CN104862276B - A kind of method that dimensional culture stimulates induction generation Activated platelets with biochemistry - Google Patents
A kind of method that dimensional culture stimulates induction generation Activated platelets with biochemistry Download PDFInfo
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Abstract
The present invention relates to a kind of method that dimensional culture and biochemistry stimulate induction generation Activated platelets, the present invention utilizes Rotary three-dimensional bioreactor RCCS and the interim micromolecular compound and cell factor Combined culture CD34 acted on+Cell, it can efficiently obtain Activated platelets (CD41a+CD42b+).Compared with prior art, the invention has the advantages that：The biologically active pdgf that is obtained of the present invention is higher, and operability and repeatability are stronger, can be achieved it is external efficiently, quickly generate feature blood platelet.
Description
Technical field
The present invention relates to one kind to induce human cord blood CD 34+The method that cell differentiation produces Activated platelets, specifically, this
Invention, which is related to a kind of dimensional culture and biochemistry, stimulates induction human cord blood CD 34+Cell high-efficient differentiation produces Activated platelets
Method.
Background technology
Blood platelet is as caused by the megacaryocyte in marrow hemopoiesis tissue, and weight is played during body hemostasis and thrombus
Act on.Hematoblastic direct infusion is the effective means for alleviating inpatient with haematological diseases thrombocytopenia, and domestic " blood is waste " and
The problems such as blood product pollution, immunological rejection, cause blood platelet source insufficient, puzzlement is brought for treating hematopathy.Therefore, seek
Blood platelet new sources and external large-scale production feature blood platelet are the Major Strategic Demands that China faces.
So far, unified method and apparatus is not yet established to be used to efficiently produce feature blood platelet in vitro.To be terrible
To enough high quality blood platelets, microenvironment as far as possible in analogue body, optimize each stage condition of culture with promote megacaryocyte into
Ripe and thrombocytopoiesis is an important research direction.Research is found, utilizes the in-vitro simulated wall shear stress of fluid means
[Nakagawa Y,Nakamura S,Nakajima M,et al.Two differential flows in a
bioreactor promoted platelet generation from human pluripotent stem cell-
derived megakaryocytes.Exp Hematol.2013；41:742-748.] or utilize fibroin micro-pipe to simulate
Blood vessel wraps up various bone marrow matrix compositions simultaneously, and bone marrow microenvironment [Isabella is further simulated by perfusion
Pallotta,Michael Lovett,David L.Kaplan,Alessandra Balduini.Three-dimensional
system for the in vitro study of megakaryocytes and functional platelet
production using silk-based vascular tubes.Tissue Engineering Part C
Methods.2011；17(12):1223-32.] etc. can effectively facilitate thrombocytopoiesis and release.This studies body for us
Efficiently producing hematoblastic method provides new approaches outside.
Chinese Patent Application No.：201410067679.6 denomination of invention：One kind structure dimensional culture system induction of cord blood is done
The method that cell breaks up to blood platelet) one kind is disclosed with homemade chitosan-gelatin support and rotary incubation system
(rotaD, cell culture system, RCCS) co-cultures bleeding of the umbilicus CD34+Cells in vitro produces hematoblastic method.The party
Method prepares chitosan-gelatin support with freeze-drying, utilizes immunological magnetic bead sorting bleeding of the umbilicus CD34+Cell is simultaneously making support by oneself
Cultivated in culture systems with reference to RCCS.The 3D systems can be adapted to navel blood stem cell growth and Induction of committed differentiation to be that blood is small
Plate, but operating procedure is relatively cumbersome, and cultivation cycle is relatively long, is difficult to realize large-scale production, and biologically active pdgf detects
(including class granule of platelet adhesion rate, CD62p the positive expression rates) is without clear superiority.In addition, the system is subtracted using OPTI-MEM
Blood serum medium, the alloplasm in animal blood serum can not be evaded, its platelet product there may be the wind of human body alloplasm repulsion
Danger.
The content of the invention
The purpose of the present invention is to explore a kind of simple, economic, safety and efficiently from bleeding of the umbilicus CD34+Cell differentiation produces work
The hematoblastic method of property；This method operating process is simple, high income, biologically active pdgf are high.This method compared with the conventional method, has
There are higher operability and repeatability, be used for biomedical and clinical treatment for large-scale production feature blood platelet in the future
Lay the foundation.
The technical solution adopted by the present invention is：
It is a kind of from bleeding of the umbilicus CD34+The method that cell differentiation produces Activated platelets, this method comprise the following steps：
1) bleeding of the umbilicus CD34+The separation of cell
Human umbilical cord blood mononuclear cell is separated first by Ficoll separating liquids, is carried out twice using CD34 immunomagnetic beadses afterwards
Positive sorting, obtains high-purity (>=90%) CD34+Cell, and carry out cell count；
2) cell culture
A. with the serum-free of addition human thrombopoietin (hTPO), stem cell factor (SCF) and interleukin-3 (IL-3)
CD34 is resuspended in culture medium StemSpan SFEM+Cell, with 1 × 105/ mL initial densities are inoculated in 12 orifice plates, in 37 DEG C, 5%CO2
Quiescent culture 3 days in incubator；
B. the 3rd day full dose changes above-mentioned nutrient solution and cell factor, and adds ATRA in original culture medium
(ATRA) quiescent culture, is continued 3 days；
C. the 6th day fresh medium for changing addition human thrombopoietin (hTPO) and interleukin-11 (IL-11)
StemSpan SFEM, while the material for promoting megakaryocyte polyploid is added, it is transferred to rotation culture apparatus RCCS and continues to train
Support；The material of the promotion megakaryocyte polyploid is Rho kinase inhibitor Y-27632, Y-27632 functional analogues
Y230141, niacinamide NIC, serine/threonine kinases Aurora B inhibitor ZM447439, Src kinase inhibitor SU6656 or
One kind in inhibitors of myosin light chain kinase MLCKI；
D. the 9th day full dose changes nutrient solution and cell factor (hTPO, IL-11), and adds Rho kinase inhibitors Y-
27632nd, Decitabine and Wnt/ β-catenin signal secreting type activator R-spondin2, partly amount changes liquid, rotating and culturing daily
To the 10-15 days, CD41a is collected+CD42b+The granule of platelet.
Preferably, the final concentration of 50ng/mL of human thrombopoietin (hTPO) in step a, stem cell factor (SCF)
The final concentration of 20ng/mL of final concentration of 20ng/mL and interleukin-3 (IL-3).
Preferably, in the step b ATRA (ATRA) final concentration of 10-10-10-11M。
Preferably, the final concentration of 50ng/mL of human thrombopoietin (hTPO) described in the step c；White Jie
- 11 (IL-11) of element final concentration of 20ng/mL；Final concentration of the 10 of the material of the promotion megakaryocyte polyploid-9M。
Preferably, the material of the promotion megakaryocyte polyploid is Rho kinase inhibitors Y-27632.
Preferably, the model RCCS-4SC of the RCCS, culture rotating speed are 20rpm.
Preferably, in the step d Rho kinase inhibitors Y-27632 final concentration of 10-9M；Decitabine (DAC)
Final concentration of 10-7-10-9M；With Wnt/ β-catenin signal secreting type activator R-spondin2 (RSPO2) it is final concentration of
50ng/mL。
Preferably, rotating and culturing to the 12-13 days, collects CD41a in the step d+CD42b+The granule of platelet.
Preferably, described step 1) comprises the following steps：
A. Ficoll separating liquid separating umbilical blood mononuclearcells are utilized, and carry out cell count, every 1 × 108Individual cell adds
300 μ l sortings buffer is resuspended, and lucifuge adds 100 μ l FcR blocking agents and 100 μ l CD34 magnetic beads, and 4 DEG C of refrigerators stand 30min；
B. sample is taken out, adds 10ml sortings buffer to wash away uncombined cell, 1200rpm, 5min；Supernatant is removed completely,
1ml sortings buffer is added to be resuspended；
C. MS sorting posts are placed in magnetic field corresponding to MACS sorters, fully soaked point with 500 μ l sortings buffer
Post is selected, cell suspension is added into sorting post through filter membrane (200 mesh), and is rinsed 3 times with buffer, collects all cells as far as possible；
D. MS sorting posts are removed into magnetic field, be placed on 15ml centrifuge tube, sorting buffer is added in sorting post, uses MS
The piston pressurization that sorting post is equipped with releases rapidly magnetic mark cell；
E. above-mentioned sorting step is repeated, the CD34 being eluted out with new MS sorting post sortings+Cell, carry out secondary point
Choosing, obtain high-purity (>=90%) CD34+Cell, and carry out cell count.
The present invention using Rotary three-dimensional bioreactor RCCS (Synthecon, Inc., Houston, TX, U.S.A) with
The micromolecular compound and cell factor Combined culture CD34 of stage effect+Cell, it can efficiently obtain Activated platelets
(CD41a+CD42b+).Compared with prior art, the invention has the advantages that：
The biologically active pdgf that the present invention is obtained is higher, and operability and repeatability are stronger, can be achieved external efficient, quick
Generating functionality blood platelet.Applicant promotes megakaryocytic maturation using rotation culture apparatus RCCS, improves platelet yield, together
When by a small-scale molecule screening, in the specific feature small molecule of addition of different differential periods, further increase work
Property blood platelet (CD41a+CD42b+) ratio.Flow cytometer showed result shows, in biophysics and the dual thorn of biochemical signals
Under swashing, single CD34+The quantity of Activated platelets caused by cell improves 3 times or so than quiescent culture, is also significantly greater than text
Offer the general range of report.This inexpensive, highly controllable serum-free, without matrix Three-Dimensional Dynamic training strategy it is big rule in the future
Mould produces lays a good foundation for the feature blood platelet of clinical treatment.
Brief description of the drawings
Fig. 1 is Activated platelets monitoring curve after cell culture.
Fig. 2 is the 13rd day platelet activation curve.
Fig. 3 is each group platelet yield statistical chart.
In Fig. 1, Fig. 2, Fig. 3
RCCS (+) group, i.e., according to 1)-the 4 of embodiment 1) step tested；Since the 7th day, it is monitored daily.
RCCS (-) group, compared with RCCS (+), ATRA, Y-27632, RSPO are not added in this group of cell cultivation process2With
DAC。
SC (+) group, compared with RCCS (+), this group of cell culture is carried out under static conditions.
SC (-) group, compared with RCCS (+), this group of cell culture is carried out under static conditions, and in cell cultivation process
Do not add ATRA, Y-27632, RSPO2And DAC.
Embodiment
The present invention is further described below by specific embodiment, but does not limit protection scope of the present invention.
It is a kind of from bleeding of the umbilicus CD34+The method that cell differentiation produces Activated platelets, this method comprise the following steps：
1) bleeding of the umbilicus CD34+The separation of cell
A. bleeding of the umbilicus is poured into plasma bottle or 50ml centrifuge tubes, according to VCord blood:VSort buffer=1:1 adds sorting buffer
(formula is shown in Table 1), along with the HES for stating mixed volume 1/4 mixes, it is stored at room temperature 45min；
B. supernatant is gone into 50ml centrifuge tubes, room temperature, 1500rpm centrifugations 10min；Supernatant is abandoned, bonus point selects buffer to be resuspended
Cell；15ml centrifuge tubes are taken, often pipe first respectively adds 4.5ml Ficoll separating liquids (VFicoll:VSort buffer=1:1), above-mentioned resuspension
Liquid is added slowly in Ficoll separating liquids, avoids damage to liquid level layering, 1800rpm, 15min (0 liter of 0 drop)；
C. upper strata water sample is discarded, careful to draw tunica albuginea layer (mononuclearcell layer), bonus point selects buffer to dilute, 1200rpm,
5min；Supernatant is abandoned, bonus point selects buffer that cell is resuspended, and takes 10 μ l (10-100 times of dilution) to be used for cell count, remaining suspension
1200rpm centrifuges 5min；
D. supernatant is abandoned, every 1 × 108Individual cell adds 300 μ l sortings buffer to be resuspended, and lucifuge adds 100 μ l FcR blocking agents
With 100 μ l CD34 magnetic beads, 4 DEG C of refrigerators stand 30min；
E. sample is taken out, adds 10ml sortings buffer to wash away uncombined cell, 1200rpm, 5min；Supernatant is removed completely,
1ml sortings buffer is added to be resuspended；
F. MS sorting posts are placed in magnetic field corresponding to MACS sorters, fully soaked point with 500 μ l sortings buffer
Post is selected, cell suspension is added into sorting post through filter membrane (200 mesh), and is rinsed 3 times with sorting buffer, is collected as far as possible all thin
Born of the same parents；
G. MS sorting posts are removed into magnetic field, be placed on 15ml centrifuge tube, sorting buffer MS are added in sorting post
The piston pressurization that sorting post is equipped with releases rapidly magnetic mark cell；
H. above-mentioned sorting step is repeated, the CD34 being eluted out with new MS sorting post sortings+Cell, carry out secondary point
Choosing, obtain the CD34 of high-purity (>=90%)+Cell, and carry out cell count.
2) quiescent culture
A. with addition human thrombopoietin (hTPO, final concentration of 50ng/mL), stem cell factor (SCF, it is final concentration of
20ng/mL) CD34 is resuspended in the serum free medium StemSpan SFEM with interleukin-3 (IL-3, final concentration of 20ng/mL)+
Cell, with 1 × 105/ mL initial densities are inoculated in 12 orifice plates, in 37 DEG C, 5%CO2Quiescent culture 3 days in incubator；
B. the 3rd day full dose changes above-mentioned nutrient solution and cell factor, and adds ATRA in original culture medium
(ATRA, final concentration of 10-11M), continue quiescent culture 3 days, promote cell amplification；
3) three-dimensional rotation culture
A. the 6th day change addition human thrombopoietin (hTPO, final concentration of 50ng/mL) and interleukin-11 (IL-11,
Final concentration of 20ng/mL) fresh medium (StemSpan SFEM), induction megacaryocyte unidirectionally breaks up, while adds Rho
Kinase inhibitor Y-27632 (final concentration of 10-9M), promote megakaryocyte polyploid, cell is transferred to rotating and culturing afterwards
Device RCCS-4SC (Synthecon, Inc., Houston, TX, U.S.A) continues to cultivate, and culture rotating speed is 20rpm；
B. the 9th day full dose changes above-mentioned nutrient solution and cell factor, and additionally adds Rho kinase inhibitors Y-27632 (eventually
Concentration is 10-9M), Decitabine (DAC, final concentration of 10-7) and Wnt/ β-catenin signal secreting type activator R- M
spondin2(RSPO2, final concentration of 50ng/mL), rotating and culturing was to the 15th day.(explanation：Here for the different culture days of acquisition
Comparing result (i.e. Fig. 1) under several, rotating and culturing was to the 15th day.Optimal embodiment be herein rotating and culturing to the 12nd day or
Person is to be collected for the 13rd day) half amount changes liquid daily, and collect detection CD41a+CD42b+The granule of platelet.
4) blood platelet purifies
A. cell suspension, 800g centrifugations 10min are collected；
B. supernatant is abandoned, 1 × CGS buffer (formula is shown in Table 2) that 1 μM of prostaglandin E1 (PGE1) is added with 2mL are resuspended；
C. BSA density gradients liquid (2%, 5%, 7%, 10%, 12%) is configured, each gradient respectively takes 1.5mL from high to low
15mL centrifuge tubes are added slowly to, form the layering of BSA density gradients；
D. 2mL 1 × CGS buffer re-suspension liquids are added slowly to BSA density gradient liquid upper strata, (0 liter of 10min of 80g centrifugations
0 drop)；
E. Xi Qi the superiors 0.5mL water layers, then 4.5mL (≤5%BSA layerings) is slowly drawn in empty centrifuge tube, add 1%
BSA dilutes, 800g centrifugations 10min；
F. supernatant is abandoned, adds 200 μ L fresh culture StemSpan SFEM to be resuspended.
5) Activated platelets streaming monitors
4 experimental groups, SC (+), SC (-), RCCS (+), RCCS (-) are set；
RCCS (+) group, i.e., according to 1)-the 4 of embodiment 1) step tested；Since the 7th day, it is monitored daily；
RCCS (-) group, compared with RCCS (+), ATRA, Y-27632, RSPO are not added in this group of cell cultivation process2With
DAC。
SC (+) group, compared with RCCS (+), this group of cell culture is carried out under static conditions.
SC (-) group, compared with RCCS (+), this group of cell culture is carried out under static conditions, and in cell cultivation process
Do not add ATRA, Y-27632, RSPO2And DAC.
B. under the conditions of lucifuge, the granule of platelet of every group of purifying is resuspended with 200 μ L fresh culture StemSpan SFEM
Afterwards plus 2 μ L anti-CD41a-APC and anti-CD42b-PE, after lucifuge is incubated 30min on horizontal shaker, upper machine (FACS
Canto II；BD Biosciences) detection.
C. positive control circle door is done with the blood platelet in normal peripheral blood source, carries out streaming interpretation of result.
D. as shown in figure 1, compared with other groups, the physical mechanics that RCCS (+) group provides three-dimensional rotation culture pierces result
Sharp and micromolecular compound use in conjunction, can effectively improve CD41a+CD42b+The ratio of Activated platelets.
6) Platelet alpha granule release test
A.4 individual experimental group, SC (+), SC (-), RCCS (+), RCCS (-), while the positive is done with normal peripheral blood blood platelet
Control.
B. every group of platelet sample is divided into 3 parts, and portion is without any processing, as negative control；Portion is not added with excitement
Agent Thrombin processing, direct labelled antibody anti-CD41a-APC and anti-CD62p-PE, as group before activation；Another
First add activator fibrin ferment (Thrombin, 2U/mL) 37 DEG C of warm bath 30min after, then labelled antibody anti-CD41a-APC and
Anti-CD62p-PE, as activation group.
C. after labelled antibody, lucifuge incubation at room temperature 30min, flow cytometer showed is carried out.
D. CD62P differential expressions can reflect platelet function activity before and after comparing activator activation.As shown in Fig. 2 result
Show, compared with other groups, RCCS (+) group i.e. by three-dimensional rotation culture and the combination application of small molecule, is greatly improved CD41a+CD42b+The yield of Activated platelets.
7) blood platelet is quantitative
A. single bleeding of the umbilicus CD34 is calculated according to flow cytometer showed result+Initiator cell produces hematoblastic quantity, and it calculates public
Formula is：Blood platelet/megacaryocyte ratio × CD41a+CD42b+-Blood platelet percentage × total number of nucleated cells；Wherein blood platelet/
Platelet count/megacaryocyte number in megacaryocyte ratio=streaming figure；Total number of nucleated cells=day (t) cell densities/day (0)
Cell density × total extension rate.
B. result of calculation is as shown in figure 3, as seen in Figure 3, compared with other groups, RCCS (+) group three-dimensional will be revolved
Turn culture and the combination application of small molecule, be greatly improved CD41a+CD42b+The yield of Activated platelets.
Can be by single CD34 using technical scheme+Initiator cell obtains~1907.67 ± 367.15 activity
The granule of platelet, its blood platelet generation efficiency be significantly larger than prior art (use same computational methods, existing literature report
Each CD34+Hematoblastic general range caused by cells in vitro induction is 40~180).
8) statistical procedures
All data are represented with x ± s, are compared between group and are carried out one-way analysis of variance using SPSS10.0 medical statisticses software
Examined with t, P ＜ 0.05 are significant difference, and P ＜ 0.0l are difference highly significant.
The above embodiments merely illustrate the technical concept and features of the present invention, and the protection model of the present invention can not be limited with this
Enclose.Any equivalent change or modification in accordance with the spirit of the invention, it should all be included within the scope of the present invention.
Claims (6)
- It is 1. a kind of from bleeding of the umbilicus CD34+The method that cell differentiation produces Activated platelets, it is characterised in that：This method includes following step Suddenly：1）Bleeding of the umbilicus CD34+The separation of cellHuman umbilical cord blood mononuclear cell is separated first by Ficoll separating liquids, is carried out afterwards using CD34 immunomagnetic beadses positive twice Sorting, obtain the CD34 of high-purity >=90%+Cell, and carry out cell count；2）Cell cultureA. with the free serum culture of addition human thrombopoietin (hTPO), stem cell factor (SCF) and interleukin-3 (IL-3) CD34 is resuspended in base StemSpan SFEM+Cell, with 1 × 105/ mL initial densities are inoculated in 12 orifice plates, in 37 DEG C, 5%CO2Incubator Middle quiescent culture 3 days；B. the 3rd day full dose changes above-mentioned nutrient solution and cell factor, and adds ATRA in original culture medium （ATRA）, continue quiescent culture 3 days；C. the 6th day fresh medium for changing addition human thrombopoietin (hTPO) and interleukin-11 (IL-11) StemSpan SFEM, while the material for promoting megakaryocyte polyploid is added, it is transferred to rotation culture apparatus RCCS and continues to train Support；The material of the promotion megakaryocyte polyploid is Rho kinase inhibitor Y-27632, Y-27632 functional analogues Y230141, niacinamide NIC, serine/threonine kinases Aurora B inhibitor ZM447439, Src kinase inhibitors SU6656 Or one kind in inhibitors of myosin light chain kinase MLCKI；D. the 9th day full dose changes nutrient solution and cell factor, and add Rho kinase inhibitors Y-27632, Decitabine and Wnt/ β-catenin signal secreting type activator R-spondin2, partly amount changes liquid daily, and rotating and culturing was collected to the 10-15 days CD41a+CD42b+The granule of platelet；The final concentration of 50ng/mL of human thrombopoietin (hTPO) in step a, stem cell factor (SCF) it is final concentration of 20ng/mL and interleukin-3 (IL-3) final concentration of 20ng/mL；The final concentration of 50ng/mL of human thrombopoietin (hTPO) described in the step c；The interleukin-11 (IL-11) Final concentration of 20ng/mL；Final concentration of the 10 of the material of the promotion megakaryocyte polyploid-9 M。
- It is 2. a kind of from bleeding of the umbilicus CD34 according to claim 1+The method that cell differentiation produces Activated platelets, it is characterised in that： ATRA in the step b（ATRA）Final concentration of 10-10-10-11 M。
- It is 3. a kind of from bleeding of the umbilicus CD34 according to claim 1+The method that cell differentiation produces Activated platelets, it is characterised in that： The material of the promotion megakaryocyte polyploid is Rho kinase inhibitors Y-27632.
- It is 4. a kind of from bleeding of the umbilicus CD34 according to claim 1+The method that cell differentiation produces Activated platelets, it is characterised in that： The model RCCS-4SC of the RCCS, culture rotating speed are 20rpm.
- It is 5. a kind of from bleeding of the umbilicus CD34 according to claim 1+The method that cell differentiation produces Activated platelets, it is characterised in that： Final concentration of the 10 of Rho kinase inhibitors Y-27632 in the step d-9 M；Final concentration of the 10 of Decitabine (DAC)-7-10-9 M；With Wnt/ β-catenin signal secreting type activator R-spondin2（RSPO2）Final concentration of 50ng/mL.
- It is 6. a kind of from bleeding of the umbilicus CD34 according to claim 1+The method that cell differentiation produces Activated platelets, it is characterised in that： Rotating and culturing was to the 12-13 days in the step d.
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