CN104928230B - The cultural method of vascular endothelial cell - Google Patents
The cultural method of vascular endothelial cell Download PDFInfo
- Publication number
- CN104928230B CN104928230B CN201510309077.1A CN201510309077A CN104928230B CN 104928230 B CN104928230 B CN 104928230B CN 201510309077 A CN201510309077 A CN 201510309077A CN 104928230 B CN104928230 B CN 104928230B
- Authority
- CN
- China
- Prior art keywords
- concentration
- cell
- growth factor
- culture medium
- vascular endothelial
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses a kind of cultural method of vascular endothelial cell, 1) multipotential stem cell is divided into mesendoderm precursors cell in culture medium A；2) mesendoderm precursors cell is divided into vascular endothelial cell progenitor cells in culture medium B；3) vascular endothelial cell progenitor cells are divided into vascular endothelial cell in culture medium C；Culture medium A culture medium containing DMEM, F12 culture mediums, sodium selenate, sodium bicarbonate, vitamin C, insulin, activin A, Bone Morphogenetic Protein 4, glycogen synthase kinase 3 inhibitor；Culture medium B culture mediums containing DMEM, F12 culture mediums, sodium selenate, sodium bicarbonate, vitamin C, insulin, vascular endothelial growth factor and transforming growth factor β signal pathway inhibitor；Culture medium C culture medium containing DMEM, F12 culture mediums, sodium selenate, sodium bicarbonate, vitamin C, insulin, vascular endothelial growth factor, epidermal growth factor and fibroblast growth factor.Pluripotent stem cell differentiation is formed vascular endothelial cell by this method.
Description
Technical field
The present invention relates to the cultures of cell, and in particular, to a kind of cultural method of vascular endothelial cell.
Background technology
Stem cell refer to can self-renewing, divide and be divided into a kind of cells of other cell populations.It is thin from doing
From the point of view of born of the same parents' stage residing in development, stem cell can be divided into embryonic stem cell (embryonic stem cell, ES cell)
With adult stem cell (adult stem cell).Embryonic stem cell is a kind of multipotential stem cell (pluripotent stem
Cell), can be broken up with infinite multiplication and the tissue and organ that break up 200 multiple types cell of adult body, form body
At the stem cell of various kinds of cell and the potential of tissue.Evans and Kaufman in 1981 is from mouse inner cell mass (inner cell
Mass, ICM) since Isolation of Embryonic Stem Cell, ES cell researches become the hot spot of scientists from all over the world's research, surround in recent years dry thin
The research that born of the same parents are directed differentiation to other cell types occurs (1981 like the mushrooms after rain；Nature292(5819):154-6).
Thomson is successfully separated, cultivates hESC within 1998, and possibility is provided for the physianthropy application of stem cell
(1998；Science 282(5391)：1145-7).Stretched in the mountain of Kyoto Univ Japan in 2006 more etc. take the lead in reporting iPS it is thin
The research (2006 of born of the same parents；Cell126(4):663-76), they in the mouse skin fibroblast to terminal differentiation by importing
Four kinds of transcription factor OSKM (Oct4, Sox2, Klf4, c-Myc), can induce the cell of terminal differentiation at stem cell state, i.e.,
Hereafter there are the Technology Ways of various manufacture iPS cells in iPS cells (induced pluripotent stem cell).People
The multipotential stem cell of work induction have differentiation capability similar with embryonic stem cell, epigenetic modification and cellular morphology etc. other
Feature provides new thinking for the research of stem cell.
Stem cell has a variety of differentiation potentials, can long-term preservation in vitro, there are self-renewing and infinite multiplication potentiality.
These features of stem cell provide good cell origin for scientific research and medical application.At present stem cell mainly study with
Application direction includes mainly：1) cell therapy, hepatoblast can be broken up by stem cell directional (notification number is by having been reported
The patent of CN102666853A) and cardiac muscle cell's patent of CN103602633A and CN104293730A (notification number be) etc. other
The cell of type provides a large amount of cell origin for having physiological function for cell therapy；2) regenerative medicine field, conventional regeneration
Medicine such as organ transplant are by the way that in donor person organ transplant to receptor body, this transplanting means will produce many problems, such as exempt from
Epidemic disease rejection and infection etc..Being divided by the external patient artificial induction stem cell directional of oneself ripe has physiology work(
The organ of energy can efficiently solve these negative effects, and new method is provided for regenerative medicine.3) drug development, it is external evoked dry
Various drugs are added after being divided into specific cell type in cell directional, and research drug saves the toxicity of specific organization or cell
Numerous studies cost provides mass data for clinical application.
Blood vessel is the important channel for being responsible for transport blood, oxygen and nutriment etc. in vivo, and blood vessel goes wrong, Ke Nengzao
At the even necrosis of tissue and organ failure, threat to life, such as vascular endothelial cell damage can be with induced hypertensions and coronary heart disease
The generation of vascular diseases.Angiocardiopathy is to threaten the common disease of human health, is located at the first place of China's disease incidence.Together
When, scientific research is also required to a large amount of blood vessels, and material is provided to vascular tissue injury's experiment for drug.In addition, external artificial tissue
Organ needs blood vessel transport nutriment to provide nutrient for organ growth.In addition, clinical and regenerative medicine field also there is an urgent need to
Various vascular tissue's cells.In view of the foregoing, how to obtain a large amount of vascular tissue's cell in vitro seems particularly important.It is more
Energy stem cell can provide a large amount of seed cell for us, can be directed differentiation to vascular endothelial cell (endothelial
Cell) and progenitor cells (endothelial progenitor cell), so as to provide a large amount of cells for repairing and treating, be
External structure tissue model provides vascular cell source, also can provide good material for drug screening and drug toxicity research
Material.
Currently, in the culture medium used in the incubation of multipotential stem cell often contain animal derived components, especially in
The culture of vascular endothelial cell progenitor cells such as uses MEF (mouse embryonic fibroblasts, Mouse Embryonic
Fibroblast) the animal blood serum in trophocyte and culture medium.Although above-mentioned culture medium can ensure stem cell and its lure
The cell for leading differentiation has excellent self-renewal capacity, but due to introducing a large amount of animal derived substances in culture medium, to dry
The cell self-renewal and atomization of cell and its induction differentiation bring uncertain factor, increase error；Simultaneously may
Introduce animal derived pollution, such as mycoplasma contamination；In addition, can also induce immune response during cell therapy.
Invention content
The object of the present invention is to provide a kind of cultural method of vascular endothelial cell, what is used in the cultural method does not contain
Animal derived components, and the living environment that the culture medium can provide enough nutriments and stablize for culture cell, to
Can multipotential stem cell be efficiently induced to differentiate to form vascular endothelial cell, and the vascular endothelial cell is formed with height class
It is similar to the vessel-like grid of Human Umbilical Cord's vascular endothelial cell.
To achieve the goals above, the present invention provides the present invention provides a kind of cultural method of vascular endothelial cell,
Including：
1) multipotential stem cell is induced to differentiate to form to mesendoderm precursors cell in culture medium A；
2) mesendoderm precursors cell is induced to differentiate to form vascular endothelial cell progenitor cells in culture medium B；
3) vascular endothelial cell progenitor cells are induced to differentiate to form vascular endothelial cell in culture medium C；
Wherein, culture medium A contain DMEM culture mediums, F12 culture mediums, sodium selenate, sodium bicarbonate, vitamin C, insulin,
Activin A, Bone Morphogenetic Protein 4, glycogen synthase kinase-3 inhibitor；Culture medium B contains DMEM culture mediums, F12 culture mediums, selenium
Sour sodium, sodium bicarbonate, vitamin C, insulin, vascular endothelial growth factor and transforming growth factor β signal pathway inhibitor；Training
It supports base C and contains DMEM culture mediums, F12 culture mediums, sodium selenate, sodium bicarbonate, vitamin C, insulin, vascular endothelial growth factor
Son, epidermal growth factor and fibroblast growth factor.
Through the above technical solutions, the culture medium in cultural method provided by the invention include culture medium A, culture medium B and
Culture medium C, each component can provide enough nutriments and steady by synergistic effect to culture cell in these three culture mediums
Fixed living environment so that cultivating cell has excellent self-renewal capacity, while animal derived components being not used in the culture medium
And then effectively evaded due to animal derived substance to cultivate cell self-renewing and atomization bring it is uncertain because
Generation plain and to cultivating the case where cell pollutes, and can avoid inducing completely during cell therapy exempting from
The generation of epidemic disease reaction.On the basis of the above, method provided by the invention can effectively lure human pluripotent stem cell
It leads and is divided into vascular endothelial cell, so that vascular endothelial cell can realize large-scale commercial Application, and the blood vessel
Endothelial cell progenitor cells are formed with the vessel-like grid that height is similar to Human Umbilical Cord's vascular endothelial cell, so that the blood vessel
Endothelial cell progenitor cells can be in cell therapy, vitro tissue structure, external graft structure, drug screening and Vascular Biology
It is able to extensive use in research.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Attached drawing is to be used to provide further understanding of the present invention, an and part for constitution instruction, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the morphological feature figure of human pluripotent stem cell；
Fig. 2 is the morphological feature figure detected based on CD31+CD34+ characterization cells D1 in example 1；
Fig. 3 is the morphological feature figure detected based on CD31+CD34- characterization cells E1 in example 1；
Fig. 4 is the cell line figure of human pluripotent stem cell；
Fig. 5 is the detection of expression result figure for detecting 5 days CD31 and CD34 after human pluripotent stem cell induction differentiation in example 1；
Fig. 6 is Human Umbilical Cord's Blood vessel endothelial cell line figure (as positive control)；
Fig. 7 is the class blood vessel network generation result figure for detecting cell D1 in example 2；
Fig. 8 is the class blood vessel network generation result figure for detecting cell E1 in example 2；
Fig. 9 is the common location testing result figure for detecting cell E1 and pericyte in example 3.
Specific implementation mode
The specific implementation mode of the present invention is described in detail below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides a kind of cultural methods of vascular endothelial cell, including：
1) multipotential stem cell is induced to differentiate to form to mesendoderm precursors cell in culture medium A；
2) mesendoderm precursors cell is induced to differentiate to form vascular endothelial cell progenitor cells in culture medium B；
3) vascular endothelial cell progenitor cells are induced to differentiate to form vascular endothelial cell in culture medium C；
Wherein, culture medium A contain DMEM culture mediums, F12 culture mediums, sodium selenate, sodium bicarbonate, vitamin C, insulin,
Activin A, Bone Morphogenetic Protein 4, glycogen synthase kinase-3 inhibitor；Culture medium B contains DMEM culture mediums, F12 culture mediums, selenium
Sour sodium, sodium bicarbonate, vitamin C, insulin, vascular endothelial growth factor and transforming growth factor β signal pathway inhibitor；Training
It supports base C and contains DMEM culture mediums, F12 culture mediums, sodium selenate, sodium bicarbonate, vitamin C, insulin, vascular endothelial growth factor
Son, epidermal growth factor and fibroblast growth factor.
In above-mentioned culture medium A, the content of each component can select in a wide range, but in order to enable culture medium A
More sufficient nutriment and more stable living environment can be provided for culture cell, it is preferable that in culture medium A,
The volume ratio of DMEM culture mediums and F12 culture mediums is 1：1-1：4, ascorbic a concentration of 1-100 μ g/ml, the concentration of insulin
For 1-100 μ g/ml, a concentration of 5-100ng/ml of activin A, a concentration of 1-100ng/ml of Bone Morphogenetic Protein 4, Glycogen synthesis
A concentration of 1-100 μM of -3 inhibitor of kinase enzyme, a concentration of 1-100 μ g/L of sodium selenate, a concentration of 100- of sodium bicarbonate
1000mg/L.It is highly preferred that in culture medium A, the volume ratio of DMEM culture mediums and F12 culture mediums is 1：1-1：4, vitamin C
A concentration of 20-100 μ g/ml, a concentration of 5-50 μ g/ml of insulin, a concentration of 5-50ng/ml of activin A, bon e formation egg
White 4 a concentration of 1-50ng/ml, a concentration of 2-40 μM of glycogen synthase kinase-3 inhibitor, a concentration of 1-50 μ of sodium selenate
G/L, a concentration of 200-700mg/L of sodium bicarbonate.It is further preferred that in culture medium A, DMEM culture mediums are cultivated with F12
The volume ratio of base is 1：1-1：4, ascorbic a concentration of 50-100 μ g/ml, a concentration of 10-30 μ g/ml of insulin, activation
A concentration of 10-30ng/ml of plain A, a concentration of 2-10ng/ml of Bone Morphogenetic Protein 4, glycogen synthase kinase-3 inhibitor it is dense
Degree is 10-30 μM, a concentration of 2-30 μ g/L of sodium selenate, a concentration of 400-600mg/L of sodium bicarbonate.
In above-mentioned culture medium B, the content of each component can select in a wide range, but in order to enable culture medium B
More sufficient nutriment and more stable living environment can be provided for culture cell, it is preferable that DMEM culture mediums and
The volume ratio of F12 culture mediums is 1：1-1：4, ascorbic a concentration of 1-100 μ g/ml, a concentration of 1-100 μ g/ of insulin
Ml, a concentration of 5-100ng/ml of vascular endothelial growth factor, a concentration of 1-50 of transforming growth factor β signal pathway inhibitor
μM, a concentration of 1-100 μ g/L of sodium selenate, a concentration of 100-1000mg/L of sodium bicarbonate.It is highly preferred that in culture medium B,
The volume ratio of DMEM culture mediums and F12 culture mediums is 1：1-1：4, ascorbic a concentration of 20-100 μ g/ml, insulin it is dense
Degree be 5-50 μ g/ml, a concentration of 5-50ng/ml of vascular endothelial growth factor, transforming growth factor β signal pathway inhibitor
A concentration of 1-20 μM, a concentration of 1-50 μ g/L of sodium selenate, a concentration of 200-700mg/L of sodium bicarbonate.It is further preferred that
In culture medium B, the volume ratio of DMEM culture mediums and F12 culture mediums is 1：1-1：4, ascorbic a concentration of 50-100 μ g/
Ml, a concentration of 10-30 μ g/ml of insulin, a concentration of 10-30ng/ml of vascular endothelial growth factor, transforming growth factor β
A concentration of 5-10 μM of signal pathway inhibitor, a concentration of 2-30 μ g/L of sodium selenate, a concentration of 400- of sodium bicarbonate
600mg/L。
In above-mentioned culture medium C, the content of each component can select in a wide range, but in order to enable culture medium C
More sufficient nutriment and more stable living environment can be provided for culture cell, it is preferable that in culture medium C,
The volume ratio of DMEM culture mediums and F12 culture mediums is 1：1-1：4, ascorbic a concentration of 1-100 μ g/ml, the concentration of insulin
For 1-100 μ g/ml, a concentration of 5-100ng/ml of vascular endothelial growth factor, a concentration of 5-100ng/ of epidermal growth factor
Ml, a concentration of 5-100ng/ml of fibroblast growth factor, a concentration of 1-100 μ g/L of sodium selenate, sodium bicarbonate it is dense
Degree is 100-1000mg/L.It is highly preferred that in culture medium C, the volume ratio of DMEM culture mediums and F12 culture mediums is 1：1-1：4,
Ascorbic a concentration of 20-100 μ g/ml, a concentration of 5-50 μ g/ml of insulin, a concentration of 5- of vascular endothelial growth factor
50ng/ml, a concentration of 5-50ng/ml of epidermal growth factor, a concentration of 5-50ng/ml of fibroblast growth factor, selenium
A concentration of 1-50 μ g/L of sour sodium, a concentration of 200-700mg/L of sodium bicarbonate.It is further preferred that in culture medium C,
The volume ratio of DMEM culture mediums and F12 culture mediums is 1：1-1：4, ascorbic a concentration of 50-100 μ g/ml, insulin it is dense
Degree is 10-30 μ g/ml, a concentration of 10-30ng/ml of vascular endothelial growth factor, a concentration of 10- of epidermal growth factor
30ng/ml, a concentration of 10-30ng/ml of fibroblast growth factor, a concentration of 2-30 μ g/L of sodium selenate, sodium bicarbonate
A concentration of 400-600mg/L.
Meanwhile the pH of above-mentioned culture medium A, culture medium B and culture medium C can selected in a wide range, still
In order to enable culture cell can have more stable living environment, it is preferable that the pH of culture medium A is 7-8, culture medium B's
PH is 7-8, and the pH of culture medium C is 7-8.
In addition, glycogen synthase kinase-3 inhibitor can be the glycogen synthase kinase of any type routine in this field
- 3 inhibitor of enzyme, such as BIO or CHIR99021, but in order to enable glycogen synthase kinase-3 inhibitor can play it is more excellent
The effect of different induction regulating controlling, it is preferable that it is BIO's and/or CHIR99021 that glycogen synthase kinase-3 inhibitor, which is selected from the trade mark,
Inhibitor；
Similarly, transforming growth factor β signal pathway inhibitor can be the conversion life of any type routine in this field
Long factor-beta signal pathway inhibitor, but in order to enable transforming growth factor β signal pathway inhibitor can play it is more excellent
Induction regulating controlling effect, it is preferable that transforming growth factor β signal pathway inhibitor is the inhibitor that the trade mark is SB431542.
Similarly, fibroblast growth factor can be the Desmocyte growth factor of any type routine in this field
Son, but in order to enable fibroblast growth factor can play the role of more excellent induction regulating controlling, it is preferable that at fibre
It ties up Porcine HGF and is selected from basic fibroblast growth factor (bFGF).
In order to enable improving the survival of mesendoderm precursors cell (mesodermal precursor cells and/or entoderm precursor)
Rate, it is preferable that culture medium A also contains Rho kinases inhibitors.Certainly, the specific type of Rho kinases inhibitors can be with
It selects in a wide range, but in order to further increase mesendoderm precursors cell, the preferably trade mark is the inhibitor of Y27632.
In addition, the dosage of Rho kinases inhibitors can select in a wide range, examined from the effect for improving cell survival rate
Consider, it is highly preferred that in culture medium A, a concentration of 1-50 μM of Rho kinases inhibitors, further preferably 2-20 μM.
On the basis of the above, cultivating the induction differentiation condition of cell can select in a wide range, but be
Culture cell is enable more rapidly to induce differentiation, it is preferable that in step 1), induction is broken up and meets the following conditions：
Cultivation temperature is 35-40 DEG C, and incubation time is 1-3 days；In step 2), induction differentiation meets the following conditions：Cultivation temperature is
35-40 DEG C, incubation time is 1-30 days；In step 3), induction differentiation meets the following conditions：Cultivation temperature is 35-40 DEG C, training
It is 1-30 days to support the time.
There is enough nutriments and in order to understand the new of cell in time in living environment in order to ensure cell
The product of old metabolism, it is preferable that replacement frequency, the replacement frequency of culture medium B and the replacement frequency of culture medium C of culture medium A
It is each independently 1 time/15-30h.
In addition, in the present invention, in order to enable before multipotential stem cell more rapidly can be induced to differentiate into mesendoderm
Body cell, it is preferable that before step 1), cultural method further includes digesting multipotential stem cell to single layer multipotential stem cell, this
It is because can more be contacted with culture medium with multipotential stem cell existing for single layer, and then promote induction differentiation.
Wherein, the method for cell dissociation can be the method for any type routine in this field, such as enzyme digestion, ion chela
Mixture method, Physical and freezing, preferably enzyme digestion specially pass through the multipotential stem cell that degrees of fusion is 70-80%
PBS buffer solution is cleaned, and then by trypsin digestion, then postdigestive cell monolayer is transferred in culture dish and is lured
Lead differentiation.The actual conditions of trypsin digestion can select in a wide range, but in order to further increase digestive efficiency,
Preferably, digestion meets the following conditions：Digestion time is 3-6min.In addition, postdigestive cell monolayer is transferred to culture dish
In process in, the dosage of cell monolayer can select in a wide range, but from induction differentiation effect on consider, preferably
Ground, on the basis of the area of the upper surface of culture dish, the footprint area of cell monolayer is 0.1-0.2.
In the present invention, in order to be more advantageous to culture cell adherency, growth, transfer and differentiation, it is preferable that in step 1)
Before, cultural method further includes that Matrigel matrigels or vitronectin are coated with culture medium.
In addition, in the present invention, the specific type of multipotential stem cell can select in a wide range, can be this field
Middle any type multipotential stem cell, but consider from the application range of vascular endothelial cell progenitor cells obtained, it is preferable that it is more
It can stem cell behaviour class multipotential stem cell.
The present invention will be described in detail by way of examples below.In following embodiment and preparation example, DMEM cultures are
The commercially available product of Thermo Fisher Scientific companies, F12 culture mediums are Thermo Fisher Scientific companies
Commercially available product, vitamin C, insulin be Sigma Aldriches commercially available product, vascular endothelial growth factor R＆D
The commercially available product of Biosystems companies, transforming growth factor β signal pathway inhibitor are that the Sigma Aldrich trades mark are
The commercially available product of SB431542, activin A are the commercially available product of R＆D Biosystems companies, and Bone Morphogenetic Protein 4 is R＆D
The commercially available product of Biosystems companies, glycogen synthase kinase-3 are that the trade mark of Sigma Aldriches is the commercially available of BIO
Product, the relevant protein kinases of Rho (ROCK inhibitor) are the commercially available product that the trade mark of Sigma Aldriches is Y27632, table
Skin growth factor is the commercially available product of R＆D Biosystems companies, and fibroblast growth factor is R＆D Biosystems companies
The trade mark be bFGF commercially available product.
Preparation example 1
1) by DMEM culture mediums, F12 culture mediums, sodium selenate, vitamin C, sodium bicarbonate, insulin, activin A, bone shape
At albumen 4, Rho kinases inhibitors (trade mark Y27632), glycogen synthase kinase-3 inhibitor, (trade mark is
CHIR99021) and glycogen synthase kinase-3 inhibitor (trade mark BIO) is mixed to prepare mixture W1；Wherein, DMEM culture mediums
Volume ratio with F12 culture mediums is 1：2, ascorbic a concentration of 50 μ g/ml, a concentration of 50 μ g/ml of insulin, activin A
A concentration of 55ng/ml, a concentration of 60ng/ml of Bone Morphogenetic Protein 4, (trade mark is glycogen synthase kinase-3 inhibitor
CHIR99021) a concentration of 30 μM, a concentration of 25 μM of glycogen synthase kinase-3 inhibitor (trade mark BIO), sodium selenate
A concentration of 20 μ g/L, a concentration of 500mg/L. of sodium bicarbonate
2) sodium hydroxide is added into said mixture W1 so that pH is adjusted to 7.8 obtained mixture W2；
3) cell culture medium A1 is made by the membrane filtration sterilizing with 0.2 μ m diameter micropore in said mixture W2.
Preparation example 2
It carries out that cell culture medium A2 is made according to the method for preparation example 1, except that Rho albumen is not used in step 1)
Kinase inhibitor, and a concentration of 10 μM of Rho kinases inhibitors.
Preparation example 3
1) by DMEM culture mediums, F12 culture mediums, sodium selenate, vitamin C, sodium bicarbonate, insulin, activin A, bone shape
At albumen 4, Rho kinases inhibitors (trade mark Y27632), glycogen synthase kinase-3 inhibitor, (trade mark is
CHIR99021) and glycogen synthase kinase-3 inhibitor (trade mark BIO) is mixed to prepare mixture W1；Wherein, DMEM culture mediums
Volume ratio with F12 culture mediums is 1：1, ascorbic a concentration of 5 μ g/ml, a concentration of 3 μ g/ml of insulin, activin A
A concentration of 7ng/ml, a concentration of 3ng/ml of Bone Morphogenetic Protein 4, (trade mark is glycogen synthase kinase-3 inhibitor
CHIR99021) a concentration of 2 μM, a concentration of 1 μM of glycogen synthase kinase-3 inhibitor (trade mark BIO), sodium selenate it is dense
Degree be 2 μ g/L, a concentration of 400mg/L of sodium bicarbonate, a concentration of 2 μM of Rho kinases inhibitors (trade mark Y27632).
2) sodium hydroxide is added into said mixture W1 so that pH is adjusted to 7.8 obtained mixture W2；
3) cell culture medium A3 is made by the membrane filtration sterilizing with 0.2 μ m diameter micropore in said mixture W2.
Preparation example 4
1) by DMEM culture mediums, F12 culture mediums, sodium selenate, vitamin C, sodium bicarbonate, insulin, activin A, bone shape
At albumen 4, Rho kinases inhibitors (trade mark Y27632), glycogen synthase kinase-3 inhibitor, (trade mark is
CHIR99021) and glycogen synthase kinase-3 inhibitor (trade mark BIO) is mixed to prepare mixture W1；Wherein, DMEM culture mediums
Volume ratio with F12 culture mediums is 1：4, ascorbic a concentration of 80 μ g/ml, a concentration of 90 μ g/ml of insulin, activin A
A concentration of 95ng/ml, a concentration of 85ng/ml of Bone Morphogenetic Protein 4, (trade mark is glycogen synthase kinase-3 inhibitor
CHIR99021) a concentration of 45 μM, a concentration of 50 μM of glycogen synthase kinase-3 inhibitor (trade mark BIO), sodium selenate
A concentration of 30 μ g/L, a concentration of 600mg/L of sodium bicarbonate, Rho kinases inhibitors (trade mark Y27632) it is a concentration of
20μM。
2) sodium hydroxide is added into said mixture W1 so that pH is adjusted to 7.8 obtained mixture W2；
3) cell culture medium A4 is made by the membrane filtration sterilizing with 0.2 μ m diameter micropore in said mixture W2.
Preparation example 5
A, by DMEM culture mediums, F12 culture mediums, vitamin C, sodium selenate, 500mg sodium bicarbonates, insulin, blood vessel endothelium
Growth factor and the transforming growth factor β signal pathway inhibitor mixing that the trade mark is SB431542 fully obtain mixture W3；Its
In, the volume ratio of DMEM culture mediums and F12 culture mediums is 1：2, ascorbic a concentration of 50 μ g/ml, a concentration of the 55 of insulin
μ g/ml, a concentration of 45ng/ml of vascular endothelial growth factor, a concentration of 25 μ of transforming growth factor β signal pathway inhibitor
M, a concentration of 20 μ g/L of sodium selenate, a concentration of 500mg/L of sodium bicarbonate.
B, sodium hydroxide is added into said mixture W3 so that pH is adjusted to 7.8 obtained mixture W4；
C, cell culture medium B1 is made by the membrane filtration sterilizing with 0.2 μ m diameter micropore in said mixture W4.
Preparation example 6
A, by DMEM culture mediums, F12 culture mediums, vitamin C, sodium selenate, 500mg sodium bicarbonates, insulin, blood vessel endothelium
Growth factor and the transforming growth factor β signal pathway inhibitor mixing that the trade mark is SB431542 fully obtain mixture W3；Its
In, the volume ratio of DMEM culture mediums and F12 culture mediums is 1：1, ascorbic a concentration of 2 μ g/ml, a concentration of 4 μ of insulin
G/ml, a concentration of 5ng/ml of vascular endothelial growth factor, a concentration of 3 μM of transforming growth factor β signal pathway inhibitor, selenium
A concentration of 2 μ g/L of sour sodium, a concentration of 200mg/L of sodium bicarbonate.
B, sodium hydroxide is added into said mixture W3 so that pH is adjusted to 7.8 obtained mixture W4；
C, cell culture medium B2 is made by the membrane filtration sterilizing with 0.2 μ m diameter micropore in said mixture W4.
Preparation example 7
A, by DMEM culture mediums, F12 culture mediums, vitamin C, sodium selenate, 500mg sodium bicarbonates, insulin, blood vessel endothelium
Growth factor and the transforming growth factor β signal pathway inhibitor mixing that the trade mark is SB431542 fully obtain mixture W3；Its
In, the volume ratio of DMEM culture mediums and F12 culture mediums is 1：4, ascorbic a concentration of 90 μ g/ml, a concentration of the 95 of insulin
μ g/ml, a concentration of 100ng/ml of vascular endothelial growth factor, a concentration of 45 μ of transforming growth factor β signal pathway inhibitor
M, a concentration of 50 μ g/L of sodium selenate, a concentration of 600mg/L of sodium bicarbonate.
B, sodium hydroxide is added into said mixture W3 so that pH is adjusted to 7.8 obtained mixture W4；
C, cell culture medium B3 is made by the membrane filtration sterilizing with 0.2 μ m diameter micropore in said mixture W4.
Preparation example 8
A, by DMEM culture mediums, F12 culture mediums, vitamin C, sodium selenate, sodium bicarbonate, insulin, vascular endothelial growth
The factor, epidermal growth factor and fibroblast growth factor (trade mark bFGF) are mixed to get mixture W5；Wherein, DMEM is trained
The volume ratio for supporting base and F12 culture mediums is 1：2, ascorbic a concentration of 55 μ g/ml, a concentration of 60 μ g/ml of insulin, blood
A concentration of 65ng/ml of endothelial tube growth factor, a concentration of 50ng/ml of epidermal growth factor, fibroblast growth factor
A concentration of 45ng/ml, a concentration of 20 μ g/L of sodium selenate, a concentration of 500mg/L of sodium bicarbonate.
B, sodium hydroxide is added into said mixture W5 so that pH is adjusted to 7.8 obtained mixture W6；
C, cell culture medium C1 is made by the membrane filtration sterilizing with 0.2 μ m diameter micropore in said mixture W6.
Preparation example 9
A, by DMEM culture mediums, F12 culture mediums, vitamin C, sodium selenate, sodium bicarbonate, insulin, vascular endothelial growth
The factor, epidermal growth factor and fibroblast growth factor (trade mark bFGF) are mixed to get mixture W5；Wherein, DMEM is trained
The volume ratio for supporting base and F12 culture mediums is 1：1, ascorbic a concentration of 2 μ g/ml, a concentration of 3 μ g/ml of insulin, blood vessel
A concentration of 7ng/ml of endothelial growth factors, a concentration of 8ng/ml of epidermal growth factor, fibroblast growth factor it is dense
Degree is 8ng/ml, a concentration of 2 μ g/L of sodium selenate, a concentration of 400mg/L of sodium bicarbonate.
B, sodium hydroxide is added into said mixture W5 so that pH is adjusted to 7.8 obtained mixture W6；
C, cell culture medium C2 is made by the membrane filtration sterilizing with 0.2 μ m diameter micropore in said mixture W6.
Preparation example 10
A, by DMEM culture mediums, F12 culture mediums, vitamin C, sodium selenate, sodium bicarbonate, insulin, vascular endothelial growth
The factor, epidermal growth factor and fibroblast growth factor (trade mark bFGF) are mixed to get mixture W5；Wherein, DMEM is trained
The volume ratio for supporting base and F12 culture mediums is 1：4, ascorbic a concentration of 90 μ g/ml, a concentration of 100 μ g/ml of insulin, blood
A concentration of 95ng/ml of endothelial tube growth factor, a concentration of 85ng/ml of epidermal growth factor, fibroblast growth factor
A concentration of 90ng/ml, a concentration of 40 μ g/L of sodium selenate, a concentration of 600mg/L of sodium bicarbonate.
B, sodium hydroxide is added into said mixture W5 so that pH is adjusted to 7.8 obtained mixture W6；
C, cell culture medium C3 is made by the membrane filtration sterilizing with 0.2 μ m diameter micropore in said mixture W6.
Embodiment 1
1) culture dish is coated with Matrigel matrigels and (connects mucoprotein Vitronection also using glass certainly
Can) 2h；Then it recovers in 37 DEG C of water-bath the human pluripotent stem cell frozen, and by the cell inoculation to above-mentioned culture medium
In, E8 multipotential stem cells culture solution (being purchased from Life Technologies companies) is then added and is cultivated at 37 DEG C, and
Culture medium is replaced daily, until when proliferation of pluripotent stem cells is to 80% degrees of fusion (confluency), then with 0.5mM EDTA
(PH=8.0, osmotic pressure (Osmolarity)=340mOsm) carries out had digestive transfer culture to maintain human pluripotent stem cells cell mass
State.When needing unicellular passage, using Trypsin enzymes (certain TrypLE Express enzymes can also) digestion, and to training
Supporting the Rock inhibitor (working concentration is 10 μM, improves the survival rate of cell) that the addition trade mark is Y-27632 in base, culture is for 24 hours.
2) culture medium is removed, is cleaned 3 times with PBS buffer solutions, is then divided with Trypsin (trypsase) vitellophag 5
Clock is to unicellular, with area than 1：8 reach new use Matrigel matrigels or the coated culture dishes of Vitronectin
In, cell culture medium A2 is added and is cultivated 1 day at 37 DEG C；Then replace cell culture medium A1 cultivate at 37 DEG C 1 day it is obtained in
Entoderm precursor.
3) at 37 DEG C, above-mentioned mesendoderm precursor is cultivated 3 days in cell culture medium B1 and obtains cell D1,
In, culture medium is replaced daily.
4) at 37 DEG C, above-mentioned cell D1 is cultivated 5 days in cell culture medium C1 and obtains cell E1, wherein daily more
Change culture medium.
Embodiment 2
It is cultivated to obtain cell E2 according to the method for embodiment 1, except that changing cell culture medium A1 into cells
Culture medium A 3.
Embodiment 3
It is cultivated to obtain cell E3 according to the method for embodiment 1, except that changing cell culture medium A1 into cells
Culture medium A 4.
Embodiment 4
It is cultivated to obtain cell E4 according to the method for embodiment 1, except that changing cell culture medium B1 into cells
Culture medium B2.
Embodiment 5
It is cultivated to obtain cell E5 according to the method for embodiment 1, except that changing cell culture medium B1 into cells
Culture medium B3.
Embodiment 6
It is cultivated to obtain cell E6 according to the method for embodiment 1, except that changing cell culture medium C1 into cells
Culture medium C 2.
Embodiment 7
It is cultivated to obtain cell E7 according to the method for embodiment 1, except that changing cell culture medium C1 into cells
Culture medium C 3.
Comparative example 1
It is cultivated to obtain cell E8 according to the method for embodiment 1, except that cell culture medium A1 is without containing activation
Plain A.
Comparative example 2
It is cultivated to obtain cell E9 according to the method for embodiment 1, except that cell culture medium A1 does not contain bone shape
At albumen 4.
Comparative example 3
It is cultivated to obtain cell E10 according to the method for embodiment 1, except that cell culture medium A1 does not contain selenic acid
Sodium.
Comparative example 4
It is cultivated to obtain cell E11 according to the method for embodiment 1, except that cell culture medium B1 does not contain blood vessel
Endothelial growth factors.
Comparative example 5
It is cultivated to obtain cell E12 according to the method for embodiment 1, except that cell culture medium B1 does not contain selenic acid
Sodium.
Comparative example 6
It is cultivated to obtain cell E13 according to the method for embodiment 1, except that cell culture medium B1 is without containing conversion
Grouth factor beta signal pathway inhibitor.
Comparative example 7
It is cultivated to obtain cell E14 according to the method for embodiment 1, except that cell culture medium C1 does not contain epidermis
Growth factor.
Comparative example 8
It is cultivated to obtain cell E15 according to the method for embodiment 1, except that cell culture medium C1 does not contain selenic acid
Sodium.
Comparative example 9
It is cultivated to obtain cell E16 according to the method for embodiment 1, except that cell culture medium C1 does not contain blood vessel
Endothelial growth factors.
Detect example 1
First, above-mentioned cell D1 or cell E1 is digested to unicellular, gently with PBS buffer solutions with trypsin
It rocks and suspends and count again.It takes the cell of 80,000 single-cell suspensions, 800g to centrifuge 3 minutes, abandons supernatant, 100ul is added and contains
The PBS buffer solutions for having 1%BSA (bovine serum albumin(BSA)), jiggle suspension cell again.Each detection antibody is added
(CD31 and CD34), gently mixing.Incubation 30 minutes is protected from light at 4 DEG C；Then 800g is centrifuged 5 minutes, abandons supernatant, is added
100ul contains the PBS of 1%BSA, jiggles suspension cell again, and last flow cytometer is detected.Testing result is shown in figure
2 and Fig. 3.
Wherein, Fig. 1 is the morphological feature figure of human pluripotent stem cell, and Fig. 2 is based on CD31+CD34+ characterization cells D1
Morphological feature figure, Fig. 3 are based on the morphological feature figure of CD31+CD34- characterization cells E1, and as seen from the figure, cell D1 is successfully lured
It leads and is divided into vascular endothelial cell progenitor cells, cell E1 is successfully induced to differentiate into vascular endothelial cell.
Fig. 4 is the cell line figure of human pluripotent stem cell, Fig. 5 be after human pluripotent stem cell induction differentiation 5 days CD31 and
The detection of expression result figure of CD34, wherein the antibody and PE fluorophors (Phycoerythrin) of CD31 is coupled, and CD34's is anti-
Body and APC protein fluorescences group (Allophycocyanin) coupling.Due to CD31 and CD34 can with vascular endothelial cell and its
Labelled protein on progenitor cell surface is coupled, so that vascular endothelial cell and its progenitor cell surface obtain fluorescence or magnetic
Property, so that vascular endothelial cell and its progenitor cells can be accredited in flow cytometer, detach or be enriched with.Pass through figure
4 and Fig. 5 it is found that differentiation after cell in, a part be CD31+CD34+ expression vascular endothelial cell progenitor cells, another part
For the vascular endothelial cell of CD31+CD34- expression.
Similarly, by detecting the method for example 1 it is found that cell E2-E7 is successfully induced to differentiate into vascular endothelial cell, and
Cell E8-E16 is induced to differentiate into vascular endothelial cell not successfully, to illustrate cell culture medium A, cell culture medium B and cell
Multipotential stem cell can be successfully induced to differentiate into vascular endothelial cell by culture medium C.
Detect example 2
First, Matrigel matrigels are incubated at 4 DEG C and become within 3 hours flowing shape semisolid, and be positioned over ice
On.Then it is laid with one layer of Matrigel matrigel in Tissue Culture Dish, its solidification is waited at 37 DEG C.By cell D1 or cell
E1 is laid on Matrigel after solidification, and culture medium is added to be generated by micro- sem observation class blood vessel network after cultivating 4 days
Situation.Testing result is shown in Fig. 7 and Fig. 8.
Wherein, Fig. 6 is Human Umbilical Cord's Blood vessel endothelial cell line figure (as positive control), and Fig. 7 is the class blood vessel of cell D1
Network generates result figure, and Fig. 8 is that the class blood vessel network of cell E1 generates result figure.Above-mentioned three figures control is it is found that cell D1 and E1
It is each formed with the grid of similar vessel-like similar with Human Umbilical Cord's vascular endothelial cell, further illustrates cell culture medium A, thin
Multipotential stem cell can be successfully induced to differentiate into vascular endothelial cell by born of the same parents' culture medium B and cell culture medium C.
Similarly, by detecting the method for example 2 it is found that cell E2-E7 is formed with and Human Umbilical Cord's vascular endothelial cell phase
As similar vessel-like grid, and similar blood vessel similar with Human Umbilical Cord's vascular endothelial cell is not formed in cell E8-E16
The grid of shape, to further illustrate cell culture medium A, cell culture medium B and cell culture medium C can by multipotential stem cell at
It is induced to differentiate into vascular endothelial cell to work(.
Detect example 3
Cell E1 and pericyte are co-cultured on matrigel matrigels jointly, as a result see Fig. 9.It can be seen that thin
Born of the same parents E1 forms the network of similar vessel-like with pericyte, and two kinds of cells have significant common location, it is shown that blood vessel endothelium is thin
Born of the same parents recruit the function of pericyte, to illustrate that vascular endothelial cell can made from cell culture medium culture provided by the invention
It is able to extensive use.
Similarly, by detecting the method for example 3 it is found that cell E2-E7 can form the net of similar vessel-like with pericyte
Network, and two kinds of cells have significant common location；And cell E8-E16 fails to form the network of similar vessel-like with pericyte.From
And extensive use can be able to by further illustrating vascular endothelial cell made from cell culture medium culture provided by the invention.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail can carry out a variety of simple variants to technical scheme of the present invention within the scope of the technical concept of the present invention, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (15)
1. a kind of cultural method of vascular endothelial cell, which is characterized in that the cultural method includes：
1) multipotential stem cell is induced to differentiate to form to mesendoderm precursors cell in culture medium A；
2) the mesendoderm precursors cell is induced to differentiate to form vascular endothelial cell progenitor cells in culture medium B；
3) the vascular endothelial cell progenitor cells are induced to differentiate to form vascular endothelial cell in culture medium C；
Wherein, the culture medium A contain DMEM culture mediums, F12 culture mediums, sodium selenate, sodium bicarbonate, vitamin C, insulin,
Activin A, Bone Morphogenetic Protein 4, glycogen synthase kinase-3 inhibitor；The culture medium B contains DMEM culture mediums, F12 cultures
Base, sodium selenate, sodium bicarbonate, vitamin C, insulin, vascular endothelial growth factor and transforming growth factor β signal path inhibit
Agent；The culture medium C contains DMEM culture mediums, F12 culture mediums, sodium selenate, sodium bicarbonate, vitamin C, insulin, intravascular
Skin growth factor, epidermal growth factor and fibroblast growth factor；
Wherein, in the culture medium A, the volume ratio of the DMEM culture mediums and F12 culture mediums is 1：1-1：4, the dimension life
A concentration of 1-100 μ g/ml of plain C, a concentration of 1-100 μ g/ml, a concentration of 5- of the activin A of the insulin
100ng/ml, a concentration of 1-100ng/ml of the Bone Morphogenetic Protein 4, the glycogen synthase kinase-3 inhibitor it is a concentration of
1-100 μM, a concentration of 1-100 μ g/L, a concentration of 100-1000mg/L of the sodium bicarbonate of the sodium selenate；
In the culture medium B, the volume ratio of the DMEM culture mediums and F12 culture mediums is 1：1-1：4, it is described ascorbic
A concentration of 1-100 μ g/ml, a concentration of 1-100 μ g/ml, a concentration of 5- of the vascular endothelial growth factor of the insulin
100ng/ml, a concentration of 1-50 μM of the transforming growth factor β signal pathway inhibitor, a concentration of 1- of the sodium selenate
100 μ g/L, a concentration of 100-1000mg/L of the sodium bicarbonate；
In the culture medium C, the volume ratio of the DMEM culture mediums and F12 culture mediums is 1：1-1：4, it is described ascorbic
A concentration of 1-100 μ g/ml, a concentration of 1-100 μ g/ml, a concentration of 5- of the vascular endothelial growth factor of the insulin
100ng/ml, a concentration of 5-100ng/ml of the epidermal growth factor, a concentration of 5- of the fibroblast growth factor
100ng/ml, a concentration of 1-100 μ g/L, a concentration of 100-1000mg/L of the sodium bicarbonate of the sodium selenate.
2. cultural method according to claim 1, wherein in the culture medium A, the DMEM culture mediums are trained with F12
The volume ratio for supporting base is 1：1-1：4, the ascorbic a concentration of 20-100 μ g/ml, a concentration of 5-50 μ of the insulin
G/ml, a concentration of 5-50ng/ml of the activin A, a concentration of 1-50ng/ml of the Bone Morphogenetic Protein 4, the glycogen close
At a concentration of 2-40 μM of -3 inhibitor of kinase enzyme, a concentration of 1-50 μ g/L of the sodium selenate, the concentration of the sodium bicarbonate
For 200-700mg/L；
In the culture medium B, the volume ratio of the DMEM culture mediums and F12 culture mediums is 1：1-1：4, it is described ascorbic
A concentration of 20-100 μ g/ml, a concentration of 5-50 μ g/ml, a concentration of 5- of the vascular endothelial growth factor of the insulin
50ng/ml, a concentration of 1-20 μM of the transforming growth factor β signal pathway inhibitor, a concentration of 1-50 μ of the sodium selenate
G/L, a concentration of 200-700mg/L of the sodium bicarbonate；
In the culture medium C, the volume ratio of the DMEM culture mediums and F12 culture mediums is 1：1-1：4, it is described ascorbic
A concentration of 20-100 μ g/ml, a concentration of 5-50 μ g/ml, a concentration of 5- of the vascular endothelial growth factor of the insulin
50ng/ml, a concentration of 5-50ng/ml of the epidermal growth factor, a concentration of 5- of the fibroblast growth factor
50ng/ml, a concentration of 1-50 μ g/L, a concentration of 200-700mg/L of the sodium bicarbonate of the sodium selenate.
3. cultural method according to claim 2, wherein in the culture medium A, the DMEM culture mediums are trained with F12
The volume ratio for supporting base is 1：1-1：4, the ascorbic a concentration of 50-100 μ g/ml, a concentration of 10-30 μ of the insulin
G/ml, a concentration of 10-30ng/ml of the activin A, a concentration of 2-10ng/ml of the Bone Morphogenetic Protein 4, the glycogen
A concentration of 10-30 μM of -3 inhibitor of synthase kinase, a concentration of 2-30 μ g/L of the sodium selenate, the sodium bicarbonate it is dense
Degree is 400-600mg/L；
In the culture medium B, the volume ratio of the DMEM culture mediums and F12 culture mediums is 1：1-1：4, it is described ascorbic
A concentration of 50-100 μ g/ml, a concentration of 10-30 μ g/ml of the insulin, the vascular endothelial growth factor it is a concentration of
10-30ng/ml, a concentration of 5-10 μM of the transforming growth factor β signal pathway inhibitor, a concentration of 2- of the sodium selenate
30 μ g/L, a concentration of 400-600mg/L of the sodium bicarbonate；
In the culture medium C, the volume ratio of the DMEM culture mediums and F12 culture mediums is 1：1-1：4, it is described ascorbic
A concentration of 50-100 μ g/ml, a concentration of 10-30 μ g/ml of the insulin, the vascular endothelial growth factor it is a concentration of
10-30ng/ml, a concentration of 10-30ng/ml of the epidermal growth factor, the fibroblast growth factor it is a concentration of
10-30ng/ml, a concentration of 2-30 μ g/L, a concentration of 400-600mg/L of the sodium bicarbonate of the sodium selenate.
4. according to the cultural method described in any one of claim 1-3, wherein the glycogen synthase kinase-3 inhibitor
Selected from the inhibitor that the trade mark is BIO and/or CHIR99021.
5. according to the cultural method described in any one of claim 1-3, wherein the transforming growth factor β signal path
Inhibitor is the inhibitor that the trade mark is SB431542.
6. according to the cultural method described in any one of claim 1-3, wherein the fibroblast growth factor is selected from
Basic fibroblast growth factor.
7. according to the cultural method described in any one of claim 1-3, wherein the culture medium A also contains Rho albumen and swashs
Enzyme inhibitor.
8. cultural method according to claim 7, wherein the Rho kinases inhibitors are the suppression of trade mark Y27632
Preparation.
9. cultural method according to claim 7, wherein a concentration of 1-50 μM of the Rho kinases inhibitors.
10. the cultural method according to any one of claim 1-3,8-9, wherein before step 1), the culture
Method further includes digesting multipotential stem cell to single layer multipotential stem cell.
11. cultural method according to claim 10, wherein digesting multipotential stem cell to the step of single layer multipotential stem cell
Suddenly it is：The multipotential stem cell that degrees of fusion is 70-80% is cleaned by PBS buffer solution, then by trypsin digestion, then
Postdigestive cell monolayer, which is transferred in culture dish, by under carries out induction differentiation.
12. cultural method according to claim 11, wherein the digestion meets the following conditions：Digestion time is 3-
6min。
13. cultural method according to claim 11, wherein on the basis of the area of the upper surface of the culture dish, institute
The footprint area for stating cell monolayer is 0.1-0.2.
14. the cultural method according to any one of claim 1-3,8-9, wherein before step 1), the culture
Method further includes that Matrigel matrigels or vitronectin are coated with culture vessel.
15. the cultural method according to any one of claim 1-3,8-9, wherein the multipotential stem cell is the mankind
Multipotential stem cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510309077.1A CN104928230B (en) | 2015-06-05 | 2015-06-05 | The cultural method of vascular endothelial cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510309077.1A CN104928230B (en) | 2015-06-05 | 2015-06-05 | The cultural method of vascular endothelial cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104928230A CN104928230A (en) | 2015-09-23 |
| CN104928230B true CN104928230B (en) | 2018-08-21 |
Family
ID=54115658
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510309077.1A Active CN104928230B (en) | 2015-06-05 | 2015-06-05 | The cultural method of vascular endothelial cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104928230B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969720B (en) * | 2016-07-29 | 2019-11-05 | 上海瑞鹿生物技术有限公司 | A kind of Human vascular endothelial's cell culture fluid and its cultural method |
| CN108570415A (en) * | 2017-03-10 | 2018-09-25 | 上海睿泰生物科技股份有限公司 | A kind of culture apparatus being used to prepare epithelial cell piece and its application |
| CN108048390B (en) * | 2017-12-21 | 2021-04-27 | 清华大学 | Method for preparing vascular endothelial cells and special kit thereof |
| CN109988744A (en) * | 2019-03-01 | 2019-07-09 | 宁波医诺生物技术有限公司 | Human pluripotent stem cells culture medium and its preparation method and application |
| CN112662627B (en) * | 2021-01-19 | 2022-09-02 | 上海爱萨尔生物科技有限公司 | Culture solution for differentiating pluripotent stem cells into natural killer cells and differentiation method |
| CN114540278B (en) * | 2022-03-25 | 2023-11-21 | 青岛大学 | Microvascular in-vitro culture method and culture solution |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102732483B (en) * | 2011-03-31 | 2014-12-03 | 北京大学 | Preparation method for hematopoietic progenitor cells and special medium for same |
-
2015
- 2015-06-05 CN CN201510309077.1A patent/CN104928230B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN104928230A (en) | 2015-09-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105062957B (en) | The cultural method of vascular endothelial cell progenitor cells | |
| CN104928230B (en) | The cultural method of vascular endothelial cell | |
| CN104204193B (en) | The cultivation of mescenchymal stem cell | |
| US6875605B1 (en) | Modular cell culture bioreactor and associated methods | |
| CN108384755A (en) | A method of efficiently, efficiently inductive pluripotent stem cells are to neural stem cell differentiating | |
| CN102787092B (en) | Culture medium, cell culture kit and cell culture processes | |
| Quattrocelli et al. | Mouse and human mesoangioblasts: isolation and characterization from adult skeletal muscles | |
| CN102643784B (en) | A kind of amplification in vitro system of hematopoietic stem/progenitor | |
| CN106834212A (en) | A kind of culture medium for lung tissue 3D cultures | |
| CN105112362B (en) | A kind of serum free medium of placenta mesenchyma stem cell and preparation method thereof | |
| CN109609441A (en) | A kind of culture medium and organoid cultural method of renal tissue organoid 3D culture | |
| JPWO2005063967A1 (en) | Induction of cardiomyocytes using mammalian bone marrow cells or cord blood-derived cells and adipose tissue | |
| CN102517251A (en) | Mesenchymal stem cells, as well as preparation method and application thereof | |
| CN104520423A (en) | Method for manufacturing stem cell having appropriate size for intravascular administration | |
| CN106987555A (en) | Efficiently induce the micromolecular compound composition of human pluripotent stem cells myocardiac differentiation | |
| CN105861428A (en) | Inducing culture medium for inducing fibroblast to trans-differentiate into cardiac muscle cells and application of inducing culture medium | |
| CN107460158A (en) | A kind of inducing umbilical cord mesenchymal stem is divided into the method to form insulin | |
| CN106929470A (en) | It is a kind of for derived mesenchymal stem cells in vitro culture and amplification serum free medium | |
| CN109988744A (en) | Human pluripotent stem cells culture medium and its preparation method and application | |
| CN112608896A (en) | NK cell culture method and application thereof | |
| CN107287156A (en) | A kind of isolated culture method of fat mesenchymal stem cell and its application | |
| CN103865873B (en) | The allochthon of Subaerial blue green algae secretion and its application | |
| CN104928244B (en) | cell culture medium and its preparation method and application | |
| CN104928231B (en) | cell culture medium and its preparation method and application | |
| CN105456293A (en) | Stem cell-based medicinal product for treating diabetes and preparing method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20170707 Address after: 241000, A-15 building, Service Outsourcing Industrial Park, 717 South Zhongshan Road, Wuhu hi tech Development Zone, Anhui, China Applicant after: Zhang Jingfang Address before: 241000, A-15 building, Service Outsourcing Industrial Park, 717 South Zhongshan Road, Wuhu hi tech Development Zone, Anhui, China Applicant before: WUHU INNO BIOTECHNOLOGY CO., LTD. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20191126 Address after: 315040 S-216, Building 9, Yunsheng Science and Technology Industrial Park, No. 2, 189 Lane, Canghai Road, Ningbo High-tech Zone, Zhejiang Province Patentee after: Ningbo Yinuo Biotechnology Co., Ltd. Address before: 241000, A-15 building, Service Outsourcing Industrial Park, 717 South Zhongshan Road, Wuhu hi tech Development Zone, Anhui, China Patentee before: Zhang Jingfang |