7A CN201510288551A CN104845935A CN 104845935 A CN104845935 A CN 104845935A CN 201510288551 A CN201510288551 A CN 201510288551A CN 104845935 A CN104845935 A CN 104845935A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture
- cultivation method
- progenitor cells
- isolation cultivation
- 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.)
- Pending
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of separation culture of endothelial progenitor cells of umbilical blood, particularly a separation culture method for endothelial progenitor cells of umbilical blood and a kit of the method. The method comprises the following steps: carrying out adherent culture on a mononuclear cell of umbilical blood in advance; and carrying out cell culture on non-adherent cells, wherein a culture medium for cell culture is a full culture medium containing fetuin. Compared to cells obtained by conventional methods, the endothelial progenitor cells of umbilical blood cultured by the method provided by the invention are relatively uniform in shape, relatively high in convergence degree, relatively great in cell harvest yield, relatively fast in cell proliferation and relatively high in antigen presentation rate on cell surface.
Description
Technical field
The present invention relates to endothelial progenitor cells separation and Culture technical field, particularly a kind of isolation cultivation method of umbilical cord blood endothelial progenitor cells and test kit thereof.
Background technology
Cardiovascular and cerebrovascular diseases is exactly that cardiovascular and cerebrovascular disease are referred to as, and makes a general reference because ischemia or hemorrhagic diseases occur for the heart that hyperlipidaemia, blood are sticky, atherosclerosis, hypertension etc. cause, brain and body tissue.A kind of serious threat mankind, the common disease that particularly more than 50 years old the elderly is healthy, even if apply treatment means most advanced, perfect at present, the cerebrovascular accident survivor of more than 50% still can be had to live can not take care of oneself completely, the number of cardiovascular and cerebrovascular diseases is died from every year up to 1,500 ten thousand people in the whole world, occupies the various cause of the death the first.Therefore, a kind of therapeutic strategy effectively can preventing and treating cardiovascular and cerebrovascular diseases is badly in need of.
Cord blood is after baby due, stays the blood in placenta and umbilical cord, containing abundant stem cell.It is mainly containing hemopoietic stem cell and mescenchymal stem cell (mesenchymal stem cells, MSCs).In addition, the mononuclearcell in bleeding of the umbilicus, through inducing culture, can also obtain endothelial progenitor cells (endothelial progenitor cells, EPCs).Endothelial progenitor cells is a kind of precursor cell that directly can be divided into vascular endothelial cell, not only participant's embryonic blood vessel generates, and research recent years finds that EPCs also participates in postnatal angiogenic process, the prompting critical treatment effect of EPCs in ischemic disease, wound healing and wide clinical application prospect.
Recent study finds, EPCs plays a significant role in the treatment of cardiovascular disorder, ischemic disease of limb, and therefore, " alternative strategy " that external EPCs can be used as angiogenesis is widely used in clinical.In addition EPCs easily obtains, simple to operate, directional implantation (going back to the nest) is in vasculogenesis position, to break up the endotheliocyte generated be one layer of cells closest to blood flow, the active substance discharged easily is diffused into whole body by blood flow, thus play and act on widely, therefore EPCs or the desirable target cell of gene therapy.EPCs can derive from bleeding of the umbilicus, marrow, peripheral blood, and Schmidt D etc. report that the EPCs of derived from cord blood has in-vitro multiplication activity, these cell continuous expression endothelial cell phenotype.The EPCs of prompting derived from cord blood can originate as the autogenous cell that organizational project is desirable.
But endothelial progenitor cells is limited contained by every part of bleeding of the umbilicus, in order to meet therapeutic engraftment requirement, need further amplification in vitro EPCs.In existing blood, endothelial progenitor cells isolation cultivation method generally adopts culture dish bag quilt, from blood, isolate adherent culture after mononuclearcell afterwards, the method efficiency is low, only can from blood separation to a small amount of endothelial progenitor cells, amplification efficiency is afterwards also lower, and cell proliferation is slower.Therefore, provide a kind of harvest yield large and the isolation cultivation method of breeding umbilical cord blood endothelial progenitor cells rapidly has important practical significance.
Summary of the invention
In view of this, the invention provides a kind of isolation cultivation method and test kit thereof of umbilical cord blood endothelial progenitor cells.The cell that the umbilical cord blood endothelial progenitor cells that method provided by the invention is cultivated obtains than traditional method, form aspect is more homogeneous, cell confluency Du Genggao, and cell harvesting amount is larger, and cell proliferation is more rapid, and cell-surface antigens expression rate is higher.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of isolation cultivation method of umbilical cord blood endothelial progenitor cells, comprise the steps:
By Cord Blood Mononuclear Cell adherent culture in advance, get not adherent cell and carry out cell cultures, the substratum of cell cultures is the perfect medium containing Pp63 glycophosphoproteins.
The present invention by after Cord Blood Mononuclear Cell in advance adherent culture, then gets not adherent cell and carries out separation and Culture EPCs, adds Pp63 glycophosphoproteins in the substratum of separation and Culture, promotes adherent, the propagation of EPCs.The cell that the umbilical cord blood endothelial progenitor cells that method provided by the invention is cultivated obtains than traditional method, form aspect is more homogeneous, cell confluency Du Genggao, and cell harvesting amount is larger, and cell proliferation is more rapid, and cell-surface antigens expression rate is higher.
As preferably, the concentration of Pp63 glycophosphoproteins is 0.5 ~ 5mg/mL.
Preferably, the concentration of Pp63 glycophosphoproteins is 1mg/mL.
In embodiments more provided by the invention, Pp63 glycophosphoproteins is Pp63 glycophosphoproteins B.
As preferably, the time of cell cultures is 5 ~ 10 days.
Preferably, the time of cell cultures is 8 days.
As preferably, the time of adherent culture is 12 ~ 36h in advance.
Preferably, the time of adherent culture is 24h in advance.
In embodiments more provided by the invention, the substratum of adherent culture is the DMEM substratum containing 10%FBS in advance.
In embodiments more provided by the invention, also containing somatomedin in the substratum of adherent culture in advance, somatomedin is one or more in VEGF, FGF, IGF or EGF.
In embodiments more provided by the invention, containing 20ng/mLVEGF, 20ng/mL FGF, 1ng/mL IGF, 10ng/mL EGF in the substratum of adherent culture in advance.
In embodiments more provided by the invention, the culture dish of adherent culture is the culture dish of gelatin bag quilt in advance.
As preferably, the cell density of adherent culture is 10 in advance
7/ mL.
In embodiments more provided by the invention, adherent culture is at 37 DEG C, 5%CO in advance
2, carry out under saturated humidity condition.
In embodiments more provided by the invention, after cell cultures, also comprise the step adopting digestive ferment digestion endotheliocyte colony, Secondary Culture.
In embodiments more provided by the invention, digestive ferment is pancreatin.
In embodiments more provided by the invention, the preparation method of Cord Blood Mononuclear Cell is: get bleeding of the umbilicus and mix with PBS, centrifugal, abandons supernatant and lipid layer, and cell precipitation PBS is resuspended, adds lymphocyte separation medium, centrifugal, obtains Cord Blood Mononuclear Cell.
Present invention also offers a kind of test kit of separation and Culture umbilical cord blood endothelial progenitor cells, comprise Pp63 glycophosphoproteins, DMEM substratum containing 10%FBS.
In embodiments more provided by the invention, Pp63 glycophosphoproteins is Pp63 glycophosphoproteins B.
In embodiments more provided by the invention, test kit is also containing somatomedin, and somatomedin is one or more in VEGF, FGF, IGF or EGF.
In embodiments more provided by the invention, test kit is also containing digestive ferment.
In embodiments more provided by the invention, digestive ferment is pancreatin.
In embodiments more provided by the invention, test kit is also containing lymphocyte separation medium.
The invention provides a kind of isolation cultivation method and test kit thereof of umbilical cord blood endothelial progenitor cells.The method comprises: by Cord Blood Mononuclear Cell adherent culture in advance, gets not adherent cell and carries out cell cultures, and the substratum of cell cultures is the perfect medium containing Pp63 glycophosphoproteins.The cell that the umbilical cord blood endothelial progenitor cells that method provided by the invention is cultivated obtains than traditional method, form aspect is more homogeneous, cell confluency Du Genggao, and cell harvesting amount is larger, and cell proliferation is more rapid, and cell-surface antigens expression rate is higher.
Accompanying drawing explanation
Fig. 1 shows that P0 is for cellular form; Wherein, A1 illustrates the EPCs that the embodiment of the present invention 1 method is cultivated 2 days, and A2 illustrates the EPCs that the embodiment of the present invention 1 method is cultivated 9 days, and A3 illustrates the EPCs that the embodiment of the present invention 1 method is cultivated 13 days; B1 illustrates the EPCs that traditional method is cultivated 2 days, and B2 illustrates the EPCs that traditional method is cultivated 9 days, and B3 illustrates the EPCs that traditional method is cultivated 13 days;
Fig. 2 shows the growth curve of cell;
Fig. 3 shows the expression rate of cell-surface antigens Vwf-cy2; Wherein scheme the surface antigen Vwf-cy2 expression rate that A shows traditional method separation and Culture EPCs; Figure B shows the expression rate of the embodiment of the present invention 1 method separation and Culture cell.
Embodiment
The invention discloses a kind of isolation cultivation method and test kit thereof of umbilical cord blood endothelial progenitor cells, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In the isolation cultivation method of umbilical cord blood endothelial progenitor cells provided by the invention and test kit thereof, agents useful for same or instrument all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
The separation and ientification of embodiment 1 bleeding of the umbilicus EPCs
Bleeding of the umbilicus takes from the umbilical cord of mature healthy newborn, adds compound Sodium Citrate (CPD-A) anti-freezing, is separated after collection in 24h.
Bleeding of the umbilicus PBS presses the dilution proportion of 1:1,20 DEG C, and the centrifugal 10min of 400g, abandons supernatant and lipid layer, and cell precipitation carefully adds isopyknic Ficoll-paque lymphocyte separation medium (density 1.077 × 10 afterwards with appropriate PBS is resuspended
3g/L), on, 20 DEG C, the centrifugal 30min of 600g, collects mononuclearcell layer.
After PBS washs 2 times, mononuclearcell is suspended from DMEM+10%FBS complete culture solution again, adjustment cell concn to 10
7/ mL, plants in the 35mm culture dish being covered with gelatin, and culture condition is the DMEM substratum containing 10%FBS, adds VEGF 20ng/mL, FGF 20ng/mL, IGF 1ng/mL, EGF10ng/mL, puts 37 DEG C, 5%CO
2, cultivate in saturated humidity incubator.
After cultivating 24h, non-adherent cell is transferred to new ware and cultivates, and add 1mg/mL Pp63 glycophosphoproteins B (Fetuin B) in the medium, within later every 3 days, change liquid once.
Cultivate 8 days, occur the endotheliocyte colony (endothelial cellcolonies, ECCs) that pebbles sample endotheliocyte forms, carry out Secondary Culture with the endotheliocyte of the trysinization composition ECCs of 0.25%.
The separation and ientification of embodiment 2 bleeding of the umbilicus EPCs
Bleeding of the umbilicus takes from the umbilical cord of mature healthy newborn, adds compound Sodium Citrate (CPD-A) anti-freezing, is separated after collection in 24h.
Bleeding of the umbilicus PBS presses the dilution proportion of 1:1,20 DEG C, and the centrifugal 10min of 400g, abandons supernatant and lipid layer, and cell precipitation carefully adds isopyknic Ficoll-paque lymphocyte separation medium (density 1.077 × 10 afterwards with appropriate PBS is resuspended
3g/L), on, 20 DEG C, the centrifugal 30min of 600g, collects mononuclearcell layer.
After PBS washs 2 times, mononuclearcell is suspended from DMEM+10%FBS complete culture solution again, adjustment cell concn to 10
7/ mL, plants in the 35mm culture dish being covered with gelatin, and culture condition is the DMEM substratum containing 10%FBS, adds VEGF 20ng/mL, FGF 20ng/mL, IGF 1ng/mL, EGF10ng/mL, puts 37 DEG C, 5%CO
2, cultivate in saturated humidity incubator.
After cultivating 12h, non-adherent cell is transferred to new ware and cultivates, and add 5mg/mL Pp63 glycophosphoproteins B (Fetuin B) in the medium, within later every 3 days, change liquid once.
Cultivate 5 days, occur the endotheliocyte colony (endothelial cellcolonies, ECCs) that pebbles sample endotheliocyte forms, carry out Secondary Culture with the endotheliocyte of the trysinization composition ECCs of 0.25%.
The separation and ientification of embodiment 3 bleeding of the umbilicus EPCs
Bleeding of the umbilicus takes from the umbilical cord of mature healthy newborn, adds compound Sodium Citrate (CPD-A) anti-freezing, is separated after collection in 24h.
Bleeding of the umbilicus PBS presses the dilution proportion of 1:1,20 DEG C, and the centrifugal 10min of 400g, abandons supernatant and lipid layer, and cell precipitation carefully adds isopyknic Ficoll-paque lymphocyte separation medium (density 1.077 × 10 afterwards with appropriate PBS is resuspended
3g/L), on, 20 DEG C, the centrifugal 30min of 600g, collects mononuclearcell layer.
After PBS washs 2 times, mononuclearcell is suspended from DMEM+10%FBS complete culture solution again, adjustment cell concn to 10
7/ mL, plants in the 35mm culture dish being covered with gelatin, and culture condition is the DMEM substratum containing 10%FBS, adds VEGF 20ng/mL, FGF 20ng/mL, IGF 1ng/mL, EGF10ng/mL, puts 37 DEG C, 5%CO
2, cultivate in saturated humidity incubator.
After cultivating 36h, non-adherent cell is transferred to new ware and cultivates, and add 0.5mg/mL Pp63 glycophosphoproteins B (Fetuin B) in the medium, within later every 3 days, change liquid once.
Cultivate 10 days, occur the endotheliocyte colony (endothelial cellcolonies, ECCs) that pebbles sample endotheliocyte forms, carry out Secondary Culture with the endotheliocyte of the trysinization composition ECCs of 0.25%.
The comparison of embodiment 4 bleeding of the umbilicus EPCs isolation cultivation method
Method: get 6 batches of bleedings of the umbilicus respectively, often criticize bleeding of the umbilicus volume the same, be 60mL.The 3 batches of bleedings of the umbilicus isolation cultivation method of the embodiment of the present invention 1; Another 3 batches with traditional isolation cultivation method, that is: after being separated mononuclearcell in bleeding of the umbilicus, cultivate in former bottle, non-rolling bottle always.Relatively its P0 for the cell harvesting amount of cellular form, cultured continuously 5 generations, P1 for cell-surface antigens expression rate.
Fresh separated Cord Blood Mononuclear Cell kind enters to be covered with in the 35mm culture dish of FN, and vitro culture is after 2 days, and cell attachment, cell is the variforms such as short fusiformis, polygon.Cultivate 8th ~ 10 days, occur the cell cluster (Fig. 1-A1) of about 20-50 pebbles like cell composition.Cell cluster increases gradually (Fig. 1-A2), and volume increases, and forms the endotheliocyte colony (ECCs) (Fig. 1-A3) being greater than 500 pebbles like cell compositions.Cultivate 14th ~ 15 days, endotheliocyte grows to 95% fusion, carries out 1:3 Secondary Culture.Result shows, and the cell that the embodiment of the present invention 1 method cultured cells form obtains than traditional method, form aspect is more homogeneous, cell confluency Du Genggao.
The EPCs cell harvesting amount that the embodiment of the present invention 1 method is cultivated is larger, and its propagation more rapid (Fig. 2) is described, the embodiment of the present invention 1 method cultured cells surface antigen expression rate also higher (Fig. 3).
The isolation cultivation method that Example 2,3 provides, observes its P0 for cellular form, and the cell harvesting amount of detection cultured continuously 5 generations and P1 are for cell-surface antigens expression rate.The result of result and embodiment 1 method is close.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. an isolation cultivation method for umbilical cord blood endothelial progenitor cells, is characterized in that, comprises the steps:
By Cord Blood Mononuclear Cell adherent culture in advance, get not adherent cell and carry out cell cultures, the substratum of described cell cultures is the perfect medium containing Pp63 glycophosphoproteins.
2. isolation cultivation method according to claim 1, is characterized in that, the concentration of described Pp63 glycophosphoproteins is 0.5 ~ 5mg/mL.
3. isolation cultivation method according to claim 1, is characterized in that, described Pp63 glycophosphoproteins is Pp63 glycophosphoproteins B.
4. isolation cultivation method according to claim 1, is characterized in that, the time of described cell cultures is 5 ~ 10 days.
5. isolation cultivation method according to claim 1, is characterized in that, the time of described adherent culture is in advance 12 ~ 36h.
6. isolation cultivation method according to claim 1, is characterized in that, the substratum of described adherent culture is in advance the DMEM substratum containing 10%FBS.
7. isolation cultivation method according to claim 1, is characterized in that, described adherent culture is in advance at 37 DEG C, 5%CO
2, carry out under saturated humidity condition.
8. isolation cultivation method according to claim 1, is characterized in that, also comprises the step adopting digestive ferment digestion endotheliocyte colony, Secondary Culture after described cell cultures.
9. isolation cultivation method according to any one of claim 1 to 8, it is characterized in that, the preparation method of described Cord Blood Mononuclear Cell is: get bleeding of the umbilicus and mix with PBS, centrifugal, abandon supernatant and lipid layer, cell precipitation PBS is resuspended, adds lymphocyte separation medium, centrifugal, obtain Cord Blood Mononuclear Cell.
10. a test kit for separation and Culture umbilical cord blood endothelial progenitor cells, is characterized in that, comprises Pp63 glycophosphoproteins, DMEM substratum containing 10%FBS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510288551.7A CN104845935A (en) | 2015-05-29 | 2015-05-29 | Separation culture method for endothelial progenitor cells and kit of method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510288551.7A CN104845935A (en) | 2015-05-29 | 2015-05-29 | Separation culture method for endothelial progenitor cells and kit of method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104845935A true CN104845935A (en) | 2015-08-19 |
Family
ID=53845925
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510288551.7A Pending CN104845935A (en) | 2015-05-29 | 2015-05-29 | Separation culture method for endothelial progenitor cells and kit of method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104845935A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574143A (en) * | 2017-10-19 | 2018-01-12 | 山东省齐鲁干细胞工程有限公司 | It is a kind of from freeze in bleeding of the umbilicus separate endothelial progenitor cells method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100015710A1 (en) * | 2008-04-25 | 2010-01-21 | Sunghoon Jung | Methods and Compositions for Isolating, Maintaining and Serially Expanding Human Mesenchymal Stem Cells |
| CN102703385A (en) * | 2012-06-26 | 2012-10-03 | 亚太干细胞科研中心有限公司 | Mesenchymal stem cell nutrient solution |
-
2015
- 2015-05-29 CN CN201510288551.7A patent/CN104845935A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100015710A1 (en) * | 2008-04-25 | 2010-01-21 | Sunghoon Jung | Methods and Compositions for Isolating, Maintaining and Serially Expanding Human Mesenchymal Stem Cells |
| CN102703385A (en) * | 2012-06-26 | 2012-10-03 | 亚太干细胞科研中心有限公司 | Mesenchymal stem cell nutrient solution |
Non-Patent Citations (5)
| Title |
|---|
| R. 弗雷谢 著: "《动物细胞培养 基本技术指南 第5版》", 29 February 2008, 北京科学出版社 * |
| 元英进 主编: "《现代制药工艺学 上》", 31 July 2004 * |
| 曹程程: "人脐血内皮祖细胞的分离、培养和鉴定及改良冻融法脱细胞血管支架的建立", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 陈常军: "人脐血内皮祖细胞移植治疗下肢缺血的实验研究", 《中国优秀硕士学位论文全文数据库（医药卫生科技辑）》 * |
| 马志成等: "脐血来源内皮祖细胞的生物学特性与安全性评价", 《中南大学学报（医学版）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107574143A (en) * | 2017-10-19 | 2018-01-12 | 山东省齐鲁干细胞工程有限公司 | It is a kind of from freeze in bleeding of the umbilicus separate endothelial progenitor cells method |
| CN107574143B (en) * | 2017-10-19 | 2020-07-07 | 山东省齐鲁干细胞工程有限公司 | Method for separating endothelial progenitor cells from cryopreserved cord blood |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106109496B (en) | Human umbilical cord mesenchymal stem cells extract freeze-drying powder and preparation method | |
| CN108823156A (en) | For the clinical grade human umbilical cord mesenchymal stem cells composite factor of reparation and the preparation method of freeze-dried powder | |
| CN104726406B (en) | It is a kind of to induce the method that dental pulp Derived from Mesenchymal Stem Cells is nerve cell | |
| CN105112362B (en) | A kind of serum free medium of placenta mesenchyma stem cell and preparation method thereof | |
| CN105132370A (en) | Clinic-level adipose-derived stem cell preparation and storage methods | |
| CN104622902B (en) | It is a kind of for treating the stem cell medicine of liver fibrosis | |
| CN105820998A (en) | Isolation extraction and culture method for human adipose-derived stem cells (ADSCs) for clinical back-transfusion grade cell therapy | |
| CN107810014A (en) | Composition comprising mescenchymal stem cell and application thereof | |
| CN104988110A (en) | Method for transforming umbilical cord mesenchymal stem cells into islet cells | |
| CN105534848A (en) | Cosmetic or pharmaceutical composition and application thereof | |
| CN103585177A (en) | Applications of mesenchymal stem cell and genetically modified mesenchymal stem cell | |
| CN106801038A (en) | A kind of utilization Three-dimensional cell culture system promotes the cell culture processes of umbilical cord blood hematopoietic stem cell fast and stable propagation | |
| Athanassopoulos et al. | Microvessel networks in pre-formed in artificial clinical grade dermal substitutes in vitro using cells from haematopoietic tissues | |
| CN104087551A (en) | Novel method for in-vitro separated culture of human epidermal cells | |
| CN107384858A (en) | A kind of preparation method and applications of hypoxic tolerance type mescenchymal stem cell | |
| CN107858329A (en) | The method of fractionation of fatty mescenchymal stem cell and the test solution used from fat | |
| CN106566803A (en) | Culture solution, application of culture solution and method for culturing umbilical cord mesenchymal stem cells | |
| CN106701670A (en) | Methods for enhancing bioactive factor secretion capacity of mesenchymal stem cells and extracting active factors in culture solution | |
| CN103497892A (en) | Cell culture substrate, and preparation method and application thereof | |
| CN105062953A (en) | Method for three-dimensional induction of transformation of mesenchymal stem cells into islet cells | |
| CN101775369B (en) | High-lung-metastasis human breast cancer cell line | |
| CN103421740A (en) | In-vitro culture and proliferation method for human mesenchymal stem cells | |
| CN101525596A (en) | Method for obtaining stem cell by using serum of same cord blood | |
| CN104845935A (en) | Separation culture method for endothelial progenitor cells and kit of method | |
| CN109749992A (en) | A kind of mesenchymal stem cell serum-free cultural method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| EXSB | Decision made by sipo to initiate substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150819 |
|
| RJ01 | Rejection of invention patent application after publication |