CN106282089B - 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 a kind of efficient amplification cultivating systems of non-human primate endothelial progenitor cell.Provide a kind of novel non-human primate endothelial progenitor cell efficient amplification cultural method, this method can efficiently obtain endothelial progenitor cells/endothelial cell with endothelial activity function, to meet the required cell quantity in the preclinical study that primate feeds back transplanting self, 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 biologies, more particularly it relates to which a kind of non-human primate is intravascular
The efficient amplification cultivating system of skin progenitor cells.
Background technique
Cell therapy is the disease treatment new technology of rising in recent years, is that the cell using certain with specific function is special
Property, after handling by amplification in vitro, specific culture etc., achieve the purpose that treat disease.Endothelial progenitor cells (Endothelial
Progenitor cells, EPCs) be vascular endothelial cell precursor, be primarily present in Cord blood, adult peripheral blood, bone
Marrow.Studies have shown that endothelial progenitor cells in recent years are in cardiovascular and cerebrovascular disease, peripheral artery disease, Tumor angiogenesis and wound
Healing etc. plays a significant role, meanwhile, because the transplanting that it expresses FVIII and migration in amplification and atomization is special
Property, it can be used as a kind of hemophilia A that cell therapy approach lacks for FVIII, title has been reported can be by syngenetic graft just
Normal endothelial progenitor cells alleviate the bleeding of hemophilia A mouse, improve survival rate.
Under normal circumstances, quantity of the endothelial progenitor cells in blood circulation system is few, only accounts for the 0.005- in peripheral blood
0.01%, also there was only 0.2-1% in Cord blood, the quantity directly acquired is unable to satisfy the needs of clinical treatment and scientific research.Closely
It is over year research shows that can by way of Gene intervention or the addition specific cells factor, small molecule compound and chemical component
Promote its amplification efficiency to a certain extent.
Currently, the in vitro culture of endothelial progenitor cell mainly pass through density-gradient centrifugation method separating umbilical blood, peripheral blood,
Mononuclearcell in marrow, further using after CD34, CD133 immunomagnetic beads positive-selecting endothelial growth factors (VEGF,
EGF, IGF etc.) it is expanded and is broken up under induction.Before this, the present inventor passes through a kind of newly-built culture technique (human vascular endothelial
The efficient amplification cultivating system 201410397090.2 of progenitor cells) have been carried out peripheral blood Vascular Endothelial after people's bleeding of the umbilicus and mobilization
The external amplification efficiently, safe of progenitor cells.
But this field currently there is no the amplification means for non-human primate origin's endothelial progenitor cell, be
Functional study in clinical precursor is carried out preferably in non-human primate level, a kind of non-human primates of optimization are dynamic
Object endothelial progenitor cell culture technique is urgently established.
Summary of the invention
The purpose of the present invention is to provide a kind of efficient amplification culture bodies of non-human primate endothelial progenitor cell
System.
In the first aspect of the present invention, provide it is a kind of cultivate non-human primate endothelial progenitor cell method (for
Non-therapeutic method), which comprises
(1) non-human primate CD34 is expanded using expansion of stem cells culture medium+Mononuclearcell；
(2) cell after step (1) amplification is transferred in endothelial progenitor cells culture medium and is cultivated, it is dynamic to obtain non-human primates
Object endothelial progenitor cell；
Wherein, the expansion of stem cells culture medium includes: stem cell basal medium and following cell factor:
Stem cell factor (SCF): 50-500ng/mL；
Flt3- ligand (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
Vascular endothelial growth factor (VEGF): 15-150ng/mL.
Wherein, the endothelial progenitor cells culture medium includes: endothelial basal medium and following component:
Vascular endothelial growth factor (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 preferred embodiment, the stem cell basal medium is selected from (but being not limited to): Modified IMDM culture
Base or Modified StemSpan culture medium；Or the endothelial basal medium is selected from (but being not limited to): EBM-2 training
Support the similar culture medium such as base, M199, M200 culture medium (preferably containing 5-25% fetal calf serum).
In another preferred example, step (1) includes: CD34+Mononuclearcell is added to the expansion of stem cells culture
In base, so that cell concentration is 3-20 × 105A cell/(preferably 6-12 × 10 mL5A cell/mL)；After culture 2-4 days
Carrying out expansion of stem cells culture medium described in point hole and addition according to cell number incrementss makes cell concentration be 3-20 × 105
A cell/(preferably 6-12 × 10 mL5A cell/mL)；Step (1) carries out 5-7 days.
In another preferred example, step (2) includes: and is transferred to the cell of acquisition described after step (1) carries out 5-7 days
Endothelial progenitor cells culture medium in, be placed in the culture plate for be coated with fibronectin and cultivate, cell density in the medium
For 0.3-3 × 106A cell/(preferably 0.5-2 × 10 mL6A cell/mL), cell is dispelled after continuing culture 2-4 days, is selected
Attached cell continues to cultivate；The 1 endothelial progenitor cells culture medium of replacement in subsequent every 2-3 days；Cell is harvested after replacement 3-6 times.
In another preferred example, the non-human primate includes: monkey.
In another aspect of this invention, it provides a kind of to expand the expansion of stem cells culture based on CD34+ mononuclearcell
Base, including: stem cell basal medium and following cell factor:
Stem cell factor (SCF): 50-500ng/mL；
Flt3- ligand (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
Vascular endothelial growth factor (VEGF): 15-150ng/mL.
In a preferred embodiment, described for expanding CD34+Include in the expansion of stem cells culture medium of mononuclearcell
Following cell factor:
Stem cell factor: 100-400ng/mL；
Flt3- ligand: 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 (VEGF): 20-100ng/mL.
In another aspect of this invention, it provides a kind of for cultivating the culture of non-human primate endothelial progenitor cell
Base, including: endothelial basal medium and following component:
Vascular endothelial growth factor (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 preferred embodiment, in the culture medium for cultivating non-human primate endothelial progenitor cell,
The component includes:
Vascular endothelial growth factor: 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, the purposes of any culture medium in front is provided, non-human primates are used to prepare
Animal blood vessels endothelial progenitor cells.
In another aspect of this invention, a kind of reagent being used to prepare non-human primate endothelial progenitor cell is provided
Box includes: in the kit
(a) described in to expand the expansion of stem cells culture medium based on CD34+ mononuclearcell；With
(b) culture medium for being used to cultivate non-human primate endothelial progenitor cell described in.
Other aspects of the invention are apparent to those skilled in the art due to this disclosure
's.
Detailed description of the invention
Fig. 1, the endothelial progenitor cells amplification situation (I) of the invention to monkey marrow CD34+ source of human stem cell.
The number of amplification of Figure 1A, endothelial progenitor cells.
The amplification times of Figure 1B, endothelial progenitor cells.
Fig. 2, the present invention see the endothelial progenitor cells amplification of monkey marrow CD34+ source of human stem cell and the morphology of differentiated result
It examines and Function Identification.
Fig. 2A, 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.
The endothelial progenitor cells amplification situation (I) of peripheral blood CD34+ source of human stem cell after Fig. 3, the present invention mobilize monkey.
The number of amplification of Fig. 3 A, endothelial progenitor cells.
The amplification times of Fig. 3 B, endothelial progenitor cells.
The endothelial progenitor cells amplification of peripheral blood CD34+ source of human stem cell and differentiated result after Fig. 4, the present invention mobilize monkey
Morphological observation and Function Identification.
The endothelial progenitor cells amplification of derived from peripheral blood and the cellular morphology figure of differentiation after Fig. 4 A, monkey are mobilized.
The endothelial progenitor cells amplification of derived from peripheral blood and the qualification figure of differentiation after Fig. 4 B, monkey are mobilized.
Specific embodiment
In order to overcome lacking without the cultivating system for non-human primate endothelial progenitor cell in the prior art
It falls into, after the present inventor's further investigation, develops a kind of novel non-human primate endothelial progenitor cell efficient amplification training
The technology of supporting, so as to greatly push progress of the endothelial progenitor cells before entering human implantation's clinical trial.
As used herein, term " containing " or " comprising " include "comprising", " mainly by ... constitute (being made) ", " base
On this by ... constitute " and " by ... constitute ".
As used herein, " non-human primate " refers to the primate other than people, comprising: monkey, orangutan,
Ape etc..
The present inventor provides different culture mediums according to the different phase of human vascular endothelial progenitor cells amplification culture, packet
It includes: expansion of stem cells culture medium and endothelial progenitor cells culture medium.
The expansion of stem cells culture medium include: stem cell factor (SCF), Flt3- ligand (FLT-3L), blood platelet because
Son (TPO), interleukin-13 (IL-3), the granular leukocyte colony stimulating organism factor (GM-CSF), granulocyte colony stimulating factor (G-
CSF), Sall sample albumen 4B (Sall4B) and vascular endothelial growth factor (VEGF).Above-mentioned each cell factor is with suitable ratio
It is added in stem cell basal medium, the culture medium of suitable isolated growth environment can be provided for CD34+ mononuclearcell, promotees
Into the growth and amplification of CD34+ mononuclearcell.As preferred embodiment of the invention, for preparing expansion of stem cells of the invention
The dosage of each component of culture medium is as shown in table 1.
Table 1
The cell factor that table 1 is formulated is added in stem cell basal medium, obtains expansion of stem cells culture medium, thus
Suitable growth and amplification environment are provided for CD34+ mononuclearcell.The cell culture medium can choose Modified
IMDM culture medium, Modified StemSpan culture medium or similar cell culture medium.
The endothelial progenitor cells culture medium includes: vascular endothelial growth factor (VEGF), insulin-like growth factor
(IGF), epithelical cell growth factor (EGF), fibroblast growth factor (b-FGF), ascorbic acid (Ascorbic
Acid), heparin (Heparin), hydrocortisone (Hydrocortisone) and L glutamine (L-glutamine).It is above-mentioned each
Component is added in endothelial basal medium with suitable ratio, obtains endothelial progenitor cells culture medium of the invention.As
Preferred embodiment of the invention, it is as shown in table 2 for preparing the dosage of each component of endothelial progenitor cells culture medium of the invention.
Table 2
| Content | Preferred amounts | More preferably amount | |
| Vascular endothelial growth factor (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 that table 2 is formulated is added in endothelial basal medium, obtains Endothelial cell culture base of the invention,
To provide preferably simplified culture medium prescription for endothelial progenitor cells.
Above-mentioned culture medium after the present inventor optimizes, containing it is enough and it is reasonable promote cell growth and amplification at
Part and the ingredient for being conducive to cell holding stemness or realization differentiation, are conducive to the culture of endothelial progenitor cells.
For prepare Sall4B, SCF of culture medium, TPO, Flt-3L, IL3, GM-CSF, G-CSF, VEGF, IGF, EGF,
The cell factors such as b-FGF are that those skilled in the art are easily obtained, such as can be bought by commercial sources, or can pass through people
Work synthesis or recombinant expression obtain.
In a preferred embodiment of the invention, the non-human primate is monkey.The present inventor extracts food first
CD34 after crab monkey femur marrow and mobilization in peripheral blood+Mononuclearcell, for the growth characteristics of monkey blood stem cell, in people
The adjustment and optimization of combination of cytokines, training method are carried out on the basis of skin progenitor cells amplification cultivating system, initiatively
The endothelial progenitor cell of non-human primate origin is expanded and broken up on a large scale, the monkey endothelium ancestral of acquisition/interior
Chrotoplast can freeze and recover in the different phase of culture, and can further expand to meet the functionality of not homologous transplantation demand
Endothelial cell, so that the syngenetic graft for being applied to a variety of disease models is treated.
Monkey marrow (the 6- of single acquisition can be made by adjusting combination of cytokines and concentration, the above method by different phase
10mL) endothelial progenitor cells are expanded by culture in 12 days to 2.33 × 10 in sample7A, amplification times are 1142 times；To the 18th
It when number of amplification can reach 1.28 × 108A, multiple reaches 6275 times；At the same time, after G-CSF/SCF can also be made to mobilize
Peripheral blood (15-20mL) sample in endothelial progenitor cells pass through the amplification of culture in 24 days to 1.83 × 107A, amplification times are
1274 times.
Method of the invention can efficiently obtain endothelial progenitor cells/endothelial cell with endothelial activity function, thus
Meet the required cell quantity in the preclinical study that primate feeds back transplanting self, for endothelial progenitor cells amplification training
The technology of supporting is applied to clinical treatment ischemic disease, hemophilia provides reliable laboratory research basis.
The present invention realizes the efficient amplification of the endothelial progenitor cell of non-human primate origin in vitro initiatively,
It is established in primate level and utilizes marrow and blood stem cell preparation safely and effectively endothelial progenitor cell/interior
The technology of chrotoplast.The present invention is endothelial progenitor cells for non-human primate autotransplantation and the experiment of homologous heteroplastic transplantation
Research provides valuable resource, while also providing preclinical data for the transplanting of people to people and supporting, will further push in source of people
Skin progenitor cells as it is a kind of can safe and efficient preparation in vitro cell preparation for ischemic class disease and hemophilia A
Clinical treatment and scientific research demand.
Main feature and advantage of the invention further include: first, the present invention is other using clinical grade in entire cultivating system
Porcine HGF is not inserted into allogenic gene, therefore does not change the Genome stability of former stem cell, no tumorigenesis risk；The
Two, after the present invention can make non-human primate marrow and mobilize peripheral blood CD34+ stem cell in vitro respectively by 12 days and
Culture in 24 days, so that the quantity of endothelial progenitor cells is more than 107, amplification times, and can be further at 1000 times or more
Exponentially growing location expands and is divided into mature endothelial cell, plays the effect at blood vessel function and secreting function albumen.The
Three, a variety of stem cell factors being added in culture early period of the invention can preferably maintain the stemness of cell, so that amplification
Endothelial progenitor cells afterwards keep high proliferation activity, facilitate migration from transplanting to lesion and Function after.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or
According to the normal condition proposed by manufacturer.
The preparation of embodiment 1, culture medium
Cultivating system of the invention includes two CD34+ expansion of stem cells, the adherent amplification differentiation of endothelial progenitor cells parts:
One, the CD34+ expansion of stem cells phase: expansion of stem cells culture medium
It is any in table 3 wherein adding using Modified IMDM culture medium (serum-free) as basic culture medium
The cell factor of formula:
Table 3 (unit: ng/mL)
| It is formulated A | It is formulated B | It is formulated 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 expand differential medium
Using EBM-2 culture medium (10% or 20% fetal calf serum) as basic culture medium, wherein adding in table 4
The cell factor and other compositions of any formula:
Table 4 (in addition in addition indicating, unit ng/mL)
| It is formulated D | It is formulated E | It is formulated 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, amplification and differentiation (I) to the endothelial progenitor cells of monkey marrow CD34+ source of human stem cell
0th day:
(1) CD34 is prepared+Expansion of stem cells culture medium
Various cell factors are added in Modified IMDM culture medium (serum-free), make the final concentration of SCF of cell factor
200ng/mL、Flt-3L 200ng/mL、IL-310ng/mL、TPO 20ng/mL、Sall4B3ng/mL、GM-CSF 12.5ng/
ML, G-CSF 12.5ng/mL, VEGF 50ng/mL (being formulated A in embodiment 1) are used as amplification culture medium.
(2) CD34 in marrow is isolated and purified+Stem cell
Femur marrow 8-10mL is taken from Healthy Youth machin anterior superior spine, PBS dilutes 10 times, using density gradient centrifugation
CD34 antibody (APC-mouse anti-human CD34) Ji Meitian Ni company of BD company is used after method acquisition mononuclearcell
MACS magnetic bead (Rat anti-mouse IgG) two step separating methods separate CD34+Mononuclearcell, obtain cell quantity be 1.4 ×
106A cell.Using CD34+Cell is resuspended expansion of stem cells culture medium, and adjusting cell concentration is 6 × 105A cell/mL is added
In 24 orifice plates, every hole 1mL, i.e., 6 × 105A cells/well.Then pipe is placed in incubator with micro- sem observation (37 DEG C, 5%
CO2)。
(3) flow cytometer detection
Respectively take 5 × 10 walked in poly- (2) after magnetic bead sorting4A cell is placed in 1.5mL EP pipe, totally 3 EP pipe (pipe 1,
2,3), PBS cleaning solution of the 1mL containing 1%BSA is added in every pipe, and 1200rpm is centrifuged 5 minutes at room temperature, discards supernatant, is contained with 100 μ L
Cell precipitation is resuspended in the PBS of 1%BSA, and 2 μ LVEGFR-2 antibody (PE-Mouseanti-humanVEGFR-2), pipe 2 is added in pipe 1
Corresponding Isotype control is added, and (2 μ LCD34 antibody (APC-Mouse are added in PE-Mouse anti-human IgG1 κ, pipe 3
Anti-human CD34), separately take the mononuclearcell 5 × 10 in (2) before CD34+ sorting4Simultaneously 2 μ L are added in a the same operation
Isotype control (APC-Mouse anti-human IgG1 κ).Room temperature is protected from light incubation 15 minutes after antibody is added, and then every pipe adds
Enter 1mL PBS washing, 1200rpm is centrifuged 5 minutes, discards supernatant, and cell is resuspended with 300 μ L PBS, uses flow cytometry analysis
The expression of cell surface CD34 and VEGFR-2.
3rd day:
(1) cell count with the expression of flow cytometry analysis cell surface CD34, VEGFR-2, and counts total thin
Born of the same parents' number, CD34+ and VEGFR-2+Cell number and amplification times.
(2) a point hole is carried out according to cell number and (controls cell density lower than 1 × 106A/mL), expansion cultivating system is simultaneously every
Fresh culture is added to 1mL (being formulated A in addition embodiment 1) in hole.
6th day:
Culture to the 6th day replacement endothelial progenitor cells culture medium carries out adhere-wall culture.
(1) cell count with the expression of flow cytometry analysis cell surface CD34 and VEGFR-2, and counts
VEGFR-2+ cell number and amplification times.
(2) endothelial progenitor cells culture medium is prepared, following component is added in EBM-2 culture medium (20% fetal calf serum), respectively
Ingredient is final concentration of: VEGF 25ng/mL, IGF20ng/mL, EGF10ng/mL, b-FGF10ng/mL, 2 μ g/mL of ascorbic acid,
Heparin 100U/mL, hydrocortisone 100ng/mL, L- glutamine 4mM (being formulated D in embodiment 1)
(3) it is coated with culture plate: 2.5 μ g/cm is pressed using people's fibronectin2Concentration 24 orifice plates are coated with, 37
It DEG C being incubated for 2 hours, PBS washes 15min, and 3 times.
(4) it cultivates: according to 1 × 106All cells after amplification are replaced to endothelial progenitor cells and are trained by the density of a cell/mL
It supports and is cultivated in base, cultivated respectively to the 18th day.
The 9-18 days:
Cell is dispelled at (1) the 9th day, suspension cell is removed, attached cell is selected to continue to cultivate.Replacement in every 3 days is primary
Fresh culture (is formulated D in embodiment 1).
(2) it respectively at the 9th, 12,15,18 day, is counted using 0.25% pancreatin digestion attached cell, counts attached cell
Absolute number, the 12nd day when, adherent endothelial progenitor cells absolute number was 2.33 × 107±2.15×106(n=3), the 18th day when
It is 1.28 × 108±1.67×107(n=3), see Figure 1A.(the VEGFR2 compared with the endothelial progenitor cells of starting in the 0th day+:
1.7%) amplification times that, the 12nd day amplification times are the 1142 ± 121, the 18th day are 6275 ± 792 (n=3), see Figure 1B.
The 3-18 days cellular morphology figures of record simultaneously, are as a result shown in Fig. 2A.
(3) it cellular identification: cultivates to the 18th day cell, by 5 × 104The density in a/hole is inoculated in 24 orifice plates, to adherent
Afterwards, with the endothelium medium treatment of serum-free 24 hours, the Dil-ac-LDL of 10 μ g/mL is added, 37 DEG C are incubated for 4 hours, and PBS is washed
3 times；4 DEG C of 4% paraformaldehyde fixes 15 minutes, and the FITC-lectin of 10 μ g/mL is added, and 37 DEG C are incubated for 2 hours, and PBS washes 3
It is taken pictures after with microscope.As shown in Figure 2 B, the DIL-ac-LDL and green of 80% or more attached cell red color visible fluorescence
The FITC-lectin double expression of fluorescence.Illustrate that it is thin to obtain endothelium ancestral/endothelium for amplification from the CD34+ stem cell of monkey derived from bone marrow
Born of the same parents have the characteristic of expression endothelial activity function, can be further used for internal transplantation experiments.
Embodiment 3, amplification and differentiation (II) to the endothelial progenitor cells of monkey marrow CD34+ source of human stem cell
0th day:
(1) CD34 is prepared+Expansion of stem cells culture medium
Various cell factors are added in Modified IMDM culture medium (serum-free), make the final concentration of SCF of cell factor
100ng/mL、Flt-3L 100ng/mL、IL-35ng/mL、TPO 10ng/mL、Sall4B2ng/mL、GM-CSF 8ng/mL、G-
CSF 8ng/mL, VEGF 25ng/mL (being formulated B in embodiment 1) are used as amplification culture medium.
(2) CD34 in marrow is isolated and purified+Stem cell
Femur marrow 6-8mL is taken from Healthy Youth machin anterior superior spine, PBS dilutes 10 times, using 2 respective party of embodiment
Method sorts CD34+Cell, obtaining cell quantity is 1.2 × 106A cell.Using CD34+Expansion of stem cells culture medium is by cell weight
Outstanding, adjusting cell concentration is 5 × 105A cell/mL is added in 24 orifice plates, every hole 1mL, i.e., 5 × 105A cells/well.Use microscope
Observation then pipe is placed in incubator (37 DEG C, 5%CO2)。
(3) flow cytometer detection: with corresponding portion in embodiment 2 (the 0th day, (3)).
3rd day:
(1) cell count with the expression of flow cytometry analysis cell surface CD34, VEGFR-2, and counts total thin
Born of the same parents' number, CD34+ and VEGFR-2+Cell number and amplification times.
(2) a point hole is carried out according to cell number and (controls cell density lower than 1 × 106A/mL), expansion cultivating system is simultaneously every
Fresh culture is added to 1mL (being formulated B in addition embodiment 1) in hole.
6th day:
Culture to the 6th day replacement endothelial progenitor cells culture medium carries out adhere-wall culture.
(1) cell count with the expression of flow cytometry analysis cell surface CD34 and VEGFR-2, and counts
VEGFR-2+ cell number and amplification times.
(2) endothelial progenitor cells culture medium is prepared, following component is added in EBM-2 culture medium (20% fetal calf serum), respectively
Ingredient is final concentration of: VEGF 10ng/mL, IGF10ng/mL, EGF 4ng/mL, b-FGF20ng/mL, 1 μ g/mL of ascorbic acid,
Heparin 50U/mL, hydrocortisone 150ng/mL, L- glutamine 2mM (being formulated E in embodiment 1)
(3) it is coated with culture plate: 2.5 μ g/cm is pressed using people's fibronectin2Concentration 24 orifice plates are coated with, 37
It DEG C being incubated for 2 hours, PBS washes 15min, and 3 times.
(4) it cultivates: according to 1 × 106All cells after amplification are replaced to endothelial progenitor cells and are trained by the density of a cell/mL
It supports and is cultivated in base, cultivated respectively to the 18th day.
The 9-18 days:
Cell is dispelled at (1) the 9th day, suspension cell is removed, attached cell is selected to continue to cultivate.Replacement in every 3 days is primary
Fresh culture (is formulated E in embodiment 1).
(2) it respectively at the 9th, 12,15,18 day, is counted using 0.25% pancreatin digestion attached cell, counts attached cell
Absolute number, the 12nd day when, adherent endothelial progenitor cells absolute number was 6.83 × 106±4.66×105(n=3), the 18th day when
It is 3.15 × 107±3.07×106(n=3).(the VEGFR2 compared with the endothelial progenitor cells of starting in the 0th day+: 1.3%), and the 12nd day
Amplification times be the 523 ± 46, the 18th day amplification times be 2423 ± 322 (n=3).
Embodiment 4, amplification and differentiation (I) to the endothelial progenitor cells of peripheral blood CD34+ source of human stem cell after monkey mobilization
Monkey mobilize after peripheral blood acquire: acquisition first 5 days, using subcutaneous injection give Healthy Youth G-CSF (100 μ g/kg)+
SCF (50 μ g/kg), it is continuous to mobilize 5 days, 15-20mL peripheral blood is acquired every time.
0th day:
(1) CD34 is prepared+Expansion of stem cells culture medium
Various cell factors are added in Modified IMDM culture medium (serum-free), keep cell factor final concentration of
SCF200ng/mL、Flt-3L200ng/mL、IL-310ng/mL、TPO 20ng/mL、Sall4B3ng/mL、GM-CSF
12.5ng/mL, G-CSF 12.5ng/mL, VEGF 50ng/mL (being formulated A in embodiment 1) are used as amplification culture medium.
(2) CD34 in peripheral blood is isolated and purified+Stem cell
5 times are diluted using peripheral blood 15-20mL after fresh mobilization, PBS, CD34 is sorted using 2 correlation method of embodiment+
Cell, obtaining cell quantity is 1.5 × 106A cell.Using CD34+Cell is resuspended expansion of stem cells culture medium, adjusts cell dense
Degree is 6 × 105A cell/mL is added in 24 orifice plates, every hole 1mL, i.e., 6 × 105A cells/well.Then will with micro- sem observation
Pipe be placed in incubator (37 DEG C, 5%CO2)。
(3) flow cytometer detection: with corresponding portion in embodiment 2 (the 0th day, (3)).
3rd day:
(1) cell count with the expression of flow cytometry analysis cell surface CD34, VEGFR-2, and counts total thin
Born of the same parents' number, CD34+ and VEGFR-2+Cell number and amplification times.
(2) a point hole is carried out according to cell number and (controls cell density lower than 1 × 106A/mL), expansion cultivating system is simultaneously every
Fresh culture is added to 1mL (being formulated A in addition embodiment 1) in hole.
6th day:
Culture to the 6th day replacement endothelial progenitor cells culture medium carries out adhere-wall culture.
(1) cell count with the expression of flow cytometry analysis cell surface CD34 and VEGFR-2, and counts
VEGFR-2+ cell number and amplification times.
(2) endothelial progenitor cells culture medium is prepared, following component is added in EBM-2 culture medium (20% fetal calf serum), respectively
Ingredient is final concentration of: VEGF 25ng/mL, IGF20ng/mL, EGF10ng/mL, b-FGF10ng/mL, 2 μ g/mL of ascorbic acid,
Heparin 100U/mL, hydrocortisone 100ng/mL, L- glutamine 4mM (being formulated D in embodiment 1)
(3) it is coated with culture plate: 2.5 μ g/cm is pressed using people's fibronectin2Concentration 24 orifice plates are coated with, 37
It DEG C being incubated for 2 hours, PBS washes 15min, and 3 times.
(4) it cultivates: according to 1 × 106All cells after amplification are replaced to endothelial progenitor cells and are trained by the density of a cell/mL
It supports and is cultivated in base, cultivated to the 24th day.
The 9-24 days:
Cell is dispelled at (1) the 9th day, suspension cell is removed, attached cell is selected to continue to cultivate.Replacement in every 3 days is primary
Fresh culture (is formulated D in embodiment 1).
(2) it respectively at the 9th, 12,15,18,21,24 day, is counted using 0.25% pancreatin digestion attached cell, statistics patch
Parietal cell absolute number, the 24th day when, adherent endothelial progenitor cells absolute number was 1.83 × 107±2.89×106(n=3), see
Fig. 3 A.(the VEGFR2 compared with the endothelial progenitor cells of starting in the 0th day+: 1.2%), the 24th day amplification times are 1274 ± 154 (n
=3), see Fig. 3 B.The 3-24 days cellular morphology figures of record simultaneously, are as a result shown in Fig. 4 A.
(3) cellular identification: as a result method is shown in Fig. 4 B with embodiment 2.85% or more attached cell red color visible fluorescence
The FITC-lectin double expression of DIL-ac-LDL and green fluorescence.Illustrate the CD34 of derived from peripheral blood after mobilizing from monkey+Stem cell
Middle amplification obtains the characteristic that endothelium ancestral/endothelial cell has expression endothelial activity function, can be further used for internal transplantation experiments.
Embodiment 5, amplification and differentiation (II) to the endothelial progenitor cells of peripheral blood CD34+ source of human stem cell after monkey mobilization
Peripheral blood acquires after monkey is mobilized: with corresponding portion in case study on implementation 4.
0th day:
(1) CD34 is prepared+Expansion of stem cells culture medium
Various cell factors are added in Modified IMDM culture medium (serum-free), keep cell factor final concentration of
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 (being formulated C in embodiment 1) are used as amplification culture medium.
(2) CD34 in peripheral blood is isolated and purified+Stem cell
5 times are diluted using peripheral blood 15-20mL (n=3) after fresh mobilization, PBS, using 2 correlation method of embodiment point
Select CD34+Cell, obtaining cell quantity is 2.1 × 106A cell.Using CD34+Cell is resuspended expansion of stem cells culture medium,
Adjusting cell concentration is 8 × 105A cell/mL is added in 24 orifice plates, every hole 1mL, i.e., 8 × 105A cells/well.It is seen with microscope
Examine then pipe is placed in incubator (37 DEG C, 5%CO2)。
(3) flow cytometer detection: with corresponding portion in embodiment 2 (the 0th day, (3)).
3rd day:
(1) cell count with the expression of flow cytometry analysis cell surface CD34, VEGFR-2, and counts total thin
Born of the same parents' number, CD34+ and VEGFR-2+Cell number and amplification times.
(2) a point hole is carried out according to cell number and (controls cell density lower than 1 × 106A/mL), expansion cultivating system is simultaneously every
Fresh culture is added to 1mL (being formulated C in addition embodiment 1) in hole.
6th day:
Culture to the 6th day replacement endothelial progenitor cells culture medium carries out adhere-wall culture.
(1) cell count with the expression of flow cytometry analysis cell surface CD34 and VEGFR-2, and counts
VEGFR-2+ cell number and amplification times.
(2) endothelial progenitor cells culture medium is prepared, following component is added in EBM-2 culture medium (20% fetal calf serum), respectively
Ingredient is final concentration of: VEGF 75ng/mL, IGF80ng/mL, EGF16ng/mL, b-FGF80ng/mL, 4 μ g/mL of ascorbic acid,
Heparin 200U/mL, hydrocortisone 250ng/mL, L- glutamine 5mM (being formulated F in embodiment 1)
(3) it is coated with culture plate: 2.5 μ g/cm is pressed using people's fibronectin2Concentration 24 orifice plates are coated with, 37
It DEG C being incubated for 2 hours, PBS washes 15min, and 3 times.
(4) it cultivates: according to 1 × 106All cells after amplification are replaced to endothelial progenitor cells and are trained by the density of a cell/mL
It supports and is cultivated in base, cultivated to the 24th day.
The 9-24 days:
Cell is dispelled at (1) the 9th day, suspension cell is removed, attached cell is selected to continue to cultivate.Replacement in every 3 days is primary
Fresh culture (is formulated F in embodiment 1).
(2) it respectively at the 9th, 12,15,18,21,24 day, is counted using 0.25% pancreatin digestion attached cell, statistics patch
Parietal cell absolute number, the 24th day when, adherent endothelial progenitor cells absolute number was 2.56 × 107±3.10×106(n=3).With
The endothelial progenitor cells of starting in 0th day compare (VEGFR2+: 1.0%), the 24th day amplification times are 1600 ± 257 (n=3).
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (9)
1. a kind of method for cultivating non-human primate endothelial progenitor cell, which is characterized in that the described method includes:
(1) non-human primate CD34 is expanded using expansion of stem cells culture medium+Mononuclearcell；
(2) cell after step (1) amplification is transferred in endothelial progenitor cells culture medium and is cultivated, obtain non-human primate
Endothelial progenitor cell；
Wherein, the expansion of stem cells culture medium is made of stem cell basal medium and following cell factor:
Stem cell factor: 50-500 ng/mL；
Flt3- ligand: 50-500 ng/mL；
Platelet factor TPO:5-200 ng/mL；
Interleukin-13: 5-50 ng/mL；
The granular leukocyte colony stimulating organism factor: 5-25ng/mL；
Granulocyte colony stimulating factor: 5-25ng/mL；
Sall sample albumen 4B:1-10 ng/mL；With
Vascular endothelial growth factor: 15-150ng/mL；
Wherein, the endothelial progenitor cells culture medium is grouped as by endothelial basal medium and following group:
Vascular endothelial growth factor: 5-100ng/mL；
Insulin-like growth factor: 5-100 ng/mL；
Epithelical cell growth factor: 2-20 ng/mL；
Fibroblast growth factor: 5-100 ng/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 as described in claim 1, which is characterized in that the endothelial basal medium is selected from: EBM-2 culture
Base, M199, M200 culture medium.
3. the method as described in claim 1, which is characterized in that step (1) includes: CD34+Mononuclearcell is added to described
In expansion of stem cells culture medium, so that cell concentration is 3-20 × 105A cell/mL；Culture increases after 2-4 days according to cell number
Dosage carries out expansion of stem cells culture medium described in point hole and addition and cell concentration is made to be 3-20 × 105A cell/mL；Step
(1) it carries out 5-7 days.
4. the method as described in claim 1, which is characterized in that step (2) includes: that after step (1) carries out 5-7 days, will obtain
Cell be transferred in the endothelial progenitor cells culture medium, be placed in the culture plate for be coated with fibronectin and cultivate, carefully
Density is 0.3-3 × 10 to born of the same parents in the medium6A cell/mL dispels cell after continuing culture 2-4 days, selection attached cell after
Continuous culture；The 1 endothelial progenitor cells culture medium of replacement in subsequent every 2-3 days；Cell is harvested after replacement 3-6 times.
5. the method as described in claim 1-4 is any, which is characterized in that the non-human primate includes: monkey.
6. a kind of kit for being used to prepare non-human primate endothelial progenitor cell, include: in the kit
Amplification culture medium is made of stem cell basal medium and following cell factor: stem cell factor: 50-500 ng/
mL；Flt3- ligand: 50-500 ng/mL；Platelet factor TPO:5-200 ng/mL；Interleukin-13: 5-50 ng/mL；Granulocyte
Colony-stimulating biotic factor: 5-25ng/mL；Granulocyte colony stimulating factor: 5-25ng/mL；Sall sample albumen 4B:1-10 ng/
ML and vascular endothelial growth factor: 15-150ng/mL；And
For cultivating the culture medium of non-human primate endothelial progenitor cell, by endothelial basal medium and as follows
Group is grouped as: vascular endothelial growth factor: 5-100ng/mL；Insulin-like growth factor: 5-100 ng/mL；Epidermal cell is raw
The long factor: 2-20 ng/mL；Fibroblast growth factor: 5-100 ng/mL；Ascorbic acid: 0.5-6 μ g/mL；Heparin: 40-
200U/mL；Hydrocortisone: 80-300ng/mL and L glutamine: 1-6mM.
7. kit as claimed in claim 6, which is characterized in that the cell factor in the amplification culture medium are as follows: dry thin
Intracellular cytokine: 100-400 ng/mL；Flt3- ligand: 100-400 ng/mL；Platelet factor TPO:10-150 ng/mL；Bai Jie
Plain 3:5-30 ng/mL；The granular leukocyte colony stimulating organism factor: 8-20 ng/mL；Granulocyte colony stimulating factor: 8-20 ng/
mL；Sall sample albumen 4B:2-8 ng/mL；And vascular endothelial growth factor: 20-100 ng/mL.
8. kit as claimed in claim 6, which is characterized in that described for cultivating non-human primate blood vessel endothelium ancestral
The culture medium of cell is grouped as by endothelial basal medium and following group: vascular endothelial growth factor: 10-80 ng/
mL；Insulin-like growth factor: 10-80 ng/mL；Epithelical cell growth factor: 4-16 ng/mL；Desmocyte growth factor
Son: 8-80 ng/mL；Ascorbic acid: 1-4 μ g/mL；Heparin: 80-200 U/mL；Hydrocortisone: 100-250 ng/mL；With
L glutamine: 2-5 mM.
9. the purposes of kit as claimed in claim 6 to 8 is used to prepare non-human primate endothelial progenitor cell.
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| 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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| 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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| Isolation and characterization of endothelialprogenitor cells from Rhesus monkeys;Wen Sun et.al.;《Regenerative Medicine Research》;20140303;第2卷;1 摘要，第5页左栏第3段、右栏第2段，第6页左栏第1-4段、右栏第1段 |
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