CN109456936A - A kind of cultural method of endothelial progenitor cells - Google Patents
A kind of cultural method of endothelial progenitor cells Download PDFInfo
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- CN109456936A CN109456936A CN201811599511.4A CN201811599511A CN109456936A CN 109456936 A CN109456936 A CN 109456936A CN 201811599511 A CN201811599511 A CN 201811599511A CN 109456936 A CN109456936 A CN 109456936A
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Abstract
The present invention provides a kind of cultural methods of endothelial progenitor cells, comprising: S1) mononuclearcell is subjected to adhere-wall culture, the cell after digestion, after being expanded；S2 the cell inoculation after the amplification is cultivated in culture medium), obtains endothelial progenitor cells；It include basic fibroblast growth factor, vascular endothelial growth factor and microcarrier in the culture medium.Compared with prior art, the present invention adds basic fibroblast growth factor in the medium and vascular endothelial growth factor promotes cell growth, the microcarrier of large specific surface area is added simultaneously, the cell yield for improving unit volume culture solution simplifies cell and grows detecting and controlling for various environmental factors, favorable reproducibility, culture medium utilization rate is high, and cell harvest process is also simplified, labor intensity is small, and culture systems occupied area is small.
Description
Technical field
The invention belongs to cell engineering field more particularly to a kind of cultural methods of endothelial progenitor cells.
Background technique
Endothelial progenitor cells are the precursors of vascular endothelial cell, are typically distributed on the peripheral blood, Cord blood and bone of people
In marrow, the formation of new vessels is participated in, vascular endothelial cell is often used to carry out injury of blood vessel in organizational engineering
It repairs.
Injured Vascular Diseases have the characteristics that high incidence and the death rate.Effective reparation of damaged endothelial and new vessels
Generation is that the treatment of this kind of disease is crucial.When endothelium integrity violations, endothelial cell (endothelial cells, ECs) is from neighbour
It is proliferated and migrates in nearby bleeding pipe, promote damaged blood vessels endothelium reparation.In the past it is thought that the sole mode of neovascularization.So
And the traditional concept of this vascularization is faced with challenge at present.Asahara etc. first describes the cell mass of derived from bone marrow
Body facilitates neovascularization, and name this cell mass be endothelial progenitor cells (endothelial progenitor cells,
EPCs).Relative to vascularization, the new blood vessel from marrow and the EPCs of proliferation and differentiation and maturation formation is defined as blood vessel
Occur.Therefore, EPCs is considered as the new method for treating vascular conditions.
Prior art culture endothelial progenitor cells be usually all by two-dimentional adhere-wall culture, that is, pass through culture dish or training
Bottle is supported to be cultivated in plane, and this cultural method cell pick-up rate first is lower, expend it is a large amount of manually and place, expend
A large amount of culture medium, and obtained cell is easy differentiation, it is second-rate.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is that providing a kind of culture side of extensive endothelial progenitor cells
Method.
The present invention provides a kind of cultural methods of endothelial progenitor cells, comprising:
S1 mononuclearcell) is subjected to adhere-wall culture, the cell after digestion, after being expanded；
S2 the cell inoculation after the amplification is cultivated in culture medium), obtains endothelial progenitor cells；The culture
It include basic fibroblast growth factor, vascular endothelial growth factor and microcarrier in base.
Preferably, the step S2) expand in culture medium after cell density be 1 × 104~1 × 106cells/ml。
Preferably, the step S2) culture medium bFGF and vascular endothelial growth factor
Concentration is each independently 10~50ng/ml.
Preferably, the step S2) culture medium bFGF and vascular endothelial growth factor
Concentration is each independently 10~30ng/ml.
Preferably, the step S2) concentration of microcarrier is 0.001~0.02g/ml in culture medium.
Preferably, the step S2) in cultivate temperature be 36 DEG C~37 DEG C, CO2Volumetric concentration be 3%~10%.
Preferably, the step S2) in culture specifically:
10~20 revs/min, 1~3h is cultivated, then 25~35 revs/min of cultures were to the 2nd day, supplemented medium, then with
40~60 revs/min are cultivated.
Preferably, the step S2) in culture specifically:
13~18 revs/min, 1.5~2.5h is cultivated, then 28~32 revs/min of cultures were to the 2nd day, supplemented medium,
It is cultivated again with 45~55 revs/min.
Preferably, the amount of the supplemented medium is the 1/10~1/5 of original culture medium volume.
Preferably, the step S2) in after culture, centrifugation is digested with trypsase-EDTA digestive juice, and termination disappears
After change, centrifugation removal supernatant, after precipitating is resuspended with phosphate buffer, filtering obtains endothelial progenitor cells.
The present invention provides a kind of cultural methods of endothelial progenitor cells, comprising: S1) mononuclearcell is subjected to adherent training
It supports, the cell after digestion, after being expanded；S2 the cell inoculation after the amplification is cultivated in culture medium), is obtained
Endothelial progenitor cells；It include basic fibroblast growth factor, vascular endothelial growth factor and microcarrier in the culture medium.With
The prior art is compared, and the present invention adds basic fibroblast growth factor in the medium and vascular endothelial growth factor promotes
Cell growth, while the microcarrier of large specific surface area is added, the cell yield of unit volume culture solution is improved, cell is simplified
Detecting and controlling for various environmental factors is grown, favorable reproducibility, culture medium utilization rate is high, and also simplifies cell and harvested
Journey, labor intensity is small, and culture systems occupied area is small.
Detailed description of the invention
Fig. 1 is the expression of endothelial progenitor cells CD133, CD309, CD34, CD31, CD105 obtained in the embodiment of the present invention 1
Analysis of spectra.
Specific embodiment
Below in conjunction with the embodiment of the present invention, technical scheme in the embodiment of the invention is clearly and completely described,
Obviously, described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based in the present invention
Embodiment, every other embodiment obtained by those of ordinary skill in the art without making creative efforts, all
Belong to the scope of protection of the invention.
The present invention provides a kind of cultural methods of endothelial progenitor cells, comprising:
S1 mononuclearcell) is subjected to adhere-wall culture, the cell after digestion, after being expanded；
S2 the cell inoculation after the amplification is cultivated in culture medium), obtains endothelial progenitor cells；The culture
It include basic fibroblast growth factor, vascular endothelial growth factor and microcarrier in base.
The present invention is not particularly limited the source of all raw materials, for commercially available or self-control.
Wherein, the preparation method of the mononuclearcell is method well known to those skilled in the art, and it is special to have no
Limitation, Ficoll density-gradient centrifugation method can be used, can also be used hydroxyethyl starch partition method, it is preferably specific in the present invention
Are as follows: the hydroxyethyl starch of bleeding of the umbilicus and 6% is mixed according to the ratio of 4:1, is stored at room temperature 30~60min, collects upper layer rich in white
The plasma layer of cell, 400~500g are centrifuged 5~15min, abandon supernatant, and cell precipitation is resuspended with phosphate buffer, obtains cell
Suspension；Cell suspension is mixed with Ficoll, using 600~800g of horizontal centrifuge be centrifuged 20~40min, collect Ficoll with
The white cellular layer of plasma layer intersection to get arrive mononuclearcell；The volume ratio of the cell suspension and Ficoll be (2~
4): 1, more preferably (2.5~3.5): 1, it is further preferably 3:1；Cell suspension preferably mixes in the following way with Ficoll: using
Cell suspension is slowly added to the upper layer of Ficoll by pasteur pipet along centrifugation tube wall；It is preferably using the revolving speed of horizontal centrifuge
650~750g, more preferably 700g, the time of centrifugation are preferably 25~35min, more preferably 30min；It is collected after centrifugation
Phosphate buffer dilution is preferably added to after the white cellular layer of Ficoll and plasma layer intersection, it is slow with phosphate again after centrifugation
Fliud flushing washing.The volume ratio of the white cellular layer and phosphate buffer is preferably 1:(3~5), more preferably 1:(3.5~
It 4.5) is further preferably, 1:4；The revolving speed of the centrifugation is preferably 300~500g, more preferably 350~450g, further preferably for
400g；The time of the centrifugation is preferably 3~10min, more preferably 3~8min, is further preferably 4~6min, most preferably
5min。
Mononuclearcell is subjected to adhere-wall culture, the cell after digestion, after being expanded；Training used in the adhere-wall culture
Supporting base is culture medium well known to those skilled in the art, has no special limitation, preferably EMG-2MV in the present invention；Institute
When stating adhere-wall culture, the density of mononuclearcell is preferably 1 × 10 in culture medium4~1 × 105Cells/ml, more preferably 3 ×
104~8 × 104Cells/ml is further preferably 5 × 104cells/ml；The condition of the adhere-wall culture be preferably humidity 90% with
On, 36 DEG C~37 DEG C, CO2Volumetric concentration be preferably 3%~10%, more preferably 3%~8%, be further preferably 4%~6%,
Most preferably 5%；Preferred supplemented medium after adhere-wall culture three days, the volume of supplemented medium preferably most start medium body
Long-pending 0.8~1.2 times, more preferably 0.9~1.1 times, most preferably 1 times；When culture was to the 6th day, preferably discard on culture medium
Clear and not adherent cell, supplemented medium, the volume of supplemented medium preferably most start the 0.8 of culture volume again
~1.2 times, more preferably 0.9~1.1 times, most preferably 1 times；It preferably changes the liquid once every three days later, until cell fusion degree
Reach 80%, discard culture medium, phosphate buffer cleaning is added, pancreatin digestion is then added, after terminating digestion, centrifugation is obtained
Cell after to amplification；The volume ratio of phosphate buffer and cell is preferably (0.9~1.2) when the cleaning: 1, more preferably
For 1:1；The concentration for digesting pancreatin used is preferably 0.2%~0.3%, and more preferably 0.25%；The amount that pancreatin is added is culture
1/6~1/3, more preferably the 1/5~1/4 of matrix product；The time of the digestion is preferably 1~2min, more preferably 1min；Institute
Termination digestion is stated preferably using 1640 culture mediums containing fetal calf serum；The quality percentage of fetal calf serum in 1640 culture medium
Content is preferably 8%~12%, and more preferably 9%~11%, it is further preferably 10%；The volume of 1640 culture medium and pancreatin
Than preferably (4~7): 1, more preferably (5~6): 1, it is further preferably 5:1；The revolving speed of the centrifugation is preferably 300~500g,
More preferably 350~450g is further preferably 400g；The time of the centrifugation is preferably 4~7min, more preferably 5~6min；From
After the heart discards supernatant, it is preferred to use the cell after amplification is resuspended in culture medium；The culture medium is preferably EMG-2MV.
By the cell inoculation after the amplification in culture medium；The culture medium is culture well known to those skilled in the art
Base has no special limitation, preferably EMG-2MV in the present invention；In the culture medium include basic fibroblast because
Son, vascular endothelial growth factor and microcarrier；The bFGF is alkalinity well known to those skilled in the art
The fibroblast factor has no special limitation, preferably hFGF-B in the present invention；The culture medium neutral and alkali is at fiber
The concentration of cell factor is preferably 10~50ng/ml, more preferably 10~30ng/ml, is further preferably 10~20ng/ml, optimal
It is selected as 10ng/ml；The concentration of the culture medium medium vascular endothelial growth factor is preferably 10~50ng/ml, more preferably 10~
30ng/ml is further preferably 10~20ng/ml, most preferably 10ng/ml；The microcarrier is well known to those skilled in the art
Microcarrier, has no special limitation, in the present invention preferably in Cytodex1,2,3, Cytopore and Cytoline one
Kind is a variety of；The concentration of microcarrier is preferably 0.001~0.02g/ml in the culture medium, more preferably 0.004~0.015g/
Ml is further preferably 0.008~0.01g/ml；The concentration of cell after the inoculation of medium amplification is preferably 1 × 104~1
×106Cells/ml, more preferably 5 × 104~5 × 105Cells/ml is further preferably 8 × 104~2 × 105Cells/ml, most
Preferably 1 × 105cells/ml。
After being inoculated in culture medium, cultivated；The culture preferably carries out in pot type bioreactor；The culture
Temperature is 36 DEG C~37 DEG C, CO2Volumetric concentration be preferably 3%~10%, more preferably 3%~8%, further preferably for 4%~
6%, most preferably 5%；The cultural method is method well known to those skilled in the art, has no special limitation,
It is preferably cultivated in accordance with the following methods in the present invention: 10~20 revs/min, cultivating 1~3h, then cultivated extremely for 25~35 revs/min
2nd day, supplemented medium, then cultivated with 40~60 revs/min；More preferably 13~18 revs/min, culture 1.5~
2.5h, then 28~32 revs/min of cultures were to the 2nd day, supplemented medium, then were cultivated with 45~55 revs/min；Further preferably
It is 15 revs/min, cultivates 2h, then 30 revs/min of cultures was to the 2nd day, supplemented medium, then were cultivated with 50 revs/min；
To the 2nd day, the amount of supplemented medium was preferably the 1/10~1/5 of original culture medium volume, more preferably 1/8~1/ for the culture
5。
It is preferably centrifuged after culture, is digested with trypsase-EDTA digestive juice, after terminating digestion, centrifugation removal supernatant,
After precipitating is resuspended with phosphate buffer, filtering obtains endothelial progenitor cells；Phosphate-buffered is preferably used after being centrifuged in the present invention
Trypsase-EDTA digestive juice digests again after liquid cleaning twice；Trypsase-EDTA the digestive juice is 0.25% tryptose
Enzyme-EDTA digestive juice；The time of the digestion is preferably 10~20min, more preferably 13~18min, is further preferably 15min；
The method for terminating digestion is terminating method well known to those skilled in the art, has no special limitation, in the present invention preferably
Using 1640 culture mediums containing fetal calf serum；In 1640 culture medium fetal calf serum-mass percentage is preferably 8%
~12%, more preferably 9%~11%, it is further preferably 10%；The filtering preferably uses 50~100 μm of the screen to filtrates, more excellent
60~90 μm are selected as, is further preferably 70~80 μm, most preferably 70 μm；The endothelial progenitor cells being obtained by filtration preferably are resuspended in physiology
In salt water, cell count and flow cytometer detection are carried out.
The present invention adds basic fibroblast growth factor in the medium and vascular endothelial growth factor promotes cell
Growth, while the microcarrier of large specific surface area is added, the cell yield of unit volume culture solution is improved, cell growth is simplified
Various environmental factors detect and control, favorable reproducibility, and culture medium utilization rate is high, and also simplify cell harvest process, labor
Fatigue resistance is small, and culture systems occupied area is small.
In order to further illustrate the present invention, with reference to embodiments to a kind of culture of endothelial progenitor cells provided by the invention
Method is described in detail.
Reagent used in following embodiment is commercially available.
Embodiment 1
Mononuclearcell separation:
Bleeding of the umbilicus 20mL is taken, according to bleeding of the umbilicus: HES=4: 1 ratio is mixed in volumetric flask, is turned upside down 20 times and is mixed；
40 minutes are stood at room temperature, collects plasma layer of the upper layer rich in leucocyte in centrifuge tube, 450g centrifugation
10min；
Liquid is discarded supernatant after centrifugation, cell precipitation is resuspended with PBS, takes a centrifuge tube to be added a certain amount of
Ficoll, according to cell suspension: cell suspension is slowly added to by Ficoll=3: 1 ratio with pasteur pipet along centrifugation tube wall
The upper layer of Ficoll；
700g is centrifuged 30min on horizontal centrifuge；
With the white cellular layer of pasteur pipet careful collection Ficoll and plasma layer intersection, as mononuclearcell, add
Enter the PBS diluting cells suspension of 4 times of volumes, 400g is centrifuged 5min；
Liquid is discarded supernatant, 20mLPBS is added and washs twice, with EGM-2MV culture solution diluting cells, is counted spare.
The preliminary culture of endothelial progenitor cells:
By the mononuclearcell isolated by 5 × 104/cm2Density be inoculated in the culture dish of 10cm respectively, add
EGM-2MV culture medium is to 10mL；
It is put into incubator, 90% or more humidity, 37 DEG C, 5%CO2Middle culture；
Culture added 10mL EGM-2MV culture medium after 3 days；
Culture medium supernatant and not adherent cell are discarded when culture was to the 6th day, adds 10mL EGM-2MV culture again
Base；
It changes the liquid once within every 3 days later, until cell fusion degree reaches 80%；
The culture medium in culture dish is discarded, equivalent PBS cleaning is added；
PBS is discarded, the pancreatin of every ware 2mL0.25% is added, digests 1min；
1640 culture mediums of 5 times of pancreatin volumes containing 10%FBS are added into culture dish and terminate digestion；
Cell suspension is transferred in 15ml centrifuge tube, 400g is centrifuged 5min on horizontal centrifuge；
It discards supernatant, EGM-2MV culture medium is added, cell adjustment density is resuspended to 5 × 104Cells/mL is spare.
The culture of endothelial progenitor cells:
With the EGM-2MV culture medium containing 10ng/mL bFGF and 10ng/mL VEGF by 8 × 107A cell adjustment is thin
Born of the same parents' density is to 1 × 105The mixing of 8g microcarrier is added in cells/ml, and the pot type bioreactor that volume is 2L is added in mixed liquor
In in 37 DEG C, 5%CO2Under the conditions of cultivate；15 revs/min are cultivated 2 hours；
Then 30 revs/min of cultures added the EGM-2MV culture medium of 200mL to the 2nd day, then cultivated extremely for 50 revs/min
4th day；
The culture medium in reactor is poured out, is centrifuged and removes supernatant, with 0.25% pancreas enzyme -EDTA digestive juice after PBS cleaning 2 times
Digestion 15 minutes, 1640 culture mediums containing 10%FBS terminate digestion, and centrifugation removal supernatant is resuspended with PBS and is precipitated, 70 μm of sieves
Net filtration collects endothelial progenitor cells, is resuspended in progress cell count and flow cytometer detection, cell counts in physiological saline and is shown in Table
1.The method of flow cytometer detection are as follows: antibody is added, analyzes CD133, CD309, CD34, CD31 and CD105 with flow cytomery
Expression analysis spectrogram, it is as shown in Figure 1 to obtain result.
Embodiment 2
Mononuclearcell separation:
Bleeding of the umbilicus 20mL is taken, according to bleeding of the umbilicus: HES=4: 1 ratio is mixed in volumetric flask, is turned upside down 20 times and is mixed；
40 minutes are stood at room temperature, collects plasma layer of the upper layer rich in leucocyte in centrifuge tube, 450g centrifugation
10min；
Liquid is discarded supernatant after centrifugation, cell precipitation is resuspended with PBS, takes a centrifuge tube to be added a certain amount of
Ficoll, according to cell suspension: cell suspension is slowly added to by Ficoll=3: 1 ratio with pasteur pipet along centrifugation tube wall
The upper layer of Ficoll；
700g is centrifuged 30min on horizontal centrifuge；
With the white cellular layer of pasteur pipet careful collection Ficoll and plasma layer intersection, as mononuclearcell, add
Enter the PBS diluting cells suspension of 4 times of volumes, 400g is centrifuged 5min；
Liquid is discarded supernatant, 20mLPBS is added and washs twice, with EGM-2MV culture solution diluting cells, is counted spare.
The preliminary culture of endothelial progenitor cells:
By the mononuclearcell isolated by 5 × 104/cm2Density be inoculated in the culture dish of 10cm respectively, add
EGM-2MV culture medium is to 10mL；
It is put into incubator, 90% or more humidity, 37 DEG C, 5%CO2Middle culture；
Culture added 10mL EGM-2MV culture medium after 3 days；
Culture medium supernatant and not adherent cell are discarded when culture was to the 6th day, adds 10mLEGM-2MV culture again
Base；
It changes the liquid once within every 3 days later, until cell fusion degree reaches 80%；
The culture medium in culture dish is discarded, equivalent PBS cleaning is added；
PBS is discarded, the pancreatin of every ware 2mL0.25% is added, digests 1min；
1640 culture mediums of 5 times of pancreatin volumes containing 10%FBS are added into culture dish and terminate digestion；
Cell suspension is transferred in 15ml centrifuge tube, 400g is centrifuged 5min on horizontal centrifuge；
It discards supernatant, EGM-2MV culture medium is added, cell adjustment density is resuspended to 5 × 104Cells/mL is spare.
The culture of endothelial progenitor cells:
With the EGM-2MV culture medium containing 10ng/mL bFGF and 10ng/mL VEGF by 8 × 107A cell adjustment is thin
Born of the same parents' density is to 1 × 105The mixing of 10g microcarrier is added in cells/ml, and the pot type bioreactor that volume is 2L is added in mixed liquor
In in 37 DEG C, 5%CO2Under the conditions of cultivate；15 revs/min are cultivated 2 hours；
Then 30 revs/min of cultures added the EGM-2MV culture medium of 200mL to the 2nd day, then cultivated extremely for 50 revs/min
4th day；
The culture medium in reactor is poured out, is centrifuged and removes supernatant, with 0.25% pancreas enzyme -EDTA digestive juice after PBS cleaning 2 times
Digestion 15 minutes, 1640 culture mediums containing 10%FBS terminate digestion, and centrifugation removal supernatant is resuspended with PBS and is precipitated, 70 μm of sieves
Net filtration collects endothelial progenitor cells, is resuspended in progress cell count detection, cell counts in physiological saline and is shown in Table 1.
Embodiment 3
Mononuclearcell separation:
Bleeding of the umbilicus 20mL is taken, according to bleeding of the umbilicus: HES=4: 1 ratio is mixed in volumetric flask, is turned upside down 20 times and is mixed；
40 minutes are stood at room temperature, collects plasma layer of the upper layer rich in leucocyte in centrifuge tube, 450g centrifugation
10min；
Liquid is discarded supernatant after centrifugation, cell precipitation is resuspended with PBS, takes a centrifuge tube to be added a certain amount of
Ficoll, according to cell suspension: cell suspension is slowly added to by Ficoll=3: 1 ratio with pasteur pipet along centrifugation tube wall
The upper layer of Ficoll；
700g is centrifuged 30min on horizontal centrifuge；
With the white cellular layer of pasteur pipet careful collection Ficoll and plasma layer intersection, as mononuclearcell, add
Enter the PBS diluting cells suspension of 4 times of volumes, 400g is centrifuged 5min；
Liquid is discarded supernatant, 20mLPBS is added and washs twice, with EGM-2MV culture solution diluting cells, is counted spare.
The preliminary culture of endothelial progenitor cells:
By the mononuclearcell isolated by 5 × 104/cm2Density be inoculated in the culture dish of 10cm respectively, add
EGM-2MV culture medium is to 10mL；
It is put into incubator, 90% or more humidity, 37 DEG C, 5%CO2Middle culture；
Culture added 10mL EGM-2MV culture medium after 3 days；
Culture medium supernatant and not adherent cell are discarded when culture was to the 6th day, adds 10mLEGM-2MV culture again
Base；
It changes the liquid once within every 3 days later, until cell fusion degree reaches 80%；
The culture medium in culture dish is discarded, equivalent PBS cleaning is added；
PBS is discarded, the pancreatin of every ware 2mL0.25% is added, digests 1min；
1640 culture mediums of 5 times of pancreatin volumes containing 10%FBS are added into culture dish and terminate digestion；
Cell suspension is transferred in 15ml centrifuge tube, 400g is centrifuged 5min on horizontal centrifuge；
It discards supernatant, EGM-2MV culture medium is added, cell adjustment density is resuspended to 5 × 104Cells/mL is spare.
The culture of endothelial progenitor cells:
With the EGM-2MV culture medium containing 10ng/mL bFGF and 10ng/mL VEGF by 8 × 107A cell adjustment is thin
Born of the same parents' density is to 1 × 105The mixing of 9g microcarrier is added in cells/ml, and the pot type bioreactor that volume is 2L is added in mixed liquor
In in 37 DEG C, 5%CO2Under the conditions of cultivate；15 revs/min are cultivated 2 hours；
Then 30 revs/min of cultures added the EGM-2MV culture medium of 200mL to the 2nd day, then cultivated extremely for 50 revs/min
4th day；
The culture medium in reactor is poured out, is centrifuged and removes supernatant, with 0.25% pancreas enzyme -EDTA digestive juice after PBS cleaning 2 times
Digestion 15 minutes, 1640 culture mediums containing 10%FBS terminate digestion, and centrifugation removal supernatant is resuspended with PBS and is precipitated, 70 μm of sieves
Net filtration collects endothelial progenitor cells, is resuspended in progress cell count detection, cell counts in physiological saline and is shown in Table 1.
1 embodiment cell counts of table
| Embodiment 1 | Embodiment 2 | Embodiment 3 | |
| Count results | 2.5×10^9 | 3.8×10^9 | 3.1×10^9 |
| Cell viability | 94.36% | 95.89% | 94.72% |
Comparative example 1
By 8 × 107A EPC cell (the tentatively culture of embodiment 1 obtains) is by -5x104The density of/mL is inoculated in respectively
In the culture dish of 15cm, EGM-2MV culture medium is added to 20mL, is put into incubator, 90% or more humidity, 37 DEG C, 5%CO2
Middle culture；
It changes the liquid once after 48 hours of incubation, every ware replaces 20mL culture medium, cultivates to the 4th day；
The culture medium in culture dish is discarded, equivalent PBS cleaning is added；
PBS is discarded, the pancreatin of every ware 2mL 0.25% is added, digests 1min；
1640 culture mediums of 5 times of pancreatin volumes containing 10%FBS are added into culture dish and terminate digestion；
Digestive juice is centrifuged removal supernatant, 10mL physiological saline is added and cleans 2 times, cell is resuspended with physiological saline and is counted
Number, obtains the results are shown in Table 2.
Comparative example 2
By 8 × 107A EPC cell (the tentatively culture of embodiment 1 obtains) is by 8x104The density of/mL is inoculated in 15cm respectively
Culture dish in, add EGM-2MV culture medium to 20mL, be put into incubator, 90% or more humidity, 37 DEG C, 5%CO2Middle training
It supports；
It changes the liquid once after 48 hours of incubation, every ware replaces 20mL culture medium, cultivates to the 4th day；
The culture medium in culture dish is discarded, equivalent PBS cleaning is added；
PBS is discarded, the pancreatin of every ware 2mL 0.25% is added, digests 1min；
1640 culture mediums of 5 times of pancreatin volumes containing 10%FBS are added into culture dish and terminate digestion；
Digestive juice is centrifuged removal supernatant, 10mL physiological saline is added and cleans 2 times, cell is resuspended with physiological saline and is counted
Number, obtains the results are shown in Table 2.
Comparative example 3
By 8 × 107A EPC cell (the tentatively culture of embodiment 1 obtains) is by 2x104The density of/mL is inoculated in 15cm respectively
Culture dish in, add EGM-2MV culture medium to 20mL, be put into incubator, 90% or more humidity, 37 DEG C, 5%CO2Middle training
It supports；
It changes the liquid once after 48 hours of incubation, every ware replaces 20mL culture medium, cultivates to the 4th day；
The culture medium in culture dish is discarded, equivalent PBS cleaning is added；
PBS is discarded, the pancreatin of every ware 2mL0.25% is added, digests 1min；
1640 culture mediums of 5 times of pancreatin volumes containing 10%FBS are added into culture dish and terminate digestion；
Digestive juice is centrifuged removal supernatant, 10mL physiological saline is added and cleans 2 times, cell is resuspended with physiological saline and is counted
Number, obtains the results are shown in Table 2.
2 cell counts of table
| Embodiment 1 | Comparative example 1 | Comparative example 2 | Comparative example 3 | |
| Cell count | 2.5×10^9 | 7.6×10^8 | 8.4×10^8 | 4.9×10^8 |
| Cell viability | 94.36% | 95.01% | 92.58% | 90.87% |
Claims (10)
1. a kind of cultural method of endothelial progenitor cells characterized by comprising
S1 mononuclearcell) is subjected to adhere-wall culture, the cell after digestion, after being expanded；
S2 the cell inoculation after the amplification is cultivated in culture medium), obtains endothelial progenitor cells；In the culture medium
Including basic fibroblast growth factor, vascular endothelial growth factor and microcarrier.
2. cultural method according to claim 1, which is characterized in that the step S2) expand in culture medium after cell
Density is 1 × 104~1 × 106cells/ml。
3. cultural method according to claim 1, which is characterized in that the step S2) culture medium neutral and alkali is at fiber finer
The concentration of the intracellular growth factor and vascular endothelial growth factor is each independently 10~50ng/ml.
4. cultural method according to claim 3, which is characterized in that the step S2) culture medium neutral and alkali is at fiber finer
The concentration of the intracellular growth factor and vascular endothelial growth factor is each independently 10~30ng/ml.
5. cultural method according to claim 1, which is characterized in that the step S2) concentration of microcarrier in culture medium
For 0.001~0.02g/ml.
6. cultural method according to claim 1, which is characterized in that the step S2) in cultivate temperature be 36 DEG C~
37 DEG C, CO2Volumetric concentration be 3%~10%.
7. cultural method according to claim 1, which is characterized in that the step S2) in culture specifically:
10~20 revs/min, 1~3h is cultivated, then 25~35 revs/min of cultures were to the 2nd day, supplemented medium, then with 40~
60 revs/min are cultivated.
8. cultural method according to claim 7, which is characterized in that the step S2) in culture specifically:
13~18 revs/min, 1.5~2.5h is cultivated, then 28~32 revs/min of cultures were to the 2nd day, supplemented medium, then with
45~55 revs/min are cultivated.
9. cultural method according to claim 7, which is characterized in that the amount of the supplemented medium is original culture medium body
Long-pending 1/10~1/5.
10. cultural method according to claim 1, which is characterized in that the step S2) in culture after, be centrifuged, use
The digestion of trypsase-EDTA digestive juice, after terminating digestion, centrifugation removal supernatant, after precipitating is resuspended with phosphate buffer, mistake
Filter, obtains endothelial progenitor cells.
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| CN112980776A (en) * | 2021-03-23 | 2021-06-18 | 上海简巨医学生物工程有限公司 | Application of niobium carbide nano material and cell culture medium for promoting cell proliferation |
| CN116515736A (en) * | 2023-06-21 | 2023-08-01 | 广州市天河诺亚生物工程有限公司 | Umbilical cord blood-derived endothelial progenitor cells and culture method thereof |
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