CN105039257A - In-vitro expansion cultivation method of hematopoietic stem cells - Google Patents
In-vitro expansion cultivation method of hematopoietic stem cells Download PDFInfo
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Abstract
The invention relates to an in-vitro expansion cultivation method of hematopoietic stem cells. The cultivation method comprises the following steps that hematopoietic stem cells and matrix cells are respectively obtained; the hematopoietic stem cells and the matrix cells are separated by culture device having biocompatibility for co-culture, wherein the culture device is provided with a plurality of through holes, and the hole diameter of the through hole is smaller than or equal to 3.0 microns. The in-vitro expansion cultivation method of the hematopoietic stem cells facilitates purification of the hematopoietic stem cells and can increase cell proliferation times of the hematopoietic stem cells and improve immunity safety.
Description
Technical field
The present invention relates to biotechnology and biomedicine field, particularly a kind of amplification in vitro cultural method of hemopoietic stem cell.
Background technology
Current hemopoietic stem cell has three sources usually: marrow, peripheral blood and Cord blood.Compare with peripheral blood hematopoietic stem cells with marrow, umbilical cord blood hematopoietic stem cell is more original, self duplication ability is the strongest, and Cord blood have be rich in more early stage hemopoietic stem cell, gather convenient, the antibody that hemopoietic stem cell immunogenicity is weak, produce heterogenetic antigen is few, do not need in migration process strict distribution type, maturity T cell less, gather and preserve easily, negative for tumor cells pollutes, donor be can't harm to the ratio comparatively advantages of higher injuring side effect, CD34+CD38-and CD34+CD33-, thus umbilical cord blood hematopoietic stem cell has great clinical value.But the hemopoietic stem cell content of single part of bleeding of the umbilicus is low, be difficult to the transplanting of normal type adult patient in clinical.Therefore, hemopoietic stem cell needs to carry out amplification in vitro before transplantation.
And the amplification in vitro of hemopoietic stem cell needs the nourishing of stroma cell, the research of associated substrate cell and hemopoietic stem cell Dual culture, in currently available technology, major part all have employed the method for directly contact Dual culture.In direct contact Dual culture, hemopoietic stem cell can stick on stroma cell or enter cell feeder layer inside, although two kinds of cells are weak binding, but realizing two kinds of cells is separated very difficult completely, and must use not containing the pure hemopoietic stem cell of stroma cell in clinical application, therefore, the method for this direct contact Dual culture also exists purifying cells difficulty or the safety issue such as immunological safety is low in clinical application.
Summary of the invention
Technical problem to be solved by this invention is, there is the defects such as purification of hematopoietic stem cells difficulty, immunological safety are low in the mode directly contacting Dual culture amplifying candidate stem cell for hemopoietic stem cell in prior art and stroma cell, provides a kind of amplification in vitro method being convenient to the safer hemopoietic stem cell of purification of hematopoietic stem cells, immunity.
The technical solution adopted for the present invention to solve the technical problems is: the amplification in vitro cultural method providing a kind of hemopoietic stem cell, and described cultural method comprises the following steps:
Obtain hemopoietic stem cell and stroma cell respectively;
Described hemopoietic stem cell and stroma cell are separated by the cultivation part with biocompatibility and carry out Dual culture;
Wherein, described cultivation part has some through holes, and the aperture of described through hole is less than or equal to 3.0 microns.
In the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, the concentration ratio of described hemopoietic stem cell and stroma cell is 5:1-3:1.
In the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, the pore size of described through hole is in 0.4-3.0 micrometer range.
In the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, described cultivation part is that described hemopoietic stem cell and stroma cell separate by tabular.
In the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, described cultivation part is opening-like and have a spatial accommodation, is set in cell culture container.
In the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, bottom described cultivation part with culture hole bottom spacing be 10-450 μm.
In the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, bottom described cultivation part with cell culture container bottom spacing be 150-350 μm.
In the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, described cultivation part is made up of polycarbonate material.
In the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, described hemopoietic stem cell is cord blood stem cell, and described stroma cell is fat stem cell.
Implement hematopoietic stem cell population cultural method provided by the invention, following beneficial effect can be reached: hemopoietic stem cell and stroma cell are separated by adopting the cultivation part with biocompatibility and permeability, and only carry out material exchange by cultivating part, hemopoietic stem cell carries out the indirect contact of kytoplasm fine hair by cultivation part and stroma cell, also can be noncontact, not only solve hemopoietic stem cell and stroma cell in prior art and directly contact the difficult problem be separated of Dual culture, and also improve the amplification times of hemopoietic stem cell, and strengthen the immunological safety of hemopoietic stem cell.
Accompanying drawing explanation
Below in conjunction with drawings and Examples, the invention will be further described, in accompanying drawing:
Fig. 1 is in hematopoietic stem cell population cultural method provided by the invention, the schematic cross-section of the first culture vessel and cultivation part;
Fig. 2 is the second culture vessel provided by the invention and the schematic cross-section cultivating part;
Fig. 3 is the structural representation of the third cultivation part provided by the invention;
Fig. 4 is the 4th kind of culture vessel provided by the invention and the schematic cross-section cultivating part.
Embodiment
There is purifying stem cell difficulty in the mode directly contacting Dual culture amplifying candidate stem cell for solving hemopoietic stem cell and stroma cell in prior art, the defects such as immunological safety is low, innovative point of the present invention is in hemopoietic stem cell and stroma cell co-culture system, a kind of cultivation part with permeability is provided, for separating hemopoietic stem cell and stroma cell, prevent both directly contacts, make hemopoietic stem cell contact indirect type with stroma cell by cell fine hair to cultivate or contactless cultivation, and the secretory product of nutrient solution and stroma cell can pass freely through and cultivates part and provide nutrition for hemopoietic stem cell and stroma cell, thus solve in prior art, be difficult to a difficult problem for isolating hematopoietic stem cells and stroma cell, also improve the immunological safety of hemopoietic stem cell.
Particularly, the amplification in vitro cultural method of a kind of hemopoietic stem cell provided by the invention, comprises the following steps:
S1, respectively acquisition hemopoietic stem cell and stroma cell;
S2, adopt the cultivation part with biocompatibility to separate hemopoietic stem cell and stroma cell, then Dual culture is carried out to hemopoietic stem cell and stroma cell;
Wherein, cultivate part and there is some through holes, and the aperture of through hole is less than or equal to 3.0 microns.
Further, in step sl, hemopoietic stem cell can derive from marrow, peripheral blood and Cord blood, compare with peripheral blood hematopoietic stem cells with marrow, umbilical cord blood hematopoietic stem cell is more original, self duplication ability is the strongest, it is convenient to gather, immunogenicity is weak, the antibody that produces heterogenetic antigen is few, do not need in migration process strict distribution type, maturity T cell less, gather and preserve easily, negative for tumor cells pollutes, donor be can't harm to the ratio comparatively advantages of higher injuring side effect, CD34+CD38-and CD34+CD33-; Therefore, in the present invention, preferentially adopt umbilical cord blood hematopoietic stem cell, particularly, the source procedure that comes of hemopoietic stem cell is:
S11a, collection Cord blood;
S12a, separation obtain human umbilical cord blood mononuclear cell;
S13a, human umbilical cord blood mononuclear cell inducing culture is become umbilical cord blood hematopoietic stem cell.
In step S11a, Cord blood is all from healthy puerpera's term birth or mature surgical neonate, and each collection capacity is 50-100mL; In step S12a, first the Cord blood gathered in step S11a and PBS damping fluid are obtained Cord blood diluent with 1:1 mixed diluting; Then, in 15mL aseptic plastic centrifuge tube, adding the Ficoll parting liquid that 4mL density is 1.077g/mL, (Ficoll parting liquid is a kind of according to cell density difference, by the universal gravity constant of centrifugal generation, carry out the common agents of the separation and purification of cell, purchased from Sigma company), then lightly Cord blood diluent is added drop-wise on Ficoll liquid level with suction pipe, with the rotating speed density gradient centrifugation 25 minutes of 2500 revs/min, wherein the ratio of Ficoll parting liquid and Cord blood diluent is 1:(1-2); After centrifugal end, tunica albuginea layer in the middle of drawing with suction pipe, obtains UCB-MNCS (human umbilical cord blood mononuclear cell); Cell after separation IMDM basic medium centrifuge washing twice (1000rpm, 5 minutes), adjustment mononuclearcell density is 2 × 10
5cells/ml is for subsequent use.Owing to containing a large amount of hemopoietic stem cells in Cord blood, therefore, obtain containing umbilical cord hemopoietic stem cell in mononuclearcell in this step, by its Dual culture together with mononuclearcell, the microenvironment of a umbilical cord hemopoietic stem cell can be simulated, more contribute to the amplification of umbilical cord hemopoietic stem cell.In step S13a, by the mononuclearcell that obtains in step S12a with 2 × 10
5it is 25cm that the density of cells/ml is seeded in floorage
2and be equipped with in the tissue culture flasks (T-flask) of nutrient solution, and at 37 DEG C, 5%CO
2, 20%O
2, cultivate 7 days under saturated humidity condition, change into hemopoietic stem cell to mononuclearcell, and adopt flow cytometric sorting to go out the hemopoietic stem cell of Cord Blood-Derived.
Wherein, nutrient solution is IMDM substratum (Iscove'sModifiedDulbecco'sMedium, Gibco company produces), and in IMDM substratum, be added with the HS (horse serum) that volume fraction is 10%, 10%FBS, 16ng/mlSCF (human stem cell factor), the FL (Flt-3 part) of 7.47ng/ml, the TPO (thrombopoietin) of 7.47ng/m1, IL-3 (the interleukin Ⅲ of 5.33ng/ml, hematopoietic stem cell growth factor), the G-CSF (granulocyte colony-stimulating factor) of 3.33ng/ml, the GM-CSF (granulocyte-macrophage colony-stimulating factor) of 2.13ng/ml.
In addition, in step sl, due to the self that fat stem cell provides fatty tissue lifelong, there is multi-lineage potential, multiple known and unidentified growth factor can be secreted, to maintain and amplifying candidate stem cell, and fat stem cell is present in fatty tissue, extract the advantages such as more convenient, therefore, the present invention preferably selects fat stem cell as stroma cell.
The acquisition process of fat stem cell is:
S11b, acquisition fatty tissue;
S12b, from fatty tissue, extract fat stem cell;
S13b, Secondary Culture is carried out to fat stem cell, and obtain third generation fat stem cell.
In step S11b, fatty tissue takes from the subcutaneous normal-fat tissue in surgery patients (age 16-60 year).
Step S12b is specially: get the subcutaneous lipids obtained in step S11b and be about 5g, with the abundant rinsing of the PBS damping fluid dual anti-containing 500U/mL 3 times, reject macroscopic blood vessel and reticular tissue, with eye scissors, it is fully shredded, repeatedly rinse with PBS again, remove red corpuscle as far as possible.Then 0.2% NTx enzyme of 2 times of volumes is added, after vibration mixing, in the constant water bath box digestion 60min of 37 DEG C, add isopyknic containing volume fraction be 10% foetal calf serum low sugar DMEM stop digestion, the centrifugal 10min of 1800r/min, is divided into 3 layers after centrifugal, upper strata is grease and does not digest fatty tissue completely, middle level is supernatant liquor, and lower floor is the precipitation of the cell mixing such as fat stem cell and red corpuscle.Abandon upper strata and middle level, (perfect medium is low sugar DMEM to add perfect medium, wherein, also containing volume fraction is 10% foetal calf serum, 100U/mL penicillin and 100U/mL Streptomycin sulphate) re-suspended cell precipitation, filtrate adjustment cell concn after filtering is 1 × 10 by 70 μm of cell screen filtrations
8cells/L is seeded in 60mm culture dish, in 37 DEG C, volume fraction 5%CO
2, cultivate under the condition of saturated humidity.The later half amount of 24h changes liquid, and after 48h, full dose changes liquid, and every 2d changes liquid 1 time later.
Step S13b is specially: original cuiture 7 ~ 9d grows fusion and reaches 90%, after 0.25% pancreatin (containing 0.2%EDTA) conventional digestion, to go down to posterity the nutrient solution be inoculated into containing serum, put into 37 DEG C, 5%CO by 1:2 or 1: 3
2carry out subculture in vitro separately amplification cultivation in incubator, obtain the 3rd fat subsitutes stem cell.
Be understandable that, in above-mentioned steps S1, the obtain manner of hemopoietic stem cell and stroma cell is only wherein a kind of embodiment provided by the invention, and the technique means for obtaining hemopoietic stem cell and stroma cell in prior art is equally applicable to the present invention.
In S2 step, when the aperture of cultivating the through hole on part is greater than 3.0 microns, stroma cell can move to hemopoietic stem cell side through through hole, directly contacts Dual culture with hemopoietic stem cell, now, substantially identical with stroma cell Dual culture mode with hemopoietic stem cell of the prior art; Therefore, in the present invention, the aperture of limited hole is less than or equal to 3 microns, and preferably, the pore size of through hole is in 0.4-3.0 micrometer range, and when aperture is 0.45 micron, the kytoplasm fine hair of stroma cell can realize indirect contact through through hole and hemopoietic stem cell; Substratum transmits the paracrine of stroma cell by through hole, promotes the amplification of hemopoietic stem cell.
Further, cultivating part is be made up of polycarbonate membrane or the semi-permeable membranes with biocompatibility, because polycarbonate has good biocompatibility, and solubility solution can freely pass through, the key component that can not discharge toxin or stick in substratum is contacted with cell culture fluid, the amplification cultivation environment of comparatively suitable hemopoietic stem cell, therefore, in the present invention, the preferential polycarbonate membrane that adopts makes cultivation part, the material of certain cultivation part is also not limited thereto, can select according to the cell type of Dual culture and the nutrient solution adopted type, be advisable not affect hematopoietic stem cell expansion effect, there is biocompatibility and permeable materials.
Further, cultivating part can be that cell culture container is divided into two or more cultivation region by tabular or the tubulose coaxial with cell culture container, only carries out material exchange by the through hole cultivated on part between different cultivation region.Because hemopoietic stem cell and stroma cell are adherent growth, therefore, when cultivation part is tabular, preferably, cultivate part and cell culture container is separated into its different cultivation regions axially, namely the different cultivation regions on vertical direction, are convenient to the distance controlling hemopoietic stem cell and stroma cell, improve stroma cell to the nutritional support effect of hemopoietic stem cell; Meanwhile, for ensureing the hypertonicity of nutrient solution, the thickness cultivating part is less than 20 microns.And cultivate part with cell culture container inwall is movable and be connected, be convenient to inoculating cell in different cultivation region, cell culture container 2 inwall arrange for lay the boss 21 (as shown in Figure 1) of cultivating part 100 or cell culture container 2 inwall offer for card establish cultivate part 110 groove 22 (as shown in Figure 2) also or as shown in Figure 3, cultivating bottom part 120 arranges for supporting the support portion 121 of cultivating part 120, the support that support portion 121 is made up of two or more supporting leg, can cultivation part 120 be directly positioned in cell culture container 2, picking and placeing of convenient cultivation part 120.In addition, migrate to two kind cells by cultivation part with the gap between cell culture container for preventing hemopoietic stem cell and stroma cell directly to contact, the surrounding of cultivating part is provided with O-ring seal, with sealing gap between the two, the material of O-ring seal is not to be advisable with amplification cultivation system generation chemical reaction.
Be understandable that, cultivate part also can be fixedly connected with cell culture container inwall, now, the sidewall of cell culture container bottom it offers inoculation mouth, and configure sealing plug, can sealing plug be extracted during inoculation, stroma cell or hemopoietic stem cell be added the cultivation region be positioned at bottom cell culture container; Inoculation completes, and will prevent nutrient solution from flowing out with sealing plug sealing inoculation mouth.
As shown in Figure 4, cultivating part 130 also can be opening-like and have a spatial accommodation, is set in cell culture container 2, and axially cell culture container 2 is divided into different cultivation regions along cell culture container 2; Preferably, cultivate part 130 cup-shaped, the bottom that cup-shaped cultivates part 130 is permeability film, and for ensureing the hypertonicity of nutrient solution and stroma cell secretory product, the thickness of film is less than 20 microns; It is identical with cell culture container 2 or to be made up of polycarbonate membrane equally that cup-shaped cultivates the side-wall material of part 130, can directly be set in cell culture container 2; Also a support platform 131 can be extended outward at cultivation part 130 opening part, cultivate part to coordinate with cell culture container 2 opening part by this support platform 131, making to cultivate part 130 can be directly sheathed or hang in cell culture container 2, be unlikely to touch bottom cell culture container 2 to avoid cultivating part 130 to the further support limit of cultivation part 130, the modes such as cultivation part 130 and cell culture container 2 shrink-fit certainly also can be made to realize cultivating the fixing of part; Inoculated stromal cell, in cell culture container 2, cultivates the outside of part 130; Hemopoietic stem cell is inoculated in be cultivated in part 130, part 130 is cultivated owing to placed in cell culture container 2, therefore the spatial accommodation cultivated in part 130 is comparatively large relative to the remaining space in cell culture container 2, is comparatively suitable for hemopoietic stem cell and provides an amplification space be relatively suitable for; The relative position of certain hemopoietic stem cell and stroma cell is also not limited thereto, and can be configured according to specific needs.Be understandable that, the shape and structure cultivating part is also not limited thereto, and also can be membrane filter or permeability support.
Further, when cultivate part cell culture container is divided into vertical on different cultivation region time, to cultivate bottom part and bottom cell culture container between spacing excessive or too small, all can affect stroma cell paracrine to the support of hemopoietic stem cell and amplification effect, therefore, in the present invention, cultivate bottom part with cell culture container bottom spacing be 10-450 μm, make stroma cell and hemopoietic stem cell by the indirect contact of cell fine hair or noncontact, simultaneously, distance between cultivating bottom part and cell culture container also determines stroma cell and closely secretes and the concentration of/paracrine, and then affect its power to the supporting function of hematopoietic stem cell expansion, and when to cultivate the spacing bottom part and cell culture container be 150-350 μm, hemopoietic stem cell and stroma cell can carry out cell signal transmission, there are paracrine and nearly secretion, and paracrine accounts for leading role, nutrient under this spacing needed for mononuclearcell by stroma cell absorb little, can keep needed for cell amplification, therefore, the expanding effect of hemopoietic stem cell is best.Be understandable that, if to cultivate bottom part and bottom cell culture container between spacing excessive or too small time, the concentration by adjustment hemopoietic stem cell and stroma cell realizes the amplification of hemopoietic stem cell, specifically can adjust according to practical situation and experiment.
Further, in step s 2, be that the hemopoietic stem cell of 5:1-3:1 and stroma cell are inoculated in cell culture well respectively by the different cultivation regions of cultivating part and being separated by concentration ratio, the DMEM nutrient solution added containing 10%FBS is cultivated hemopoietic stem cell and stroma cell simultaneously, remains that the total cell concentration in culture system is (1.0-1.5) x10 in Dual culture process
6individual/mL; If cell concn is more than 2 × 10
6during cells/ml, part cell suspension can be taken out from system, add fresh medium simultaneously, make the cell concn in substratum system keep (1.0-1.5) x10
6individual/mL.
These are only wherein a kind of hemopoietic stem cell provided by the invention and stroma cell Dual culture process, on improvement basis of the present invention, in prior art, hemopoietic stem cell and stroma cell Dual culture process are equally applicable to the present invention.
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Embodiment is:
Embodiment 1
The amplification in vitro cultural method of hemopoietic stem cell provided by the invention is:
1, obtain umbilical cord blood hematopoietic stem cell and third generation fat stem cell respectively, and to adjust hemopoietic stem cell concentration be 1 × 10
6individual/m1, fat stem cell concentration is 2 × l0
5individual/m1;
2, adopt cultivation part as shown in Figure 1, the culture hole of culture plate is divided into two cultivation regions in vertical direction; Wherein, this cultivation part is opening cup-shaped and has a spatial accommodation, and its opening part extends a support platform laterally, for coordinating with section in culture hole, provides support position-limiting action to cultivation part; Cultivating bottom part is polycarbonate membrane, and the aperture of polycarbonate membrane is 0.4 micron, and thickness is 10 microns, and the distance bottom this polycarbonate membrane and culture hole is 350 microns.
First, be 2 × l0 by concentration
5the fat stem cell of individual/m1 is inoculated in pretreated 6 orifice plates, cultivation part is put into the culture hole of 6 orifice plates after most cell attachment merges, then inoculum density is 1 × 10 in the spatial accommodation cultivating part
6individual/m1 hemopoietic stem cell, and the DMEM substratum added containing 10%FBS carries out Dual culture.In Dual culture process, when cultivating the total cell density of the mononuclearcell in part more than 2 × 10
6individual/ml time, from system, take out part cell suspension, add fresh medium simultaneously, make cell density in system be reduced to (1.0-1.5) × 10
6in the scope of individual/m1, dilution is namely adopted to change the method for liquid.
For verifying the unusual effect of hematopoietic stem cell population cultural method provided by the invention further, carry out detection validation by following experiment.
In this experimentation, control group is Co-culture group, by 2 kinds of direct mixed culture of cell; Remaining 6 groups of experimental group be respectively cultivate part through-hole aperture at 0.45 micron and cultivate bottom part with the indirect contact culture experiment of culture hole bottom space within the scope of 10-450 μm, namely cultivate in 6 groups of experimental group bottom part and be respectively 10 μm, 50 μm, 150 μm, 250 μm, 350 μm, 450 μm with culture hole bottom space, often organize co-culture experiments and carry out 7 days.
1.CD34+ the flow cytometry analysis of cell
Application BectonDickinson type flow cytometer carries out CD34-FITC antibody test to the cell before and after amplification, and cell marking method is: once, add CD34-FITC antibody 10 μ L, 4 DEG C of lucifuges hatch 30 minutes to the washing of cell PBS buffer salt solution; Hatch after terminating and wash again once with PBS buffer salt solution, use the content of flow cytometry analysis CD34+ cell, as following table 1.
2. amplification times calculates
In Dual culture process, the total cell density of the mononuclearcell cultivated in part when experimental group is more than 2 × 10
6individual/ml time, from system, take out part cell suspension, add fresh medium simultaneously, make cell density in system be reduced to (1.0-1.5) × 10
6in the scope of individual/m1, dilution is namely adopted to change the method for liquid.When calculating cells expanded, think that the cell be removed has the amplification ability the same with the cell stayed in system, as following table 1.
Test-results:
Table 1
As can be seen from the above results, the amplification in vitro cultural method of hemopoietic stem cell provided by the invention obtains hematopoietic stem cell expansion multiple and significantly improves; And can find out in Electronic Speculum display, when being 350 microns with culture hole bottom space bottom cultivation part, the cell fine hair of Electronic Speculum display hemopoietic stem cell and fat stem cell can contact with each other, and the amplification times of hemopoietic stem cell is the highest.
In sum, the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, hemopoietic stem cell and stroma cell are cultivated part and are separated and carry out Dual culture, by the indirect contact of cell fine hair between two kinds of cells, or noncontact, thus the step be separated with stroma cell after saving hematopoietic stem cell expansion, solve in prior art and hemopoietic stem cell is directly contacted Dual culture with stroma cell, be difficult to the problems such as separation, improve the immunological safety of hemopoietic stem cell, simultaneously, by the amplification in vitro cultural method of hemopoietic stem cell provided by the invention, substantially increase the amplification times of hemopoietic stem cell.
Below by reference to the accompanying drawings embodiments of the invention are described; but the present invention is not limited to above-mentioned embodiment; above-mentioned embodiment is only schematic; instead of it is restrictive; those of ordinary skill in the art is under enlightenment of the present invention; do not departing under the ambit that present inventive concept and claim protect, also can make a lot of form, these all belong within protection of the present invention.
Claims (9)
1. an amplification in vitro cultural method for hemopoietic stem cell, is characterized in that: described cultural method comprises the following steps:
Obtain hemopoietic stem cell and stroma cell respectively;
Described hemopoietic stem cell and stroma cell are separated by the cultivation part with biocompatibility and carry out Dual culture;
Wherein, described cultivation part has some through holes, and the aperture of described through hole is less than or equal to 3.0 microns.
2. the amplification in vitro cultural method of hemopoietic stem cell according to claim 1, is characterized in that, the concentration ratio of described hemopoietic stem cell and stroma cell is 5:1-3:1.
3. the amplification in vitro cultural method of hemopoietic stem cell according to claim 1, is characterized in that, the pore size of described through hole is in 0.4-3.0 micrometer range.
4. the amplification in vitro cultural method of hemopoietic stem cell according to claim 1, is characterized in that, described cultivation part is that described hemopoietic stem cell and stroma cell separate by tabular.
5. the amplification in vitro cultural method of hemopoietic stem cell according to claim 1, is characterized in that, described cultivation part is opening-like and have a spatial accommodation, is set in cell culture container.
6. the amplification in vitro cultural method of hemopoietic stem cell according to claim 1, is characterized in that, and bottom described cultivation part with cell culture container bottom spacing be 10-450 μm.
7. the amplification in vitro cultural method of hemopoietic stem cell according to claim 6, is characterized in that, bottom described cultivation part with cell container bottom spacing be 150-350 μm.
8. the amplification in vitro cultural method of hemopoietic stem cell according to claim 1, is characterized in that, described cultivation part is made up of polycarbonate material.
9. the amplification in vitro cultural method of the hemopoietic stem cell according to any one of claim 1-8, is characterized in that, described hemopoietic stem cell is cord blood stem cell, and described stroma cell is fat stem cell.
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