CN108384746A - A kind of method that inductive pluripotent stem cells efficiently break up to mature endothelial cell - Google Patents
A kind of method that inductive pluripotent stem cells efficiently break up to mature endothelial cell Download PDFInfo
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- C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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Abstract
The invention discloses a kind of methods that inductive pluripotent stem cells efficiently break up to mature endothelial cell, belong to cell engineering field.It includes the following steps：Induced multi-potent stem cell pretreatment culture；Induced synthesis lateral plate mesoderm cell；The endothelial progenitor cells of induced synthesis endothelial cell tendency；Induced synthesis mature endothelial cell.The present invention passes through increase and decrease collocation various kinds of cell inducible factor, and rationally upgraded on abductive approach, it can efficiently induce induced multi-potent stem cell to endothelial cell directed differentiation, using a small amount of induced multi-potent stem cell and under conditions of do not need purification process, you can obtain that a large amount of purity are high, mature endothelial cell of perfect in shape and function.
Description
Technical field
The present invention relates to a kind of methods that inductive pluripotent stem cells efficiently break up to mature endothelial cell, belong to cell work
Journey technical field.
Background technology
Vascular endothelial cell (Vascular Endothelial Cells, vEC) is that one layer of composition blood vessel is special
Property flat epithelial cell, be maintain systemic blood circulation system stable state important leverage.In diabetes, hypertension and other
In chronic pathology, it is often accompanied by inner skin cell function imbalance or damage, easily induces atherosclerosis, even myocardial infarction
Etc. angiocardiopathies, seriously threaten patient vitals health.Therefore, the blood vessel endothelium repaired or replace damage is adjusted, for preventing
There is important clinical value with treatment angiocardiopathy.Research points out that the ripe vascular endothelial cell for having consummating function can
To re-form vascular tissue in body blood vessel infarcted region, ischemic conditions are effectively solved.In addition, by ripe blood vessel endothelium
The blood vessel structure that cell is formed in vitro also has the conventional vein separation means of very high potential substitution, is carried out for cardiovascular patient
Bypass surgery provides the stronger alternative materials of applicability.But how to obtain a large amount of ripe blood vessel endotheliums for having into pipe ability
Cell, at present still in the research stage of fumbling, this also just directly limits its smooth development in clinical application.
As the most important thing in regenerative medicine evolution, inductive pluripotent stem cells (induced pluripotent
Stem cells, iPSC) appearance, provide a tool for the clinical application of endothelial cell (Endothelial Cells, EC)
There is the scheme of high feasibility.Induced multi-potent stem cell has successfully got around asking for immunogenicity and two most criticals of moral check
Topic, and derives from a wealth of sources, and can complete individualized treatment by establishing patient's specific induced multi-potent stem cell.Using trouble
Induced amplification obtains a large amount of ripe vascular endothelial cells to the induced multi-potent stem cell in person source in vitro, contribute to smoothly across
The high threshold of EC clinical applications, while the gene therapy being alternatively in the future for endothelial cell is ready.
Related Research Domain was quickly grown in recent years, and it is thin that current a variety of induced multi-potent stem cells are induced to differentiate into ripe endothelium
The method of born of the same parents is proposed in succession, includes the 3D embryoid body revulsions of early stage[1]With the recent direct revulsions of 2D[2-4], it is all made of one
Needed for serial endothelial differentiation inducible factor carry out Multiple Combination, can induce to a certain extent induced multi-potent stem cell at
Ripe endothelial cell differentiation.But these abductive approach also have certain limitation, for example induction duration length (generally needs 9-20
It), transformation efficiency low (20%-60%), purification process cumbersome (need to be screened through MACS or streaming), endothelial function it is not perfect
(being short of in vitro or in vivo at pipe ability) etc..
In consideration of it, to really obtain the endothelial cell of clinical application rank, it is also necessary to it is more further to grope optimization induction
The method of the inside chrotoplast induction differentiation of energy stem cell.
Invention content
It is thin to ripe endothelium the purpose of the present invention is overcoming the deficiencies of the prior art and provide a kind of inductive pluripotent stem cells
The method that born of the same parents efficiently break up.The present invention is rationally risen by increase and decrease collocation various kinds of cell inducible factor on abductive approach
Grade can efficiently induce induced multi-potent stem cell to endothelial cell directed differentiation, using a small amount of induced multi-potent stem cell and not
Under conditions of needing purification process, you can obtain that a large amount of purity are high, mature endothelial cell of perfect in shape and function.Meanwhile entire induction
Process take it is short, it is easy to operate, be not related to animal derived cell and ingredient, substantially increase the interior of induced multi-potent stem cell source
The high efficiency of chrotoplast, functionality, safety, to mass produce clinical application using the induced multi-potent stem cell of patient itself
The mature endothelial cell of rank provides a kind of new approaches and methods.
The technical solution that the present invention solves above-mentioned technical problem is as follows：A kind of inductive pluripotent stem cells are thin to ripe endothelium
The method that born of the same parents efficiently break up, includes the following steps：
Step 1：Inductive pluripotent stem cells are pre-processed, cell re-suspension liquid is obtained；
Step 2：The cell re-suspension liquid that step 1 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 20,000-5 ten thousand
The ratio of a cell is moved into the tissue culture plate of the mixture containing TeSR-E8 culture mediums and ROCK inhibitor Y27632, is set
It is cultivated 16-24 hours in cell incubator；
Step 3：Old culture medium in tissue culture plate is removed in step 2, according to every 10cm2Tissue culture plate floor space add
Liquid measure is the ratio of 4mL, and the first N2B27 insulin deficiency culture mediums are added, is placed in cell incubator and cultivates 16-24 hours；
Step 4：The old culture medium in tissue culture plate in step 3 is removed, PBS is used in combination to rinse tissue culture plate, by every
10cm2Tissue culture plate floor space liquid volume added be 6mL ratio, the 2nd N2B27 insulin deficiency culture mediums are added, are placed in thin
It is cultivated 48 hours in born of the same parents' incubator, lateral plate mesoderm cell is obtained in tissue culture plate；
Step 5：The old culture medium in the tissue culture plate containing lateral plate mesoderm cell in step 4 is removed, PBS is used in combination to rush
Tissue culture plate is washed, according to every 10cm2Tissue culture plate floor space liquid volume added be 4mL ratio, be added the first StemPro-
34 culture mediums are subsequently placed in cell incubator and cultivate 16-24 hours, repeat step 5 and operate once, are lured in tissue culture plate
It leads to obtain the endothelial progenitor cells being inclined to containing endothelial cell；
Step 6：Remove the old culture in the tissue culture plate for the endothelial progenitor cells being inclined to containing endothelial cell in step 5
Base is used in combination PBS to rinse tissue culture plate, according to every 10cm2Tissue culture plate floor space liquid volume added be 0.5mL ratio, add
Enter cell disintegration enzyme mixation, is digested, the endothelial progenitor cells containing endothelial cell tendency after being digested；
Step 7：The endothelial progenitor cells being inclined to containing endothelial cell after the enzymolysis that step 6 is obtained, according to every 10cm2It is thin
Born of the same parents' culture plate floor space liquid volume added is the ratio of 1mL, and the 2nd StemPro-34 culture mediums are added, and obtains being inclined to containing endothelial cell
Endothelial progenitor cells re-suspension liquid；
Step 8：The endothelial progenitor cells re-suspension liquid being inclined to containing endothelial cell that step 7 is obtained,
According to every 1cm230,000-6 ten thousand cells of tissue culture plate floor space cover plant ratio, immigration contain second
In the cell differentiation culture plate of StemPro-34 culture mediums, it is placed in cell incubator and cultivates 16-24 hours；
Step 9：The old culture medium in tissue culture plate in step 8 is removed, is used in combination PBS to rinse tissue culture plate, according to every
10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added the 2nd StemPro-34 culture mediums, be placed in cell training
It supports in case, culture breaks up endothelial cell in 48 hours to get to Maturation induction.
Compared with prior art, method of the invention, it is no longer necessary to which purification step, gained mature endothelial cell purity are up to
96% or more, operation difficulty is reduced, while reducing interference of the purification step to cell state.By taking 6 porocyte culture plates as an example,
Cover plant ten thousand induced multi-potent stem cells of 30-35 are originated per hole, and ten thousand mature endothelial cells of 400-500 can be harvested within the 9th day in induction,
It is 2-3 times of prior art endothelial cell yield.Method using the present invention induces 9 days, and endothelial cell/starting of acquisition lures
Lead the 2-3 that multipotential stem cell conversion ratio converts ratio up to 15/1 and prior art endothelial cell/induced multi-potent stem cell
Times.Gained mature endothelial cell of the invention has significant in vitro at pipe ability.
Wherein, in step 3, remove in step 2 that old culture medium, specific operating method are in tissue culture plate：Tilt training
Plate is supported, old culture medium is sucked out with pipettor.
In step 4, in step 5, in step 6, in step 9, the method for removing the old culture medium in tissue culture plate, ibid
State the method that the old culture medium in tissue culture plate is removed in step 3.
In step 5, endothelial progenitor cells, i.e. Endothelial progenitor cell, abbreviation EPC.
Based on the above technical solution, the present invention can also be improved as follows.
Further, the method that is described to pre-process inductive pluripotent stem cells in step 1, obtaining cell re-suspension liquid
For：
In tissue culture plate, after inductive pluripotent stem cells recovery, passage, rinsed 2 times with PBS, according still further to every
10cm2Tissue culture plate floor space liquid volume added be 0.5mL ratio, be added 37 DEG C of preheated cell dissociation buffers digested
Digestion, obtains enzymolysis, digestion liquid；When the inductive pluripotent stem cells of 70-80% start shedding off, above-mentioned enzymolysis, digestion liquid 5 is added
The DMEM/F12 culture mediums of times volume are neutralized, and then inductive pluripotent stem cells are transferred in centrifuge tube, are centrifuged, are removed
TeSR-E8 culture mediums are added in supernatant, and cell precipitation is resuspended, obtains cell re-suspension liquid.
Wherein, above-mentioned DMEM/F12 culture mediums are purchased from Life Technologies companies, model 11039021.
Further, the inductive pluripotent stem cells recovery, the method passed on are：
Recovery the previous day gets out the coated tissue culture plate of basement membrane matrix, is placed in cell incubator, is incubated overnight,
The tissue culture plate that basement membrane matrix after being incubated overnight was coated with；
The induced multi-potent stem cell frozen is taken out from liquid nitrogen, is inoculated in the coating of the basement membrane matrix after above-mentioned be incubated overnight
It in the tissue culture plate crossed, is placed in cell incubator, is incubated 16-24 hours；
The preheated TeSR-E8 culture mediums of fresh 37 DEG C are then replaced, repeat to change liquid daily until cell aggregation degree increases
To 75-85% to get to the inductive pluripotent stem cells after recovery；
By the induced multi-potent stem cell passage after recovery, the preheated TeSR-E8 cultures of fresh 37 DEG C are replaced after one day
Base repeats to change liquid daily until cell aggregation degree increases to 75-85% to get to the inductive pluripotent stem cells after passage.
Further, the condition of the centrifugation is room temperature, and 200g centrifuges 5min.
Further, in step 2, in the TeSR-E8 culture mediums and ROCK inhibitor Y27632 mixtures, ROCK inhibitor
A concentration of 10 μM/L of Y27632.
Further, in step 3, the first N2B27 insulin deficiency culture mediums are in N2B27 insulin deficiency culture mediums
Middle addition BMP4, CHIR-99021 and Activin A, a concentration of 20-30ng/mL of wherein BMP4, the concentration of CHIR-99021
For 6-10 μM/L, a concentration of 30-70ng/mL of Activin A.
Above-mentioned BMP4, CHIR-99021 and Activin A is induction small molecule.Wherein, BMP4, full name Bone
Morphogenetic Protein4, the entitled bone morphogenetic protein 4 of Chinese, wither with the proliferation of cell and differentiation, cell
Die, form occur when cell citation etc. cell developments have direct or indirect relationship.CHIR-99021 is GSK inhibitor, high
Specificity inhibits GSK3 β and GSK3 α.Activin A are activin A, be participate in the multiple-effect cells of a variety of important biomolecule processes because
Son, including maintain multipotential stem cell.
It is using above-mentioned further advantageous effect：BMP4 and CHIR-99021 can start induced multi-potent stem cell to side plate
Mesoblastemic induction differentiation process, and Activin A can further promote induced multi-potent stem cell on this basis
Differentiation process improves final endothelial converting efficiency.Activin A processing times can cause induced multi-potent stem cell final too long
It is divided into endoderm cell, and endothelial cell is only derived from mesoblastema, so the processing time of Activin A needs to control
Within 24 hours.
Further, in the first N2B27 insulin deficiency culture mediums BMP4 a concentration of 25ng/mL, CHIR-
99021 a concentration of 8 μM/L, a concentration of 50ng/mL of Activin A.
Further, in step 4, the 2nd N2B27 insulin deficiency culture mediums are in N2B27 insulin deficiency culture mediums
A concentration of 6-10 μM/L of a concentration of 20-30ng/mL of middle addition BMP4 and CHIR-99021, wherein BMP4, CHIR-99021.
It is using above-mentioned further advantageous effect：BMP4 and CHIR-99021 can start induced multi-potent stem cell to side plate
Mesoblastemic induction differentiation process.
Further, in the 2nd N2B27 insulin deficiency culture mediums BMP4 a concentration of 25ng/mL, CHIR-
99021 a concentration of 8 μM/L.
Further, in step 5, the first StemPro-34 culture mediums are added in StemPro-34 culture mediums
VEGFA, Forskolin and SB431542, a concentration of 1-4 μ of a concentration of 100-300ng/mL of wherein VEGFA, Forskolin
5-15 μM of the concentration of M/L, SB431542/L.
Above-mentioned VEGFA, Forskolin and SB431542 are induction small molecule.Wherein, VEGFA, full name Vascular
EndothelialGrowth Factor A, the entitled Vascular endothelial growth factor A of Chinese, is a kind of rush blood vessel of high degree of specificity
Endothelial growth factor has and promotes vasopermeability increase, extra cellular matrix degeneration, migration of vascular endothelial cells, proliferation
And the effects that vascularization.Forskolin, i.e. forskolin are adenyl cyclase activator.SB431542 is a kind of potent
Micromolecular inhibitor, selective depression transforming growth factor β (TGF-β) I receptor Activin receptor-like kinases ALK5 (IC50
=94nM), ALK4 (IC50=140nM) and ALK7.
It is using above-mentioned further advantageous effect：VEGFA, Forskolin and SB431542 are combined can be high
Effect rapidly from the inside skin direction of mesoblastema break up by guiding induced multi-potent stem cell, improves endothelial converting efficiency.
Further, in the first StemPro-34 culture mediums VEGFA a concentration of 200ng/mL, Forskolin's
A concentration of 2 μM/L, 10 μM/L of the concentration of SB431542.
Further, in step 6, the cell disintegration enzyme mixation is 1 by volume by cell dissociation buffer and trypsase:
1 composition.
Further, in step 7, the 2nd StemPro-34 culture mediums are added in StemPro-34 culture mediums
VEGFA, wherein a concentration of 30-70ng/mL of VEGFA.
It is using above-mentioned further advantageous effect：The 2nd StemPro-34 culture mediums containing VEGFA can be used for luring
It leads that endothelial cell is further ripe, while the normal growth of endothelial cell can also be maintained to be proliferated.
Further, in the 2nd StemPro-34 culture mediums VEGFA a concentration of 50ng/mL.
Further, in step 8, the cell differentiation culture plate containing the 2nd StemPro-34 culture mediums is by the following method
It obtains：According to every 10cm2Tissue culture plate floor space liquid volume added be 2ml ratio, 10 μ g/mL are added in tissue culture plate
Fibronectin working solution, be stored at room temperature coating 1 hour, fibronectin working solution is removed, according still further to every 10cm2's
Tissue culture plate floor space liquid volume added is the ratio of 2mL, and the 2nd StemPro-34 culture mediums are added and contain second to get to described
The cell differentiation culture plate of StemPro-34 culture mediums.
Further, the condition of culture of the cell incubator is 37 DEG C, 5%v/v CO2。
Further, further include step 10 after step 9, i.e., to obtained Maturation induction differentiation endothelial cell carry out amplification and
It freezes.
Wherein, breaking up the specific method that endothelial cell is expanded to obtained Maturation induction is：
When cell aggregation degree has been expanded to 80-90%, you can proceed by passage operation.
Tissue culture plate is taken out, sucks old culture medium, PBS is washed 2 times at room temperature, then according to every 10cm2Cell training
Support the ratio that board bottom area liquid volume added is 0.5mL, be added 37 DEG C it is preheated by cell dissociation buffer and trypsase by volume
It is 1:The cell disintegration enzyme mixation of 1 composition, is placed in 37 DEG C, 5%v/v CO22-3min in cell incubator, until major part
Cell starts to fall off with lumps floating.
According to every 10cm2Tissue culture plate floor space liquid volume added be 1mL ratio, be added 37 DEG C preheated first
StemPro-34 culture mediums neutralize the enzymolysis of cell dissociation buffer and trypsase, cast-off cells are gently blown and beaten with liquid-transfering gun
Agglomerate is resuspended, and is subsequently transferred in 15mL centrifuge tubes.
Under room temperature, 200g centrifuges 5min.Supernatant is removed with liquid-transfering gun, is then added 3mL37 DEG C preheated the
Cell precipitation is fully resuspended in two StemPro-34 culture mediums, gently piping and druming.
According to every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, in tissue culture plate be added 37 DEG C
The 2nd preheated StemPro-34 culture mediums.Then according to 1:3 or 1:2 passage ratio divides 3mL cell re-suspension liquids equally
In tissue culture plate, it is put into 37 DEG C, 5%v/v CO2Cell incubator, subsequent stationary culture.
Wherein, breaking up the specific method that endothelial cell is frozen to obtained Maturation induction is：
Microscopic observation cell determines that EC cells are pollution-free, in good condition, and the degree of polymerization when reaching 80-90% can to its into
Row freezes operation.
Tissue culture plate is taken out, sucks old culture medium, PBS is washed 2 times at room temperature, then according to every 10cm2Cell training
Support the ratio that board bottom area liquid volume added is 0.5mL, be added 37 DEG C it is preheated by cell dissociation buffer and trypsase by volume
It is 1:The cell disintegration enzyme mixation of 1 composition, is placed in 37 DEG C, 5%v/v CO22-3min in cell incubator, until major part
Cell starts to fall off with lumps floating.
According to every 10cm2Tissue culture plate floor space liquid volume added be 1mL ratio, be added 37 DEG C preheated second
StemPro-34 culture mediums neutralize the enzymolysis of cell dissociation buffer and trypsase, cast-off cells are gently blown and beaten with liquid-transfering gun
Agglomerate is resuspended, and is subsequently transferred in 15mL centrifuge tubes.
200g centrifuges 5min under room temperature.Period prepares cryopreservation tube, and be marked (culture people's name, freezes the time,
Cultivate algebraically etc.).
Supernatant is sucked with liquid-transfering gun, 2mLCryo-SFM frozen stock solutions are then added, it is heavy that cell is fully resuspended in gently piping and druming
It forms sediment, is then dispensed into cryopreservation tube and (suggests according to 1 when freezing by every pipe 1mL:2 ratios carry out).
Cryopreservation tube is put into the program temperature reduction box of 4 DEG C of precoolings, is placed in -80 DEG C of refrigerators, is transferred to liquid nitrogen container memory within second day
Storage.
The specific preparation method of used medium of the present invention：
1, TeSR-E8 culture mediums：According to specification ratio matching while using shown in table 1, longest preserves 1 month under the conditions of 4 DEG C.
The preparation of 1 TeSR-E8 culture mediums of table
2, N2B27 insulin deficiencies culture medium：According to specification ratio matching while using shown in table 2, longest preserves under the conditions of 4 DEG C
1 month.
The preparation of 2 N2B27 insulin deficiency culture mediums of table
3, StemPro-34 culture mediums：According to specification ratio matching while using shown in table 3, longest preserves 1 under the conditions of 4 DEG C
Month.
The preparation of 3 StemPro-34 culture mediums of table
The beneficial effects of the invention are as follows：
1, entire Induction Process of the invention takes short, easy to operate, is not related to animal derived cell and ingredient, carries significantly
The high high efficiency of the endothelial cell in inductive pluripotent stem cells sources, functionality, safety, to utilize the induction of patient itself
The mature endothelial cell of multipotential stem cell large-scale production clinical application rank provides a kind of new approaches and methods.
2, the present invention is arranged in pairs or groups various kinds of cell inducible factor by increase and decrease, and is rationally upgraded on abductive approach, can be with
Efficiently induction induced multi-potent stem cell is using a small amount of induced multi-potent stem cell and need not purify to endothelial cell directed differentiation
Under conditions of operation, you can obtain that a large amount of purity are high, mature endothelial cell of perfect in shape and function.
3, method of the invention also has following remarkable advantage and good effect：
(1) short-time characteristic：The method of the present invention only needs 8-9 days, takes and has reached the most fast level in presently relevant field, is answered in clinic
Using has very high application timeliness.
(2) convenience：The method of the present invention, which is simplified, optimizes inducible factor combination, and simple to operation, repeatability is stablized.
(3) high-purity：Cell purity is high, is not required to extra purification steps, you can in the maturation for obtaining 96% or more purity
Chrotoplast.
(4) high efficiency：Cell yield is notable, and it is thin to can be obtained a large amount of ripe endotheliums using a small amount of induced multi-potent stem cell
Born of the same parents, endothelial cell/inductive pluripotent that 8-9 days time finally obtained do conversion ratio up to 15/1.
(5) functional：The mature endothelial cell of acquisition has significantly into pipe ability, perfect in shape and function.
(6) safety：The transparent easy monitoring of Induction Process, is not related to animal derived cell and ingredient；Induction is thorough, without dry thin
Born of the same parents retain, and eliminate tumor formation risk.
Description of the drawings
Fig. 1 is the overall flow figure of the present invention.
Fig. 2 be lateral plate mesoderm cell induced synthesis during the 1st day cell morphological characteristic figure (10X microscopes regard
It is wild).
Fig. 3 be lateral plate mesoderm cell induced synthesis during the 2nd day cell morphological characteristic figure (10X microscopes regard
It is wild).
Fig. 4 be lateral plate mesoderm cell induced synthesis during the 4th day cell morphological characteristic figure (10X microscopes regard
It is wild).
Fig. 5 be the present invention method in the endothelial progenitor cells induced synthesis that high endothelial cell is inclined to the 6th day cell shape
State characteristic pattern (10X fields of microscope).
Fig. 6 is that (10X is aobvious for the 6th day cell morphological characteristic figure in endothelial progenitor cells induced synthesis for the method for the prior art
Microscope fields).
Fig. 7 for method of the invention, exempt from by the 6th day cell in the endothelial progenitor cells induced synthesis that high endothelial cell is inclined to
Epidemic disease label figure (10X fields of microscope).
Fig. 8 is that (10X is aobvious for the 6th day cellular immunity label figure in endothelial progenitor cells induced synthesis for the method for the prior art
Microscope fields).
Fig. 9 be mature endothelial cell induced synthesis during the 7th day cell morphological characteristic figure (10X microscopes regard
It is wild).
Figure 10 be mature endothelial cell induced synthesis during the 9th day cell morphological characteristic figure (10X microscopes regard
It is wild).
Figure 11 is that the external of mature endothelial cell identifies (10X fields of microscope) at pipe ability.
Specific implementation mode
Principles and features of the present invention are described below in conjunction with specific embodiment, example is served only for explaining this hair
It is bright, it is not intended to limit the scope of the present invention.
Embodiment 1
The method that the inductive pluripotent stem cells of the present embodiment efficiently break up to mature endothelial cell, detailed process such as Fig. 1
It is shown, include the following steps：
Step 1：Inductive pluripotent stem cells are pre-processed, cell re-suspension liquid is obtained.Its specific method is：
In tissue culture plate, after inductive pluripotent stem cells recovery, passage, rinsed 2 times with PBS, according still further to every
10cm2Tissue culture plate floor space liquid volume added be 0.5mL ratio, be added 37 DEG C of preheated cell dissociation buffers digested
Digestion, obtains enzymolysis, digestion liquid；When the inductive pluripotent stem cells of 70-80% start shedding off, above-mentioned enzymolysis, digestion liquid 5 is added
The DMEM/F12 culture mediums of times volume are neutralized, and then inductive pluripotent stem cells are transferred in centrifuge tube.Wherein, above-mentioned
DMEM/F12 culture mediums are purchased from Life Technologies companies, model 11039021.
Room temperature, 200g centrifuge 5min, remove supernatant, and TeSR-E8 culture mediums are added, and cell precipitation is resuspended, obtains cell weight
Suspension.
Wherein, the inductive pluripotent stem cells recovery, the method passed on are：
Recovery the previous day gets out the coated tissue culture plate of basement membrane matrix, is placed in 37 DEG C, 5%v/v CO2Cell is trained
It supports in case, is incubated overnight, the tissue culture plate that the basement membrane matrix after being incubated overnight was coated with.
Inductive pluripotent stem cells used are using induction agent box inducing somatic, induction agent box used
Epi5TMEpisomal iPSC Reprogramming Kit, purchased from the silent winged generation that science and technology of match, MAN0008352.
The induced multi-potent stem cell frozen is taken out from liquid nitrogen, is inoculated in the coating of the basement membrane matrix after above-mentioned be incubated overnight
In the tissue culture plate crossed, it is placed in 37 DEG C, 5%v/v CO2In cell incubator, it is incubated 16-24 hours.
The preheated TeSR-E8 culture mediums of fresh 37 DEG C are then replaced, repeat to change liquid daily until cell aggregation degree increases
To 80% to get to the inductive pluripotent stem cells after recovery.
By the induced multi-potent stem cell passage after recovery, the preheated TeSR-E8 cultures of fresh 37 DEG C are replaced after one day
Base repeats to change liquid daily until cell aggregation degree increases to 80% to get to the inductive pluripotent stem cells after passage.
Step 2：The cell re-suspension liquid that step 1 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 3.5 ten thousand
The ratio of cell moves into the tissue culture plate containing TeSR-E8 culture mediums and ROCK inhibitor Y27632 mixtures, is placed in 37
DEG C, 5%v/v CO2It is cultivated 20 hours in cell incubator.Wherein, the TeSR-E8 culture mediums and ROCK inhibitor Y27632
In mixture, a concentration of 10 μM/L of ROCK inhibitor Y27632.Induce the 1st day cellular morphology as shown in Figure 2.
Step 3：Old culture medium in tissue culture plate is removed in step 2, according to every 10cm2Tissue culture plate floor space add
Liquid measure is the ratio of 4mL, and the first N2B27 insulin deficiency culture mediums are added, are placed in 37 DEG C, 5%v/v CO2In cell incubator
Culture 20 hours.The first N2B27 insulin deficiency culture mediums be in N2B27 insulin deficiency culture mediums add BMP4,
A concentration of 8 μM/L, Activin of a concentration of 25ng/mL of CHIR-99021 and Activin A, wherein BMP4, CHIR-99021
A concentration of 50ng/mL of A.Induce the 2nd day cellular morphology as shown in Figure 3.
Step 4：The old culture medium in tissue culture plate in step 3 is removed, PBS is used in combination to rinse tissue culture plate, by every
10cm2Tissue culture plate floor space liquid volume added be 6mL ratio, be added the 2nd N2B27 insulin deficiency culture mediums, be placed in 37
DEG C, 5%v/v CO2It is cultivated 48 hours in cell incubator, lateral plate mesoderm cell is obtained in tissue culture plate.Wherein, institute
It is the addition BMP4 and CHIR-99021 in N2B27 insulin deficiency culture mediums to state the 2nd N2B27 insulin deficiency culture mediums,
A concentration of 8 μM/L of a concentration of 25ng/mL of middle BMP4, CHIR-99021.Induce the 4th day cellular morphology as shown in Figure 4.
Step 5：The old culture medium in the tissue culture plate containing lateral plate mesoderm cell in step 4 is removed, PBS is used in combination to rush
Tissue culture plate is washed, according to every 10cm2Tissue culture plate floor space liquid volume added be 4mL ratio, be added the first StemPro-
34 culture mediums are subsequently placed in 37 DEG C, 5%v/v CO2It is cultivated 20 hours in cell incubator, repeats step 5 once, trained in cell
It supports induction in plate and obtains the endothelial progenitor cells being inclined to containing endothelial cell.Wherein, the first StemPro-34 culture mediums be
Addition VEGFA, Forskolin and SB431542, a concentration of 200ng/mL of wherein VEGFA in StemPro-34 culture mediums,
A concentration of 2 μM/L of Forskolin, 10 μM/L of the concentration of SB431542.
Step 6：Remove the old culture in the tissue culture plate for the endothelial progenitor cells being inclined to containing endothelial cell in step 5
Base is used in combination PBS to rinse tissue culture plate, according to every 10cm2Tissue culture plate floor space liquid volume added be 0.5mL ratio, add
It is 1 by volume to enter by cell dissociation buffer and trypsase:The cell disintegration enzyme mixation of 1 composition, is digested, is digested
Endothelial progenitor cells afterwards containing endothelial cell tendency.It induces the 6th day, i.e. induced synthesis has higher endothelial cell compared to original method
The endothelial progenitor cells of tendency, state under cell mirror compared to original method as shown in figure 5, no longer have significant " crater " structure
(as shown in Figure 6)；It can see by the way that cellular immunity label is (as shown in Figure 7,8), method of the invention obtained mixed at the 6th day
Closing cell has the later stage endothelial progenitor cells (shown in black arrow) of smaller scale, and with more nearly mature endothelial cells
(shown in white arrow).
Step 7：The endothelial progenitor cells being inclined to containing endothelial cell after the enzymolysis that step 6 is obtained, according to every 10cm2It is thin
Born of the same parents' culture plate floor space liquid volume added is the ratio of 1mL, and the 2nd StemPro-34 culture mediums are added, and obtains being inclined to containing endothelial cell
Endothelial progenitor cells re-suspension liquid.Wherein, the 2nd StemPro-34 culture mediums are added in StemPro-34 culture mediums
VEGFA, wherein a concentration of 50ng/mL of VEGFA.
Step 8：The endothelial progenitor cells re-suspension liquid being inclined to containing endothelial cell that step 7 is obtained, according to every 1cm2It is thin
The ratio of born of the same parents' culture plate floor space 4.5 ten thousand cells of cover plant moves into the cell differentiation training containing the 2nd StemPro-34 culture mediums
It supports in plate, is placed in 37 DEG C, 5%v/v CO2It is cultivated 20 hours in cell incubator.Wherein, described to contain the 2nd StemPro-34
The cell differentiation culture plate of culture medium obtains by the following method：According to every 10cm2Tissue culture plate floor space liquid volume added be 2ml
Ratio, the fibronectin working solution of 10 μ g/mL is added in tissue culture plate, be stored at room temperature coating 1 hour, remove fibre
Dimension connection albumen working solution, according still further to every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added second
StemPro-34 culture mediums are to get to the cell differentiation culture plate containing the 2nd StemPro-34 culture mediums.After 20 hours,
State is as shown in Figure 9 under cell mirror.
Step 9：The old culture medium in tissue culture plate in step 8 is removed, is used in combination PBS to rinse tissue culture plate, according to every
10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added the 2nd StemPro-34 culture mediums, be placed in 37 DEG C,
5%v/v CO2In cell incubator, culture breaks up endothelial cell in 48 hours to get to Maturation induction.State is as schemed under cell mirror
Shown in 10.Obtained ripe endothelial cell has significantly in vitro at pipe ability, as shown in figure 11.
It can be seen that the present invention is arranged in pairs or groups various kinds of cell inducible factor by increase and decrease, and rationally risen on abductive approach
Grade can efficiently induce induced multi-potent stem cell to endothelial cell directed differentiation, using a small amount of induced multi-potent stem cell and not
Under conditions of needing purification process, you can obtain that a large amount of purity are high, mature endothelial cell of perfect in shape and function.Meanwhile entire induction
Process take it is short, it is easy to operate, be not related to animal derived cell and ingredient, substantially increase the interior of induced multi-potent stem cell source
The high efficiency of chrotoplast, functionality, safety, to mass produce clinical application using the induced multi-potent stem cell of patient itself
The mature endothelial cell of rank provides a kind of new approaches and methods.
Embodiment 2
The method that the inductive pluripotent stem cells of the present embodiment efficiently break up to mature endothelial cell, detailed process such as Fig. 1
It is shown, include the following steps：
Step 1：Inductive pluripotent stem cells are pre-processed, cell re-suspension liquid is obtained.Its specific method is：
In tissue culture plate, after inductive pluripotent stem cells recovery, passage, rinsed 2 times with PBS, according still further to every
10cm2Tissue culture plate floor space liquid volume added be 0.5mL ratio, be added 37 DEG C of preheated cell dissociation buffers digested
Digestion, obtains enzymolysis, digestion liquid；When 70% inductive pluripotent stem cells start shedding off, 5 times of above-mentioned enzymolysis, digestion liquid is added
The DMEM/F12 culture mediums of volume are neutralized, and then inductive pluripotent stem cells are transferred in centrifuge tube.Wherein, above-mentioned
DMEM/F12 culture mediums are purchased from Life Technologies companies, model 11039021.
Room temperature, 200g centrifuge 5min, remove supernatant, and TeSR-E8 culture mediums are added, and cell precipitation is resuspended, obtains cell weight
Suspension.
Wherein, the inductive pluripotent stem cells recovery, the method passed on are：
Recovery the previous day gets out the coated tissue culture plate of basement membrane matrix, is placed in 37 DEG C, 5%v/v CO2Cell is trained
It supports in case, is incubated overnight, the tissue culture plate that the basement membrane matrix after being incubated overnight was coated with.
Inductive pluripotent stem cells used are using induction agent box inducing somatic, induction agent box used
Epi5TMEpisomal iPSC Reprogramming Kit, purchased from the silent winged generation that science and technology of match, MAN0008352.
The induced multi-potent stem cell frozen is taken out from liquid nitrogen, is inoculated in the coating of the basement membrane matrix after above-mentioned be incubated overnight
In the tissue culture plate crossed, it is placed in 37 DEG C, 5%v/v CO2In cell incubator, it is incubated 16 hours.
The preheated TeSR-E8 culture mediums of fresh 37 DEG C are then replaced, repeat to change liquid daily until cell aggregation degree increases
To 75% to get to the inductive pluripotent stem cells after recovery.
By the induced multi-potent stem cell passage after recovery, the preheated TeSR-E8 cultures of fresh 37 DEG C are replaced after one day
Base repeats to change liquid daily until cell aggregation degree increases to 75% to get to the inductive pluripotent stem cells after passage.
Step 2：The cell re-suspension liquid that step 1 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 20,000 it is thin
The ratio of born of the same parents moves into the tissue culture plate containing TeSR-E8 culture mediums and ROCK inhibitor Y27632 mixtures, is placed in 37
DEG C, 5%v/v CO2It is cultivated 24 hours in cell incubator.Wherein, the TeSR-E8 culture mediums and ROCK inhibitor Y27632
In mixture, a concentration of 10 μM/L of ROCK inhibitor Y27632.
Step 3：Old culture medium in tissue culture plate is removed in step 2, according to every 10cm2Tissue culture plate floor space add
Liquid measure is the ratio of 4mL, and the first N2B27 insulin deficiency culture mediums are added, are placed in 37 DEG C, 5%v/v CO2In cell incubator
Culture 24 hours.The first N2B27 insulin deficiency culture mediums be in N2B27 insulin deficiency culture mediums add BMP4,
A concentration of 6 μM/L, Activin of a concentration of 20ng/mL of CHIR-99021 and Activin A, wherein BMP4, CHIR-99021
A concentration of 30ng/mL of A.
Step 4：The old culture medium in tissue culture plate in step 3 is removed, PBS is used in combination to rinse tissue culture plate, by every
10cm2Tissue culture plate floor space liquid volume added be 6mL ratio, be added the 2nd N2B27 insulin deficiency culture mediums, be placed in 37
DEG C, 5%v/v CO2It is cultivated 48 hours in cell incubator, lateral plate mesoderm cell is obtained in tissue culture plate.Wherein, institute
It is the addition BMP4 and CHIR-99021 in N2B27 insulin deficiency culture mediums to state the 2nd N2B27 insulin deficiency culture mediums,
A concentration of 6 μM/L of a concentration of 20ng/mL of middle BMP4, CHIR-99021.
Step 5：The old culture medium in the tissue culture plate containing lateral plate mesoderm cell in step 4 is removed, PBS is used in combination to rush
Tissue culture plate is washed, according to every 10cm2Tissue culture plate floor space liquid volume added be 4mL ratio, be added the first StemPro-
34 culture mediums are subsequently placed in 37 DEG C, 5%v/v CO2It is cultivated 24 hours in cell incubator, repeats step 5 once, trained in cell
It supports induction in plate and obtains the endothelial progenitor cells being inclined to containing endothelial cell.Wherein, the first StemPro-34 culture mediums be
Addition VEGFA, Forskolin and SB431542, a concentration of 100ng/mL of wherein VEGFA in StemPro-34 culture mediums,
A concentration of 1 μM/L of Forskolin, 5 μM/L of the concentration of SB431542.
Step 6：Remove the old culture in the tissue culture plate for the endothelial progenitor cells being inclined to containing endothelial cell in step 5
Base is used in combination PBS to rinse tissue culture plate, according to every 10cm2Tissue culture plate floor space liquid volume added be 0.5mL ratio, add
It is 1 by volume to enter by cell dissociation buffer and trypsase:The cell disintegration enzyme mixation of 1 composition, is digested, is digested
Endothelial progenitor cells afterwards containing endothelial cell tendency.
Step 7：The endothelial progenitor cells being inclined to containing endothelial cell after the enzymolysis that step 6 is obtained, according to every 10cm2It is thin
Born of the same parents' culture plate floor space liquid volume added is the ratio of 1mL, and the 2nd StemPro-34 culture mediums are added, and obtains being inclined to containing endothelial cell
Endothelial progenitor cells re-suspension liquid.Wherein, the 2nd StemPro-34 culture mediums are added in StemPro-34 culture mediums
VEGFA, wherein a concentration of 30ng/mL of VEGFA.
Step 8：The endothelial progenitor cells re-suspension liquid being inclined to containing endothelial cell that step 7 is obtained, according to every 1cm2It is thin
The ratio of born of the same parents' culture plate floor space 30,000 cells of cover plant moves into the cell differentiation culture containing the 2nd StemPro-34 culture mediums
In plate, it is placed in 37 DEG C, 5%v/v CO2It is cultivated 16-24 hours in cell incubator.Wherein, described to contain the 2nd StemPro-34
The cell differentiation culture plate of culture medium obtains by the following method：According to every 10cm2Tissue culture plate floor space liquid volume added be 2mL
Ratio, the fibronectin working solution of 10 μ g/mL is added in tissue culture plate, be stored at room temperature coating 1 hour, remove fibre
Dimension connection albumen working solution, according still further to every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added second
StemPro-34 culture mediums are to get to the cell differentiation culture plate containing the 2nd StemPro-34 culture mediums.
Step 9：The old culture medium in tissue culture plate in step 8 is removed, is used in combination PBS to rinse tissue culture plate, according to every
10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added the 2nd StemPro-34 culture mediums, be placed in 37 DEG C,
5%v/v CO2In cell incubator, culture breaks up endothelial cell in 48 hours to get to Maturation induction.Obtained ripe endothelium
Cell has significant in vitro at pipe ability.
Embodiment 3
The method that the inductive pluripotent stem cells of the present embodiment efficiently break up to mature endothelial cell, detailed process such as Fig. 1
It is shown, include the following steps：
Step 1：Inductive pluripotent stem cells are pre-processed, cell re-suspension liquid is obtained.Its specific method is：
In tissue culture plate, after inductive pluripotent stem cells recovery, passage, rinsed 2 times with PBS, according still further to every
10cm2Tissue culture plate floor space liquid volume added be 0.5mL ratio, be added 37 DEG C of preheated cell dissociation buffers digested
Digestion, obtains enzymolysis, digestion liquid；When 80% inductive pluripotent stem cells start shedding off, 5 times of above-mentioned enzymolysis, digestion liquid is added
The DMEM/F12 culture mediums of volume are neutralized, and then inductive pluripotent stem cells are transferred in centrifuge tube.Wherein, above-mentioned
DMEM/F12 culture mediums are purchased from Life Technologies companies, model 11039021.
Room temperature, 200g centrifuge 5min, remove supernatant, and TeSR-E8 culture mediums are added, and cell precipitation is resuspended, obtains cell weight
Suspension.
Wherein, the inductive pluripotent stem cells recovery, the method passed on are：
Recovery the previous day gets out the coated tissue culture plate of basement membrane matrix, is placed in 37 DEG C, 5%v/v CO2Cell is trained
It supports in case, is incubated overnight, the tissue culture plate that the basement membrane matrix after being incubated overnight was coated with.
Inductive pluripotent stem cells used are using induction agent box inducing somatic, induction agent box used
Epi5TMEpisomal iPSC Reprogramming Kit, purchased from the silent winged generation that science and technology of match, MAN0008352.
The induced multi-potent stem cell frozen is taken out from liquid nitrogen, is inoculated in the coating of the basement membrane matrix after above-mentioned be incubated overnight
In the tissue culture plate crossed, it is placed in 37 DEG C, 5%v/v CO2In cell incubator, it is incubated 24 hours.
The preheated TeSR-E8 culture mediums of fresh 37 DEG C are then replaced, repeat to change liquid daily until cell aggregation degree increases
To 85% to get to the inductive pluripotent stem cells after recovery.
By the induced multi-potent stem cell passage after recovery, the preheated TeSR-E8 cultures of fresh 37 DEG C are replaced after one day
Base repeats to change liquid daily until cell aggregation degree increases to 85% to get to the inductive pluripotent stem cells after passage.
Step 2：The cell re-suspension liquid that step 1 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 50,000 it is thin
The ratio of born of the same parents moves into the tissue culture plate containing TeSR-E8 culture mediums and ROCK inhibitor Y27632 mixtures, is placed in 37
DEG C, 5%v/v CO2It is cultivated 16 hours in cell incubator.Wherein, the TeSR-E8 culture mediums and ROCK inhibitor Y27632
In mixture, a concentration of 10 μM/L of ROCK inhibitor Y27632.
Step 3：Old culture medium in tissue culture plate is removed in step 2, according to every 10cm2Tissue culture plate floor space add
Liquid measure is the ratio of 4mL, and the first N2B27 insulin deficiency culture mediums are added, are placed in 37 DEG C, 5%v/v CO2In cell incubator
Culture 16 hours.The first N2B27 insulin deficiency culture mediums be in N2B27 insulin deficiency culture mediums add BMP4,
A concentration of 10 μM/L of a concentration of 30ng/mL of CHIR-99021 and Activin A, wherein BMP4, CHIR-99021,
A concentration of 70ng/mL of Activin A.
Step 4：The old culture medium in tissue culture plate in step 3 is removed, PBS is used in combination to rinse tissue culture plate, by every
10cm2Tissue culture plate floor space liquid volume added be 6mL ratio, be added the 2nd N2B27 insulin deficiency culture mediums, be placed in 37
DEG C, 5%v/v CO2It is cultivated 48 hours in cell incubator, lateral plate mesoderm cell is obtained in tissue culture plate.Wherein, institute
It is the addition BMP4 and CHIR-99021 in N2B27 insulin deficiency culture mediums to state the 2nd N2B27 insulin deficiency culture mediums,
A concentration of 10 μM/L of a concentration of 30ng/mL of middle BMP4, CHIR-99021.
Step 5：The old culture medium in the tissue culture plate containing lateral plate mesoderm cell in step 4 is removed, PBS is used in combination to rush
Tissue culture plate is washed, according to every 10cm2Tissue culture plate floor space liquid volume added be 4mL ratio, be added the first StemPro-
34 culture mediums are subsequently placed in 37 DEG C, 5%v/v CO2It is cultivated 16 hours in cell incubator, repeats step 5 once, trained in cell
It supports induction in plate and obtains the endothelial progenitor cells being inclined to containing endothelial cell.Wherein, the first StemPro-34 culture mediums be
Addition VEGFA, Forskolin and SB431542, a concentration of 300ng/mL of wherein VEGFA in StemPro-34 culture mediums,
A concentration of 4 μM/L of Forskolin, 15 μM/L of the concentration of SB431542.
Step 6：Remove the old culture in the tissue culture plate for the endothelial progenitor cells being inclined to containing endothelial cell in step 5
Base is used in combination PBS to rinse tissue culture plate, according to every 10cm2Tissue culture plate floor space liquid volume added be 0.5mL ratio, add
It is 1 by volume to enter by cell dissociation buffer and trypsase:The cell disintegration enzyme mixation of 1 composition, is digested, is digested
Endothelial progenitor cells afterwards containing endothelial cell tendency.
Step 7：The endothelial progenitor cells being inclined to containing endothelial cell after the enzymolysis that step 6 is obtained, according to every 10cm2It is thin
Born of the same parents' culture plate floor space liquid volume added is the ratio of 1mL, and the 2nd StemPro-34 culture mediums are added, and obtains being inclined to containing endothelial cell
Endothelial progenitor cells re-suspension liquid.Wherein, the 2nd StemPro-34 culture mediums are added in StemPro-34 culture mediums
VEGFA, wherein a concentration of 70ng/mL of VEGFA.
Step 8：The endothelial progenitor cells re-suspension liquid being inclined to containing endothelial cell that step 7 is obtained, according to every 1cm2It is thin
The ratio of born of the same parents' culture plate floor space 60,000 cells of cover plant moves into the cell differentiation culture containing the 2nd StemPro-34 culture mediums
In plate, it is placed in 37 DEG C, 5%v/v CO2It is cultivated 16 hours in cell incubator.Wherein, described to contain the 2nd StemPro-34 trainings
The cell differentiation culture plate for supporting base obtains by the following method：According to every 10cm2Tissue culture plate floor space liquid volume added be 2mL
The fibronectin working solution of 10 μ g/mL is added in ratio in tissue culture plate, is stored at room temperature coating 1 hour, removes fiber
Albumen working solution is connected, according still further to every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added second
StemPro-34 culture mediums are to get to the cell differentiation culture plate containing the 2nd StemPro-34 culture mediums.
Step 9：The old culture medium in tissue culture plate in step 8 is removed, is used in combination PBS to rinse tissue culture plate, according to every
10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, be added the 2nd StemPro-34 culture mediums, be placed in 37 DEG C,
5%v/v CO2In cell incubator, culture breaks up endothelial cell in 48 hours to get to Maturation induction.Obtained ripe endothelium
Cell has significant in vitro at pipe ability.
Step 10：Obtained Maturation induction differentiation endothelial cell is expanded and frozen.
Wherein, breaking up the specific method that endothelial cell is expanded to obtained Maturation induction is：
When cell aggregation degree has been expanded to 80-90%, you can proceed by passage operation.
Tissue culture plate is taken out, sucks old culture medium, PBS is washed 2 times at room temperature, then according to every 10cm2Cell training
Support the ratio that board bottom area liquid volume added is 0.5mL, be added 37 DEG C it is preheated by cell dissociation buffer and trypsase by volume
It is 1:The cell disintegration enzyme mixation of 1 composition, is placed in 37 DEG C, 5%v/v CO22-3min in cell incubator, until major part
Cell starts to fall off with lumps floating.
According to every 10cm2Tissue culture plate floor space liquid volume added be 1mL ratio, be added 37 DEG C preheated first
StemPro-34 culture mediums neutralize the enzymolysis of cell dissociation buffer and trypsase, cast-off cells are gently blown and beaten with liquid-transfering gun
Agglomerate is resuspended, and is subsequently transferred in 15mL centrifuge tubes.
Under room temperature, 200g centrifuges 5min.Supernatant is removed with liquid-transfering gun, is then added 3mL37 DEG C preheated the
Cell precipitation is fully resuspended in two StemPro-34 culture mediums, gently piping and druming.
According to every 10cm2Tissue culture plate floor space liquid volume added be 2mL ratio, in tissue culture plate be added 37 DEG C
The 2nd preheated StemPro-34 culture mediums.Then according to 1:3 or 1:2 passage ratio divides 3mL cell re-suspension liquids equally
In tissue culture plate, it is put into 37 DEG C, 5%v/v CO2Cell incubator, subsequent stationary culture.
Wherein, breaking up the specific method that endothelial cell is frozen to obtained Maturation induction is：
Microscopic observation cell determines that EC cells are pollution-free, in good condition, and the degree of polymerization when reaching 80-90% can to its into
Row freezes operation.
Tissue culture plate is taken out, sucks old culture medium, PBS is washed 2 times at room temperature, then according to every 10cm2Cell training
Support the ratio that board bottom area liquid volume added is 0.5mL, be added 37 DEG C it is preheated by cell dissociation buffer and trypsase by volume
It is 1:The cell disintegration enzyme mixation of 1 composition, is placed in 37 DEG C, 5%v/v CO22-3min in cell incubator, until major part
Cell starts to fall off with lumps floating.
According to every 10cm2Tissue culture plate floor space liquid volume added be 1mL ratio, be added 37 DEG C preheated second
StemPro-34 culture mediums neutralize the enzymolysis of cell dissociation buffer and trypsase, cast-off cells are gently blown and beaten with liquid-transfering gun
Agglomerate is resuspended, and is subsequently transferred in 15mL centrifuge tubes.
200g centrifuges 5min under room temperature.Period prepares cryopreservation tube, and be marked (culture people's name, freezes the time,
Cultivate algebraically etc.).
Supernatant is sucked with liquid-transfering gun, 2mLCryo-SFM frozen stock solutions are then added, it is heavy that cell is fully resuspended in gently piping and druming
It forms sediment, is then dispensed into cryopreservation tube and (suggests according to 1 when freezing by every pipe 1mL:2 ratios carry out).
Cryopreservation tube is put into the program temperature reduction box of 4 DEG C of precoolings, is placed in -80 DEG C of refrigerators, is transferred to liquid nitrogen container memory within second day
Storage.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of method that inductive pluripotent stem cells efficiently break up to mature endothelial cell, which is characterized in that including walking as follows
Suddenly：
Step 1：Inductive pluripotent stem cells are pre-processed, cell re-suspension liquid is obtained；
Step 2：The cell re-suspension liquid that step 1 is obtained, according to every 1cm2Tissue culture plate floor space cover plant 20,000-5 ten thousand it is thin
The ratio of born of the same parents moves into the tissue culture plate of the mixture containing TeSR-E8 culture mediums and ROCK inhibitor Y27632, is placed in thin
It is cultivated 16-24 hours in born of the same parents' incubator；
Step 3：Old culture medium in tissue culture plate is removed in step 2, according to every 10cm2Tissue culture plate floor space liquid volume added
For the ratio of 4mL, the first N2B27 insulin deficiency culture mediums are added, is placed in cell incubator and cultivates 16-24 hours；
Step 4：The old culture medium in tissue culture plate in step 3 is removed, PBS is used in combination to rinse tissue culture plate, by per 10cm2's
Tissue culture plate floor space liquid volume added is the ratio of 6mL, and the 2nd N2B27 insulin deficiency culture mediums are added, are placed in cell culture
It is cultivated 48 hours in case, lateral plate mesoderm cell is obtained in tissue culture plate；
Step 5：The old culture medium in the tissue culture plate containing lateral plate mesoderm cell in step 4 is removed, is used in combination PBS to rinse thin
Born of the same parents' culture plate, according to every 10cm2Tissue culture plate floor space liquid volume added be 4mL ratio, be added the first StemPro-34 training
Base is supported, is subsequently placed in cell incubator and cultivates 16-24 hours, step 5 is repeated and operates once, induced in tissue culture plate
To the endothelial progenitor cells being inclined to containing endothelial cell；
Step 6：The old culture medium in the tissue culture plate for the endothelial progenitor cells being inclined to containing endothelial cell in step 5 is removed, and
Tissue culture plate is rinsed with PBS, according to every 10cm2Tissue culture plate floor space liquid volume added be 0.5mL ratio, be added cell
Enzyme mixation is decomposed, is digested, the endothelial progenitor cells containing endothelial cell tendency after being digested；
Step 7：The endothelial progenitor cells being inclined to containing endothelial cell after the enzymolysis that step 6 is obtained, according to every 10cm2Cell training
The ratio that board bottom area liquid volume added is 1mL is supported, the 2nd StemPro-34 culture mediums are added, is obtained containing in endothelial cell tendency
Skin progenitor cells re-suspension liquid；
Step 8：The endothelial progenitor cells re-suspension liquid being inclined to containing endothelial cell that step 7 is obtained, according to every 1cm2Cell culture
The ratio of 30,000-6 ten thousand cells of board bottom area cover plant moves into the cell differentiation culture plate containing the 2nd StemPro-34 culture mediums
In, it is placed in cell incubator and cultivates 16-24 hours；
Step 9：The old culture medium in tissue culture plate in step 8 is removed, is used in combination PBS to rinse tissue culture plate, according to every 10cm2
Tissue culture plate floor space liquid volume added be 2mL ratio, be added the 2nd StemPro-34 culture mediums, be placed in cell incubator
In, culture breaks up endothelial cell in 48 hours to get to Maturation induction.
2. the method that a kind of inductive pluripotent stem cells according to claim 1 efficiently break up to mature endothelial cell,
It is characterized in that, described to pre-process inductive pluripotent stem cells in step 1, the method for obtaining cell re-suspension liquid is：
In tissue culture plate, after inductive pluripotent stem cells recovery, passage, rinsed 2 times with PBS, according still further to every 10cm2's
Tissue culture plate floor space liquid volume added is the ratio of 0.5mL, and 37 DEG C of preheated cell dissociation buffers are added and carry out enzymolysis, digestion, obtain
To enzymolysis, digestion liquid；When the inductive pluripotent stem cells of 70-80% start shedding off, 5 times of volumes of above-mentioned enzymolysis, digestion liquid are added
DMEM/F12 culture mediums neutralized, then inductive pluripotent stem cells are transferred in centrifuge tube, centrifuge, remove supernatant,
TeSR-E8 culture mediums are added, cell precipitation is resuspended, obtains cell re-suspension liquid.
3. the method that a kind of inductive pluripotent stem cells according to claim 1 efficiently break up to mature endothelial cell,
It is characterized in that, in step 2, in the mixture of the TeSR-E8 culture mediums and ROCK inhibitor Y27632, ROCK inhibitor
A concentration of 10 μM/L of Y27632.
4. the method that a kind of inductive pluripotent stem cells according to claim 1 efficiently break up to mature endothelial cell,
It is characterized in that, in step 3, the first N2B27 insulin deficiency culture mediums are added in N2B27 insulin deficiency culture mediums
A concentration of 20-30ng/mL of BMP4, CHIR-99021 and Act i v i n A, wherein BMP4, CHIR-99021's is a concentration of
6-10 μM/L, a concentration of 30-70ng/mL of Act i v i n A.
5. the method that a kind of inductive pluripotent stem cells according to claim 1 efficiently break up to mature endothelial cell,
It is characterized in that, in step 4, the 2nd N2B27 insulin deficiency culture mediums are added in N2B27 insulin deficiency culture mediums
A concentration of 6-10 μM/L of a concentration of 20-30ng/mL of BMP4 and CHIR-99021, wherein BMP4, CHIR-99021.
6. the method that a kind of inductive pluripotent stem cells according to claim 1 efficiently break up to mature endothelial cell,
Be characterized in that, in step 5, the first StemPro-34 culture mediums be in StemPro-34 culture mediums add VEGFA,
A concentration of 1-4 μM/L of a concentration of 100-300ng/mL of Forskolin and SB431542, wherein VEGFA, Forsko lin,
5-15 μM of the concentration of SB431542/L.
7. the method that a kind of inductive pluripotent stem cells according to claim 1 efficiently break up to mature endothelial cell,
It is characterized in that, in step 6, the cell disintegration enzyme mixation is 1 by volume by cell dissociation buffer and trypsase:1 composition.
8. the method that a kind of inductive pluripotent stem cells according to claim 1 efficiently break up to mature endothelial cell,
It being characterized in that, in step 7, the 2nd StemPro-34 culture mediums are that VEGFA is added in StemPro-34 culture mediums,
In, a concentration of 30-70ng/mL of VEGFA.
9. the method that a kind of inductive pluripotent stem cells according to claim 1 efficiently break up to mature endothelial cell,
It is characterized in that, in step 8, the cell differentiation culture plate containing the 2nd StemPro-34 culture mediums obtains by the following method：
According to every 1cm2Tissue culture plate floor space liquid volume added be 2mL ratio, the fiber of 10 μ g/mL is added in tissue culture plate
Albumen working solution is connected, is stored at room temperature coating 1 hour, fibronectin working solution is removed, according still further to every 10cm2Cell training
The ratio that board bottom area liquid volume added is 2mL is supported, the 2nd StemPro-34 culture mediums are added and contain second to get to described
The cell differentiation culture plate of StemPro-34 culture mediums.
10. efficiently being broken up to mature endothelial cell according to a kind of inductive pluripotent stem cells of claim 1-9 any one of them
Method, which is characterized in that the condition of culture of the cell incubator is 37 DEG C, 5%v/v CO2。
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438065A (en) * | 2019-07-25 | 2019-11-12 | 中山大学 | A method of induction people's inductive pluripotent stem cells are divided into endothelial progenitor cells |
| CN112725261A (en) * | 2021-01-19 | 2021-04-30 | 上海爱萨尔生物科技有限公司 | Culture solution for differentiation culture of endothelial cells by pluripotent stem cells and differentiation method |
| CN113633663A (en) * | 2021-10-12 | 2021-11-12 | 呈诺再生医学科技(北京)有限公司 | Application of EPC derived from induced pluripotent stem cell differentiation in preparation of cerebral apoplexy therapeutic agent |
| CN113846052A (en) * | 2021-10-12 | 2021-12-28 | 呈诺再生医学科技(北京)有限公司 | Application of endothelial cells and precursor cells thereof in treating demyelinating diseases |
| CN114231481A (en) * | 2021-12-21 | 2022-03-25 | 中国人民解放军总医院 | Chemical induction method for reprogramming dermal fibroblasts into endothelial progenitor cells |
| CN114561356A (en) * | 2022-03-09 | 2022-05-31 | 北京呈诺医学科技有限公司 | Method for influencing cytokine secretion and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103620024A (en) * | 2011-06-09 | 2014-03-05 | 弗·哈夫曼-拉罗切有限公司 | Method for differentiation of pluripotent stem cells into vascular bed cells |
| CN103627671A (en) * | 2007-01-30 | 2014-03-12 | 佐治亚大学研究基金会 | Method for generating endoderm and mesoderm lineages and multipotent migratory cells (MMC), and cell population and use |
| CN103834613A (en) * | 2012-11-27 | 2014-06-04 | 中国科学院上海生命科学研究院 | Methods for preparing pleuripotent cardiovascular progenitor cells and maintaining cardiovascular differentiation capacity |
-
2018
- 2018-02-08 CN CN201810128553.3A patent/CN108384746B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103627671A (en) * | 2007-01-30 | 2014-03-12 | 佐治亚大学研究基金会 | Method for generating endoderm and mesoderm lineages and multipotent migratory cells (MMC), and cell population and use |
| CN103620024A (en) * | 2011-06-09 | 2014-03-05 | 弗·哈夫曼-拉罗切有限公司 | Method for differentiation of pluripotent stem cells into vascular bed cells |
| CN103834613A (en) * | 2012-11-27 | 2014-06-04 | 中国科学院上海生命科学研究院 | Methods for preparing pleuripotent cardiovascular progenitor cells and maintaining cardiovascular differentiation capacity |
Non-Patent Citations (1)
| Title |
|---|
| LIN Y等: "Differentiation, Evaluation, and Application of Human Induced讱? Pluripotent Stem Cell-Derived Endothelial Cells.", 《ARTERIOSCLER THROMB VASC BIOL.》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438065A (en) * | 2019-07-25 | 2019-11-12 | 中山大学 | A method of induction people's inductive pluripotent stem cells are divided into endothelial progenitor cells |
| CN110438065B (en) * | 2019-07-25 | 2020-06-05 | 中山大学 | Method for inducing human induced pluripotent stem cells to differentiate into endothelial progenitor cells |
| CN112725261A (en) * | 2021-01-19 | 2021-04-30 | 上海爱萨尔生物科技有限公司 | Culture solution for differentiation culture of endothelial cells by pluripotent stem cells and differentiation method |
| CN112725261B (en) * | 2021-01-19 | 2022-09-02 | 上海爱萨尔生物科技有限公司 | Culture solution for differentiation culture of endothelial cells by pluripotent stem cells and differentiation method |
| CN113633663A (en) * | 2021-10-12 | 2021-11-12 | 呈诺再生医学科技(北京)有限公司 | Application of EPC derived from induced pluripotent stem cell differentiation in preparation of cerebral apoplexy therapeutic agent |
| CN113846052A (en) * | 2021-10-12 | 2021-12-28 | 呈诺再生医学科技(北京)有限公司 | Application of endothelial cells and precursor cells thereof in treating demyelinating diseases |
| CN114231481A (en) * | 2021-12-21 | 2022-03-25 | 中国人民解放军总医院 | Chemical induction method for reprogramming dermal fibroblasts into endothelial progenitor cells |
| CN114561356A (en) * | 2022-03-09 | 2022-05-31 | 北京呈诺医学科技有限公司 | Method for influencing cytokine secretion and application |
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