CN106282089A - A kind of efficient amplification cultivating system of non-human primate endothelial progenitor cell - Google Patents
A kind of efficient amplification cultivating system of non-human primate endothelial progenitor cell Download PDFInfo
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Abstract
The present invention relates to the efficient amplification cultivating system of a kind of non-human primate endothelial progenitor cell.Provide a kind of novel non-human primate endothelial progenitor cell efficient amplification cultural method, the method can obtain the endothelial progenitor cells/endotheliocyte with endothelial activity function efficiently, thus meet cell quantity required in the autologous preclinical study feeding back transplanting of primate, it is applied to clinical treatment for endothelial progenitor cells amplification cultivation technology and provides effective way.
Description
Technical field
The invention belongs to cell biology, more particularly it relates to an non-human primates moves
The efficient amplification cultivating system of thing endothelial progenitor cell.
Background technology
Cell therapy is the disease treatment new technique of rising in recent years, utilizes some to have specific function
Cell characteristics, after being processed by amplification in vitro, specific culture etc., reaches to treat the purpose of disease.Endothelium
CFU-GM (Endothelial progenitor cells, EPCs) is the precursor of vascular endothelial cell, mainly
It is present in Cord blood, adult peripheral blood, bone marrow.Research in recent years shows, endothelial progenitor cells is at heart and brain
The aspects such as angiopathy, peripheral blood vessel, Tumor angiogenesis and wound healing all play a significant role,
Meanwhile, because it expresses FVIII and the transplanting characteristic of migration in amplification and atomization, can be as one
Plant the cell therapy approach hemophilia A for FVIII shortage, have been reported title and can pass through syngenetic graft
Normal endothelial progenitor cells alleviates the bleeding of hemophilia A mice, improves survival rate.
Under normal circumstances, endothelial progenitor cells quantity in blood circulation is few, only accounts in peripheral blood
0.005-0.01%, Cord blood also only has 0.2-1%, the quantity directly obtained cannot meet clinic and control
Treat and the needs of scientific research.Recent studies indicate that can by Gene intervention or add the specific cells factor,
The mode of micromolecular compound and chemical composition promotes its amplification efficiency to a certain extent.
At present, the In vitro culture of endothelial progenitor cell is mainly by density－gradient centrifuga－tion method separation umbilicus
Mononuclearcell in blood, peripheral blood, bone marrow, uses CD34, CD133 immunomagnetic beads sun further
Property screening after expand and break up under endothelial cell growth factor (ECGF) (VEGF, EGF, IGF etc.) is induced.This
Before, by newly-built culture technique, (efficient amplification of a kind of human vascular endothelial CFU-GM cultivates body to the present inventor
Be 201410397090.2) have been carried out people's umbilical blood and mobilize after the body of peripheral blood Vascular Endothelial CFU-GM
The most efficient, safe amplification.
But, this area currently there is no the amplification for non-human primate origin's endothelial progenitor cell
Means, in order to preferably carry out functional study in clinical precursor in non-human primate level, one
Plant the non-human primate endothelial progenitor cell culture technique optimized urgently to set up.
Summary of the invention
It is an object of the invention to provide the efficient amplification of a kind of non-human primate endothelial progenitor cell
Cultivating system.
In a first aspect of the present invention, it is provided that a kind of non-human primate endothelial progenitor cell cultivated
Method (for non-therapeutic method), described method includes:
(1) expansion of stem cells culture medium is utilized to expand non-human primate CD34+Mononuclearcell；
(2) cell after step (1) being expanded is transferred to cultivate in endothelial progenitor cells culture medium, it is thus achieved that inhuman
Primate endothelial progenitor cell；
Wherein, described expansion of stem cells culture medium includes: stem cell basal medium and following cell
The factor:
Stem cell factor (SCF): 50-500ng/mL；
Flt3-part (FLT-3L): 50-500ng/mL；
Platelet factor (TPO): 5-200ng/mL；
Interleukin-13 (IL-3): 5-50ng/mL；
The granular leukocyte colony stimulating organism factor (GM-CSF): 5-25ng/mL；
Granulocyte colony-stimulating factor (G-CSF): 5-25ng/mL
Sall sample albumen 4B (Sall4B): 1-10ng/mL；With
VEGF (VEGF): 15-150ng/mL.
Wherein, described endothelial progenitor cells culture medium includes: endothelial basal medium and following group
Point:
VEGF (VEGF): 5-100ng/mL；
Insulin like growth factor (IGF): 5-100ng/mL；
Epithelical cell growth factor (EGF): 2-20ng/mL；
Fibroblast growth factor (b-FGF): 5-100ng/mL；
Ascorbic acid (Ascorbic Acid): 0.5-6 μ g/mL；
Heparin (Heparin): 40-200U/mL；
Hydrocortisone (Hydrocortisone): 80-300ng/mL；With
L glutamine (L-glutamine): 1-6mM.
In a preference, described stem cell basal medium is selected from (but not limited to): Modified
IMDM culture medium or Modified StemSpan culture medium；Or described endothelial basal medium
Selected from (but not limited to): the similar culture medium such as EBM-2 culture medium, M199, M200 culture medium are (preferably
Containing 5-25% hyclone).
In another preference, step (1) including: CD34+Mononuclearcell joins described stem cell
In amplification culture medium so that cell concentration is 3-20 × 105Individual cell/mL (preferably 6-12 × 105Individual carefully
Born of the same parents/mL)；The stem cell carried out described in point hole interpolation according to cell number increments after cultivating 2-4 days is expanded
Increasing culture medium makes cell concentration be 3-20 × 105Individual cell/mL (preferably 6-12 × 105Individual cell
/mL)；Step (1) is carried out 5-7 days.
In another preference, step (2) including: after step (1) is carried out 5-7 days, is turned by the cell of acquisition
Move on in described endothelial progenitor cells culture medium, be placed in and be coated with in the culture plate of fibronectin cultivation,
Cell density in the medium is 0.3-3 × 106Individual cell/mL (preferably 0.5-2 × 106Individual cell/mL),
Dispel cell after continuing to cultivate 2-4 days, select attached cell to continue to cultivate；Within follow-up every 2-3 days, change 1
Secondary endothelial progenitor cells culture medium；Harvesting after changing 3-6 time.
In another preference, described non-human primate includes: monkey.
In another aspect of this invention, it is provided that a kind of to expand the stem cell of CD34+ mononuclearcell
Amplification culture medium, including: stem cell basal medium and following cytokine:
Stem cell factor (SCF): 50-500ng/mL；
Flt3-part (FLT-3L): 50-500ng/mL；
Platelet factor (TPO): 5-200ng/mL；
Interleukin-13 (IL-3): 5-50ng/mL；
The granular leukocyte colony stimulating organism factor (GM-CSF): 5-25ng/mL；
Granulocyte colony-stimulating factor (G-CSF): 5-25ng/mL
Sall sample albumen 4B (Sall4B): 1-10ng/mL；With
VEGF (VEGF): 15-150ng/mL.
In a preference, described being used for expands CD34+The expansion of stem cells of mononuclearcell is cultivated
Base includes following cytokine:
Stem cell factor: 100-400ng/mL；
Flt3-part: 100-400ng/mL；
Platelet factor: 10-150ng/mL；
Interleukin-13: 5-30ng/mL；
The granular leukocyte colony stimulating organism factor: 8-20ng/mL；
Granulocyte colony-stimulating factor: 8-20ng/mL
Sall sample albumen 4B:2-8ng/mL；With
VEGF (VEGF): 20-100ng/mL.
In another aspect of this invention, it is provided that it is thin that one is used for cultivating non-human primate blood vessel endothelium ancestral
The culture medium of born of the same parents, including: endothelial basal medium and following component:
VEGF (VEGF): 5-100ng/mL；
Insulin like growth factor (IGF): 5-100ng/mL；
Epithelical cell growth factor (EGF): 2-20ng/mL；
Fibroblast growth factor (b-FGF): 5-100ng/mL；
Ascorbic acid (Ascorbic Acid): 0.5-6 μ g/mL；
Heparin (Heparin): 40-200U/mL；
Hydrocortisone (Hydrocortisone): 80-300ng/mL；With
L glutamine (L-glutamine): 1-6mM.
In a preference, the described training for cultivating non-human primate endothelial progenitor cell
Supporting in base, described component includes:
VEGF: 10-80ng/mL；
Insulin like growth factor: 10-80ng/mL；
Epithelical cell growth factor: 4-16ng/mL；
Fibroblast growth factor: 8-80ng/mL；
Ascorbic acid: 1-4 μ g/mL；
Heparin: 80-200U/mL；
Hydrocortisone: 100-250ng/mL；With
L glutamine: 2-5mM.
In another aspect of this invention, it is provided that the purposes of arbitrary described culture medium, it is used for preparing non-
People's primate endothelial progenitor cell.
In another aspect of this invention, it is provided that it is thin that one is used for preparing non-human primate blood vessel endothelium ancestral
The test kit of born of the same parents, described test kit includes:
The expansion of stem cells culture medium to expand CD34+ mononuclearcell described in (a)；With
The culture medium for cultivating non-human primate endothelial progenitor cell described in (b).
The other side of the present invention, due to this disclosure, is aobvious to those skilled in the art
And be clear to.
Accompanying drawing explanation
Fig. 1, the present invention expand situation (I) to the endothelial progenitor cells of Os Macaca mulatta marrow CD34+ source of human stem cell.
Figure 1A, the number of amplification of endothelial progenitor cells.
Figure 1B, the amplification times of endothelial progenitor cells.
The endothelial progenitor cells of Os Macaca mulatta marrow CD34+ source of human stem cell is expanded and differentiated result by Fig. 2, the present invention
Morphological observation and Function Identification.
Fig. 2 A, the endothelial progenitor cells amplification of monkey derived from bone marrow and the cellular morphology figure of differentiation.
Fig. 2 B, the endothelial progenitor cells amplification of monkey derived from bone marrow and the qualification figure of differentiation.
Fig. 3, the present invention are to the endothelial progenitor cells amplification feelings of peripheral blood CD34+ source of human stem cell after monkey mobilization
Condition (I).
Fig. 3 A, the number of amplification of endothelial progenitor cells.
Fig. 3 B, the amplification times of endothelial progenitor cells.
Fig. 4, the present invention to monkey mobilize after peripheral blood CD34+ source of human stem cell endothelial progenitor cells amplification and
The morphological observation of differentiated result and Function Identification.
After Fig. 4 A, monkey mobilization, the endothelial progenitor cells of derived from peripheral blood expands and the cellular morphology figure of differentiation.
After Fig. 4 B, monkey mobilization, the endothelial progenitor cells of derived from peripheral blood expands and the qualification figure of differentiation.
Detailed description of the invention
In order to overcome in prior art not for the cultivation body of non-human primate endothelial progenitor cell
The defect of system, after the present inventor's further investigation, develops a kind of novel non-human primate Ink vessel transfusing
Skin CFU-GM efficient amplification culture technique, thus can greatly promote endothelial progenitor cells entering human implantation
Progress before clinical experiment.
As used herein, term " contain " or " including " include " comprising ", " mainly by ...
Constitute (making) ", " substantially by ... constitute " and " by ... composition ".
As used herein, described " non-human primate " refers to the primate beyond people, bag
Include: monkey, orangutan, ape etc..
The different phase that the present inventor cultivates according to human vascular endothelial progenitor cells amplification, it is provided that different trainings
Support base, including: expansion of stem cells culture medium and endothelial progenitor cells culture medium.
Described expansion of stem cells culture medium includes: stem cell factor (SCF), Flt3-part (FLT-3L),
Platelet factor (TPO), interleukin-13 (IL-3), the granular leukocyte colony stimulating organism factor (GM-CSF), grain
Colony-stimulating factor (G-CSF), Sall sample albumen 4B (Sall4B) and VEGF
(VEGF).Above-mentioned each cytokine is added in stem cell basal medium with applicable ratio, can be
CD34+ mononuclearcell provides the culture medium of the isolated growth environment being suitable for, and promotes that CD34+ single core is thin
The growth of born of the same parents and amplification.As the optimal way of the present invention, for preparing the expansion of stem cells training of the present invention
The consumption of each component supporting base is as shown in table 1.
Table 1
The cytokine of table 1 formula is added in stem cell basal medium, obtains expansion of stem cells training
Support base, thus provide suitable growth and amplification environment for CD34+ mononuclearcell.Described cell training
Foster base can select Modified IMDM culture medium, Modified StemSpan culture medium or similar
Cell culture medium.
Described endothelial progenitor cells culture medium includes: VEGF (VEGF), Insulin-Like are raw
The long factor (IGF), epithelical cell growth factor (EGF), fibroblast growth factor (b-FGF), anti-bad
Hematic acid (Ascorbic Acid), heparin (Heparin), hydrocortisone (Hydrocortisone) and L paddy amine
Amide (L-glutamine).Above-mentioned each component is added in endothelial basal medium with applicable ratio,
Obtain the endothelial progenitor cells culture medium of the present invention.As the optimal way of the present invention, it is used for preparing the present invention
The consumption of each component of endothelial progenitor cells culture medium as shown in table 2.
Table 2
| Content | Preferred amounts | More preferably measure | |
| VEGF (VEGF) | 5-100ng/mL | 10-80 ng/mL | 25 ng/mL |
| Insulin like growth factor (IGF) | 5-100ng/mL | 10-80ng/mL | 20ng/mL |
| Epithelical cell growth factor (EGF) | 2-20ng/mL | 4-16ng/mL | 10ng/mL |
| Fibroblast growth factor (b-FGF) | 5-100ng/mL | 8-80ng/mL | 10ng/mL |
| Ascorbic acid (Ascorbic Acid) | 0.5-6μg/mL | 1-4μg/mL | 2μg/mL |
| Heparin (Heparin) | 40-200U/mL | 80-200U/mL | 100U/mL |
| Hydrocortisone (Hydrocortisone) | 80-300ng/mL | 100-250ng/mL | 100ng/mL |
| L glutamine (L-Glutamine) | 1-6mM | 2-5mM | 4mM |
The component of table 2 formula is added in endothelial basal medium, and the endothelium obtaining the present invention is thin
Born of the same parents' culture medium, thus the culture medium prescription of preferably simplification is provided for endothelial progenitor cells.
Above-mentioned culture medium after the present inventor optimizes, containing enough and reasonably promoting cell growth and expanding
The composition and the beneficially cell that increase keep dryness or realize the composition of differentiation, beneficially endothelial progenitor cells
Cultivation.
For prepare the Sall4B of culture medium, SCF, TPO, Flt-3L, IL3, GM-CSF, G-CSF,
The cytokines such as VEGF, IGF, EGF, b-FGF are all that those skilled in the art are easily obtained, example
Buy such as by commercial sources, maybe can pass through synthetic or recombinant expressed acquisition.
In a preferred embodiment of the invention, described non-human primate is monkey.The present inventor is first
CD34 in peripheral blood after first extracting machin femur marrow and mobilizing+Mononuclearcell, does for monkey blood
The growth characteristics of cell, have carried out cytokine group on the basis of people's endothelial progenitor cells amplification cultivation system
Conjunction, the adjustment of training method and optimization, the blood vessel endothelium ancestral to non-human primate origin initiatively
Cell expands on a large scale and breaks up, it is thus achieved that monkey endothelium ancestral/endotheliocyte can cultivate not same order
Section is frozen and recovers, and can expand the functional endothelial cells into meeting not homologous transplantation demand further,
Thus it is applied to the syngenetic graft treatment of multiple disease model.
Adjusting combination of cytokines and concentration by different phase, said method can make the monkey of single acquisition
In bone marrow (6-10mL) sample, endothelial progenitor cells expands 2.33 × 10 by the cultivation of 12 days7Individual, amplification times
Number is 1142 times；Can reach 1.28 × 10 to number of amplification when the 18th day8Individual, multiple reaches 6275 times；
Meanwhile, it is also possible to the endothelium ancestral in peripheral blood (15-20mL) sample after making G-CSF/SCF mobilize
Cell expands 1.83 × 10 by the cultivation of 24 days7Individual, amplification times is 1274 times.
The method of the present invention can obtain that to have the endothelial progenitor cells/endothelium of endothelial activity function thin efficiently
Born of the same parents, thus meet cell quantity required in the autologous preclinical study feeding back transplanting of primate,
Be applied to clinical treatment ischemic diseases for endothelial progenitor cells amplification cultivation technology, hemophilia provides can
The laboratory research basis leaned on.
The present invention realizes the endothelial progenitor cell of non-human primate origin initiatively in vitro
Efficient amplification, establishes in primate level and utilizes bone marrow and blood stem cell preparation safety to have
The technology of the endothelial progenitor cell/endotheliocyte of effect.The present invention is that endothelial progenitor cells is for non-human primates
The experimentation of animal autotransplantation and homology heteroplastic transplantation provides valuable resource, the most also for people to people
Transplanting preclinical data support is provided, will promote the people source endothelial progenitor cells can be as one further
The cell preparation of external safe efficient preparation for ischemia class disease and hemophilia A clinical treatment and
Scientific research demand.
Main feature and the advantage of the present invention also include: first, and the present invention uses at whole cultivating system and faces
The cell growth factor of bed rank, is not inserted into allogenic gene, does not the most change the genome of former stem cell
Stability, without tumorigenesis risk；Second, the present invention can make periphery after non-human primate bone marrow and mobilization
Blood CD34+ stem cell is the most respectively by 12 days and the cultivation of 24 days so that the number of endothelial progenitor cells
Amount is more than 107, amplification times is all more than 1000 times, and can expand the most vegetatively
Be divided into maturation endotheliocyte, play into blood vessel function and the effect of secretory function albumen.3rd,
The multiple stem cell factor that early stage of the present invention adds in cultivating can preferably maintain the dryness of cell,
Make the endothelial progenitor cells after amplification keep high proliferation activity, contribute to transplanting migration and the merit of backward focus
Can play.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are only used for
The present invention is described rather than limits the scope of the present invention.The reality of unreceipted actual conditions in the following example
Proved recipe method, generally writes according to normal condition such as J. Pehanorm Brooker etc., Molecular Cloning: A Laboratory guide, the
Three editions, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.
Embodiment 1, the preparation of culture medium
The cultivating system of the present invention comprises CD34+ expansion of stem cells, endothelial progenitor cells adherent amplification differentiation two
Individual part:
One, the CD34+ expansion of stem cells phase: expansion of stem cells culture medium
Use culture medium based on Modified IMDM culture medium (serum-free), add wherein and be selected from
The cytokine of arbitrary formula in table 3:
Table 3 (unit: ng/mL)
| Formula A | Formula B | Formula C | |
| SCF | 200 | 100 | 400 |
| FLT-3L | 200 | 100 | 400 |
| TPO | 20 | 10 | 150 |
| IL-3 | 10 | 5 | 20 |
| GM-CSF | 12.5 | 8 | 20 |
| G-CSF | 12.5 | 8 | 20 |
| Sall4B | 3 | 2 | 8 |
| VEGF | 50 | 25 | 75 |
Two, the adherent amplification phase: endothelial progenitor cells amplification division culture medium
Use culture medium based on EBM-2 culture medium (10% or 20% hyclone), add wherein
The cytokine of arbitrary formula and other compositions in table 4:
Table 4 (in addition to additionally indicating, unit is ng/mL)
| Formula D | Formula E | Formula F | |
| VEGF | 25 | 10 | 75 |
| IGF | 20 | 10 | 80 |
| EGF | 10 | 4 | 16 |
| b-FGF | 10 | 20 | 80 |
| Ascorbic acid | 2μg/mL | 1μg/mL | 4μg/mL |
| Heparin | 100U/mL | 50U/mL | 200U/mL |
| Hydrocortisone | 100ng/mL | 150ng/mL | 250ng/mL |
| L glutamine | 4mM | 2mM | 5mM |
Embodiment 2, to the amplification of the endothelial progenitor cells of Os Macaca mulatta marrow CD34+ source of human stem cell and differentiation (I)
0th day:
(1) preparation CD34+Expansion of stem cells culture medium
In Modified IMDM culture medium (serum-free), add various cytokines, make cytokine eventually
Concentration is SCF 200ng/mL, Flt-3L 200ng/mL, IL-310ng/mL, TPO 20ng/mL, Sall4B
3ng/mL, GM-CSF 12.5ng/mL, G-CSF 12.5ng/mL, VEGF 50ng/mL (i.e. implements
Formula A in example 1) as amplification culture medium.
(2) CD34 in isolated and purified bone marrow+Stem cell
At Healthy Youth machin anterior superior iliac spine, take femur marrow 8-10mL, PBS dilute 10 times, use
Density－gradient centrifuga－tion method uses the CD34 antibody (APC-mouse of BD company after obtaining mononuclearcell
Anti-human CD34) and Mei Tian Ni company MACS magnetic bead (Rat anti-mouse IgG) two step separating methods
Separate CD34+Mononuclearcell, it is thus achieved that cell quantity is 1.4 × 106Individual cell.Use CD34+Dry thin
Born of the same parents' amplification culture medium is resuspended by cell, and adjusting cell concentration is 6 × 105Individual cell/mL, adds in 24 orifice plates,
Every hole 1mL, i.e. 6 × 105Individual cells/well.With microscope observe then pipe is placed in incubator (37 DEG C,
5%CO2)。
(3) flow cytometer detection
Respectively take 5 × 10 in step poly-(2) after magnetic bead sorting4Individual cell is placed in 1.5mL EP pipe, totally 3
EP manages (pipe 1,2,3), and often pipe adds the 1mL PBS cleaning mixture containing 1%BSA, 1200rpm under room temperature
Centrifugal 5 minutes, supernatant discarded, by the 100 μ L PBS re-suspended cell precipitation containing 1%BSA, pipe 1 added
Entering 2 μ LVEGFR-2 antibody (PE-Mouseanti-humanVEGFR-2), pipe 2 adds corresponding homotype
(PE-Mouse anti-human IgG1 κ, pipe 3 adds 2 μ LCD34 antibody (APC-Mouse in comparison
Anti-human CD34), separately take the mononuclearcell 5 × 10 before CD34+ sorting in (2)4Individual the same behaviour
Make and add 2 μ L Isotype control (APC-Mouse anti-human IgG1 κ).Room temperature lucifuge after antibody addition
Hatching 15 minutes, the most often pipe adds 1mL PBS washing, and 1200rpm is centrifuged 5 minutes, discards
Clearly, with 300 μ L PBS re-suspended cells, with flow cytometry analysis cell surface CD34 and VEGFR-2
Expression.
3rd day:
(1) cell counting, with the expression of flow cytometry analysis cell surface CD34, VEGFR-2,
And add up total cell number, CD34+ and VEGFR-2+Cell number and amplification times.
(2) carry out a point hole according to cell number and (control cell density less than 1 × 106Individual/mL), amplification culture body
Fresh culture is added to 1mL (adding formula A in embodiment 1) in system every hole.
6th day:
Cultivation was changed endothelial progenitor cells culture medium to the 6th day and is carried out adhere-wall culture.
(1) cell counting, by the expression feelings of flow cytometry analysis cell surface CD34 and VEGFR-2
Condition, and add up VEGFR-2+ cell number and amplification times.
(2) preparation endothelial progenitor cells culture medium, add in the EBM-2 culture medium (20% hyclone) with
Lower composition, each composition is final concentration of: VEGF 25ng/mL, IGF20ng/mL, EGF10ng/mL,
B-FGF10ng/mL, ascorbic acid 2 μ g/mL, heparin 100U/mL, hydrocortisone 100ng/mL,
L-glutamine 4mM (i.e. formula D in embodiment 1)
(3) it is coated culture plate: use people's fibronectin by 2.5 μ g/cm2Concentration 24 orifice plates are entered
Row is coated, and hatches 2 hours for 37 DEG C, and PBS washes 15min, 3 times.
(4) cultivate: according to 1 × 106All cells after amplification is changed to endothelium by the density of individual cell/mL
CFU-GM culture medium is cultivated, cultivates respectively to the 18th day.
The 9-18 days:
Dispel cell when (1) the 9th day, suspension cell is removed, select attached cell to continue to cultivate.Every 3
It changes a fresh culture (i.e. formula D in embodiment 1).
(2) respectively at the 9th, 12,15,18 day, the trypsinization attached cell counting of 0.25% is used,
Statistics attached cell absolute number, endothelial progenitor cells absolute number adherent when the 12nd day is
2.33×107±2.15×106(n=3), the 18th day time be 1.28 × 108±1.67×107(n=3), Figure 1A is seen.
(VEGFR2 compared with the endothelial progenitor cells initial with the 0th day+: 1.7%), the amplification times of the 12nd day is
The amplification times of the 1142 ± 121, the 18th day is 6275 ± 792 (n=3), sees Figure 1B.Record 3-18 simultaneously
It cellular morphology figure, result is shown in Fig. 2 A.
(3) cellular identification: cultivate the cell to the 18th day, by 5 × 104The density in individual/hole is inoculated in 24
Orifice plate, after adherent, with the endothelium medium treatment 24 hours of serum-free, adds 10 μ g/mL's
Dil-ac-LDL, hatches 4 hours for 37 DEG C, and PBS washes 3 times；4% paraformaldehyde of 4 DEG C fixes 15 minutes,
Adding the FITC-lectin of 10 μ g/mL, hatch 2 hours for 37 DEG C, PBS takes pictures with microscope after washing 3 times.
As shown in Figure 2 B, the DIL-ac-LDL of the attached cell red color visible fluorescence of more than 80% and green fluorescence
FITC-lectin double expression(DE).Illustrate that amplification obtains endothelium ancestral from the CD34+ stem cell of monkey derived from bone marrow
/ endotheliocyte has the characteristic expressing endothelial activity function, can be further used for internal transplantation experiments.
Embodiment 3, to the amplification of the endothelial progenitor cells of Os Macaca mulatta marrow CD34+ source of human stem cell and differentiation (II)
0th day:
(1) preparation CD34+Expansion of stem cells culture medium
In Modified IMDM culture medium (serum-free), add various cytokines, make cytokine eventually
Concentration is SCF 100ng/mL, Flt-3L 100ng/mL, IL-35ng/mL, TPO 10ng/mL, Sall4B
2ng/mL, GM-CSF 8ng/mL, G-CSF 8ng/mL, VEGF 25ng/mL is (i.e. in embodiment 1
Formula B) as amplification culture medium.
(2) CD34 in isolated and purified bone marrow+Stem cell
At Healthy Youth machin anterior superior iliac spine, take femur marrow 6-8mL, PBS dilute 10 times, use real
Execute example 2 correlation method sorting CD34+Cell, it is thus achieved that cell quantity is 1.2 × 106Individual cell.Use CD34+
Expansion of stem cells culture medium is resuspended by cell, and adjusting cell concentration is 5 × 105Individual cell/mL, adds 24 holes
In plate, every hole 1mL, i.e. 5 × 105Individual cells/well.Observe with microscope and then pipe is placed in incubator
(37 DEG C, 5%CO2)。
(3) flow cytometer detection: with appropriate section in embodiment 2 (the 0th day, (3)).
3rd day:
(1) cell counting, with the expression of flow cytometry analysis cell surface CD34, VEGFR-2,
And add up total cell number, CD34+ and VEGFR-2+Cell number and amplification times.
(2) carry out a point hole according to cell number and (control cell density less than 1 × 106Individual/mL), amplification culture body
Fresh culture is added to 1mL (adding formula B in embodiment 1) in system every hole.
6th day:
Cultivation was changed endothelial progenitor cells culture medium to the 6th day and is carried out adhere-wall culture.
(1) cell counting, by the expression feelings of flow cytometry analysis cell surface CD34 and VEGFR-2
Condition, and add up VEGFR-2+ cell number and amplification times.
(2) preparation endothelial progenitor cells culture medium, add in the EBM-2 culture medium (20% hyclone) with
Lower composition, each composition is final concentration of: VEGF 10ng/mL, IGF10ng/mL, EGF 4ng/mL,
B-FGF20ng/mL, ascorbic acid 1 μ g/mL, heparin 50U/mL, hydrocortisone 150ng/mL,
L-glutamine 2mM (i.e. formula E in embodiment 1)
(3) it is coated culture plate: use people's fibronectin by 2.5 μ g/cm2Concentration 24 orifice plates are carried out
Being coated, hatch 2 hours for 37 DEG C, PBS washes 15min, 3 times.
(4) cultivate: according to 1 × 106All cells after amplification is changed to endothelium by the density of individual cell/mL
CFU-GM culture medium is cultivated, cultivates respectively to the 18th day.
The 9-18 days:
Dispel cell when (1) the 9th day, suspension cell is removed, select attached cell to continue to cultivate.Every 3
It changes a fresh culture (i.e. formula E in embodiment 1).
(2) respectively at the 9th, 12,15,18 day, the trypsinization attached cell counting of 0.25% is used,
Statistics attached cell absolute number, endothelial progenitor cells absolute number adherent when the 12nd day is
6.83×106±4.66×105(n=3), the 18th day time be 3.15 × 107±3.07×106(n=3).Rose with the 0th day
The endothelial progenitor cells begun compares (VEGFR2+: 1.3%), the amplification times of the 12nd day is the 523 ± 46, the 18th
It amplification times is 2423 ± 322 (n=3).
Embodiment 4, to monkey mobilize after peripheral blood CD34+ source of human stem cell endothelial progenitor cells amplification and
Differentiation (I)
Peripheral blood collection after monkey mobilization: gather first 5 days, uses subcutaneous injection to give Healthy Youth
G-CSF (100 μ g/kg)+SCF (50 μ g/kg), mobilizes 5 days continuously, gathers 15-20mL peripheral blood every time.
0th day:
(1) preparation CD34+Expansion of stem cells culture medium
In Modified IMDM culture medium (serum-free), add various cytokines, make cytokine eventually
Concentration is SCF200ng/mL, Flt-3L200ng/mL, IL-310ng/mL, TPO 20ng/mL, Sall4B
3ng/mL, GM-CSF 12.5ng/mL, G-CSF 12.5ng/mL, VEGF 50ng/mL (i.e. implements
Formula A in example 1) as amplification culture medium.
(2) CD34 in isolated and purified peripheral blood+Stem cell
Use peripheral blood 15-20mL, PBS after fresh mobilization to dilute 5 times, use embodiment 2 corresponding
Method sorting CD34+Cell, it is thus achieved that cell quantity is 1.5 × 106Individual cell.Use CD34+Stem cell
Amplification culture medium is resuspended by cell, and adjusting cell concentration is 6 × 105Individual cell/mL, adds in 24 orifice plates,
Every hole 1mL, i.e. 6 × 105Individual cells/well.With microscope observe then pipe is placed in incubator (37 DEG C,
5%CO2)。
(3) flow cytometer detection: with appropriate section in embodiment 2 (the 0th day, (3)).
3rd day:
(1) cell counting, with the expression of flow cytometry analysis cell surface CD34, VEGFR-2,
And add up total cell number, CD34+ and VEGFR-2+Cell number and amplification times.
(2) carry out a point hole according to cell number and (control cell density less than 1 × 106Individual/mL), amplification culture body
Fresh culture is added to 1mL (adding formula A in embodiment 1) in system every hole.
6th day:
Cultivation was changed endothelial progenitor cells culture medium to the 6th day and is carried out adhere-wall culture.
(1) cell counting, by the expression feelings of flow cytometry analysis cell surface CD34 and VEGFR-2
Condition, and add up VEGFR-2+ cell number and amplification times.
(2) preparation endothelial progenitor cells culture medium, add in the EBM-2 culture medium (20% hyclone) with
Lower composition, each composition is final concentration of: VEGF 25ng/mL, IGF20ng/mL, EGF10ng/mL,
B-FGF10ng/mL, ascorbic acid 2 μ g/mL, heparin 100U/mL, hydrocortisone 100ng/mL,
L-glutamine 4mM (i.e. formula D in embodiment 1)
(3) it is coated culture plate: use people's fibronectin by 2.5 μ g/cm2Concentration 24 orifice plates are carried out
Being coated, hatch 2 hours for 37 DEG C, PBS washes 15min, 3 times.
(4) cultivate: according to 1 × 106All cells after amplification is changed to endothelium by the density of individual cell/mL
CFU-GM culture medium is cultivated, cultivates to the 24th day.
The 9-24 days:
Dispel cell when (1) the 9th day, suspension cell is removed, select attached cell to continue to cultivate.Every 3
It changes a fresh culture (i.e. formula D in embodiment 1).
(2) respectively at the 9th, 12,15,18,21,24 day, the trypsinization of employing 0.25% is adherent
Cell counting, adds up attached cell absolute number, and endothelial progenitor cells absolute number adherent when the 24th day is
1.83×107±2.89×106(n=3) Fig. 3 A, is seen.Compared with the endothelial progenitor cells initial with the 0th day
(VEGFR2+: 1.2%), the amplification times of the 24th day is 1274 ± 154 (n=3), sees Fig. 3 B.Remember simultaneously
Recording the cellular morphology figure of the 3-24 days, result is shown in Fig. 4 A.
(3) cellular identification: method is with embodiment 2, and result is shown in Fig. 4 B.The attached cell of more than 85% can
See the DIL-ac-LDL of red fluorescence and the FITC-lectin double expression(DE) of green fluorescence.Illustrate to mobilize from monkey
The CD34 of rear derived from peripheral blood+In stem cell amplification obtain endothelium ancestral/endotheliocyte have expression endothelium live
The characteristic of sexual function, can be further used for internal transplantation experiments.
Embodiment 5, to monkey mobilize after peripheral blood CD34+ source of human stem cell endothelial progenitor cells amplification and
Differentiation (II)
Peripheral blood collection after monkey mobilization: with appropriate section in case study on implementation 4.
0th day:
(1) preparation CD34+Expansion of stem cells culture medium
In Modified IMDM culture medium (serum-free), add various cytokines, make cytokine eventually
Concentration be SCF400ng/mL, Flt-3L400ng/mL, IL-3200ng/mL, TPO 150ng/mL,
Sall4B 8ng/mL, GM-CSF 20ng/mL, G-CSF 20ng/mL, VEGF 75ng/mL are (the most real
Execute formula C in example 1) as amplification culture medium.
(2) CD34 in isolated and purified peripheral blood+Stem cell
Use peripheral blood 15-20mL (n=3), PBS after fresh mobilization to dilute 5 times, use embodiment 2
Correlation method sorting CD34+Cell, it is thus achieved that cell quantity is 2.1 × 106Individual cell.Use CD34+Dry
Cell amplification culture medium is resuspended by cell, and adjusting cell concentration is 8 × 105Individual cell/mL, adds 24 orifice plates
In, every hole 1mL, i.e. 8 × 105Individual cells/well.Observe with microscope and then pipe is placed in incubator
(37 DEG C, 5%CO2)。
(3) flow cytometer detection: with appropriate section in embodiment 2 (the 0th day, (3)).
3rd day:
(1) cell counting, with the expression of flow cytometry analysis cell surface CD34, VEGFR-2,
And add up total cell number, CD34+ and VEGFR-2+Cell number and amplification times.
(2) carry out a point hole according to cell number and (control cell density less than 1 × 106Individual/mL), amplification culture body
Fresh culture is added to 1mL (adding formula C in embodiment 1) in system every hole.
6th day:
Cultivation was changed endothelial progenitor cells culture medium to the 6th day and is carried out adhere-wall culture.
(1) cell counting, by the expression feelings of flow cytometry analysis cell surface CD34 and VEGFR-2
Condition, and add up VEGFR-2+ cell number and amplification times.
(2) preparation endothelial progenitor cells culture medium, add in the EBM-2 culture medium (20% hyclone) with
Lower composition, each composition is final concentration of: VEGF 75ng/mL, IGF80ng/mL, EGF16ng/mL,
B-FGF80ng/mL, ascorbic acid 4 μ g/mL, heparin 200U/mL, hydrocortisone 250ng/mL,
L-glutamine 5mM (i.e. formula F in embodiment 1)
(3) it is coated culture plate: use people's fibronectin by 2.5 μ g/cm2Concentration 24 orifice plates are entered
Row is coated, and hatches 2 hours for 37 DEG C, and PBS washes 15min, 3 times.
(4) cultivate: according to 1 × 106All cells after amplification is changed to endothelium by the density of individual cell/mL
CFU-GM culture medium is cultivated, cultivates to the 24th day.
The 9-24 days:
Dispel cell when (1) the 9th day, suspension cell is removed, select attached cell to continue to cultivate.Every 3
It changes a fresh culture (i.e. formula F in embodiment 1).
(2) respectively at the 9th, 12,15,18,21,24 day, the trypsinization of employing 0.25% is adherent
Cell counting, adds up attached cell absolute number, and endothelial progenitor cells absolute number adherent when the 24th day is
2.56×107±3.10×106(n=3).(VEGFR2 compared with the endothelial progenitor cells initial with the 0th day+: 1.0%),
The amplification times of the 24th day is 1600 ± 257 (n=3).
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each literary composition
Offer and be individually recited as with reference to like that.In addition, it is to be understood that reading the above-mentioned teachings of the present invention
Afterwards, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values are same
Fall within the application appended claims limited range.
Claims (10)
1. the method cultivating non-human primate endothelial progenitor cell, it is characterised in that described
Method includes:
(1) expansion of stem cells culture medium is utilized to expand non-human primate CD34+Mononuclearcell；
(2) cell after step (1) being expanded is transferred to cultivate in endothelial progenitor cells culture medium, it is thus achieved that inhuman
Primate endothelial progenitor cell；
Wherein, described expansion of stem cells culture medium includes: stem cell basal medium and following cell
The factor:
Stem cell factor: 50-500ng/mL；
Flt3-part: 50-500ng/mL；
Platelet factor: 5-200ng/mL；
Interleukin-13: 5-50ng/mL；
The granular leukocyte colony stimulating organism factor: 5-25ng/mL；
Granulocyte colony-stimulating factor: 5-25ng/mL
Sall sample albumen 4B:1-10ng/mL；With
VEGF: 15-150ng/mL.
Wherein, described endothelial progenitor cells culture medium includes: endothelial basal medium and following group
Point:
VEGF: 5-100ng/mL；
Insulin like growth factor: 5-100ng/mL；
Epithelical cell growth factor: 2-20ng/mL；
Fibroblast growth factor: 5-100ng/mL；
Ascorbic acid: 0.5-6 μ g/mL；
Heparin: 40-200U/mL；
Hydrocortisone: 80-300ng/mL；With
L glutamine: 1-6mM.
2. the method for claim 1, it is characterised in that described stem cell basal medium choosing
From: Modified IMDM culture medium or Modified StemSpan culture medium；Or
Described endothelial basal medium is selected from: EBM-2 culture medium, M199, M200 culture medium
Etc. similar culture medium.
3. the method for claim 1, it is characterised in that step (1) including: CD34+Single core
Cell joins in described expansion of stem cells culture medium so that cell concentration is 3-20 × 105Individual cell
/mL；The expansion of stem cells training described in point hole interpolation is carried out according to cell number increments after cultivating 2-4 days
Foster base makes cell concentration be 3-20 × 105Individual cell/mL；Step (1) is carried out 5-7 days.
4. the method for claim 1, it is characterised in that step (2) including: step (1) is carried out
After 5-7 days, the cell of acquisition transferred in described endothelial progenitor cells culture medium, be placed in and be coated with fibre
Cultivating in the culture plate of dimension connection albumen, cell density in the medium is 0.3-3 × 106Individual cell/mL,
Dispel cell after continuing to cultivate 2-4 days, select attached cell to continue to cultivate；Within follow-up every 2-3 days, change 1
Secondary endothelial progenitor cells culture medium；Harvesting after changing 3-6 time.
5. the method as described in claim 1-4 is arbitrary, it is characterised in that described non-human primates moves
Thing includes: monkey.
6., to expand an expansion of stem cells culture medium for CD34+ mononuclearcell, its feature exists
In, including: stem cell basal medium and following cytokine:
Stem cell factor: 50-500ng/mL；
Flt3-part: 50-500ng/mL；
Platelet factor: 5-200ng/mL；
Interleukin-13: 5-50ng/mL；
The granular leukocyte colony stimulating organism factor: 5-25ng/mL；
Granulocyte colony-stimulating factor: 5-25ng/mL
Sall sample albumen 4B:1-10ng/mL；With
VEGF: 15-150ng/mL.
7. culture medium as claimed in claim 6, it is characterised in that described being used for expands CD34+
The expansion of stem cells culture medium of mononuclearcell includes following cytokine:
Stem cell factor: 100-400ng/mL；
Flt3-part: 100-400ng/mL；
Platelet factor: 10-150ng/mL；
Interleukin-13: 5-30ng/mL；
The granular leukocyte colony stimulating organism factor: 8-20ng/mL；
Granulocyte colony-stimulating factor: 8-20ng/mL
Sall sample albumen 4B:2-8ng/mL；With
Vascular endothelial growth factor: 20-100ng/mL.
8., for cultivating a culture medium for non-human primate endothelial progenitor cell, its feature exists
In, including: endothelial basal medium and following component:
VEGF: 5-100ng/mL；
Insulin like growth factor: 5-100ng/mL；
Epithelical cell growth factor: 2-20ng/mL；
Fibroblast growth factor: 5-100ng/mL；
Ascorbic acid: 0.5-6 μ g/mL；
Heparin: 40-200U/mL；
Hydrocortisone: 80-300ng/mL；With
L glutamine: 1-6mM.
9. the purposes of the arbitrary described culture medium of claim 6-8, is used for preparing non-human primate blood
Endothelial tube CFU-GM.
10. for preparing a test kit for non-human primate endothelial progenitor cell, described reagent
Box includes:
The expansion of stem cells to expand CD34+ mononuclearcell described in (a) claim 6 or 7
Culture medium；With
The cultivation for cultivating non-human primate endothelial progenitor cell described in (b) claim 8
Base.
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| CN103124784A (en) * | 2010-08-23 | 2013-05-29 | 纽约州立大学研究基金会 | Method for expansion of stem cells and the use of such cells |
| CN105331575A (en) * | 2014-08-13 | 2016-02-17 | 苏州方舟基因药业有限公司 | Efficient amplification culture system for human vascular endothelial progenitor cells |
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| CN111424014A (en) * | 2019-11-22 | 2020-07-17 | 上海交通大学医学院附属第九人民医院 | Culture medium of immortalized cell strain of human jugular auxiliary nerve ganglionic tumor |
| CN111424014B (en) * | 2019-11-22 | 2022-03-29 | 上海交通大学医学院附属第九人民医院 | Culture medium of immortalized cell strain of human jugular auxiliary nerve ganglionic tumor |
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