CN102643784B - A kind of amplification in vitro system of hematopoietic stem/progenitor - Google Patents
A kind of amplification in vitro system of hematopoietic stem/progenitor Download PDFInfo
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Abstract
The invention provides a kind of soluble fusion protein people Dll1-RGD(hDll1-RGD of Notch part of vascular endothelial cell target) the amplification in vitro system of the hematopoietic stem/progenitor of amplification in vitro people bleeding of the umbilicus, it is the supportint cell using Human umbilical vein endothelial cells as cultivating system, use in conjunction exogenous growth factor S CF, TPO, FL, IL-6, IL-3 and the hDll1-RGD researching and developing voluntarily, cultivate altogether with hematopoietic stem/progenitor, formed a kind of effective system of amplification in vitro. Be incorporated into huve cell surface by hDll1-RGD target in the present invention, provide for the activated form of Notch acceptor and stimulate to candidate stem cell, maintain self and the propagation of candidate stem cell, thereby obtained satisfied expanding effect and good implantation capability.
Description
Technical field
The present invention relates to a kind of extracorporeal culturing method of hematopoietic stem/progenitor, especially a kind of vascular endothelial cell targetThe amplification in vitro body of hematopoietic stem/progenitor of soluble fusion protein hDll1-RGD amplification in vitro people bleeding of the umbilicus of Notch partSystem, belongs to biological technical field.
Background technology
HSCT at present has become one of important means of clinical treatment blood disease and malignant tumour, due toDerived from cord blood is abundant, it is convenient to gather, a little less than immunogenicity, transplant and have more advantages compared with marrow and peripheral blood. But, hematopoiesis in bleeding of the umbilicusAncestral cells content is lower, is difficult to meet adult and the larger patient's of body weight needs, and after transplanting, hematopoietic reconstitution postpones, and is difficult to receiptsTo satisfied curative effect, seriously limit its application in clinical. How to obtain foot by the method for amplification in vitro expeditiouslyThe candidate stem cell of amount, to meet clinical needs, has become a problem demanding prompt solution. Previously studies show that: traditional cellThe amplification in vitro system of combinations of factors, in promoting hematopoietic stem/progenitor propagation, has also been accelerated the process of its differentiation and maturation,Lose the self of hematopoietic stem/progenitor and the potential of hematopoietic reconstitution, cannot maintain long-term hematopoiesis; Along with to the micro-ring of hematopoiesisThe understanding that regulates and controls the molecular mechanism of hemopoietic stem cell proliferation and self in border provides new for amplifying candidate stem cell in vitroThinking. In vivo, hematopoieticmicroenviron-ment maintains candidate stem cell by iuntercellular close contact and all kinds of hematopoietic regulation factorThe potential of self and polyphyly differentiation. Therefore, the microenvironment of simulation hematopoietic stem cell growth, becomes and improves amplification in vitro effectOne of rate important method.
Quantity research shows greatly, and Notch signal pathway is that regulation and control stem cells hyperplasia is broken up one of important signal path, hematopoiesisNotch acceptor and part are expressed respectively in the stroma cell surface of stem cell and hematopoieticmicroenviron-ment, and activating Notch signal path canPromote the self of HSC, and suppress undifferentiated. There are in recent years multiple seminar successively to express the DSL domain sheet of Dll1Section, Activation Activity is not good, and expanding effect is very limited, and its main cause is that Notch acceptor and part are all cell surface proteins,Only have Notch ligand expression could effectively be brought into play and stimulated Notch to be subject at cell surface or immobilization on culture medium surfaceThe effect of body. The hDll1-RGD of research and development is the solubility fusion eggs with vascular endothelial cell targeting proteins matter motif RGD voluntarilyIn vain, wherein RGD is the polypeptide taking arginine (R)-glycine (G)-L-aminobutanedioic acid (D) as core, can specifically identifying blood vessel inThe integrin molecule on chrotoplast surface, thus the protein molecular merging is with it anchored to Surface of Vascular Endothelial Cells. With HUVECFor supportint cell, not only can effectively activate Notch signal path, and simulate umbilical hemopoietic ancestral cells and grown micro-Environment, has best amplification efficiency. Have no at present the report of this amplification system.
Summary of the invention
The object of the invention is to make up the deficiencies in the prior art, provide a kind of cultural method of hematopoietic stem/progenitor, especiallyRelate to a kind of hematopoietic stem/progenitor external of soluble fusion protein Notch part hDll1-RGD amplification in vitro people bleeding of the umbilicusAmplification system.
What the present invention adopted is the supportint cell using Human umbilical vein endothelial cells as cultivating system, and use in conjunction is exogenousGrowth factor SCF, TPO, FL, IL-6, IL-3 and the hDll1-RGD researching and developing voluntarily, cultivate with hematopoietic stem/progenitor, altogether for carryingThe efficiency of high hematopoietic stem/progenitor amplification in vitro provides a kind of new method.
Object of the present invention realizes by following scheme: a kind of amplification in vitro system of hematopoietic stem/progenitor, its featureBe: be incorporated into huve cell Humanumbilicalvein by soluble fusion protein hDll1-RGD targetEndothelialcells, HUVEC surface, activates Notch acceptor, and amplification people umbilical hemopoietic ancestral cells, comprises following stepRapid:
1) after huve cell merges 90%, process 2.5h through mitomycin C, washing;
2) will in the huve cell after step 1), add serum-free medium;
3) toward step 2) nutrient solution in add five kinds of exogenous growth factor 5GF and soluble fusion protein hDll1-RGD, sets up the amplification in vitro system of hematopoietic stem/progenitor;
4) separation, purification of hematopoietic ancestral cells CD34 from bleeding of the umbilicus+, the amplification system that plantation is set up to step 3), 37℃、5%CO2In complete wet incubator, cultivate 7 days;
5) adopt the people umbilical hemopoietic ancestral cells of NOD/SCID mouse transplantation model assessment after amplification;
6) form the people umbilical hemopoietic ancestral cells of experimental evaluation after amplification to many by hematopoietic stem/progenitor colonyThe ability of system's differentiation.
Step 2) described serum-free medium is StemSpanTMSerum-free medium.
Exogenous growth factor 5GF described in step 3) comprises SCF, TPO, FL, IL-6, IL-3, soluble fusion proteinhDll1-RGD。
The nutrient solution of the huve cell described in step 1) is M199,20% hyclone, 30 μ g/mLECGS.
The concentration of the mitomycin C described in 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.
NOD/SCID mouse transplantation model assessment hematopoietic stem/progenitor method described in step 5): adopt sublethal doseIrradiate NOD/SCID mouse, the people's umbilical hemopoietic ancestral cells in 4h after tail vein injection amplification, 4w, 8w inspection after transplantingSurvey the ability that humanized's hematopoietic cell is implanted.
Hematopoietic stem/progenitor colony described in step 6) forms experiment, adopt people's umbilical hemopoietic after amplification dry/ancestral is thinBorn of the same parents are inoculated in methylcellulose semisolid culturemedium, count colony forming unit in the 14th day under inverted microscope, and assessment is through expandingPeople's umbilical hemopoietic ancestral cells after increasing is to the ability of polyphyly differentiation.
Feature of the present invention is: by the microenvironment of hematopoietic stem/progenitor growth in analogue body, application hDll1-RGD canBe incorporated into huve cell surface with target, provide for the activated form of Notch acceptor and stimulate to hematopoietic stem/progenitor,Thereby reach the effect of desirable proliferation of hematopoietic stem/progenitor cell in vitro, and hematopoietic cell after amplification has good hematopoiesisReconstruction ability, has illustrated that the cell after amplification is still keeping the characteristic of candidate stem cell, has wide potential applicability in clinical practice.
Brief description of the drawings
For the ease of understanding, describe the present invention by Figure of description:
Fig. 1: bleeding of the umbilicus CD34 increases under different condition of culture+Total cell number;
Fig. 2: CD34 in the amplifying cells that different condition of culture downflow system cell instruments detect+The ratio of cell;
Fig. 3: flow cytometer detects the rear cell surface marker CD34 of hDll1-RGD amplification;
Fig. 4: bleeding of the umbilicus CD34 under different condition of culture+The amplification times of cell;
Fig. 5: the plastidogenetic hematopoiesis colony amplification times increasing under different condition of culture;
Fig. 6: NOD/SCID mouse is transplanted after the cell of cultivating through different condition, the 4th week mouse peripheral blood humanized CD45+The ratio of cell;
Fig. 7: NOD/SCID mouse is transplanted after the cell of cultivating through different condition, the 8th week mouse peripheral blood humanized CD45+The ratio of cell;
Fig. 8, NOD/SCID mouse are transplanted after the cell of cultivating through different condition, and within the 8th week, flow cytometer is to mouse peripheryBlood humanized CD45+Cell detects.
Detailed description of the invention
Embodiment
(1) experiment material
(1) Human umbilical vein endothelial cells is cultivated as follows
(2) Cord Blood-Derived is in all obstetrics and gynecology hospitals of Tang of The Fourth Military Medical University
(3) reagent:
M199, hyclone: Gibco company
StemSpan serum-free medium, MethoCult GFH4434:Stemcell company
ECGS EndothelialCellGrowthSupplement (ECGS): Sigma company
Bovine serum albumin(BSA) BovineSerumAlbumin(BSA): Sigma company
II Collagenase Type: Sigma company
Mitomycin C: Sigma company
CD34MicroBeadKit:Miltenyi company
Separate buffer:PBS+2mMEDTA+0.5%BSA
Lymphocyte separation medium: TBD company
PBSA：PBS+2mMEDTA
SCF(stemcellfactor): recombinant human scf, Peprotech company
TPO(thrombopoietin): restructuring hTPO, Peprotech company
FL(flt3ligand): restructuring hFL, Peprotech company
IL-6(interleukin-6): recombined human IL-6, Peprotech company
IL-3(interleukin-3): recombined human IL-3, Peprotech company
HDll1-RGD albumen: this research department researches and develops voluntarily
FITC mark antihuman CD 34 monoclonal antibody: BDBiosciences company
The anti-human CD45 monoclonal antibody of APC mark: BDBiosciences company
The anti-mouse CD45 of FITC mark monoclonal antibody: BDBiosciences company
NOD/SCID mouse is purchased from Beijing HFK Bio-Technology Co., Ltd.
(2) experimental technique
1, the cultivation of Human umbilical vein endothelial cells
Get human umbilical vein, separate, turn out huve cell; Huve cell nutrient solution is M199,20%Hyclone, 30 μ g/mLECGS.
Concrete operations are: under aseptic condition, get the umbilical cord in 2h after healthy parturient childbirth, be about 20cm, cut offClamp place, umbilical cord two ends. Insert syringe needle in umbilical vein one end, rinse completely to without after bloodstain with PBSA, haemostatic clamp clamps navelVenal distal, is full of vein with 0.2% collagenase solution 10mL, hatches after 20min for 37 DEG C, massages gently umbilical cord, by digestive juiceBe collected into centrifuge tube, rinse umbilical vein 2 times with 30mLPBSA, flushing liquor is together collected into centrifuge tube, 1500rpm is centrifugal10min, abandons supernatant, adds huve cell complete culture solution re-suspended cell, after counting, with 1 × 105/ mL cell connectsPlant in 24 orifice plates, every hole adds complete culture solution, and 24h removes not adherent cell, within every 3 days, changes fresh medium, straightTo Growth of Cells to approximately 90% merge, carry out passage, get 3rd ~ 5 generation huve cell as supportint cell, carry outSubsequent experimental.
2, bleeding of the umbilicus CD34+Cell separation purifying
The bleeding of the umbilicus of anticoagulant heparin is pressed 1:1 dilution proportion with PBSA, and the bleeding of the umbilicus of dilution is slowly added to lymphocyte separation mediumUpper, centrifugal 15 minutes of 20 ° of C, 1500rpm, on lymphocyte separation medium liquid level, obviously visible ring-type milky cellular layer, littleThe heart is drawn cellular layer cell, PBSA washing 2 times, PBS suspension cell counting. Every 108Mononuclearcell separates with 300 μ LBuffer is resuspended, and cell suspension adds respectively FcR blocking agent, the each 100 μ L of CD34 magnetic bead, fully mixes, 4 ° of C refrigerators are hatched 30 pointsClock, separates buffer washing, abandons supernatant. With 500 μ L separation buffer suspension cells. MS splitter is fixed on to MACS to be dividedIn the magnetic field of instrument, with the moistening splitter of 500 μ L separation buffer, by the slowly adherent splitter that adds of cell suspension, make not tieThe cell closing passes through splitter. Unconjugated cell is washed away to (500 μ L × 3 time) with separating buffer, splitter is shifted out to magnetic, with 1mL separation buffer pressurization wash-out, PBSA washs CD34+Cell, and count for subsequent use.
3, the amplification in vitro of umbilical hemopoietic ancestral cells
The Human umbilical vein endothelial cells of cultivating with M199 nutrient solution (containing 20%FBS, 30 μ g/mLECGS), plants into 24 orifice plates,After merging approximately 90%, process 2.5h through mitomycin C, PBS adds StemSpan after washing 3 timesTMSerum-free medium, then pastIn cultivating system, add SCF, TPO, FL, IL-6, IL-3, soluble fusion protein hDll1-RGD(concentration to be respectively: 120ng/ML, 20ng/mL, 50ng/mL, 5ng/mL, 5ng/mL, 2.5ug/mL), the cultivating system of setting up new hematopoietic stem/progenitor is standbyWith. By the CD34 of separation and purification+Cell, plantation, to the above-mentioned serum free culture system of supporting containing huve cell, puts 37℃、5%CO2In complete wet incubator, cultivate 7 days. Suspension cell in centrifugal collection cultivating system, Trypan Blue calculates living cellsNumber, flow cytometer detects CD34+The ratio of cell, calculates CD34+The amplification times of cell, inoculation people colony formation experimentIn culture medium, and prepare to transplant to NOD/SCID mouse.
4, the detection of hematopoietic stem/progenitor
Suspension cell in centrifugal collection cultivating system, with PBS washing 2 times, and is made into 1 × 107/ mL cell concentration, gets50 μ L cell suspensions are centrifugal, then add 0.5 μ LFITC mark antihuman CD 34 monoclonal antibody, and lucifuge, hatches 30min for 4 DEG C,Then add 1mL streaming liquid (to contain 5% hyclone, 0.2%NaN3PBS) cell is hanged, centrifugal 1300rpm, 5min, heavyAfter backwashing is washed one time. Up flow type cell instrument FACScalibur detects, and obtains and analyzes with Cellquest software.
5, hematopoietic stem/progenitor colony forms experiment
Collect suspension cell in centrifugal cultivating system, PBS washes twice, with 103It is solid that/mL is suspended from 1.lmL methylcellulose halfBody culture medium H4434 kind is in diameter 35mm culture dish, and each sample is done 1 multiple hole. When cultivation by the little training of 2 identical samplesSupport ware, with 1 little culture dish that only adds sterilized water and uncovered, with the large culture dish that is placed in 1 diameter 10cm. Be placed in 37 DEG C,5%CO2Complete wet incubator is cultivated after 14 days and is observed under inverted microscope, and the cell mass that is greater than 50 cells is counted 1 colonyForm unit (Colony-formingunit, CFU). Under inverted microscope, counted the red assembly of the quick-fried formula of the CFU(shape that falls in the 14th dayBecoming the BFU-E of unit, grain is colony forming unit CFU-GM, mixed stocker colony forming unit CFU-Mix).
6, NOD/SCID mouse transplantation model
NOD/SCID mouse, female, 6-8W, raises in aseptic cage, raises the diet with high pressure sterile, 300cGy before transplantingSublethal dose radiation ray whole body irradiates, and in 4 hours, carries out transplantation experiments through tail vein injection. Experiment grouping: 1,5GF group: movePlant through using Human umbilical vein endothelial cells as supportint cell, five kinds of growth factors of use in conjunction are cultivated the cell of 7 days, average everyMouse transplants 2 × 107; 2, hDll1-RGD group: transplant warp using Human umbilical vein endothelial cells as supportint cell, use in conjunction fivePlant the serum-free co-culture system of growth factor and hDll1-RGD and cultivate the cell of 7 days, average every mouse transplants 2 × 107，Every group of 8 mouse. After transplanting, observe mouse humanized CD45+The situation of the implantation of cell. Transplant latter the 4th week, cut Mouse Tail-tipGet blood, flow cytometer detects mouse peripheral blood humanized CD45+The ratio of cell. Transplant latter the 8th week, pluck eyeball and get peripheryBlood, cervical vertebra dislocation method is put to death mouse, gets bone marrow cells in mice, and inoculation people colony forms in the culture medium of experiment, detects humanizedHematopoietic cell colony forms ability.
(1), NOD/SCID mouse peripheral blood humanized CD45+Cell detection
After transplanting, 4w, 8w gather mouse peripheral blood through heparin anti-coagulating, get 50 μ L whole bloods, after erythrocyte splitting, usePBS washing 2 times, and be made into 1 × 107/ mL cell concentration, gets 50 μ L cell suspensions centrifugal, then adds 0.5 μ LFITC markAnti-human CD45 monoclonal antibody and the anti-mouse CD45 of 0.5 μ LAPC mark monoclonal antibody, lucifuge, hatches 30min, then adds for 4 DEG CEnter 1mL streaming liquid and (contain 5% hyclone, 0.2%NaN3PBS) cell is hanged to centrifugal 1300rpm, 5min. Repeated washingOne time. Up flow type cell instrument FACScalibur detects, and obtains and analyzes, with humanized with Cellquest softwareCD45+Cell > 1% is for to transplant successfully.
(2), NOD/SCID mouse humanized hematopoietic cell colony forms experiment
After transplanting the 8th week, cervical vertebra dislocation method was put to death mouse, gets bone marrow cells in mice, after erythrocyte splitting, washes with PBSWash 2 times, with 1 × 105/ mL is suspended from 1.lmL methylcellulose semisolid culturemedium H4434 kind in diameter 35mm culture dish, everyIndividual sample is done 1 multiple hole. When cultivation by the little culture dish of 2 identical samples, with 1 little cultivation that only adds sterilized water and uncoveredWare, with the large culture dish that is placed in 1 diameter 10cm. Be placed in 37 DEG C, 5%CO2Complete wet incubator is cultivated after 14 days micro-in being invertedMicroscopic observation, the cell mass that is greater than 50 cells is counted 1 colony forming unit (Colony-formingunit, CFU).Counted under inverted microscope in the 14th day CFU(burst forming unit erythroid（BFU E） BFU-E, grain be colony forming unit CFU-GM,Mixed stocker colony forming unit CFU-Mix).
With mean+SD, (χ ± s) represent adopts SPSS17.0 software to carry out statistical analysis, two groups to experimental dataBetween relatively adopt t inspection, group difference adopts variance analysis, P < 0.05 is thought statistical significance.
Effect of the present invention is further described by following detection and verification method:
1, hematopoietic stem/progenitor CD34 under amplification system+Amplification
Separation and purification CD34 from bleeding of the umbilicus+, with 2 × 103/ mL kind was cultivated in each group of different amplification systems, in the 7th dayCollect centrifugal suspension cell, carry out cell count, CD34+The expanding effect of cell, referring to table 1, is respectively organized CD34+Cell obtains a large amount ofAmplification, and with hDll1-RGD group expanding effect the best (see Fig. 1,**P<0.01），CD34+Increase approximately 67.50 ± 3.03 times and (see figure4，**P < 0.01). And keep candidate stem cell characteristic effect to have a clear superiority in compared with other groups with hDll1-RGD group, approximately 66.12%Still keep CD34+Phenotype (seeing Fig. 2, Fig. 3). 5GF group increases for alone five kinds of growth factors; HUVEC group forHUVEC is as supportint cell, adds and increases by five kinds of growth factors; HDll1-RGD group is using HUVEC as supportint cell,Add simultaneously and increase by five kinds of growth factors and hDll1-RGD).
2, hematopoietic stem/progenitor colony forms experimental analysis
Form experiment by colony the cell after cultivating has been carried out to Function detection, organize CFU number compared with other with hDll1-RGDGroup showed increased, result is referring to table 2, and cell forms colony with the highest (see figure 5) of CFU-GM ratio.
3, NOD/SCID mouse is transplanted humanized's hematopoietic stem/progenitor Colony-forming assay in rear marrow
NOD/SCID mouse is put to death after transplanting on the 8th week, and extracting marrow cell, with 1 × 105It is solid that/mL is suspended from methylcellulose halfIn body culture medium, cultivate after two weeks, all find that there is hematopoiesis colony and form. Through using HUVEC as supportint cell, add with five kinds simultaneouslyAfter the cell transplantation mouse that growth factor and hDll1-RGD increase, the hematopoiesis colony of each system form ability all apparently higher thanThe situation of transplanting after the amplification of 5GF group, result is referring to table 3.
The result of NOD/SCID mouse transplantation model shows, is migrated in Mice Body through the cell of cultivating system amplificationMerit, and Humanized cell implant ratio apparently higher than 5GF group, hematopoietic reconstitution ability obviously strengthens in vivo. (see Fig. 6,7,8）
The part that the present embodiment does not describe in detail and english abbreviation belong to the common practise of the industry, can search on the netRope arrives, here not narration one by one.
Claims (1)
1. an amplification in vitro method for hematopoietic stem/progenitor, is characterized in that: by soluble fusion protein hDll1-RGD targetTo being incorporated into huve cell surface, activate Notch acceptor, amplification people umbilical hemopoietic ancestral cells, comprises following stepRapid:
1) after huve cell merges 90%, process 2.5h through mitomycin C, washing; Described huve cellNutrient solution is M199,20% hyclone, 30 μ g/mLECGS; The concentration of described mitomycin C: 10 μ g/mL;
2) will in the huve cell after step 1), add serum-free medium; Described serum-free medium isStemSpanTMSerum-free medium;
3) toward step 2) nutrient solution in add five kinds of exogenous growth factor 5GF and soluble fusion protein hDll1-RGD, buildThe amplification in vitro system of vertical hematopoietic stem/progenitor; Described exogenous growth factor 5GF is SCF, TPO, FL, IL-6, IL-3;
4) separation, purification of hematopoietic ancestral cells CD34 from bleeding of the umbilicus+, the amplification system that plantation is set up to step 3), 37 DEG C, 5%CO2In complete wet incubator, cultivate 7 days;
5) adopt the people umbilical hemopoietic ancestral cells of NOD/SCID mouse transplantation model assessment after amplification; Described NOD/SCID mouse transplantation model assessment hematopoietic stem/progenitor method: adopt Sublethal Doses NOD/SCID mouse, 4h is interior through tailPeople's umbilical hemopoietic ancestral cells after intravenous injection amplification, after transplanting, 4w, 8w detect the energy that humanized's hematopoietic cell is implantedPower;
6) forming the people umbilical hemopoietic ancestral cells of experimental evaluation after amplification by hematopoietic stem/progenitor colony divides to polyphylyThe ability of changing; Described hematopoietic stem/progenitor colony forms experiment, adopts the people's umbilical hemopoietic ancestral cells after amplification to connectPlant in methylcellulose semisolid culturemedium, counted colony forming unit in the 14th day under inverted microscope, assessment is after amplificationPeople's umbilical hemopoietic ancestral cells to the ability of polyphyly differentiation;
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: 2.5 μ g/mL;
Described soluble fusion protein hDll1-RGD melts with the solubility of vascular endothelial cell targeting proteins matter motif RGDHop protein, wherein RGD is the polypeptide taking arginine (R)-glycine (G)-L-aminobutanedioic acid (D) as core, can specifically identifying blood vesselThe integrin molecule of endothelial cell surface, thus the protein molecular merging is with it anchored to Surface of Vascular Endothelial Cells.
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| JP6104312B2 (en) * | 2014-06-19 | 2017-03-29 | 日東電工株式会社 | Tissue regeneration promoter |
| AU2016297523B2 (en) * | 2015-07-20 | 2022-07-28 | Angiocrine Bioscience, Inc. | Methods and compositions for stem cell transplantation |
| 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 |
| CN109722415B (en) * | 2017-10-27 | 2021-01-26 | 博雅辑因（北京）生物科技有限公司 | Hematopoietic stem cell culture composition, culture medium and hematopoietic stem cell culture method |
| 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 |
| 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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