CN102643784A - Expansion system in vitro for hematopoietic stem/progenitor cell - Google Patents
Expansion system in vitro for hematopoietic stem/progenitor cell Download PDFInfo
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- CN102643784A CN102643784A CN2012100872275A CN201210087227A CN102643784A CN 102643784 A CN102643784 A CN 102643784A CN 2012100872275 A CN2012100872275 A CN 2012100872275A CN 201210087227 A CN201210087227 A CN 201210087227A CN 102643784 A CN102643784 A CN 102643784A
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Abstract
The invention provides an expansion system in vitro for hematopoietic stem/progenitor cells used for expanding human umbilical cord blood in vitro of soluble fusion protein Dll1-RGD (Hdll1-RGD) of notch ligand of a vascular endothelial cell target. The effective expansion system in vitro is formed by taking human umbilical vein endothelial cells as supporting cells of a cultivation system, jointly applying exogenous growth factors SCF, TOP, FL, IL-6, IL-3 and own developed hDLL1-RGD, and co-cultivating with the hematopoietic stem/progenitor cells. According to the expansion system, the hDll1-RGD target is combined to the surface of a human umbilical vein endothelial cell, activated stimulation aiming at a notch receptor is provided for the hematopoietic stem cells, and self-renewal and propagation of the hematopoietic stem cells are maintained, so that a satisfactory expansion effect and favorable implantation capacity are obtained.
Description
Technical field
The present invention relates to a kind of extracorporeal culturing method of hematopoietic stem; Especially a kind of amplification in vitro system of hematopoietic stem of soluble fusion protein hDll1-RGD amplification in vitro people bleeding of the umbilicus of Notch part of vascular endothelial cell target belongs to biological technical field.
Background technology
HSCT has at present become one of important means of clinical treatment hemopathy and malignant tumour since the bleeding of the umbilicus source abundant, gather a little less than convenient, the immunogenicity, than marrow and peripheral blood transplanting more advantages are arranged.But hematopoietic stem content is lower in the bleeding of the umbilicus, is difficult to satisfy grow up reach the big patient's of body weight needs, transplants back hematopoietic reconstitution delay, is difficult to receive satisfied curative effect, seriously limits its application in clinical.The hemopoietic stem cell that how obtains capacity through the method for amplification in vitro has expeditiously become a problem demanding prompt solution to meet clinical needs.Previously research shows: the amplification in vitro system of traditional combination of cytokines is when promoting hematopoietic stem propagation; Also accelerated the process of its differentiation and maturation; Lose the self of hematopoietic stem and the potential of hematopoietic reconstitution, can't keep long-term hematopoiesis; Along with the understanding of molecular mechanism to regulation and control hemopoietic stem cell proliferation and self in the hematopoieticmicroenviron-ment is that amplifying candidate stem cell in vitro provides new thinking.In vivo, hematopoieticmicroenviron-ment is kept the potential of hemopoietic stem cell self and polyphyly differentiation through close contact of iuntercellular and all kinds of hematopoiesis regulatory factor.Therefore, the microenvironment of simulation hematopoietic stem cell growth becomes and improves one of amplification in vitro efficient important method.
Big quantity research shows; The Notch signal pathway is one of important signal path of regulation and control stem cells hyperplasia differentiation; The stroma cell surface of hemopoietic stem cell and hematopoieticmicroenviron-ment is expressed Notch acceptor and part respectively, activates the self that the Notch signal path can promote HSC, and suppresses not break up.There are a plurality of study group successively to express the DSL structural domain fragment of Dll1 in recent years; It is not good to activate activity; Expanding effect is very limited; Its major cause is that Notch acceptor and part all are cell surface proteins, has only the Notch ligand expression could effectively be brought into play the effect that stimulates the Notch acceptor at cell surface or immobilization on the developing medium surface.The hDll1-RGD of research and development is soluble fusion proteins of band vascular endothelial cell targeting proteins matter motif RGD voluntarily; Wherein RGD is to be the polypeptide of core with l-arginine (R)-glycocoll (G)-Aspartic Acid (D); Can specifically identifying blood vessel endothelial cell the plain molecule of integration on surface, thereby the protein molecular that will merge with it is anchored to the vascular endothelial cell surface.With HUVEC is sustenticular cell, not only can effectively activate the Notch signal path, and has simulated umbilical hemopoietic ancestral cells growth microenvironment, has best amplification efficiency.Do not see at present the report of this amplification system.
Summary of the invention
The objective of the invention is to remedy the deficiency of prior art, a kind of cultural method of hematopoietic stem is provided, especially relate to a kind of amplification in vitro system of hematopoietic stem of soluble fusion protein Notch part hDll1-RGD amplification in vitro people bleeding of the umbilicus.
What the present invention adopted is with the sustenticular cell of Human umbilical vein endothelial cells as culture system; Combined utilization exogenous growth factor S CF, TPO, FL, IL-6, IL-3 and the hDll1-RGD that researches and develops voluntarily; Cultivate altogether with hematopoietic stem, for the efficient that improves the hematopoietic stem amplification in vitro provides a kind of new method.
The object of the invention is realized through following scheme: a kind of amplification in vitro system of hematopoietic stem; It is characterized in that: be incorporated into huve cell Human umbilical vein endothelial cells through soluble fusion protein hDll1-RGD target; The HUVEC surface; Activate the Notch acceptor, amplification people umbilical hemopoietic ancestral cells comprises the steps:
1) after huve cell merges 90%, handles 2.5h through MTC C, washing;
2) will add serum-free medium in the huve cell after step 1);
3) toward step 2) nutrient solution in add five kinds of exogenous growth factor 5GF and soluble fusion protein hDll1-RGD, set up the amplification in vitro system of hematopoietic stem;
4) separation, purification of hematopoietic ancestral cells CD34 from bleeding of the umbilicus
+, plant the amplification system that step 3) is set up, 37 ℃, 5%CO
2Cultivated 7 days in the complete wet incubator;
5) adopt the people umbilical hemopoietic ancestral cells of NOD/SCID mouse transplantation model assessment after amplification;
6) form the ability of the people umbilical hemopoietic ancestral cells of experimental evaluation after amplification through the hematopoietic stem colony to the polyphyly differentiation.
Step 2) described serum-free medium is StemSpan
TMSerum-free medium.
The described exogenous growth factor of step 3) 5GF comprises SCF, TPO, FL, IL-6, IL-3, soluble fusion protein hDll1-RGD.
The nutrient solution of the described huve cell of step 1) is M199,20% foetal calf serum, 30 μ g/mL ECGS.
The concentration of the described ametycin of step 1): 10 μ g/mL.
The concentration of described exogenous growth factor S CF, TPO, FL, IL-6, IL-3 is respectively: 120ng/ mL, 20ng/mL, 50ng/ mL, 5ng/mL, 5ng/mL, the concentration of soluble fusion protein hDll1-RGD: 2.5ug/mL.
The described NOD/SCID mouse of step 5) transplantation model assessment hematopoietic stem method: adopt sublethal dose irradiation NOD/SCID mouse; People's umbilical hemopoietic ancestral cells in the 4h after the tail vein injection amplification, 4w, 8w detect the ability that humanized's hematopoietic cell is implanted after transplanting.
The described hematopoietic stem colony of step 6) forms experiment; Adopt the people's umbilical hemopoietic ancestral cells after amplification to be inoculated in the methylcellulose gum semisolid medium; Count colony forming unit down in the 14th day inverted microscope, the people umbilical hemopoietic ancestral cells of assessment after amplification is to the ability of polyphyly differentiation.
Characteristics of the present invention are: through the microenvironment of hematopoietic stem growth in the analogue body; Use hDll1-RGD and can be incorporated into the huve cell surface by target; Provide activated form to stimulate to hematopoietic stem, thereby reach the effect of ideal amplification in vitro hematopoietic stem, and the hematopoietic cell after the amplification has good hematopoietic reconstitution ability to the Notch acceptor; Explained that the cell after the amplification is still keeping the characteristic of hemopoietic stem cell, has broad clinical application prospect.
Description of drawings
For the ease of understanding, describe the present invention through Figure of description:
Fig. 1: different culture condition are amplification bleeding of the umbilicus CD34 down
+Total cell count;
Fig. 2: CD34 in the amplifying cells that different culture condition downflow system cell instruments detect
+The ratio of cell;
Fig. 3: flow cytometer detects hDll1-RGD amplification back cell surface marker CD34;
Fig. 4: bleeding of the umbilicus CD34 under the different culture condition
+The amplification times of cell;
Fig. 5: the hematopoiesis colony amplification times that expanded cells forms under the different culture condition;
Fig. 6: the NOD/SCID mouse is transplanted after the different condition cultured cells, the 4th all mouse peripheral blood humanized CD45
+The ratio of cell;
Fig. 7: the NOD/SCID mouse is transplanted after the different condition cultured cells, the 8th all mouse peripheral blood humanized CD45
+The ratio of cell;
Fig. 8, NOD/SCID mouse are transplanted after the different condition cultured cells, and the 8th all streaming cell instruments are to mouse peripheral blood humanized CD45
+Cell detects.
Embodiment
Embodiment
(1) experiment material
(1) Human umbilical vein endothelial cells is cultivated as follows
(2) Cord blood derives from all obstetrics and gynecology hospitals of Tang of The Fourth Military Medical University
(3) reagent:
M199, foetal calf serum: Gibco company
StemSpan serum-free medium, MethoCult GF H4434:Stemcell company
ECGS Endothelial Cell Growth Supplement (ECGS): Sigma company
Bovine serum albumin Bovine Serum Albumin (BSA): Sigma company
II Collagen Type VI enzyme: Sigma company
Ametycin: Sigma company
CD34 MicroBead Kit:Miltenyi company
Separate buffer:PBS+2mM EDTA+0.5% BSA
Lymphocyte separation medium: TBD company
PBSA：PBS?+?2mM?EDTA
SCF (stem cell factor): recombinant human scf, Peprotech company
TPO (thrombopoietin): recombinant human TPO, Peprotech company
FL (flt3 ligand): recombinant human FL, Peprotech company
IL-6 (interleukin-6): recombinant human IL-6, Peprotech company
IL-3 (interleukin-3): recombinant human IL-3, Peprotech company
HDll1-RGD albumen: this research department researches and develops voluntarily
FITC mark antihuman CD 34 monoclonal antibody: BD Biosciences company
The anti-people CD45 of APC mark monoclonal antibody: BD Biosciences company
The anti-mouse CD45 of FITC mark monoclonal antibody: BD Biosciences company
The NOD/SCID mouse is purchased China's Fukang biotech inc in Beijing
(2) experimental technique
1, the cultivation of Human umbilical vein endothelial cells
Get the human umbilical vein, separate, turn out huve cell; The huve cell nutrient solution is M199,20% foetal calf serum, 30 μ g/mL ECGS.
Concrete operations are: under aseptic condition, get the umbilical cord in the 2h behind the healthy parturient childbirth, be about 20 cm, cut off clamp place, umbilical cord two ends.Insert syringe needle in umbilical vein one end, after do not have bloodstain, mosquito forceps clamping umbilical vein far-end is full of vein with 0.2% collagenase solution 10mL with the PBSA complete flushing; 37 ℃ hatch 20min after, massage umbilical cord gently, Digestive system is collected into centrifuge tube, with 30mL PBSA flushing umbilical vein 2 times; Washing fluid together is collected into centrifuge tube, and the centrifugal 10min of 1500rpm abandons supernatant; Add huve cell complete culture solution re-suspended cell, behind the counting, with 1 * 10
5/ mL cell inoculation is in 24 orifice plates, and every hole adds complete culture solution, and 24h removes not adherent cell; Change fresh medium in per 3 days, grow to about 90 % until cell and merge, carry out passage; Get the 3rd ~ 5 generation huve cell as sustenticular cell, carry out subsequent experimental.
2, bleeding of the umbilicus CD34
+The cellular segregation purifying
The bleeding of the umbilicus of anticoagulant heparin is pressed the 1:1 dilution proportion with PBSA; Slowly add the bleeding of the umbilicus of dilution on the lymphocyte separation medium; Centrifugal 15 minutes of 20 ° of C, 1500rpm, obviously visible ring-type oyster white cellular layer is carefully drawn the cellular layer cell on the lymphocyte separation medium liquid level; PBSA washing 2 times, PBS suspension cell counting.Per 10
8It is resuspended that mononuclearcell separates buffer with 300 μ L, and cell suspension adds FcR blocker, each 100 μ L of CD34 magnetic bead respectively, fully mixing, 4 ° of C refrigerators were hatched 30 minutes, separates the buffer washing, abandons supernatant.Separate the buffer suspension cell with 500 μ L.The MS separator column is fixed in the magnetic field of MACS separometer, separates the moistening separator column of buffer,, make unconjugated cell pass through separator column the slow adherent adding separator column of cell suspension with 500 μ L.With separating buffer with unconjugated cell flush away (500 μ L * 3 time), separator column is shifted out magnetic field, separate buffer pressurization wash-out with 1mL, PBSA washs CD34
+Cell, and count subsequent use.
3, the amplification in vitro of umbilical hemopoietic ancestral cells
With the Human umbilical vein endothelial cells that M199 nutrient solution (containing 20%FBS, 30 μ g/mL ECGS) is cultivated, plant 24 orifice plates, merge about 90% after, handle 2.5h through MTC C, add StemSpan after the PBS washing 3 times
TMSerum-free medium; In culture system, add SCF, TPO, FL, IL-6, IL-3, soluble fusion protein hDll1-RGD (concentration is respectively: 120ng/mL, 20ng/mL, 50ng/mL, 5ng/mL, 5ng/mL, 2.5ug/mL) then, the culture system of setting up new hematopoietic stem is subsequent use.CD34 with separation and purification
+Cell is planted the above-mentioned serum free culture system that huve cell is supported that contains, and puts 37 ℃, 5%CO
2Cultivated 7 days in the complete wet incubator.Suspension cell in the centrifugal collection culture system, viable count is calculated in trypan blue dyeing, and flow cytometer detects CD34
+The ratio of cell is calculated CD34
+The amplification times of cell, inoculation people colony forms in the substratum of experiment, and prepares transplanting and give the NOD/SCID mouse.
4, the detection of hematopoietic stem
Suspension cell in the centrifugal collection culture system with PBS washing 2 times, and is made into 1 * 10
7/ mL cell concn, it is centrifugal to get 50 μ L cell suspensions, adds 0.5 μ L FITC mark antihuman CD 34 monoclonal antibody then, and lucifuge is hatched 30min for 4 ℃, adds 1mL streaming liquid then and (contains 5% foetal calf serum, 0.2%NaN
3PBS) cell is hanged centrifugal 1300rpm, 5min, repeated washing one time.Upflowing cell instrument FACScalibur detects, and obtains and analyzes with Cellquest software.
5, the hematopoietic stem colony forms experiment
Collect suspension cell in the centrifugal culture system, PBS washes twice, with 10
3/ mL is suspended from 1.lmL methylcellulose gum semisolid medium H4434 kind in diameter 35mm petridish, and each sample is done 1 multiple hole.With the little petridish of 2 identical samples,, place the big petridish of 1 diameter 10cm together during cultivation with 1 little petridish that only adds sterilized water and uncovered.Place 37 ℃, 5%CO
2Complete wet incubator is cultivated after 14 days and is observed down in inverted microscope, greater than the cell mass of 50 cells counted 1 colony forming unit (Colony-forming unit, CFU).Count CFU (burst forming unit erythroid BFU-E, grain are colony forming unit CFU-GM, mixed stocker colony forming unit CFU-Mix) down in the 14th day inverted microscope.
6, NOD/SCID mouse transplantation model
The NOD/SCID mouse, female, 6-8W raises in aseptic cage, raises the diet with high pressure sterile, and the irradiation of 300cGy sublethal dose radiation ray whole body was carried out transplantation experiments through tail vein injection in 4 hours before transplanting.Experiment is divided into groups: 1,5GF group: as sustenticular cell, five kinds of growth factors of combined utilization are cultivated 7 days cell to the transplanting warp with Human umbilical vein endothelial cells, and average every mouse transplants 2 * 10
72, hDll1-RGD group: as sustenticular cell, the serum-free co-culture system of five kinds of growth factors of combined utilization and hDll1-RGD is cultivated 7 days cell to the transplanting warp with Human umbilical vein endothelial cells, and average every mouse transplants 2 * 10
7, every group of 8 mouse.Transplant the back and observe mouse humanized CD45
+The situation of the implantation of cell.Transplant the 4th week of back, cut the mouse tail point and get blood, flow cytometer detects mouse peripheral blood humanized CD45
+The ratio of cell.Transplant the 8th week of back, pluck eyeball and get peripheral blood, the cervical vertebra dislocation method is put to death mouse, gets bone marrow cells in mice, and inoculation people colony forms in the substratum of experiment, detects humanized's hematopoietic cell colony and forms ability.
(1), NOD/SCID mouse peripheral blood humanized CD45
+Cell detection
In transplanting back 4w, 8w collection mouse peripheral blood through heparin anti-coagulating, get 50 μ L whole bloods, behind the erythrocyte splitting,, and be made into 1 * 10 with PBS washing 2 times
7/ mL cell concn, it is centrifugal to get 50 μ L cell suspensions, adds 0.5 μ L FITC mark anti-people CD45 monoclonal antibody and the anti-mouse CD45 of 0.5 μ L APC mark monoclonal antibody then; Lucifuge; Hatch 30min for 4 ℃, add 1mL streaming liquid then and (contain 5% foetal calf serum, 0.2%NaN
3PBS) cell is hanged centrifugal 1300rpm, 5min.Repeated washing one time.Upflowing cell instrument FACScalibur detects, and obtains and analyzes with Cellquest software, with humanized CD45
+Cell＞1% is a successful transplantation.
(2), NOD/SCID mouse humanized hematopoietic cell colony forms experiment
In transplanting the 8th week of back, the cervical vertebra dislocation method is put to death mouse, gets bone marrow cells in mice, behind the erythrocyte splitting, with PBS washing 2 times, with 1 * 10
5/ mL is suspended from 1.lmL methylcellulose gum semisolid medium H4434 kind in diameter 35mm petridish, and each sample is done 1 multiple hole.With the little petridish of 2 identical samples,, place the big petridish of 1 diameter 10cm together during cultivation with 1 little petridish that only adds sterilized water and uncovered.Place 37 ℃, 5%CO
2Complete wet incubator is cultivated after 14 days and is observed down in inverted microscope, greater than the cell mass of 50 cells counted 1 colony forming unit (Colony-forming unit, CFU).Count CFU (burst forming unit erythroid BFU-E, grain are colony forming unit CFU-GM, mixed stocker colony forming unit CFU-Mix) down in the 14th day inverted microscope.
Experimental data with mean+SD (expression of χ ± s) adopts SPSS17.0 software to carry out statistical study, relatively adopts the t check between two groups, and group difference adopts variance analysis,
P<0.05 think statistical significance arranged.
Effect of the present invention further specifies through following detection and verification method:
1, hematopoietic stem CD34 under the amplification system
+Amplification
Separation and purification CD34 from bleeding of the umbilicus
+, with 2 * 10
3/ mL kind is organized different amplification systems and is cultivated in each, collects centrifugal suspension cell in the 7th day, carries out cell counting, CD34
+The expanding effect of cell is referring to table 1, and each organizes CD34
+Cell obtains a large amount of amplifications, and with hDll1-RGD group expanding effect best (see Fig. 1,
* P<0.01), CD34
+Increase about 67.50 ± 3.03 times (see Fig. 4,
* P<0.01).And keep hemopoietic stem cell characteristic effect to have a clear superiority in than other groups with hDll1-RGD group, about 66.12% still keeps CD34
+Phenotype (seeing Fig. 2, Fig. 3).The 5GF group increases with five kinds of growth factors for single; HUVEC group for HUVEC as sustenticular cell, add with five kinds of growth factors and increase; HDll1-RGD group for HUVEC as sustenticular cell, add simultaneously with five kinds of growth factors and hDll1-RGD and increase).
2, the hematopoietic stem colony forms experimental analysis
Form experiment through colony the cell after cultivating has been carried out Function detection, organize showed increased with hDll1-RGD group CFU number than other, the result is referring to table 2, and cell forms colony with the highest (see figure 5) of CFU-GM ratio.
3, the NOD/SCID mouse is transplanted humanized's hematopoietic stem colony formation analysis in the marrow of back
The NOD/SCID mouse is transplanted the 8th week of back and puts to death, and extracting marrow cell is with 1 * 10
5/ mL is suspended from the methylcellulose gum semisolid medium, cultivated for two weeks after, all find to have the hematopoiesis colony to form.Through with HUVEC as sustenticular cell, add simultaneously with five kinds of growth factors and hDll1-RGD and carry out after expanded cells transplants mouse, the hematopoiesis colony that respectively is forms all situation of transplanting after the amplification of 5GF group of ability, the result is referring to table 3.
The result of NOD/SCID mouse transplantation model shows, through culture system expanded cells successful transplantation in the mouse body, and the ratio that Humanized cell is implanted organizes apparently higher than 5GF, and the hematopoietic reconstitution ability obviously strengthens in vivo.(seeing Fig. 6,7,8)
Part that present embodiment is not described in detail and english abbreviation belong to the common practise of the industry, can search on the net, here not narration one by one.
Claims (8)
1. the amplification in vitro system of a hematopoietic stem; It is characterized in that: be incorporated into huve cell Human umbilical vein endothelial cells through soluble fusion protein hDll1-RGD target; The HUVEC surface; Activate the Notch acceptor, amplification people umbilical hemopoietic ancestral cells comprises the steps:
1) after huve cell merges 90%, handles 2.5h through MTC C, washing;
2) will add serum-free medium in the huve cell after step 1);
3) toward step 2) nutrient solution in add five kinds of exogenous growth factor 5GF and soluble fusion protein hDll1-RGD, set up the amplification in vitro system of hematopoietic stem;
4) separation, purification of hematopoietic ancestral cells CD34 from bleeding of the umbilicus
+, plant the amplification system that step 3) is set up, 37 ℃, 5%CO
2Cultivated 7 days in the complete wet incubator;
5) adopt the people umbilical hemopoietic ancestral cells of NOD/SCID mouse transplantation model assessment after amplification;
6) form the ability of the people umbilical hemopoietic ancestral cells of experimental evaluation after amplification through the hematopoietic stem colony to the polyphyly differentiation.
2. the amplification in vitro system of hematopoietic stem according to claim 1 is characterized in that: step 2) described serum-free medium is StemSpan
TMSerum-free medium.
3. the amplification in vitro system of hematopoietic stem according to claim 1, it is characterized in that: the described exogenous growth factor of step 3) 5GF comprises SCF, TPO, FL, IL-6, IL-3, soluble fusion protein hDll1-RGD.
4. the amplification in vitro system of hematopoietic stem according to claim 1, it is characterized in that: the nutrient solution of the described huve cell of step 1) is M199,20% foetal calf serum, 30 μ g/mL ECGS.
5. the amplification in vitro system of hematopoietic stem according to claim 1 is characterized in that: the concentration of the described ametycin of step 1): 10 μ g/mL.
6. the amplification in vitro system of hematopoietic stem according to claim 3; It is characterized in that: the concentration of described exogenous growth factor S CF, TPO, FL, IL-6, IL-3 is respectively: 120ng/ mL, 20ng/mL, 50ng/ mL, 5ng/mL, 5ng/mL, the concentration of soluble fusion protein hDll1-RGD: 2.5ug/mL.
7. the amplification in vitro system of hematopoietic stem according to claim 1; It is characterized in that: the described NOD/SCID mouse of step 5) transplantation model assessment hematopoietic stem method: adopt sublethal dose irradiation NOD/SCID mouse; People's umbilical hemopoietic ancestral cells in the 4h after the tail vein injection amplification, 4w, 8w detect the ability that humanized's hematopoietic cell is implanted after transplanting.
8. the amplification in vitro system of hematopoietic stem according to claim 1; It is characterized in that: the described hematopoietic stem colony of step 6) forms experiment; Adopt the people's umbilical hemopoietic ancestral cells after amplification to be inoculated in the methylcellulose gum semisolid medium; Count colony forming unit down in the 14th day inverted microscope, the people umbilical hemopoietic ancestral cells of assessment after amplification is to the ability of polyphyly differentiation.
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| CN106244545A (en) * | 2016-08-19 | 2016-12-21 | 中国人民解放军军事医学科学院野战输血研究所 | Secrete outward body purposes in promoting hematopoietic stem/progenitor propagation |
| CN106459902A (en) * | 2014-06-19 | 2017-02-22 | 日东电工株式会社 | Tissue regeneration promoter |
| CN108350419A (en) * | 2015-07-20 | 2018-07-31 | 安吉克莱茵生物科学有限公司 | stem cell transplantation method and stem cell transplantation composition |
| CN108676777A (en) * | 2018-04-03 | 2018-10-19 | 华南生物医药研究院 | Application of the tire sinusoidal endothelial cell strain in hematopoietic stem cell expansion and differentiation |
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| CN108350419A (en) * | 2015-07-20 | 2018-07-31 | 安吉克莱茵生物科学有限公司 | stem cell transplantation method and stem cell transplantation composition |
| CN105886464A (en) * | 2016-06-07 | 2016-08-24 | 广东万海细胞生物科技有限公司 | Serum-free culture medium for umbilical cord blood mesenchymal stem cells |
| CN106244545A (en) * | 2016-08-19 | 2016-12-21 | 中国人民解放军军事医学科学院野战输血研究所 | Secrete outward body purposes in promoting hematopoietic stem/progenitor propagation |
| CN109722415A (en) * | 2017-10-27 | 2019-05-07 | 博雅辑因（北京）生物科技有限公司 | A kind of cultural method for cultivating composition, culture medium and candidate stem cell of candidate stem cell |
| CN108676777A (en) * | 2018-04-03 | 2018-10-19 | 华南生物医药研究院 | Application of the tire sinusoidal endothelial cell strain in hematopoietic stem cell expansion and differentiation |
| CN111321118A (en) * | 2020-01-14 | 2020-06-23 | 河南省银丰生物工程技术有限公司 | Method for in-vitro amplification of cord blood hematopoietic stem cells |
| CN112210527A (en) * | 2020-09-04 | 2021-01-12 | 北京昱龙盛世生物科技有限公司 | Separation culture method of vascular endothelial progenitor cells |
| CN112961821A (en) * | 2021-02-24 | 2021-06-15 | 四川大学华西医院 | Method for efficiently three-dimensionally culturing vascular endothelial cells |
| CN112961821B (en) * | 2021-02-24 | 2023-05-30 | 四川大学华西医院 | Method for three-dimensional culture of vascular endothelial cells |
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