CN106754717A - A kind of inducing embryo stem cell is divided into the method and inducing culture of endothelial cell - Google Patents
A kind of inducing embryo stem cell is divided into the method and inducing culture of endothelial cell Download PDFInfo
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Abstract
The invention provides a kind of inducing culture for inducing and producing endothelial cell, the culture medium based on DMEM/F12 culture mediums contains following components：Molar concentration is that the β mercaptoethanols of 0.05~0.15mmol/L, molar concentration are that 0.05~0.15mmol/L glutamine, mass concentration are that the bFGF of 4~8ng/ml, molar concentration are that the NEAA of 0.05~0.15mmol/L, the KSR that volume fraction is 18~25% and molar concentration are 0.5~2 μm of ol/L RA.Present invention also offers a kind of method that inducing embryo stem cell is divided into endothelial cell, the endothelial cell form rule feature that induction is obtained is obvious, methods described is not related to feeder layer, avoid other animal derived pollutions, biological safety is high, as tissue engineering comea endothelium seed cell, for the transplanting of clinical corneal endothelium provides qualified material source.
Description
Technical field
The invention belongs to organizational project and field of ophthalmology, and in particular to a kind of inducing embryo stem cell is divided into corneal endothelium
The method and inducing culture of cell.
Background technology
Corneal endothelium is the cell monolayer for being located at cornea most inner side, and corneal transparency is maintained by its active liquid pump function.People
Endothelial cell multiplication capacity is extremely limited, and impaired rear main being divided a word with a hyphen at the end of a line by the extension of damage zone periphery cell is repaired.Respectively
Planting reason such as Fuch Fuch's dystrophys, wound and operation etc. can cause endothelial cell to damage, and work as cell density
Less than 500-1500/mm2When there is corneal endothelium function decompensation, cause corneal edema muddy, bleb type cornea is presented
Lesion, cornea and its composition transplanting are that this kind of patient rebuilds bright hope, but deficient serious the constraining in corneal donor source is faced
The development of bed corneal graft.Therefore, a large amount of and with normal function human corneal endothelial cells how are obtained in vitro has weight
The clinical meaning wanted.
In recent years, embryonic stem cell (Embryonic Stem cell, ESC) and induced multi-potent stem cell (induced
Pluripotent Stem Cell, iPSC) research in field advances by leaps and bounds.Used as all-round/multipotential stem cell, they have nothing
Limit self-renewing and triploblastica differentiation capability, can break up as various cells in theory.Training altogether is used conventional abductive approach more
Support or CMC model system carries out induction differentiation to embryonic stem cell or iPS cells, for example, Publication No. CN 102952777
Patent uses the generation of original cuiture MEC 3~4 as feeder layer, using human embryonic stem cell medium routine
Culture.But feeder layer co-culture method is cumbersome, time blunt length, and extracorporeal procedures requirement try one's best simple and fast can be more preferable
Meet future clinical application.
The content of the invention
In view of this, it is an object of the invention to a kind of inducing embryo stem cell be divided into endothelial cell method and
Inducing culture, makes methods described that inducing embryo stem cell can be completed on the premise of without feeder cells and is divided into angle
Film endothelial cell.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of inducing culture for inducing and producing endothelial cell, with DMEM/F12 culture mediums as base
Basal culture medium, contains following components：Molar concentration be the beta -mercaptoethanol of 0.05~0.15mmol/L, molar concentration be 0.05~
0.15mmol/L glutamine, mass concentration are that the bFGF of 4~8ng/ml, molar concentration are 0.05~0.15mmol/L's
NEAA, volume fraction are 18~25% KSR and molar concentration is 0.5~2 μm of ol/L RA.
Preferably, molar concentration of the beta -mercaptoethanol in DMEM/F12 culture mediums is 0.1mmol/L.
Preferably, molar concentration of the glutamine in DMEM/F12 culture mediums is 0.1mmol/L.
Preferably, mass concentration 4ng/mLs of the bFGF in DMEM/F12 culture mediums.
Preferably, molar concentrations of the NEAA in DMEM/F12 culture mediums is 0.1mmol/L.
Preferably, volume fractions of the KSR in DMEM/F12 culture mediums is 20%.
Preferably, molar concentrations of the RA in DMEM/F12 culture mediums is 1 μm of ol/L.
The present invention provides a kind of method that inducing embryo stem cell is divided into endothelial cell, comprises the following steps：
1) human embryo stem cell after washing is suspended with the mTeSR1 culture mediums containing ROCK inhibitor, the people's embryo that will be suspended
Tire stem cell is seeded to carries out basic culture on the culture medium for overlay matrigel；
2) by the step 1) carry out the Fiber differentiation of the human embryo stem cell described in claim 1 or 2 of basic culture
5~6d of base continuous induction culture；
3) by the step 2) cell that obtains of Fiber differentiation is resuspended after being digested, by it is described it is resuspended after Human embryo do
Cell is seeded on gelatin or laminin, and the gelatin or Laminin lens that will be inoculated with human embryo stem cell are placed in asepsis ring
Differentiation culture is carried out in border, human corneal endothelial cells are obtained.
Preferably, the step 1) in human embryo stem cell carry out pre-processing before inoculation and obtain unicellular or less than 2mm
Small lumps；The method of the pretreatment includes dispase digestion method or mechanical dissection method.
Preferably, the step 1) in washing cleaning solution be DMEM/F12 culture mediums.
Preferably, the step 1) described in be inoculated with density be 1.2~3.0 × 103Individual cell mass or cell/cm2.
Preferably, the step 1) described in ROCK inhibitor molar concentration be 8~12 μm of ol/L；The ROCK suppresses
The addition of agent is 10 μm of ol/L.
Preferably, the step 1) described in overlay matrigel the preparation method of culture dish specifically include following steps：
Bed board is carried out after being diluted to matrigel with DMEM/F12 culture mediums；
Culture dish after bed board is placed in temperature for 37 DEG C, CO2Volumetric concentration be 5% gnotobasis in place 1~2h
Carry out gel；
DMEM/F12 culture mediums after gel are replaced by mTeSR1 culture mediums, obtain overlaying the culture medium of matrigel.
Preferably, the step 1) in basis culture time be 3-4 days.
Preferably, the step 2) in Fiber differentiation temperature be 35~38 DEG C；
The CO of the Fiber differentiation2Volumetric concentration is 4~6%.
Preferably, the step 3) in resuspended solvent be the hyclone volume fraction containing ROCK inhibitor for 4~
6% DKSFM culture mediums.
Preferably, the step 3) in overlay gelatin mass concentration be 0.08~0.2%；
The step 3) in overlay laminin mass concentration be 1~5 μ g/ml.
Preferably, the step 3) in gnotobasis temperature be 35~38 DEG C；The volume of the CO2 of the gnotobasis is dense
Spend is 4~6%.
Preferably, the step 3) in differentiation culture time be 6~8d.
A kind of induction that the present invention is provided produces the inducing culture of endothelial cell, with DMEM/F12 culture mediums as base
Basal culture medium, contains following components：Molar concentration be the beta -mercaptoethanol of 0.05~0.15mmol/L, molar concentration be 0.05~
0.15mmol/L glutamine, mass concentration are that the bFGF of 4~8ng/ml, molar concentration are 0.05~0.15mmol/L's
NEAA, volume fraction are 18~25% KSR and molar concentration is 0.5~2 μm of ol/L RA.The inducing culture is in DMEM/
Also contain certain density beta -mercaptoethanol, glutamine, bFGF, NEAA, KSR and RA on the basis of F12 culture mediums, pass through
DMEM/F12 culture mediums and above-mentioned additive act synergistically, and specifically, beta -mercaptoethanol, glutamine, NEAA, KSR are cell
Nutritional support effect is provided；RA is main neural crest direction induction reagent, and bFGF collaborations RA promotes cell to neural crest direction
Differentiation, induction is oriented to human embryo stem cell, further break up the human corneal endothelial cells for obtaining have morphosis and
The similar human corneal endothelial cells of Normal Human Corneal Endothelium cell, express human corneal endothelial cells critical function albumen, and can
The characteristics of long-term in vitro culture.
The method that a kind of inducing embryo stem cell that the present invention is provided is divided into endothelial cell, methods described is a set of
Efficiently, easy, feasible abductive approach, the endothelial cell form rule that induction is obtained, feature is obvious.And this method is not related to
And feeder layer, therefore other animal derived pollutions can be avoided, biological safety is high, careful as tissue engineering comea endothelium kind
Born of the same parents, can provide qualified material source for clinical corneal endothelium transplanting.
Brief description of the drawings
Fig. 1 is undifferentiated human embryo stem cell clone picture in embodiment 1；
Fig. 2 is human embryo stem cell clone's picture of induction 5 days in embodiment 1；
Fig. 3 is that human embryo stem cell is induced to differentiate into the form picture of human corneal endothelial cells in embodiment 1；
The picture of human corneal endothelial cells when Fig. 4 is cultivated 3 weeks to break up in embodiment 1；
Fig. 5 is noble cells expression human corneal endothelial important symbol Na in embodiment 4+-K+The immunofluorescence figure of-ATPase
Piece；
Fig. 6 is normal endothelial cell expression Na in embodiment 4+-K+The immunofluorescence picture of-ATPase；
Fig. 7 is the immune picture of noble cells expression human corneal endothelial important symbol ZO-1 in embodiment 4；
Fig. 8 is the immunofluorescence picture of normal endothelial cell expression ZO-1 in embodiment 4.
Specific embodiment
The invention provides a kind of inducing culture for inducing and producing endothelial cell, with DMEM/F12 culture mediums as base
Basal culture medium, contains following components：Molar concentration be the beta -mercaptoethanol of 0.05~0.15mmol/L, molar concentration be 0.05~
0.15mmol/L glutamine, mass concentration are that the bFGF of 4~8ng/ml, molar concentration are 0.05~0.15mmol/L's
NEAA, volume fraction are 18~25% KSR and molar concentration is 0.5~2 μm of ol/L RA.
In the present invention, the inducing culture is the culture medium based on DMEM/F12 culture mediums.It is described in the present invention
DMEM/F12 culture mediums are preferably knockout DMEM/F12.The source of the knockout DMEM/F12 culture mediums is without spy
Different limitation, using source well-known to those skilled in the art.Knockout DMEM/F12 are purchased from the embodiment of the present invention
Gibco companies.
In the present invention, the inducing culture includes beta -mercaptoethanol.The beta -mercaptoethanol is in DMEM/F12 culture mediums
In molar concentration be preferably 0.1mmol/L.The source of the beta -mercaptoethanol is not particularly limited and uses people in the art
Beta -mercaptoethanol known to member.
In the present invention, the inducing culture includes glutamine.The glutamine is in DMEM/F12 culture mediums
Molar concentration be 0.1mmol/L.The source of the glutamine is not particularly limited using well known to those skilled in the art
Glutamine.
In the present invention, the inducing culture includes bFGF (basic fibroblast growth factor).The bFGF exists
Mass concentration 4ng/mL in DMEM/F12 culture mediums.The source of the bFGF is not particularly limited and uses those skilled in the art
Known bFGF.
In the present invention, the inducing culture includes NEAA (nonessential amino acid).The NEAA is trained in DMEM/F12
It is 0.1mmol/L to support the molar concentration in base.The source of the NEAA is not particularly limited ripe using those skilled in the art
The NEAA for knowing.
In the present invention, the inducing culture includes KSR (serum substitute).The KSR is in DMEM/F12 culture mediums
In volume fraction be 20%.The source of the KSR is not particularly limited using KSR well-known to those skilled in the art i.e.
Can.KSR is purchased from Gibco companies in the embodiment of the present invention.
In the present invention, the inducing culture includes RA (vitamin Class A chemical families vitamin A acid).The RA exists
Molar concentration in DMEM/F12 culture mediums is 1 μm of ol/L.The source of the RA is not particularly limited and uses people in the art
RA known to member.RA is purchased from Sigma companies in the embodiment of the present invention.
The present invention provides a kind of method that inducing embryo stem cell is divided into endothelial cell, comprises the following steps：
1) human embryo stem cell after washing is suspended with the mTeSR1 culture mediums containing ROCK inhibitor, the people that will be suspended
Embryonic stem cell is seeded to carries out basic culture on the culture medium for overlay matrigel；
2) by step 1) the basis human embryo stem cell that obtains of culture with described inducing culture continuous induction culture 5~
6d, changes an inducing culture daily；
3) by the step 2) cell that obtains of Fiber differentiation is resuspended after being digested, will be resuspended after human embryo stem cell
It is seeded to and overlays on gelatin or laminin, the gelatin or Laminin lens that will be inoculated with human embryo stem cell is placed in asepsis ring
Differentiation culture is carried out in border, human corneal endothelial cells are obtained.
The present invention suspends the human embryo stem cell after washing with the mTeSR1 culture mediums containing ROCK inhibitor, will suspend
Human embryo stem cell be seeded to basic culture carried out on the culture medium for overlay matrigel.
In the present invention, the human embryo stem cell preferably pre-process and obtains unicellular or less than 2mm before inoculation
Small lumps；The method of the pretreatment preferably includes dispase digestion method or mechanical dissection method.The dispase digestion method does not have
Have specifically limited, using cell dissociation method well-known to those skilled in the art；The species of the dispase is preferably and disappears
Change liquid Accutase.The source of the digestive juice Accutase is preferably purchased from Sigma companies, article No. A6964.The mechanical dissection
Method is not particularly limited, using mechanical dissection method well-known to those skilled in the art.
The present invention is preferably washed pretreated human embryo stem cell.In the present invention, the washing cleaning solution
It is DMEM/F12 culture mediums.The number of times of the washing is preferably 1~3 time, more preferably 2 times.
In the present invention, the preparation method of the matrigel for overlaying preferably includes specific following steps：
Bed board is carried out after being diluted to matrigel with DMEM/F12 culture mediums；
Culture dish after bed board is placed in 1~2h of placement in the gnotobasis that temperature is 37 DEG C, CO2 volumetric concentrations are 5%
Carry out gel；
DMEM/F12 culture mediums after gel are replaced by mTeSR1 culture mediums, obtain overlaying the culture medium of matrigel.Institute
The source for stating mTeSR1 culture mediums is preferably purchased from Stem Cell companies.
In the present invention, the time of the basis culture is 3~4 days.Phase is being inverted through basis culture human embryo stem cell
Difference microscope is issued to about 20~100 cell/clones, and clones uniform in size.The purpose of the basis culture is dry thin to make
Born of the same parents' clonal growth is to the size for reaching inductive condition.
The human embryo stem cell of basis culture is obtained, be seeded to human embryo stem cell on inducing culture by the present invention, even
5~6d of continuous Fiber differentiation.
In the present invention, the method for the inoculation is not particularly limited, using inoculation side well-known to those skilled in the art
Method.The density of the inoculation is preferably 1.2~3.0 × 103Individual cell mass or cell/cm2；More preferably 1.5~2.5 ×
103Individual cell mass or cell/cm2；Most preferably 2.0 × 103Individual cell mass or cell/cm2。
In the present invention, the molar concentration of the ROCK inhibitor is preferably 8~12 μm of ol/L, more preferably 10 μm ol/L.
The present invention is not particularly limited using ROCK inhibitor well-known to those skilled in the art to the source of the ROCK inhibitor
.In the embodiment of the present invention, the source of the ROCK inhibitor is purchased from Sigma companies.
In the present invention, the time of the Fiber differentiation is preferably 5d.The temperature of the Fiber differentiation is preferably 35~38 DEG C,
More preferably 37 DEG C；The CO of the Fiber differentiation2Volumetric concentration is preferably 4~6%, more preferably 5%.
In the present invention, an inducing culture is preferably changed during the Fiber differentiation daily.The replacing culture medium
Method is not particularly limited, using the method for replacing culture medium well-known to those skilled in the art.
Treat that visible human embryo stem cell expands and connects under the microscope for the human embryo stem cell of Fiber differentiation, the present invention will
The cell is resuspended after being digested, will be resuspended after human embryo stem cell be seeded on the gelatin or laminin for overlaying,
The culture plate is placed in carries out differentiation culture in gnotobasis, obtain human corneal endothelial cells.
In the present invention, the cell that opportunity of the digestion is preferably Fiber differentiation becomes big, cellular morphology by circle in volume
Or oval is changed into polygon, arrangement is tight, is carried out when clone's periphery cell is in fusiformis threadiness.
In the present invention, the method for digestion preferably removes inducing culture, adds 2ml without Ca2+、Mg2+PBS wash
Wash 2 times, add containing the EDTA 1.5ml that 0.25% pancreatin mass concentration is 0.02%, after removal fibrous cell and pancreatin, remain
Remaining cell clone places incubator and continues to digest.
In the present invention, the resuspended solvent is that, containing ROCK inhibitor, hyclone mass concentration is 4~6% DKSFM
Culture medium, the hyclone mass concentration of the DKSFM culture mediums is more preferably 5%.The tire ox blood containing ROCK inhibitor
Clear volume fraction adds FBS and ROCK inhibitor is made for the culture medium of 5%DKSFM in DKSFM basal mediums.Institute
It is 4~6%, more preferably 5% to state FBS and account for total liquid volume percent；The molar concentration of the ROCK inhibitor is preferably 5~
20 μm of ol/L, more preferably 10 μm ol/L.
In the present invention, the mass concentration for overlaying gelatin is preferably 0.08~0.2%, more preferably 0.1%；It is described pre-
The mass concentration of laying Fibronectin (LN) is preferably 1~5 μ g/ml, more preferably 3 μ g/ml.
In the present invention, the temperature of the gnotobasis is preferably 35~38 DEG C, more preferably 37 DEG C；The gnotobasis
CO2Volumetric concentration be 4~6%, more preferably 5%.
In the present invention, the time of the differentiation culture is preferably 6~8d, more preferably 7d.
The endothelial cell for obtaining is polygon cell, and iuntercellular sharpness of border, monolayer alignment are aobvious with reference to external optics
It is thin with Normal Human Corneal Endothelium that micro mirror observation, real-time quantitative PCR, immunofluorescence etc. confirm that noble cells has
Born of the same parents similar form and phenotype, show as cell in approximate hexagon is regularly arranged, iuntercellular sharpness of border, express Na+-K+-
The cell markings such as ATPase, ZO-1, and cultivate to 3 pericytes without obvious aging apoptosis.
The side of endothelial cell is divided into a kind of inducing embryo stem cell that the present invention is provided with reference to embodiment
Method and inducing culture are described in detail, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
The culture of human embryo stem cell：A Matrigel matrigel (being purchased from BD companies, article No. 354277) for packing is taken,
4 DEG C of thawed on ice.Precooling pipette tips, culture dish and centrifuge tube.(Invitrogen is purchased from using cold DMEM/F12 culture mediums public
Department, article No. 11330) after Matrigel matrigels are diluted to concentration 1-2% by 25ml, to add 1- in every diameter 60mm culture dishes
2ml is coated with, and culture dish is moved into 37 DEG C, 5%CO2 incubators after matrigel is completely covered culture face, is changed after 1 hour
For fresh human embryonic stem cell medium mTeSR1 (being purchased from Stem Cell companies, article No. 05850) is standby.Human embryo stem cell
Cultivate to morphological state is (as shown in Figure 1) when good and washed 2 times using warm DMEM/F12 culture mediums 3ml, per 60mm culture dishes
Middle addition digestive juice Accutase (being purchased from Sigma companies, article No. A6964) about 1-1.5ml, is placed in 37 DEG C, 5%CO2Incubator
Middle digestion 4~7 minutes, observes clone edge and tilts under inverted phase contrast microscope, terminate digestion, careful piping and druming embryonic stem cell gram
Grand and collection, using brief centrifugation, is done after removing supernatant to conical centrifuge tube using the fresh resuspended Human embryo of mTeSR1 culture mediums
Cell, careful soft piping and druming to cell mass is settled without obvious, with 1.2-3 × 10 after counting3Individual cell mass/cm2Density inoculation is pre-
Coated culture dish, fluid infusion to the total amount of liquid 3ml of every ware, passage inoculation 10 μM of ROCK inhibitors of same day addition are inoculated with second day
Removed when changing liquid, liquid is changed in daily observation.
The Matrigel matrigels are the branch for being dispensed by 270-350 μ l in advance, and -80 DEG C save backup.
The brief centrifugation refer to centrifugation reach setting speed stop centrifugation.
The good human embryo stem cell of the morphological state refers to：Clone rounded or similar round, the smooth of the edge, cell row
Arrange tight homogeneous, the human embryo stem cell without differentiation.
2. the induction differentiation of human embryo stem cell：The human embryo stem cell culture of cellar culture to suitable size is after passage
(human embryo stem cell of the suitable size is cloned to start induction；About 60-100 cell under inverted phase contrast microscope/gram
It is grand, clone uniform in size, medium density), it is continuous using the inducing culture containing RA (being purchased from Sigma companies, article No. R2625)
Induction 5 days, the inducing culture containing RA is to add following material system on knockout DMEM/F12 basal mediums
Into：Beta -mercaptoethanol, glutamine, bFGF, NEAA, KSR, RA.After addition, beta -mercaptoethanol concentration is 0.1mM, glutamine
Concentration is for 0.1mM, bFGF concentration for 4ng/ml, NEAA are 20% (volume fraction), the RA that 0.1mM, KSR concentration are total amount of liquid
Concentration is 1 μM.Visible human embryo stem cell clone expands under mirror, adjacent clone is merged, and clone's inner cell volume becomes big, thin
Born of the same parents' form is changed into polygon from circular or oval, and arrangement is tight, and clone's periphery cell is in fusiformis threadiness (as shown in Figure 2).
Terminate induction, remove inducing culture, add 2ml without Ca2+、Mg2+PBS wash 2 times, add 0.25% pancreatin-
0.02%EDTA 1.5ml, after removal fibrous cell and pancreatin, remaining cell clone places incubator and continues to digest, and treats mostly
During cell detachment using the 5%DKSFM culture mediums (being purchased from Invitrogen companies, article No. 10744019) containing ROCK inhibitor eventually
Only, it is resuspended, blow and beat to unicellular rear counting, by 1.5-2.5 × 104Individual cell/cm2Density is inoculated in 1 hour in advance and overlays
(0.1% gelatin refers to addition 0.1g gelatin in every 100ml water to 0.1% gelatin, and autoclaving is standby after dissolving.) culture
Ware, noble cells is placed in 37 DEG C, 5%CO2Incubator culture, changes the 5%DKSFM culture mediums (institute containing ROCK inhibitor every other day
State that the 5%DKSFM culture mediums containing ROCK inhibitor add FBS in DKSFM basal mediums and ROCK inhibitor is made.
Its concentration be respectively total amount of liquid 5% (volume fraction) and 10 μM).Cell is covered with substantially within about 1 week or so, under inverted microscope
It can be seen that noble cells gradually becomes polygon, iuntercellular sharpness of border (as shown in Figure 3), and culture to 3 pericytes by polygonal
Without obvious aging apoptosis (as shown in Figure 4).
Embodiment 4
Immunofluorescence test：
1.4% paraformaldehyde fixes cell 15 minutes；2.PBS is washed 3 times；3.0.1%TritonX-100 saturatingization 15 minutes
(membranous antigen can omit this step)；4. add 5%BSA room temperatures to close 1 hour；5. add corresponding primary antibody to be incubated at room temperature 2h or 4 DEG C to incubate
Educate overnight；6. the fluorescence secondary antibody corresponding with primary antibody source is added, 1h is incubated at room temperature；7.DAPI is dyeed, under fluorescence microscope
Take pictures.The fluorescence immunoassay picture of cell sign thing is as shown in Figure 5 and Figure 6.Fig. 5 is that noble cells expresses endothelial cell liquid pump
Function mark Na+-K+The immunofluorescence picture of-ATPase marks；Fig. 6 is positive control picture, is normal human corneal endothelial
Cell expression endothelial cell liquid pump function mark Na+-K+The immunofluorescence picture of-ATPase marks；Fig. 7 is thin for differentiation
The tight albumen ZO-1 marks immunofluorescence picture of cellular expression endothelial cell structure mark；Fig. 8 is positive control picture, is
The tight albumen ZO-1 marks immunofluorescence picture of Normal Human Corneal Endothelium cell endothelial cell structure mark.From Na+-K+-
As can be seen that breaking up the cell behaviour angle for obtaining in the immunofluorescence picture of ATPase marks and tight albumen ZO-1 marks
Film endothelial cell.
As seen from the above embodiment, the inducing culture and abductive approach that the present invention is provided establish it is a set of efficiently, it is easy,
Feasible human embryo stem cell or iPS cell directionals is divided into the abductive approach of human corneal endothelial cells, is obtained in that form knot
The structure human corneal endothelial cells similar to Normal Human Corneal Endothelium cell, express human corneal endothelial cells critical function albumen, and
Can long-term in vitro culture, be that experiment basis have been established in further clinical practice.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (14)
1. it is a kind of to induce the inducing culture for producing endothelial cell, it is characterised in that based on DMEM/F12 culture mediums
Culture medium, contains following components：Molar concentration be the beta -mercaptoethanol of 0.05~0.15mmol/L, molar concentration be 0.05~
0.15mmol/L glutamine, mass concentration are that the bFGF of 4~8ng/ml, molar concentration are 0.05~0.15mmol/L's
NEAA, volume fraction are 18~25% KSR and molar concentration is 0.5~2 μm of ol/L RA.
2. inducing culture according to claim 1, it is characterised in that the beta -mercaptoethanol is in DMEM/F12 culture mediums
In molar concentration be 0.1mmol/L；
Molar concentration of the glutamine in DMEM/F12 culture mediums is 0.1mmol/L；
Mass concentration 4ng/mLs of the bFGF in DMEM/F12 culture mediums；
Molar concentrations of the NEAA in DMEM/F12 culture mediums is 0.1mmol/L；
Volume fractions of the KSR in DMEM/F12 culture mediums is 20%；
Molar concentrations of the RA in DMEM/F12 culture mediums is 1 μm of ol/L.
3. a kind of method that inducing embryo stem cell is divided into endothelial cell, it is characterised in that comprise the following steps：
1) human embryo stem cell after washing is suspended with the mTeSR1 culture mediums containing ROCK inhibitor, the Human embryo that will be suspended
Stem cell is seeded to carries out basic culture on the culture medium for overlay matrigel；
2) by the step 1) basis culture after human embryo stem cell it is continuous with the inducing culture described in claim 1 or 2
5~6d of Fiber differentiation；
3) by the step 2) cell that obtains of Fiber differentiation is resuspended after being digested, by it is described it is resuspended after human embryo stem cell
It is seeded to and overlays on gelatin or laminin, the gelatin or Laminin lens that will be inoculated with human embryo stem cell is placed in asepsis ring
Differentiation culture is carried out in border, human corneal endothelial cells are obtained.
4. method according to claim 3, it is characterised in that the step 1) in human embryo stem cell carried out before inoculation
Pretreatment obtains small lumps unicellular or less than 2mm；The method of the pretreatment includes dispase digestion method or machinery point
Cut method.
5. method according to claim 3, it is characterised in that the step 1) in washing cleaning solution be DMEM/F12 trainings
Support base.
6. method according to claim 4, it is characterised in that the step 1) in the density of inoculation be 1.2~3.0 × 103
Individual cell mass or cell/cm2。
7. method according to claim 3, it is characterised in that the step 1) in the molar concentration of ROCK inhibitor be 8
~12 μm of ol/L；The addition of the ROCK inhibitor is 10 μm of ol/L.
8. method according to claim 3, it is characterised in that the step 1) in overlay matrigel preparation method it is specific
Comprise the following steps：
Bed board is carried out after being diluted to matrigel with DMEM/F12 culture mediums；
Culture dish after bed board is placed in temperature for 37 DEG C, CO2Volumetric concentration be 5% gnotobasis in place 1~2h bags
Quilt；
DMEM/F12 culture mediums after coating are replaced by mTeSR1 culture mediums, obtain overlaying the culture medium of matrigel.
9. method according to claim 3, it is characterised in that the step 1) in time of basis culture be 3-4 days.
10. method according to claim 3, it is characterised in that the step 2) in the temperature of Fiber differentiation be 35~38
℃；
The CO of the Fiber differentiation2Volumetric concentration is 4~6%.
11. methods according to claim 3, it is characterised in that the step 3) in resuspended solvent be contain ROCK suppression
Preparation, the DKSFM culture mediums that hyclone volume fraction is 4~6%.
12. methods according to claim 3, it is characterised in that the step 3) in gelatin mass concentration for 0.08~
0.2%；
The step 3) Laminin mass concentration be 1~5 μ g/ml.
13. methods according to claim 3, it is characterised in that the step 3) in gnotobasis temperature be 35~38
℃；The CO of the gnotobasis2Volumetric concentration be 4~6%.
14. method according to claim 3,12 or 13, it is characterised in that the step 3) in differentiation culture time be
6~8d.
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| WO2023093084A1 (en) * | 2021-11-26 | 2023-06-01 | 内蒙古大学 | Composition and method for inducing differentiation of neural stem cells, and use thereof |
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| WO2024128696A1 (en) * | 2022-12-13 | 2024-06-20 | 재단법인 아산사회복지재단 | Method for inducing differentiation of stem cells into corneal endothelial cells and use thereof |
| WO2024210619A1 (en) * | 2023-04-06 | 2024-10-10 | 재단법인 아산사회복지재단 | Composition for inducing direct differentiation of stem cells into corneal endothelial cells and method for inducing direct differentiation using same |
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