CN101525596A - Method for obtaining stem cell by using serum of same cord blood - Google Patents
Method for obtaining stem cell by using serum of same cord blood Download PDFInfo
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- CN101525596A CN101525596A CN200910301568A CN200910301568A CN101525596A CN 101525596 A CN101525596 A CN 101525596A CN 200910301568 A CN200910301568 A CN 200910301568A CN 200910301568 A CN200910301568 A CN 200910301568A CN 101525596 A CN101525596 A CN 101525596A
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- cord blood
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Abstract
The invention discloses a method for obtaining stem cell by using serum of the same cord blood, belonging to the field of biotechnology and tissue engineering. Serum of the same cord blood is used to replace fetal calf serum, no cell factor or stroma cell support is added, and the strategy of combining a microcarrier and a bioreactor is adopted, thus realizing co-culture of cord blood source hemopoietic stem cell and mesenchymal stem cell. The method of the invention can support fine augmentation of UCB-HSCs and UCB-MSCs under three-dimensional conditions, completely replace the role of fetal calf serum and avoid immunological rejection among variants; cytodex-s microcarrier can provide adhesion surface for UCB-MSCs with adherence growth characteristics, so that the UCB-MSCs can realize possible adherence suspension culture in a three-dimensional dynamic environment; the natural settling method is adopted to separate and obtain the suspending UCB-HSCs and the UCB-MSCs which adheres to the microcarrier.
Description
Technical field
The invention belongs to biotechnology and field of tissue engineering technology, relate to a kind of serum of same cord blood that uses and gather in the crops the method for stem cell, specially refer to the method that a kind of serum that uses same cord blood obtains umbilical cord blood hematopoietic stem cell and mescenchymal stem cell simultaneously.
Technical background
(Hematopoietic stem cells HSCs) can be widely used in aspect (Bellantuono I.The International Journal of Biochemstry﹠amp such as gene therapy, tumour purification and bone marrow transplantation to hemopoietic stem cell; CellBiology, 2004,36 (4): 607-620), especially hematopoietic stem cell transplantation has become the ordinary method of hematopoietic reconstitution behind cancer patients's chemicotherapy, has greatly limited its application clinically but cell quantity is limited.The effective ways of head it off are the external extensive amplifications of implementing HSCs.Although hematopoietic stem cell transplantation can be improved the quality of life after cancer patients's chemicotherapy significantly, but the serious neutrophilic granulocyte that most patients inevitably will bear in a short time reduces the misery of disease and thrombocytopenia, so is necessary to propose the generation that new treatment plan reduces these complication.
Mescenchymal stem cell (Mesenchymal stem cells, MSCs) be proved to be the function that has the hematopoiesis support generation external, in in NOD/SCID mouse body, testing simultaneously, also proved and transplanted reconstruction (the Kim D H that HSCs and MSCs have quickened the mouse hemopoietic function simultaneously, et al.Journal of Korean Medical Science, 2006,21 (6): 1000-1004).And for the hematopoietic stem cell transplantation after patient with breast cancer's chemotherapy clinically, combined transplantation goes into from the MSCs of body to have the function that promotes hematopoietic reconstitution equally, has reduced the sickness rate of following the hematopoietic stem cell transplantation complication simultaneously.In addition, MSCs also can be used as the seed cell and the Vectors in Gene Therapy cell of cell therapy.Therefore, can in same culture system, cultivate simultaneously and results HSCs and two kinds of stem cells of MSCs, have important clinical application value.
(Umbilical cord blood UCB) as one of source of HSCs, contains MSCs to Cord blood simultaneously.With respect to marrow and peripheral blood, umbilical hemopoietic stem cell (CBSC) have be rich in that more early stage hematopoietic stem, immunogenicity antibody weak, that heterogenetic antigen is produced is few, maturity T cell is less, gather and preserve easily, negative for tumor cells pollutes, to harmless side effect, the CD34 of injuring of donor
+CD38
-With CD34
+CD33
-Ratio higher, in migration process, can reduce series of advantages such as graft versus host disease (GVH disease) (GVHD) incidence, wide material sources, make that the hematopoietic stem research relevant with Cord blood becomes focus.
Yet HSCs and MSCs have the biological characteristics of suspension growth and adherent growth respectively, externally must provide suspension culture environment and dynamic wall-attached surface simultaneously, just might be in analogue body realize the common cultivation of HSCs and MSCs under the condition of microenvironment.It is the feasible method of head it off that microcarrier combines with suitable bio-reactor.Sephadex (cytodex-3) microcarrier of denatured collagen bag quilt attaches characteristic with its surface of good, is receiving much attention aspect the cultivation of attached cell Three-Dimensional Dynamic.
In addition,, use the autoserum in the same cord blood to provide better nutritive substance as cell with respect to the foetal calf serum of widespread usage, simultaneously, in the later clinical application of cell, the also immune response that can avoid foetal calf serum to bring.
Therefore, the serum with same cord blood replaces foetal calf serum, does not add cytokine and do not have the stroma cell support, the strategy that adopts microcarrier to combine with bio-reactor, the common cultivation of realization Cord blood source hemopoietic stem cell and mescenchymal stem cell.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of method of turning out umbilical cord blood hematopoietic stem cell and mescenchymal stem cell simultaneously.
Technical scheme of the present invention may further comprise the steps:
1, separation of human Cord blood monocyte: Cord blood is from healthy puerpera's term birth or mature surgical neonate, each Cord blood collection capacity average out to 50～100mL.After Cord blood is gathered, mixing with the PBS buffer salt solution in 1: 1 by volume, is 1.077g.mL with density
-1Ficoll cellular segregation liquid with 2500rpm, (Mononuclear cells, MNCs), the volume ratio of Ficoll cellular segregation liquid and Cord blood is 1: 1～1: 2 to obtain monocyte after 25 minutes density gradient centrifugations.Cell after separation IMDM twice of centrifuge washing of basic medium (1000rpm, 5 minutes), it is standby to adjust cell density.
2, obtain same cord blood serum: the employed people's autoserum of this patent be and the Cord blood serum in the same source of cell that its preparation method is: after Cord blood was gathered, 1: 1 by volume and PBS buffer salt solution mixed, and are 1.077g.mL with density
-1Ficoll cellular segregation liquid with 2500rpm, 25 minutes density gradient centrifugation, results monocyte; Draw the first layer serum solution with dropper then, insert in the aseptic centrifuge tube standby.
3, the pre-treatment of cytodex-3 microcarrier: exsiccant cytodex-3 microcarrier is placed the vial of siliconizing, and with not containing Ca
2+, Mg
2+The PBS buffer salt solution at room temperature soak 4 hours (50～100mL
-1The cytodex-3 microcarrier) more than, to mix up once in a while therebetween.With the fresh Ca that do not contain
2+, Mg
2+Twice of PBS buffer salt solution flushing after, autoclaving (115 ℃, 15 minutes, 1 normal atmosphere) is standby.
4, cultivate UCB-HSCs and UCB-MSCs altogether: the UCB-MNCs that fresh separated is obtained is with 2 * 10
6Cells.mL
-1Density be inoculated in 24 orifice plates that are paved with the cytodex-3 microcarrier in advance, add 20% Cord blood serum.The method that adopts natural subsidence is separated the UCB-HSCs that suspends and is gathered in the crops with UCB-MSCs on sticking to the GCSC microcarrier.
Effect of the present invention and benefit are:
(1) utilizes people's autoserum, not only can support UCB-HSCs, UCB-MSCs well amplification under three-dimensional condition, substitute the effect of foetal calf serum fully, can also avoid the immunological rejection between allosome.
(2) the cytodex-3 microcarrier can provide for the UCB-MSCs of adherent growth characteristic and stick the surface, makes UCB-MSCs realize that in the Three-Dimensional Dynamic environment adherent suspension culture becomes possibility.
(3) adopt the method for free setting, the UCB-HSCs that suspends is separated easily with two kinds of stem cells of UCB-MSCs on being attached on the GCSC microcarrier and gather in the crops.
(4) utilize method of the present invention, adding Cord blood serum, do not adding under the condition of cytokine and trophocyte's support, the strategy that adopts microcarrier to cultivate is cultivated UCB-HSCs and UCB-MSCs altogether in orifice plate.The result shows, the serum in same cord blood source can well be supported the amplification in vitro of UCB-HSCs and UCB-MSCs, and the attaching growth conditions of UCB-MSCs on the cytodex-3 microcarrier is good.
Description of drawings
Fig. 1 is the schema that UCB-HSCs of the present invention and UCB-MSCs cultivate altogether.
Fig. 2 is the layering synoptic diagram after the density gradient centrifugation among the embodiment 1.
Fig. 3 is UCB-MSCs adherent om observation result on cytodex-3 microcarrier surface among the embodiment 2.
Fig. 4 be among the embodiment 3 after the cytodex-3 microcarrier natural subsidence with the layering synoptic diagram of cell suspension.
Embodiment
Be described in detail specific embodiments of the invention below in conjunction with technical scheme and accompanying drawing.
Embodiment 1
Present embodiment is to obtain simultaneously to cultivate with serum and mononuclearcell in same cord blood.
Cord blood is from healthy puerpera's term birth or mature surgical neonate, Cord blood collection capacity 120mL.After Cord blood is gathered, mixing with PBS in 1: 1 by volume, is 1.077g.mL with density
-1Ficoll cellular segregation liquid with 2500rpm, 25 minutes density gradient centrifugation, the volume ratio of Ficoll parting liquid and Cord blood is 1: 1～1: 2.After centrifugal, as shown in Figure 2, the upper strata is a serum, inserts in the aseptic centrifuge tube standby after the collection; Tunica albuginea layer acquisition monocyte in the middle of getting (Mononuclearcells, MNCs), IMDM twice of the centrifuge washing of basic medium (1000rpm, 5 minutes) of the cell after the separation, it is standby to adjust cell density.
Embodiment 2
Present embodiment is UCB-HSCs and the common cultivation of UCB-MSCs under static conditions.
The UCB-MNCs that fresh separated is obtained is with 2 * 10
6Cells.mL
-1Density be inoculated in 6 orifice plates that are paved with the cytodex-3 microcarrier in advance, add the serum in 20% same cord blood source, at 37 ℃, 5%CO
2, 20%O
2And cultivated altogether 12 days under the saturated humidity condition.Every 24 hours countings, om observation.
Experimental result: the serum in same cord blood source, can well support the amplification in vitro of UCB-HSCs and UCB-MSCs, the attaching growth conditions of UCB-MSCs on the cytodex-3 microcarrier is good as seen from Figure 2.
After present embodiment is UCB-HSCs and the common cultivation of UCB-MSCs under static conditions, the separation of two kinds of cells.
After cultivate finishing, the cytodex-3 microcarrier is blown afloat into suspension, the suspension sucking-off is placed aseptic centrifuge tube with dropper, leave standstill 5 minutes after, the cytodex-3 microcarrier is deposited in bottom, as shown in Figure 4.Sucking-off upper strata cell suspension with 5mLIMDM flushing cytodex-3 microcarrier, leaves standstill three times repeatedly once more.
The upper strata cell suspension of separating out can obtain UCB-HSCs after centrifugal, will be attached at UCB-MSCs on the cytodex-3 microcarrier with 0.125% pancreatin and 0.02%EDTA digestion results.
Claims (1)
1. one kind is used the serum of same cord blood to gather in the crops the method for stem cell, it is characterized in that following steps:
(1) separation of human Cord blood monocyte: Cord blood is from healthy puerpera's term birth or mature surgical neonate, each Cord blood collection capacity average out to 50～100mL; After Cord blood is gathered, mixed with the PBS buffer salt solution in 1: 1 by volume, with density be the Ficoll cellular segregation liquid of 1.077g.mL-1 with 2500rpm, obtained monocyte after the density gradient centrifugation in 25 minutes, the volume ratio of Ficoll cellular segregation liquid and Cord blood is 1: 1～1: 2; Cell after separation IMDM basic medium 1000rpm, 5 minutes centrifuge washings twice, it is standby to adjust cell density;
(2) obtain same cord blood serum: after Cord blood is gathered, mixed with the PBS buffer salt solution in 1: 1 by volume, with density be the Ficoll cellular segregation liquid of 1.077g.mL-1 with 2500rpm, 25 minutes density gradient centrifugation after, gather in the crops monocyte; Draw the first layer serum solution with dropper then, insert in the aseptic centrifuge tube standby;
(3) pre-treatment of cytodex-3 microcarrier: exsiccant cytodex-3 microcarrier is placed the vial of siliconizing, and with do not contain Ca2+, Mg2+ ionic PBS buffer salt solution at room temperature soaked more than 4 hours, stir once in a while; With new do not contain twice of Ca2+, the flushing of Mg2+ ionic PBS buffer salt solution after, autoclaving is standby;
(4) cultivate UCB-HSCs and UCB-MSCs altogether: be inoculated in 24 orifice plates that are paved with the cytodex-3 microcarrier in advance separating the UCB-MNCs that obtains density, add 20% Cord blood serum with 2 * 106cells.mL-1; The method that adopts natural subsidence is separated the UCB-HSCs that suspends and is gathered in the crops with UCB-MSCs on sticking to the GCSC microcarrier.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747032A (en) * | 2011-04-19 | 2012-10-24 | 财团法人佛教慈济综合医院 | Non-tumorigenic expansion of pluripotent stem cells |
| CN103998048A (en) * | 2011-07-11 | 2014-08-20 | 车比奥及戴奥斯泰有限公司 | Method for manufacturing umbilical cord extract and usage of same |
| CN106801038A (en) * | 2015-11-26 | 2017-06-06 | 中国人民解放军军事医学科学院基础医学研究所 | 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 |
| CN107446891A (en) * | 2017-09-30 | 2017-12-08 | 吉林省太阳鸟再生医学工程有限责任公司 | A kind of method that human umbilical cord's blood candidate stem cell is expanded using itself umbilical cord mesenchymal stem cells as stroma cell |
| CN109355257A (en) * | 2018-11-29 | 2019-02-19 | 云南研灵生物科技有限公司 | The mescenchymal stem cell mixed culturing method of different tissue sources |
-
2009
- 2009-04-15 CN CN200910301568A patent/CN101525596A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102747032A (en) * | 2011-04-19 | 2012-10-24 | 财团法人佛教慈济综合医院 | Non-tumorigenic expansion of pluripotent stem cells |
| CN103998048A (en) * | 2011-07-11 | 2014-08-20 | 车比奥及戴奥斯泰有限公司 | Method for manufacturing umbilical cord extract and usage of same |
| CN106801038A (en) * | 2015-11-26 | 2017-06-06 | 中国人民解放军军事医学科学院基础医学研究所 | 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 |
| CN107446891A (en) * | 2017-09-30 | 2017-12-08 | 吉林省太阳鸟再生医学工程有限责任公司 | A kind of method that human umbilical cord's blood candidate stem cell is expanded using itself umbilical cord mesenchymal stem cells as stroma cell |
| CN107446891B (en) * | 2017-09-30 | 2019-01-18 | 吉林省太阳鸟再生医学工程有限责任公司 | A method of expanding human umbilical cord's blood candidate stem cell using itself umbilical cord mesenchymal stem cells as stroma cell |
| CN109355257A (en) * | 2018-11-29 | 2019-02-19 | 云南研灵生物科技有限公司 | The mescenchymal stem cell mixed culturing method of different tissue sources |
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Open date: 20090909 |