CN106479976A - A kind of method of Cord blood megakaryoblast In vitro culture - Google Patents
A kind of method of Cord blood megakaryoblast In vitro culture Download PDFInfo
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Abstract
The present invention discloses a kind of method of Cord blood megakaryoblast In vitro culture.The method is cord blood cells to be inoculated in specific megakaryoblast culture medium cultivated.Described specific megakaryoblast culture medium is the serum-free medium containing volume fraction 40～50% hematopoietic stem cell culture supernatant, 50 ± 10ng/mL SCF and 50 ± 10ng/mL TPO.Described hematopoietic stem cell culture supernatant is that a point umbilical cord blood hematopoietic stem cell is cultivated in containing IL 6, IL 3, the serum-free medium of SCF, Flt 3L and TPO acquisition in 3～4 days.The present invention effectively increases the CFU-GM characteristic of the umbilical blood megakaryoblast of amplification in vitro, increased the efficiency obtaining megakaryoblast, thus improve megakaryoblast to be divided into hematoblastic ability.The present invention expands the stage in megakaryoblast, decreasing the consumption adding cytokine, thus decreasing material consumption, reducing cost.
Description
Technical field
The invention belongs to cell technology field is and in particular to a kind of method of Cord blood megakaryoblast In vitro culture.
Background technology
After carrying out within 1989 umbilical cord blood stem cell transplantation in the world for the first time and succeeding, umbilical cord blood hematopoietic is done thin
The clinical practice of born of the same parents is just more and more extensive, and through the development of more than 20 years, current Cord blood became together with bone marrow, peripheral blood
The three of hematopoietic stem cell originate greatly.
Due to before hematopoietic stem cell transplantation, receptor's Large Dose Irradiation/chemotherapy generally to be carried out to reach clear marrow effect, and
Radiotherapy/chemotherapy can make the hemopoietic system of receptor be badly damaged, and leads to platelet famine in a period of time, easy bleeding is even
Cause death, so needing to be transfused the platelet of a large amount of external sources.With respect to bone marrow, hematoblastic resume speed after Umbilical Cord Blood Transplantation
Slightly slow, generally believe that reason is that megakaryoblast quantity in Cord blood is not abundant and differentiation and maturation is slow at present.Therefore,
The quantity increasing every part of umbilical blood megakaryoblast is the key of solve problem.Megakaryoblast amplification in vitro can be very good to solve
The few problem of cell quantity.As long as giving enough dosage, they can provide sufficient amount of required for being quickly temporarily implanted
CFU-GM, reduces the time required for realizing being temporarily implanted.Currently, the method for external megakaryoblast directed expansion, generally adopts
With combination of cytokines expanded, mainly have IL-3, IL-6, SCF, TPO and FLT-3L etc..Although prior art is in macronucleus
CFU-GM amplification in vitro aspect achieves certain effect, but the CFU-GM characteristic of megakaryoblast is not in incubation
Can maintain well, thus reducing megakaryoblast long term to be divided into hematoblastic ability.
Content of the invention
In order to overcome shortcoming and the deficiency of prior art, it is an object of the invention to provide a kind of Cord blood megakaryoblast
The method of In vitro culture.Using the method increasing Cord blood megakaryoblast quality and quantity.The method is adopted using Cord blood
Remove blood plasma and erythrocyte after collection, obtain the total nucleated cell (TNC) rich in hematopoietic stem cell.By CD34+Magnetic bead sorting can
Obtaining purer hematopoietic stem cell, the hematopoietic stem cell sub-electing being cultivated three to four days in serum-free medium, thus obtaining
Obtain hematopoietic stem cell culture supernatant.By, in cultivating system, adding hematopoietic stem cell culture supernatant to carry out megakaryoblast
Amplification method, while relatively traditional method decreases the addition of cytokine, the megakaryoblast of acquisition maintains preferably
CFU-GM characteristic, increased the efficiency obtaining megakaryoblast.
The purpose of the present invention is achieved through the following technical solutions：
A kind of method of Cord blood megakaryoblast In vitro culture, comprises the steps of：
Cord blood cells are inoculated in specific megakaryoblast culture medium and are cultivated.
The time of described culture is 7～14 days.
Described specific megakaryoblast culture medium is the training of the serum-free containing hematopoietic stem cell culture supernatant, SCF and TPO
Foster base；
The final concentration of described SCF and TPO is respectively 50 ± 10ng/mL and 50 ± 10ng/mL；
The interpolation volume fraction of described hematopoietic stem cell culture supernatant be specific megakaryoblast culture medium 40～
50%；It is preferably 50%.
The preparation method of described hematopoietic stem cell culture supernatant, comprises the steps：
Hematopoietic stem cell is seeded in hematopoietic stem cell culture medium and is cultivated, it is thin that centrifugation method collects Hematopoietic Stem
Born of the same parents' culture supernatant.
Described hematopoietic stem cell culture medium is the serum-free medium containing IL-6, IL-3, SCF, Flt-3L and TPO.
The final concentration of described IL-6, IL-3, SCF, Flt-3L and TPO be respectively 20ng/mL, 20ng/mL, 50ng/mL,
50ng/mL and 20ng/mL.
The time of described culture is 3～4 days.
Described serum-free medium is Stemspan culture medium (STEMCELL company).
Described cord blood cells separate as follows and obtain：Gather fresh human cord blood, in people's umbilicuss of fresh and healthy
Obtain in band blood and be rich in CD34+The mononuclearcell layer of hematopoietic stem cell, adds Cell protective solutions, Programmed freezing, is frozen in liquid
Preserve in nitrogen；Before megakaryoblast amplification, take cord blood cells to thaw, plus the resuspended washing of normal saline is collected by centrifugation, obtains relatively again
Pure cord blood cells；
Described Cell protective solutions are to be that 55% dimethyl sulfoxide (DMSO) and percent by volume are containing percent by volume
The aqueous solution for injection of 5% low molecular dextran (Dextran).
Described condition of culture is preferably temperature and is 37 DEG C, and gas concentration lwevel is 5%.
The method of described raising Cord blood megakaryoblast cultured and amplified in vitro, specifically comprises the steps of：
(1) preparation of hematopoietic stem cell culture supernatant
Obtain mononuclearcell from the fresh and healthy people's umbilical blood in 24 hours, be then passed through immunological magnetic bead sorting and obtain
CD34+Hematopoietic stem cell, will sorting after hematopoietic stem cell add Stem Span culture medium culturing amplification, also need to simultaneously
Add 20ng/mL IL-6,20ng/mL IL-3,50ng/mL SCF, 50ng/mL Flt-3L and five kinds of cells of 20ng/mL TPO
The factor, cultivates to the 3rd～4 day about, collects hematopoietic stem cell culture supernatant；
Detailed process is as follows：
1) the fresh umbilical blood in collection 24 hours in healthy subjects
2) normal saline and fresh umbilical blood 2:1 dilution proportion, after mix homogeneously, is added on lymphocyte separation medium, enters
The separation of row mononuclearcell, centrifugation time 25 minutes, centrifugal force 500g, centrifugal acceleration 1, deceleration 1,20 DEG C of temperature；
3), after centrifugation terminates, take tunica albuginea layer (tunica albuginea layer in plasma layer and separates between liquid layer).Add the physiology of 3 times of volumes
Saline, washed once, centrifugal force 800g, is centrifuged 10min；
4) collect cell precipitation, washed again once with the PBS containing EDTA and human serum albumin, centrifugal force
200g, centrifugation time 10 minutes；
5), after being centrifuged, collect cell precipitation, resuspended with the PBS containing EDTA and human serum albumin,
MiniMACS immunomagnetic beadses method obtains the high CD34 of purity+Hematopoietic stem cell；
6) hematopoietic stem cell of acquisition is seeded in T25 culture bottle, add appropriate culture medium, simultaneously add cell because
Son；Culture collects cell supernatant in 3～4 days, and -20 DEG C save backup；
(2) configuration of specific megakaryoblast culture medium
Specific megakaryoblast culture medium is to add hematopoietic stem cell culture supernatant, human stem cell in serum-free medium
Somatomedin (Stem cell factor, SCF) and the culture medium of thrombopoietin (Thrombopoietin, TPO)；
(3) amplification of megakaryoblast
Cord blood cells after will be frozen are inoculated into culture in specific megakaryoblast culture medium, and during inoculation, cell density is 3
×105Individual/mL, three and half amounts change liquid once, cultivate to inverted microscope observation of cell state when the 7th day and 14 days, and cell
Count；Flow cytomery cell CD41+And CD34+The positive rate of surface antigen, cultivates to 14 days, harvesting.
Cord blood cells described in step (3) separate as follows and obtain：Gather fresh human cord blood, fresh strong
Obtain in the human cord blood of health and be rich in CD34+The mononuclearcell layer of hematopoietic stem cell, addition Cell protective solutions, Programmed freezing,
It is frozen in liquid nitrogen and preserve；Before megakaryoblast amplification, take cord blood cells to thaw, plus the resuspended washing of normal saline is centrifuged receipts again
Collection, obtains purer cord blood cells；
Described Cell protective solutions are to be that 55% dimethyl sulfoxide (DMSO) and percent by volume are containing percent by volume
The aqueous solution for injection of 5% low molecular dextran (Dextran).
Condition of culture described in step (3) is preferably temperature and is 37 DEG C, and gas concentration lwevel is 5%；
The present invention, with respect to prior art, has such advantages as and effect：
(1) present invention effectively increases the CFU-GM characteristic of the umbilical blood megakaryoblast of amplification in vitro.Thus improve huge
Core CFU-GM is divided into hematoblastic ability.
(2) present invention expands the stage in megakaryoblast, decreases the consumption adding cytokine, thus decreasing material
Consume, reduce cost.
Brief description
Fig. 1 be in embodiment 1 experimental group and matched group umbilical blood megakaryoblast the cell of the 1st, 7,14 days is total after inoculation
The result figure of quantity.
Fig. 2 is the experimental group and matched group umbilical blood megakaryoblast sample CD41 of the 7th, 14 days after inoculation in embodiment 1+
The result figure of surface antigen expression.
Fig. 3 is the experimental group and matched group umbilical blood megakaryoblast sample CD34 of the 7th, 14 days after inoculation in embodiment 1+
The result figure of cell surface antigen expression.
Fig. 4 is the experimental group and matched group sample megakaryoblast aspect graph of the 2nd, 7 days after inoculation in embodiment 1.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention do not limit
In this.
Cytoprotective is to be the injection that 55%DMSO and percent by volume are 5%Dextran containing percent by volume
Aqueous solution；
Specific megakaryoblast culture medium：Add SCF (final concentration in the Stemspan culture medium of STEMCELL company
50ng/mL) and TPO (final concentration 50ng/mL) cytokine and hematopoietic stem cell culture supernatant that volume fraction is 50%.
Embodiment 1
Cord blood picks up from the pregnant baby of healthy puerpera, after testing hepatitis B, hepatitis C, syphilis, AIDS, cytomegaloviruses,
Mycoplasma, chlamydia, G-DPD and ground lean are feminine gender.Specimen is maintained at 4～8 DEG C from collecting the transport temperature transporting blood bank back,
Cord Blood Bank is transported in 24 hours.Carry out megakaryoblast amplification operation by the following method：
(1) preparation of hematopoietic stem cell culture supernatant
Obtain mononuclearcell from the fresh and healthy people's umbilical blood in 24 hours, be then passed through immunological magnetic bead sorting and obtain
CD34+Hematopoietic stem cell, will sorting after hematopoietic stem cell add Stem Span culture medium culturing amplification.Also need to simultaneously
Add 20ng/mL IL-6,20ng/mL IL-3,50ng/mL SCF, 50ng/mL Flt-3L and five kinds of cells of 20ng/mL TPO
The factor.Cultivate to the 3rd day about, collect hematopoietic stem cell culture supernatant.Detailed process is as follows：
1. the fresh umbilical blood in collection 24 hours in healthy subjects
2. normal saline and fresh umbilical blood 2:1 dilution proportion, after mix homogeneously, is added on lymphocyte separation medium, enters
The separation of row mononuclearcell, centrifugation time 25 minutes, centrifugal force 500g, centrifugal acceleration 1, deceleration 1,20 DEG C of temperature.
3., after centrifugation terminates, take tunica albuginea layer (tunica albuginea layer in plasma layer and separates between liquid layer).Add the physiology of 3 times of volumes
Saline, washed once, centrifugal force 800g, is centrifuged 10min.
4. collect cell precipitation, washed again once with the PBS containing EDTA and human serum albumin, centrifugal force
200g, centrifugation time 10 minutes.
5., after being centrifuged, collect cell precipitation, resuspended with the PBS containing EDTA and human serum albumin,
MiniMACS immunomagnetic beadses method obtains the high CD34 of purity+Hematopoietic stem cell.
6. the hematopoietic stem cell of acquisition is seeded in T25 culture bottle, add appropriate culture medium, simultaneously add cell because
Son.Culture collects cell supernatant in 3～4 days, and -20 DEG C save backup.
(2) configuration of specific megakaryoblast culture medium
Specific megakaryoblast culture medium is to add hematopoietic stem cell culture supernatant, human stem cell in serum-free medium
Somatomedin (Stem cell factor, SCF) and the culture medium of thrombopoietin (Thrombopoietin, TPO)；
The interpolation volume fraction of described hematopoietic stem cell culture supernatant is the 50% of specific megakaryoblast culture medium.
In described specific megakaryoblast culture medium, the final concentration of SCF and TPO is respectively 50ng/mL and 50ng/mL；
Described serum-free medium is Stemspan culture medium (STEMCELL company)；
(3) amplification of megakaryoblast
Cord blood cells after will be frozen are inoculated into culture in defined medium, and during inoculation, cell density is 3 × 105Individual/mL,
Three and half amounts change liquid once, cultivate to inverted microscope observation of cell state when the 7th day and 14 days, and cell counting.Streaming is thin
Born of the same parents' instrument detects cell CD41+And CD34+The positive rate of surface antigen, cultivates to 14 days, harvesting.
Cord blood cells described in step (3) separate as follows and obtain：Gather fresh human cord blood, fresh strong
Obtain in the human cord blood of health and be rich in CD34+The mononuclearcell layer of hematopoietic stem cell, addition Cell protective solutions, Programmed freezing,
It is frozen in liquid nitrogen and preserve；Before megakaryoblast amplification, take cord blood cells to thaw, plus the resuspended washing of normal saline is centrifuged receipts again
Collection, obtains purer cord blood cells；Described Cell protective solutions be containing percent by volume be 55% dimethyl sulfoxide
(DMSO) and percent by volume be 5% low molecular dextran (Dextran) aqueous solution for injection.
Condition of culture described in step (3) is preferably temperature and is 37 DEG C, and gas concentration lwevel is 5%；
(4) identification of megakaryoblast
The identification of megakaryoblast is by the following method：
1. the morphological analysis of megakaryoblast and cell counting：By the cell of culture in inverted microscope observation of cell shape
State is simultaneously taken pictures；
2. the phenotype test of flow cytometer：Take the 7th day after culture respectively, the cell of 14 days, carry out CD41+And CD34+Inspection
Survey；
(5) megakaryoblast qualification result and cell activation assay
1. observe the cord blood cells state of the 2nd day after inoculating under inverted microscope：Observe the 2nd after inoculation under inverted microscope
It cord blood cells, cell quantity is sufficient, and in dispersed, form is more single, and cell body is bright and justifies (Fig. 4 A).Cytometer
Number result, experimental group and cellular control unit quantity are about：3.4×105Individual (Fig. 1)；
2. observe the cord blood cells state of the 7th day after inoculating under inverted microscope：Compared with the 1st day, cell quantity is slightly
Change, form starts differentiation, and central major part is bulk cord blood cells, and fraction starts to break up, clustering, after birth
Unintelligible, arrangement is in closely lumps (Fig. 4 B).Cell counts, experimental group and matched group quantity are respectively：8.46×105
Individual, 7.6 × 105Individual (Fig. 1).
Flow cytomery result：In CD41+The numerical value of cell proportion aspect, experimental group and matched group is respectively：
9.9%th, 5.4% (Fig. 2), CD34+The numerical value of cell proportion aspect, experimental group and matched group is respectively：8.47%th, 2.78%
(Fig. 3).
3. observe the umbilical blood megakaryocytopoiesis situation of the 14th day after inoculating under inverted microscope：Compared with the 7th day, carefully
Born of the same parents' quantity amplification is more obvious, and form is also substantially broken up.Cell counts：Experimental group and matched group cord blood cells quantity are respectively
For 27.5 × 105Individual/mL, 24.6 × 105Individual/mL (Fig. 1).
Flow cytomery result：In CD41+The numerical value of cell proportion aspect, experimental group and matched group is respectively：
14.6%th, 10.83% (Fig. 2), CD34+The numerical value of cell proportion aspect, experimental group and matched group is respectively：1.06%th,
0.55% (Fig. 3).
After culture in 14 days, cell counts show：Experimental group cell quantity than the 7th day when have obvious rising, carefully
Born of the same parents' total amount is slightly above matched group；In terms of streaming result, treatment group CD41+And CD34+Cell proportion is all far above matched group.
In sum, in umbilical blood megakaryoblast amplification procedure, experimental group is slightly above matched group situation in cell quantity
Under, CD41+% and CD34+% cell proportion, apparently higher than matched group, drastically increases the amplification effect of umbilical blood megakaryoblast
Rate, thus increased the growing amount of megakaryoblast and preferably maintaining the CFU-GM potential of megakaryoblast.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not subject to above-described embodiment
Limit, other any spirit without departing from the present invention and the change made under principle, modification, replacement, combine, simplify,
All should be equivalent substitute mode, be included within protection scope of the present invention.
Claims (9)
1. a kind of method of Cord blood megakaryoblast In vitro culture is it is characterised in that comprise the steps of：
Cord blood cells are inoculated in specific megakaryoblast culture medium and are cultivated；
Described specific megakaryoblast culture medium is the serum-free culture containing hematopoietic stem cell culture supernatant, SCF and TPO
Base.
2. Cord blood megakaryoblast In vitro culture according to claim 1 method it is characterised in that：
The final concentration of described SCF and TPO is respectively 50 ± 10ng/mL and 50 ± 10ng/mL.
3. Cord blood megakaryoblast In vitro culture according to claim 1 and 2 method it is characterised in that：
The interpolation volume fraction of described hematopoietic stem cell culture supernatant is the 40～50% of specific megakaryoblast culture medium.
4. Cord blood megakaryoblast In vitro culture according to claim 3 method it is characterised in that：
Hematopoietic stem cell is seeded in hematopoietic stem cell culture medium and is cultivated, centrifugation method collects hematopoietic stem cell training
Foster supernatant.
5. Cord blood megakaryoblast In vitro culture according to claim 4 method it is characterised in that：
Described hematopoietic stem cell culture medium is the serum-free medium containing IL-6, IL-3, SCF, Flt-3L and TPO.
6. Cord blood megakaryoblast In vitro culture according to claim 5 method it is characterised in that：
The final concentration of described IL-6, IL-3, SCF, Flt-3L and TPO be respectively 20ng/mL, 20ng/mL, 50ng/mL,
50ng/mL and 20ng/mL.
7. the Cord blood megakaryoblast In vitro culture according to any one of claim 4～6 method it is characterised in that：
The time of described culture is 3～4 days.
8. the Cord blood megakaryoblast In vitro culture according to claim 1 or 2 or 5 or 6 method it is characterised in that：
Described serum-free medium is Stemspan culture medium.
9. Cord blood megakaryoblast In vitro culture according to claim 1 method it is characterised in that：
The time of described culture is 7～14 days.
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| CN111053788A (en) * | 2019-12-25 | 2020-04-24 | 广州市天河诺亚生物工程有限公司 | Composition for adjuvant therapy of chronic hemorrhage and application thereof |
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| CN101864396A (en) * | 2010-05-17 | 2010-10-20 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing megakaryoblast and megakaryocyte in vitro |
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| CN111053788A (en) * | 2019-12-25 | 2020-04-24 | 广州市天河诺亚生物工程有限公司 | Composition for adjuvant therapy of chronic hemorrhage and application thereof |
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Application publication date: 20170308 |