CN104928230A - Vascular endothelial cell culture method - Google Patents
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Abstract
The invention discloses a vascular endothelial cell culture method. The method comprises the following steps: 1) differentiating pluripotent stem cells into mesendoderm precursor cells in a culture medium A; 2) differentiating the mesendoderm precursor cells into progenitor cells of vascular endothelial cells in a culture medium B; 3) differentiating the progenitor cells of the vascular endothelial cells into the vascular endothelial cells in a culture medium C. The culture medium A contains a DMEM, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, an activin A , bone morphogenetic protein-4 and a glycogen synthase kinase-3 inhibitor; the culture medium B contains a DMEM, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, a vascular endothelial growth factor and a transforming growth factor beta signaling pathway inhibitor; the culture medium C contains a DMEM, an F12 culture medium, sodium selenate, sodium bicarbonate, vitamin C, insulin, a vascular endothelial growth factor, an epidermal growth factor and a fibroblast growth factor. According to the method, the pluripotent stem cells can be differentiated into the vascular endothelial cells.
Description
Technical field
The present invention relates to the cultivation of cell, particularly, relate to a kind of cultural method of vascular endothelial cell.
Background technology
Stem cell refer to can self, divide and be divided into a kind of cell of other cell population.From the stage that stem cell is residing growth, stem cell can be divided into embryonic stem cell (embryonic stem cell, ES cell) and adult stem cell (adult stem cell).Embryonic stem cell is a kind of multipotential stem cell (pluripotent stem cell), can infinite multiplication be divided into human body 200 broad variety cell, form the tissue of body and organ, the stem cell of the potential of various kinds of cell and tissue can be divided into.Within 1981, Evans and Kaufman is from mouse inner cell mass (inner cell mass, ICM) since Isolation of Embryonic Stem Cell, ES cell research becomes the focus of various countries scientist research, and the research being divided into other cell type around stem cell directional in recent years occurs (1981 like the mushrooms after rain; Nature292 (5819): 154-6).Thomson in 1998 is successfully separated, cultivator embryonic stem cell-like, and the physianthropy application for stem cell provides may (1998; Science 282 (5391): 1145-7).The research (2006 more etc. taking the lead in reporting iPS cell is stretched in the mountain of Kyoto Univ Japan in 2006; Cell126 (4): 663-76), they by importing four kinds of transcription factor OSKM (Oct4 in the mouse skin inoblast of terminal differentiation, Sox2, Klf4, c-Myc), the cell of terminal differentiation can be induced to become stem cell state, i.e. iPS cell (induced pluripotent stem cell), after this occur the Technology Ways of various manufacture iPS cell.The multipotential stem cell of artificial induction has other features such as the differentiation capability similar to embryonic stem cell, epigenetic modification and cellular form, and the research for stem cell provides new thinking.
Stem cell has multiple differentiation potential, can preserve for a long time in vitro, has self and infinite multiplication potentiality.These features of stem cell are that scientific research and medical use provide good cell derived.The main research and apply direction of current stem cell mainly comprises: 1) cell therapy, have been reported the cell that stem cell directional can be divided into the other types such as liver cell (notification number is the patent of CN102666853A) and myocardial cell's (notification number is the patent of CN103602633A and CN104293730A), for cell therapy provides a large amount of cell deriveds having physiological function; 2) regenerative medicine field, such as the organ transplantation of conventional regeneration medical science is by donor person organ transplantation in acceptor body, and this transplanting means can produce a lot of problem, as immunological rejection and infection etc.Be divided into the ripe organ with physiological function by the stem cell directional of external patient artificial induction oneself and can effectively solve these negative effects, for regenerative medicine provides novel method.3) drug development, external evoked stem cell directional adds various medicine after being divided into specific cell type, and drugs, to the toxicity of particular organization or cell, saves a large amount of research cost, for clinical application provides mass data.
Blood vessel is the important channel being responsible for transport blood, oxygen and nutritive substance etc. in body, blood vessel goes wrong, tissue and organ failure may be caused even downright bad and threat to life, as vascular endothelial cell damage can the generation of the vascular disease such as induced hypertension and coronary heart disease.Cardiovascular disorder is the common disease threatening human health, is positioned at the first place of China's disease incidence.Meanwhile, scientific research also needs a large amount of blood vessel, for medicine provides material to vascular tissue injury's experiment.In addition, external artificial histoorgan needs blood vessel to transport nutritive substance provides nutrient for organ growth.In addition, clinical and regenerative medicine field is also in the urgent need to various vascular tissues cell.In view of the foregoing, how to obtain a large amount of vascular tissue's cells in vitro and seem very important.Multipotential stem cell can provide a large amount of seed cells for us, directed differentiation can become vascular endothelial cell (endothelial cell) and progenitor cell (endothelial progenitor cell), thus a large amount of cell can be provided, for external structure organize models provides vascular cell to originate, also can provide good material for drug screening and drug toxicity research for repairing and treating.
At present, often containing animal source composition in the substratum used in the culturing process of multipotential stem cell, especially the cultivation of Endothelial Cell progenitor cell, as used the animal serum in MEF (mouse embryo fibroblasts, Mouse Embryonic Fibroblast) trophocyte and substratum.Although above-mentioned substratum can ensure that stem cell and differentiation-inducing cell thereof have excellent self-renewal capacity, but owing to introducing a large amount of animal derived material in substratum, bring uncertain factor to stem cell and differentiation-inducing cell self-renewal thereof and atomization, add error; Animal derived pollution may be introduced, as mycoplasma contamination etc. simultaneously; In addition, in the process of cell therapy, also immune response can be brought out.
Summary of the invention
The object of this invention is to provide a kind of cultural method of vascular endothelial cell, what use in this cultural method does not contain animal source composition, and this substratum can provide enough nutritive substances and stable living environment for culturing cell, thus can efficiently by differentiation-inducing for multipotential stem cell formation vascular endothelial cell, and this vascular endothelial cell be formed with the vessel-like grid being highly similar to Human Umbilical Cord's vascular endothelial cell.
To achieve these goals, the invention provides a kind of cultural method of vascular endothelial cell, comprising:
1) by multipotential stem cell differentiation-inducing formation mesendoderm precursors cell in culture medium A;
2) by mesendoderm precursors cell differentiation-inducing formation vascular endothelial cell progenitor cell in substratum B;
3) by vascular endothelial cell progenitor cell differentiation-inducing formation vascular endothelial cell in culture medium C;
Wherein, culture medium A contains DMEM substratum, F12 substratum, sodium selenate, sodium bicarbonate, vitamins C, Regular Insulin, activin A, bone morphogenetic protein 4, GSK-3 inhibitor; Substratum B contains DMEM substratum, F12 substratum, sodium selenate, sodium bicarbonate, vitamins C, Regular Insulin, vascular endothelial growth factor and transforming growth factor-beta signal pathway inhibitor; Culture medium C contains DMEM substratum, F12 substratum, sodium selenate, sodium bicarbonate, vitamins C, Regular Insulin, vascular endothelial growth factor, Urogastron and fibroblast growth factor.
Pass through technique scheme, substratum in cultural method provided by the invention comprises culture medium A, substratum B and culture medium C, in these three kinds of substratum, each component can provide enough nutritive substances and stable living environment to make culturing cell have excellent self-renewal capacity to culturing cell by synergy, do not use animal source composition simultaneously in this substratum and then effectively evaded because animal derived material brings uncertain factor and the generation to the situation that culturing cell pollutes to the self of culturing cell and atomization, and can avoid completely bringing out immunoreactive generation in the process of cell therapy.On the basis of the above, human pluripotent stem cell can be induced to differentiate into vascular endothelial cell by method provided by the invention effectively, and then make vascular endothelial cell can realize large-scale industrial application, and this vascular endothelial cell progenitor cell is formed with the vessel-like grid being highly similar to Human Umbilical Cord's vascular endothelial cell, and then making this vascular endothelial cell progenitor cell can at cell therapy, vitro tissue builds, external graft builds, be able to widespread use in drug screening and Vascular Biology research.
Other features and advantages of the present invention are described in detail in embodiment part subsequently.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, is used from explanation the present invention, but is not construed as limiting the invention with embodiment one below.In the accompanying drawings:
Fig. 1 is the morphological specificity figure of human pluripotent stem cell;
Fig. 2 is the morphological specificity figure characterizing cell D1 in test example 1 based on CD31+CD34+;
Fig. 3 is the morphological specificity figure characterizing cell E1 in test example 1 based on CD31+CD34-;
Fig. 4 is the clone figure of human pluripotent stem cell;
Fig. 5 is the detection of expression result figure of human pluripotent stem cell differentiation-inducing latter 5 days CD31 and CD34 in test example 1;
Fig. 6 is Human Umbilical Cord's Blood vessel endothelial cell line figure (as positive control);
Fig. 7 is that the class blood vessel network of cell D1 in test example 2 generates result figure;
Fig. 8 is that the class blood vessel network of cell E1 in test example 2 generates result figure;
Fig. 9 is the common detection and localization result figure of cell E1 and pericyte in test example 3.
Embodiment
Below the specific embodiment of the present invention is described in detail.Should be understood that, embodiment described herein, only for instruction and explanation of the present invention, is not limited to the present invention.
The invention provides a kind of cultural method of vascular endothelial cell, comprising:
1) by multipotential stem cell differentiation-inducing formation mesendoderm precursors cell in culture medium A;
2) by mesendoderm precursors cell differentiation-inducing formation vascular endothelial cell progenitor cell in substratum B;
3) by vascular endothelial cell progenitor cell differentiation-inducing formation vascular endothelial cell in culture medium C;
Wherein, culture medium A contains DMEM substratum, F12 substratum, sodium selenate, sodium bicarbonate, vitamins C, Regular Insulin, activin A, bone morphogenetic protein 4, GSK-3 inhibitor; Substratum B contains DMEM substratum, F12 substratum, sodium selenate, sodium bicarbonate, vitamins C, Regular Insulin, vascular endothelial growth factor and transforming growth factor-beta signal pathway inhibitor; Culture medium C contains DMEM substratum, F12 substratum, sodium selenate, sodium bicarbonate, vitamins C, Regular Insulin, vascular endothelial growth factor, Urogastron and fibroblast growth factor.
In above-mentioned culture medium A, the content of each component can be selected in wide scope, but sufficient nutrient material and more stable living environment more can be provided for culturing cell to make culture medium A, preferably, in culture medium A, the volume ratio of DMEM substratum and F12 substratum is 1:1-1:4, ascorbic concentration is 1-100 μ g/ml, the concentration of Regular Insulin is 1-100 μ g/ml, the concentration of activin A is 5-100ng/ml, the concentration of bone morphogenetic protein 4 is 1-100ng/ml, the concentration of GSK-3 inhibitor is 1-100 μM, the concentration of sodium selenate is 1-100 μ g/L, the concentration of sodium bicarbonate is 100-1000mg/L.More preferably, in culture medium A, the volume ratio of DMEM substratum and F12 substratum is 1:1-1:4, ascorbic concentration is 20-100 μ g/ml, and the concentration of Regular Insulin is 5-50 μ g/ml, and the concentration of activin A is 5-50ng/ml, the concentration of bone morphogenetic protein 4 is 1-50ng/ml, the concentration of GSK-3 inhibitor is 2-40 μM, and the concentration of sodium selenate is 1-50 μ g/L, and the concentration of sodium bicarbonate is 200-700mg/L.Further preferably, in culture medium A, the volume ratio of DMEM substratum and F12 substratum is 1:1-1:4, ascorbic concentration is 50-100 μ g/ml, and the concentration of Regular Insulin is 10-30 μ g/ml, and the concentration of activin A is 10-30ng/ml, the concentration of bone morphogenetic protein 4 is 2-10ng/ml, the concentration of GSK-3 inhibitor is 10-30 μM, and the concentration of sodium selenate is 2-30 μ g/L, and the concentration of sodium bicarbonate is 400-600mg/L.
In above-mentioned substratum B, the content of each component can be selected in wide scope, but sufficient nutrient material and more stable living environment more can be provided for culturing cell to make substratum B, preferably, the volume ratio of DMEM substratum and F12 substratum is 1:1-1:4, ascorbic concentration is 1-100 μ g/ml, the concentration of Regular Insulin is 1-100 μ g/ml, the concentration of vascular endothelial growth factor is 5-100ng/ml, the concentration of transforming growth factor-beta signal pathway inhibitor is 1-50 μM, the concentration of sodium selenate is 1-100 μ g/L, the concentration of sodium bicarbonate is 100-1000mg/L.More preferably, in substratum B, the volume ratio of DMEM substratum and F12 substratum is 1:1-1:4, ascorbic concentration is 20-100 μ g/ml, the concentration of Regular Insulin is 5-50 μ g/ml, and the concentration of vascular endothelial growth factor is 5-50ng/ml, and the concentration of transforming growth factor-beta signal pathway inhibitor is 1-20 μM, the concentration of sodium selenate is 1-50 μ g/L, and the concentration of sodium bicarbonate is 200-700mg/L.Further preferably, in substratum B, the volume ratio of DMEM substratum and F12 substratum is 1:1-1:4, ascorbic concentration is 50-100 μ g/ml, the concentration of Regular Insulin is 10-30 μ g/ml, and the concentration of vascular endothelial growth factor is 10-30ng/ml, and the concentration of transforming growth factor-beta signal pathway inhibitor is 5-10 μM, the concentration of sodium selenate is 2-30 μ g/L, and the concentration of sodium bicarbonate is 400-600mg/L.
In above-mentioned culture medium C, the content of each component can be selected in wide scope, but sufficient nutrient material and more stable living environment more can be provided for culturing cell to make culture medium C, preferably, in culture medium C, the volume ratio of DMEM substratum and F12 substratum is 1:1-1:4, ascorbic concentration is 1-100 μ g/ml, the concentration of Regular Insulin is 1-100 μ g/ml, the concentration of vascular endothelial growth factor is 5-100ng/ml, the concentration of Urogastron is 5-100ng/ml, the concentration of fibroblast growth factor is 5-100ng/ml, the concentration of sodium selenate is 1-100 μ g/L, the concentration of sodium bicarbonate is 100-1000mg/L.More preferably, in culture medium C, the volume ratio of DMEM substratum and F12 substratum is 1:1-1:4, ascorbic concentration is 20-100 μ g/ml, and the concentration of Regular Insulin is 5-50 μ g/ml, and the concentration of vascular endothelial growth factor is 5-50ng/ml, the concentration of Urogastron is 5-50ng/ml, the concentration of fibroblast growth factor is 5-50ng/ml, and the concentration of sodium selenate is 1-50 μ g/L, and the concentration of sodium bicarbonate is 200-700mg/L.Further preferably, in culture medium C, the volume ratio of DMEM substratum and F12 substratum is 1:1-1:4, ascorbic concentration is 50-100 μ g/ml, and the concentration of Regular Insulin is 10-30 μ g/ml, and the concentration of vascular endothelial growth factor is 10-30ng/ml, the concentration of Urogastron is 10-30ng/ml, the concentration of fibroblast growth factor is 10-30ng/ml, and the concentration of sodium selenate is 2-30 μ g/L, and the concentration of sodium bicarbonate is 400-600mg/L.
Meanwhile, the pH of above-mentioned culture medium A, substratum B and culture medium C all can select in wide scope, but in order to make culturing cell can have more stable living environment, preferably, the pH of culture medium A is 7-8, and the pH of substratum B is 7-8, and the pH of culture medium C is 7-8.
In addition, GSK-3 inhibitor can be the GSK-3 inhibitor of any one routine in this area, as BIO or CHIR99021, but in order to the effect making GSK-3 inhibitor can play more excellent induction regulating controlling, preferably, GSK-3 inhibitor is selected from the inhibitor that the trade mark is BIO and/or CHIR99021;
Similarly, transforming growth factor-beta signal pathway inhibitor can be the transforming growth factor-beta signal pathway inhibitor of any one routine in this area, but in order to the effect making transforming growth factor-beta signal pathway inhibitor can play more excellent induction regulating controlling, preferably, the inhibitor of transforming growth factor-beta signal pathway inhibitor to be the trade mark be SB431542.
In like manner, fibroblast growth factor can be the fibroblast growth factor of any one routine in this area, but in order to the effect making fibroblast growth factor can play more excellent induction regulating controlling, preferably, fibroblast growth factor is selected from Prostatropin (bFGF).
In order to make the survival rate improving mesendoderm precursors cell (mesodermal precursor cells and/or entoderm precursor cell), preferably, culture medium A is also containing Rho kinases inhibitor.Certainly, the concrete kind of Rho kinases inhibitor can be selected in wide scope, but in order to improve mesendoderm precursors cell further, the preferred trade mark is the inhibitor of Y27632.In addition, the consumption of Rho kinases inhibitor can be selected in wide scope, and consider from the effect improving cell survival rate, more preferably, in culture medium A, the concentration of Rho kinases inhibitor is 1-50 μM, more preferably 2-20 μM.
On the basis of the above, the differentiation-inducing condition of culturing cell can be selected in wide scope, but in order to make culturing cell can be differentiation-inducing more rapidly, preferably, in step 1) in, differentiation-inducingly to meet the following conditions: culture temperature is 35-40 DEG C, and incubation time is 1-3 days; In step 2) in, differentiation-inducingly to meet the following conditions: culture temperature is 35-40 DEG C, and incubation time is 1-30 days; In step 3) in, differentiation-inducingly to meet the following conditions: culture temperature is 35-40 DEG C, and incubation time is 1-30 days.
In order to ensure cell living environment in there is enough nutritive substances and the metabolic product in order to know cell in time, preferably, the replacement frequency of the replacement frequency of culture medium A, the replacement frequency of substratum B and culture medium C is 1 time/15-30h independently of one another.
In addition, in the present invention, mesendoderm precursors cell can be induced to differentiate into more rapidly to make multipotential stem cell, preferably, in step 1) before, cultural method also comprises by multipotential stem cell digestion to individual layer multipotential stem cell, this is because can contact with substratum more with the multipotential stem cell of individual layer existence, and then promotes differentiation-inducing.
Wherein, the method of cell dissociation can be the method for any one routine in this area, as enzyme digestion, ion chelating agent method, Physical and cold method, be preferably enzyme digestion, be specially degrees of fusion is that the multipotential stem cell of 70-80% is by PBS buffer solution for cleaning, then by tryptic digestion, then postdigestive monolayer cell is transferred to carry out in culture dish differentiation-inducing.The actual conditions of tryptic digestion can be selected in wide scope, but in order to improve digestive efficiency further, preferably, digestion meets the following conditions: digestion time is 3-6min.In addition, be transferred to by postdigestive monolayer cell in the process in culture dish, the consumption of monolayer cell can be selected in wide scope, but consider from differentiation-inducing effect, preferably, with the area of the upper surface of culture dish for benchmark, the footprint area of monolayer cell is 0.1-0.2.
In the present invention, in order to more be conducive to the adhesion of culturing cell, growth, transfer and differentiation, preferably, in step 1) before, cultural method also comprises Matrigel matrigel or vitronectin bag by substratum.
In addition, in the present invention, the concrete kind of multipotential stem cell can be selected in wide scope, it can be any one multipotential stem cell in this area, but consider from the range of application of obtained vascular endothelial cell progenitor cell, preferably, multipotential stem cell is human pluripotent stem cell.
Below will be described the present invention by embodiment.In following examples and preparation example, DMEM cultivates the commercially available product for Thermo Fisher Scientific company, F12 substratum is the commercially available product of ThermoFisher Scientific company, vitamins C, Regular Insulin is the commercially available product of Sigma Aldrich company, vascular endothelial growth factor is the commercially available product of R & D Biosystems company, transforming growth factor-beta signal pathway inhibitor is the Sigma Aldrich company trade mark is the commercially available product of SB431542, activin A is the commercially available product of R & D Biosystems company, bone morphogenetic protein 4 is the commercially available product of R & D Biosystems company, GSK-3 is the trade mark of Sigma Aldrich company is the commercially available product of BIO, the trade mark that the protein kinase (ROCK inhibitor) that Rho is relevant is Sigma Aldrich company is the commercially available product of Y27632, Urogastron is the commercially available product of R & D Biosystems company, fibroblast growth factor is the trade mark of R & D Biosystems company is the commercially available product of bFGF.
Preparation example 1
1) by obtained for the mixing of DMEM substratum, F12 substratum, sodium selenate, vitamins C, sodium bicarbonate, Regular Insulin, activin A, bone morphogenetic protein 4, Rho kinases inhibitor (trade mark is Y27632), GSK-3 inhibitor (trade mark is CHIR99021) and GSK-3 inhibitor (trade mark is BIO) mixture W1; Wherein, the volume ratio of DMEM substratum and F12 substratum is 1:2, ascorbic concentration is 50 μ g/ml, the concentration of Regular Insulin is 50 μ g/ml, the concentration of activin A is 55ng/ml, the concentration of bone morphogenetic protein 4 is 60ng/ml, the concentration of GSK-3 inhibitor (trade mark is CHIR99021) is 30 μMs, the concentration of GSK-3 inhibitor (trade mark is BIO) is 25 μMs, the concentration of sodium selenate is 20 μ g/L, and the concentration of sodium bicarbonate is 500mg/L.
2) sodium hydroxide is added in said mixture W1 pH to be adjusted to 7.8 obtained mixture W2;
3) said mixture W2 is obtained cell culture medium A1 by the membrane filtration sterilizing with 0.2 μm of diameter micropore.
Preparation example 2
Carry out obtained cell culture medium A2 according to the method for preparation example 1, difference is step 1) in do not use Rho kinases inhibitor, and the concentration of Rho kinases inhibitor is 10 μMs.
Preparation example 3
1) by obtained for the mixing of DMEM substratum, F12 substratum, sodium selenate, vitamins C, sodium bicarbonate, Regular Insulin, activin A, bone morphogenetic protein 4, Rho kinases inhibitor (trade mark is Y27632), GSK-3 inhibitor (trade mark is CHIR99021) and GSK-3 inhibitor (trade mark is BIO) mixture W1; Wherein, the volume ratio of DMEM substratum and F12 substratum is 1:1, ascorbic concentration is 5 μ g/ml, the concentration of Regular Insulin is 3 μ g/ml, the concentration of activin A is 7ng/ml, the concentration of bone morphogenetic protein 4 is 3ng/ml, the concentration of GSK-3 inhibitor (trade mark is CHIR99021) is 2 μMs, the concentration of GSK-3 inhibitor (trade mark is BIO) is 1 μM, the concentration of sodium selenate is 2 μ g/L, the concentration of sodium bicarbonate is the concentration of 400mg/L, Rho kinases inhibitor (trade mark is Y27632) is 2 μMs.
2) sodium hydroxide is added in said mixture W1 pH to be adjusted to 7.8 obtained mixture W2;
3) said mixture W2 is obtained cell culture medium A3 by the membrane filtration sterilizing with 0.2 μm of diameter micropore.
Preparation example 4
1) by obtained for the mixing of DMEM substratum, F12 substratum, sodium selenate, vitamins C, sodium bicarbonate, Regular Insulin, activin A, bone morphogenetic protein 4, Rho kinases inhibitor (trade mark is Y27632), GSK-3 inhibitor (trade mark is CHIR99021) and GSK-3 inhibitor (trade mark is BIO) mixture W1, wherein, the volume ratio of DMEM substratum and F12 substratum is 1:4, ascorbic concentration is 80 μ g/ml, the concentration of Regular Insulin is 90 μ g/ml, the concentration of activin A is 95ng/ml, the concentration of bone morphogenetic protein 4 is 85ng/ml, the concentration of GSK-3 inhibitor (trade mark is CHIR99021) is 45 μMs, the concentration of GSK-3 inhibitor (trade mark is BIO) is 50 μMs, the concentration of sodium selenate is 30 μ g/L, the concentration of sodium bicarbonate is 600mg/L, the concentration of Rho kinases inhibitor (trade mark is Y27632) is 20 μMs.
2) sodium hydroxide is added in said mixture W1 pH to be adjusted to 7.8 obtained mixture W2;
3) said mixture W2 is obtained cell culture medium A4 by the membrane filtration sterilizing with 0.2 μm of diameter micropore.
Preparation example 5
A, be that the transforming growth factor-beta signal pathway inhibitor mixing of SB431542 fully obtains mixture W3 by DMEM substratum, F12 substratum, vitamins C, sodium selenate, 500mg sodium bicarbonate, Regular Insulin, vascular endothelial growth factor and the trade mark; Wherein, the volume ratio of DMEM substratum and F12 substratum is 1:2, ascorbic concentration is 50 μ g/ml, the concentration of Regular Insulin is 55 μ g/ml, the concentration of vascular endothelial growth factor is 45ng/ml, the concentration of transforming growth factor-beta signal pathway inhibitor is 25 μMs, and the concentration of sodium selenate is 20 μ g/L, and the concentration of sodium bicarbonate is 500mg/L.
B, sodium hydroxide to be added in said mixture W3 pH to be adjusted to 7.8 obtained mixture W4;
C, said mixture W4 is obtained cell culture medium B1 by the membrane filtration sterilizing with 0.2 μm of diameter micropore.
Preparation example 6
A, be that the transforming growth factor-beta signal pathway inhibitor mixing of SB431542 fully obtains mixture W3 by DMEM substratum, F12 substratum, vitamins C, sodium selenate, 500mg sodium bicarbonate, Regular Insulin, vascular endothelial growth factor and the trade mark; Wherein, the volume ratio of DMEM substratum and F12 substratum is 1:1, ascorbic concentration is 2 μ g/ml, the concentration of Regular Insulin is 4 μ g/ml, the concentration of vascular endothelial growth factor is 5ng/ml, the concentration of transforming growth factor-beta signal pathway inhibitor is 3 μMs, and the concentration of sodium selenate is 2 μ g/L, and the concentration of sodium bicarbonate is 200mg/L.
B, sodium hydroxide to be added in said mixture W3 pH to be adjusted to 7.8 obtained mixture W4;
C, said mixture W4 is obtained cell culture medium B2 by the membrane filtration sterilizing with 0.2 μm of diameter micropore.
Preparation example 7
A, be that the transforming growth factor-beta signal pathway inhibitor mixing of SB431542 fully obtains mixture W3 by DMEM substratum, F12 substratum, vitamins C, sodium selenate, 500mg sodium bicarbonate, Regular Insulin, vascular endothelial growth factor and the trade mark; Wherein, the volume ratio of DMEM substratum and F12 substratum is 1:4, ascorbic concentration is 90 μ g/ml, the concentration of Regular Insulin is 95 μ g/ml, the concentration of vascular endothelial growth factor is 100ng/ml, the concentration of transforming growth factor-beta signal pathway inhibitor is 45 μMs, and the concentration of sodium selenate is 50 μ g/L, and the concentration of sodium bicarbonate is 600mg/L.
B, sodium hydroxide to be added in said mixture W3 pH to be adjusted to 7.8 obtained mixture W4;
C, said mixture W4 is obtained cell culture medium B3 by the membrane filtration sterilizing with 0.2 μm of diameter micropore.
Preparation example 8
A, DMEM substratum, F12 substratum, vitamins C, sodium selenate, sodium bicarbonate, Regular Insulin, vascular endothelial growth factor, Urogastron and fibroblast growth factor (trade mark is bFGF) are mixed to get mixture W5; Wherein, the volume ratio of DMEM substratum and F12 substratum is 1:2, ascorbic concentration is 55 μ g/ml, the concentration of Regular Insulin is 60 μ g/ml, the concentration of vascular endothelial growth factor is 65ng/ml, and the concentration of Urogastron is 50ng/ml, and the concentration of fibroblast growth factor is 45ng/ml, the concentration of sodium selenate is 20 μ g/L, and the concentration of sodium bicarbonate is 500mg/L.
B, sodium hydroxide to be added in said mixture W5 pH to be adjusted to 7.8 obtained mixture W6;
C, said mixture W6 is obtained cell culture medium C1 by the membrane filtration sterilizing with 0.2 μm of diameter micropore.
Preparation example 9
A, DMEM substratum, F12 substratum, vitamins C, sodium selenate, sodium bicarbonate, Regular Insulin, vascular endothelial growth factor, Urogastron and fibroblast growth factor (trade mark is bFGF) are mixed to get mixture W5; Wherein, the volume ratio of DMEM substratum and F12 substratum is 1:1, ascorbic concentration is 2 μ g/ml, the concentration of Regular Insulin is 3 μ g/ml, the concentration of vascular endothelial growth factor is 7ng/ml, and the concentration of Urogastron is 8ng/ml, and the concentration of fibroblast growth factor is 8ng/ml, the concentration of sodium selenate is 2 μ g/L, and the concentration of sodium bicarbonate is 400mg/L.
B, sodium hydroxide to be added in said mixture W5 pH to be adjusted to 7.8 obtained mixture W6;
C, said mixture W6 is obtained cell culture medium C2 by the membrane filtration sterilizing with 0.2 μm of diameter micropore.
Preparation example 10
A, DMEM substratum, F12 substratum, vitamins C, sodium selenate, sodium bicarbonate, Regular Insulin, vascular endothelial growth factor, Urogastron and fibroblast growth factor (trade mark is bFGF) are mixed to get mixture W5; Wherein, the volume ratio of DMEM substratum and F12 substratum is 1:4, ascorbic concentration is 90 μ g/ml, the concentration of Regular Insulin is 100 μ g/ml, the concentration of vascular endothelial growth factor is 95ng/ml, and the concentration of Urogastron is 85ng/ml, and the concentration of fibroblast growth factor is 90ng/ml, the concentration of sodium selenate is 40 μ g/L, and the concentration of sodium bicarbonate is 600mg/L.
B, sodium hydroxide to be added in said mixture W5 pH to be adjusted to 7.8 obtained mixture W6;
C, said mixture W6 is obtained cell culture medium C3 by the membrane filtration sterilizing with 0.2 μm of diameter micropore.
Embodiment 1
1) culture dish Matrigel matrigel is carried out bag by (certainly use glass connect Saliva Orthana Vitronection also can) 2h; Then to recover in the water-bath of 37 DEG C frozen human pluripotent stem cell, and this cell is seeded in above-mentioned substratum, then add E8 multipotential stem cell nutrient solution (purchased from LifeTechnologies company) to cultivate at 37 DEG C, and every day replaced medium, until when proliferation of pluripotent stem cells is to 80% degrees of fusion (confluency), then had digestive transfer culture is carried out to maintain the state of human pluripotent stem cells cell mass with 0.5mM EDTA (PH=8.0, osmotic pressure (Osmolarity)=340mOsm).When needs are unicellular go down to posterity time, application Trypsin enzyme (certain TrypLE Express enzyme also can) digestion, and in substratum, add Rock inhibitor that the trade mark is Y-27632 (working concentration is 10 μMs, improves the survival rate of cell) cultivate 24h.
2) substratum is removed, 3 times are cleaned with PBS buffered soln, then use Trypsin (trypsinase) peptic cell 5 minutes to unicellular, reach new using in the culture dish of Matrigel matrigel or Vitronectin bag quilt with area ratio 1:8, add cell culture medium A2 and cultivate 1 day at 37 DEG C; Then change cell culture medium A1 at 37 DEG C, cultivate 1 day obtained mesendoderm precursors cell.
3) at 37 DEG C, above-mentioned mesendoderm precursors cell is cultivated in cell culture medium B1 and within 3 days, obtains cell D1, wherein, every day replaced medium.
4) at 37 DEG C, above-mentioned cell D1 is cultivated in cell culture medium C1 and within 5 days, obtains cell E1, wherein, every day replaced medium.
Embodiment 2
Carry out cultivation according to the method for embodiment 1 and obtain cell E2, difference changes cell culture medium A1 into cell culture medium A3.
Embodiment 3
Carry out cultivation according to the method for embodiment 1 and obtain cell E3, difference changes cell culture medium A1 into cell culture medium A4.
Embodiment 4
Carry out cultivation according to the method for embodiment 1 and obtain cell E4, difference changes cell culture medium B1 into cell culture medium B2.
Embodiment 5
Carry out cultivation according to the method for embodiment 1 and obtain cell E5, difference changes cell culture medium B1 into cell culture medium B3.
Embodiment 6
Carry out cultivation according to the method for embodiment 1 and obtain cell E6, difference changes cell culture medium C1 into cell culture medium C2.
Embodiment 7
Carry out cultivation according to the method for embodiment 1 and obtain cell E7, difference changes cell culture medium C1 into cell culture medium C3.
Comparative example 1
Carry out cultivation according to the method for embodiment 1 and obtain cell E8, difference is that cell culture medium A1 is not containing activin A.
Comparative example 2
Carry out cultivation according to the method for embodiment 1 and obtain cell E9, difference is that cell culture medium A1 is not containing bone morphogenetic protein 4.
Comparative example 3
Carry out cultivation according to the method for embodiment 1 and obtain cell E10, difference is that cell culture medium A1 is not containing sodium selenate.
Comparative example 4
Carry out cultivation according to the method for embodiment 1 and obtain cell E11, difference is that cell culture medium B1 is not containing vascular endothelial growth factor.
Comparative example 5
Carry out cultivation according to the method for embodiment 1 and obtain cell E12, difference is that cell culture medium B1 is not containing sodium selenate.
Comparative example 6
Carry out cultivation according to the method for embodiment 1 and obtain cell E13, difference is that cell culture medium B1 is not containing transforming growth factor-beta signal pathway inhibitor.
Comparative example 7
Carry out cultivation according to the method for embodiment 1 and obtain cell E14, difference is that cell culture medium C1 is not containing Urogastron.
Comparative example 8
Carry out cultivation according to the method for embodiment 1 and obtain cell E15, difference is that cell culture medium C1 is not containing sodium selenate.
Comparative example 9
Carry out cultivation according to the method for embodiment 1 and obtain cell E16, difference is that cell culture medium C1 is not containing vascular endothelial growth factor.
Test example 1
First, digest above-mentioned cell D1 or cell E1 to unicellular with trypsin, jiggle Eddy diffusion with PBS buffered soln and count.Get the cell of 80,000 single-cell suspensions, centrifugal 3 minutes of 800g, abandons supernatant liquor, adds the PBS buffered soln that 100ul contains 1%BSA (bovine serum albumin), jiggles Eddy diffusion cell.Add each and detect antibody (CD31 and CD34), mix gently.At 4 DEG C, lucifuge hatches 30 minutes; Then centrifugal 5 minutes of 800g, abandon supernatant, add the PBS that 100ul contains 1%BSA, jiggle Eddy diffusion cell, last flow cytometer detects.Detected result is shown in Fig. 2 and Fig. 3.
Wherein, Fig. 1 is the morphological specificity figure of human pluripotent stem cell, Fig. 2 is the morphological specificity figure characterizing cell D1 based on CD31+CD34+, Fig. 3 is the morphological specificity figure characterizing cell E1 based on CD31+CD34-, as seen from the figure, cell D1 is successfully induced to differentiate into vascular endothelial cell progenitor cell, and cell E1 is successfully induced to differentiate into vascular endothelial cell.
Fig. 4 is the clone figure of human pluripotent stem cell, Fig. 5 is the detection of expression result figure of human pluripotent stem cell differentiation-inducing latter 5 days CD31 and CD34, wherein, the antibody of CD31 and PE fluorophor (Phycoerythrin) coupling, the antibody of CD34 and APC protein fluorescence group (Allophycocyanin) coupling.Because CD31 and CD34 can carry out coupling with the labelled protein on vascular endothelial cell and progenitor cell surface thereof, and then make vascular endothelial cell and progenitor cell surface thereof obtain fluorescence or magnetic, thus make vascular endothelial cell and progenitor cell thereof can identified in flow cytometer, be separated or enrichment.Known by Fig. 4 and Fig. 5, in the cell after differentiation, the vascular endothelial cell progenitor cell that a part is expressed for CD31+CD34+, another part is the vascular endothelial cell that CD31+CD34-expresses.
Similarly, known by the method for test example 1, cell E2-E7 is successfully induced to differentiate into vascular endothelial cell, and the unsuccessful vascular endothelial cell that is induced to differentiate into of cell E8-E16, thus multipotential stem cell successfully can be induced to differentiate into vascular endothelial cell by explanation cell culture medium A, cell culture medium B and cell culture medium C.
Test example 2
First, Matrigel matrigel is hatched at 4 DEG C and within 3 hours, becomes flowing shape semi-solid state, and be positioned on ice.Then in Tissue Culture Dish, lay one deck Matrigel matrigel, at 37 DEG C, treat that it solidifies.Cell D1 or cell E1 is laid on Matrigel after solidification, adds substratum and generate situation by microscopic examination class blood vessel network after cultivating 4 days.Detected result is shown in Fig. 7 and Fig. 8.
Wherein, the class blood vessel network of Fig. 6 to be Human Umbilical Cord's Blood vessel endothelial cell line figure (as positive control), Fig. 7 be cell D1 generates result figure, Fig. 8 is that the class blood vessel network of cell E1 generates result figure.Above-mentioned three figure contrast known, cell D1 with E1 is all formed with the similar angioid grid similar to Human Umbilical Cord's vascular endothelial cell, further illustrates cell culture medium A, cell culture medium B and cell culture medium C and multipotential stem cell successfully can be induced to differentiate into vascular endothelial cell.
Similarly, known by the method for test example 2, cell E2-E7 is formed with the similar angioid grid similar to Human Umbilical Cord's vascular endothelial cell, and cell E8-E16 is not formed with the similar angioid grid similar to Human Umbilical Cord's vascular endothelial cell, thus further illustrates cell culture medium A, cell culture medium B and cell culture medium C and multipotential stem cell successfully can be induced to differentiate into vascular endothelial cell.
Test example 3
By cell E1 and pericyte Dual culture on matrigel matrigel jointly, the results are shown in Figure 9.As known in the figure, cell E1 and pericyte form similar angioid network, and two kinds of cells have significant location altogether, show the function that vascular endothelial cell recruits pericyte, thus the vascular endothelial cell describing cell culture medium provided by the invention cultivation obtained can be able to widespread use.
Similarly, known by the method for test example 3, cell E2-E7 can form similar angioid network with pericyte, and two kinds of cells have significant location altogether; And cell E8-E16 fails to form similar angioid network with pericyte.Thus the vascular endothelial cell further illustrating cell culture medium provided by the invention cultivation obtained can be able to widespread use.
More than describe the preferred embodiment of the present invention in detail; but the present invention is not limited to the detail in above-mentioned embodiment, within the scope of technical conceive of the present invention; can carry out multiple simple variant to technical scheme of the present invention, these simple variant all belong to protection scope of the present invention.
It should be noted that in addition, each concrete technical characteristic described in above-mentioned embodiment, in reconcilable situation, can be combined by any suitable mode, in order to avoid unnecessary repetition, the present invention illustrates no longer separately to various possible array mode.
In addition, also can carry out arbitrary combination between various different embodiment of the present invention, as long as it is without prejudice to thought of the present invention, it should be considered as content disclosed in this invention equally.
Claims (10)
1. a cultural method for vascular endothelial cell, is characterized in that, described cultural method comprises:
1) by multipotential stem cell differentiation-inducing formation mesendoderm precursors cell in culture medium A;
2) by described mesendoderm precursors cell differentiation-inducing formation vascular endothelial cell progenitor cell in substratum B;
3) by described vascular endothelial cell progenitor cell differentiation-inducing formation vascular endothelial cell in culture medium C;
Wherein, described culture medium A contains DMEM substratum, F12 substratum, sodium selenate, sodium bicarbonate, vitamins C, Regular Insulin, activin A, bone morphogenetic protein 4, GSK-3 inhibitor; Described substratum B contains DMEM substratum, F12 substratum, sodium selenate, sodium bicarbonate, vitamins C, Regular Insulin, vascular endothelial growth factor and transforming growth factor-beta signal pathway inhibitor; Described culture medium C contains DMEM substratum, F12 substratum, sodium selenate, sodium bicarbonate, vitamins C, Regular Insulin, vascular endothelial growth factor, Urogastron and fibroblast growth factor.
2. cultural method according to claim 1, wherein, in described culture medium A, the volume ratio of described DMEM substratum and F12 substratum is 1:1-1:4, described ascorbic concentration is 1-100 μ g/ml, the concentration of described Regular Insulin is 1-100 μ g/ml, the concentration of described activin A is 5-100ng/ml, the concentration of described bone morphogenetic protein 4 is 1-100ng/ml, the concentration of described GSK-3 inhibitor is 1-100 μM, the concentration of described sodium selenate is 1-100 μ g/L, and the concentration of described sodium bicarbonate is 100-1000mg/L;
In described substratum B, the volume ratio of described DMEM substratum and F12 substratum is 1:1-1:4, described ascorbic concentration is 1-100 μ g/ml, the concentration of described Regular Insulin is 1-100 μ g/ml, the concentration of described vascular endothelial growth factor is 5-100ng/ml, the concentration of described transforming growth factor-beta signal pathway inhibitor is 1-50 μM, and the concentration of described sodium selenate is 1-100 μ g/L, and the concentration of described sodium bicarbonate is 100-1000mg/L;
In described culture medium C, the volume ratio of described DMEM substratum and F12 substratum is 1:1-1:4, described ascorbic concentration is 1-100 μ g/ml, the concentration of described Regular Insulin is 1-100 μ g/ml, the concentration of described vascular endothelial growth factor is 5-100ng/ml, the concentration of described Urogastron is 5-100ng/ml, the concentration of described fibroblast growth factor is 5-100ng/ml, the concentration of described sodium selenate is 1-100 μ g/L, and the concentration of described sodium bicarbonate is 100-1000mg/L.
3. cultural method according to claim 2, wherein, in described culture medium A, the volume ratio of described DMEM substratum and F12 substratum is 1:1-1:4, described ascorbic concentration is 20-100 μ g/ml, the concentration of described Regular Insulin is 5-50 μ g/ml, the concentration of described activin A is 5-50ng/ml, the concentration of described bone morphogenetic protein 4 is 1-50ng/ml, the concentration of described GSK-3 inhibitor is 2-40 μM, the concentration of described sodium selenate is 1-50 μ g/L, and the concentration of described sodium bicarbonate is 200-700mg/L;
In described substratum B, the volume ratio of described DMEM substratum and F12 substratum is 1:1-1:4, described ascorbic concentration is 20-100 μ g/ml, the concentration of described Regular Insulin is 5-50 μ g/ml, the concentration of described vascular endothelial growth factor is 5-50ng/ml, the concentration of described transforming growth factor-beta signal pathway inhibitor is 1-20 μM, and the concentration of described sodium selenate is 1-50 μ g/L, and the concentration of described sodium bicarbonate is 200-700mg/L;
In described culture medium C, the volume ratio of described DMEM substratum and F12 substratum is 1:1-1:4, described ascorbic concentration is 20-100 μ g/ml, the concentration of described Regular Insulin is 5-50 μ g/ml, the concentration of described vascular endothelial growth factor is 5-50ng/ml, and the concentration of described Urogastron is 5-50ng/ml, and the concentration of described fibroblast growth factor is 5-50ng/ml, the concentration of described sodium selenate is 1-50 μ g/L, and the concentration of described sodium bicarbonate is 200-700mg/L.
4. cultural method according to claim 3, wherein, in described culture medium A, the volume ratio of described DMEM substratum and F12 substratum is 1:1-1:4, described ascorbic concentration is 50-100 μ g/ml, the concentration of described Regular Insulin is 10-30 μ g/ml, the concentration of described activin A is 10-30ng/ml, the concentration of described bone morphogenetic protein 4 is 2-10ng/ml, the concentration of described GSK-3 inhibitor is 10-30 μM, the concentration of described sodium selenate is 2-30 μ g/L, and the concentration of described sodium bicarbonate is 400-600mg/L;
In described substratum B, the volume ratio of described DMEM substratum and F12 substratum is 1:1-1:4, described ascorbic concentration is 50-100 μ g/ml, the concentration of described Regular Insulin is 10-30 μ g/ml, the concentration of described vascular endothelial growth factor is 10-30ng/ml, the concentration of described transforming growth factor-beta signal pathway inhibitor is 5-10 μM, and the concentration of described sodium selenate is 2-30 μ g/L, and the concentration of described sodium bicarbonate is 400-600mg/L;
In described culture medium C, the volume ratio of described DMEM substratum and F12 substratum is 1:1-1:4, described ascorbic concentration is 50-100 μ g/ml, the concentration of described Regular Insulin is 10-30 μ g/ml, the concentration of described vascular endothelial growth factor is 10-30ng/ml, and the concentration of described Urogastron is 10-30ng/ml, and the concentration of described fibroblast growth factor is 10-30ng/ml, the concentration of described sodium selenate is 2-30 μ g/L, and the concentration of described sodium bicarbonate is 400-600mg/L.
5. according to the cultural method in claim 1-3 described in any one, wherein, described GSK-3 inhibitor is selected from the inhibitor that the trade mark is BIO and/or CHIR99021;
Preferably, the inhibitor of described transforming growth factor-beta signal pathway inhibitor to be the trade mark be SB431542;
More preferably, described fibroblast growth factor is selected from Prostatropin.
6. according to the cultural method in claim 1-3 described in any one, wherein, described culture medium A is also containing Rho kinases inhibitor, and the preferred trade mark is the inhibitor of Y27632.
7. cultural method according to claim 6, wherein, the concentration of described Rho kinases inhibitor is 1-50 μM.
8. according to the cultural method in claim 1-3 described in any one, wherein, in step 1) before, described cultural method also comprises multipotential stem cell digestion to individual layer multipotential stem cell;
Preferably, by degrees of fusion be the multipotential stem cell of 70-80% by PBS buffer solution for cleaning, then by tryptic digestion, then lower postdigestive monolayer cell is transferred to carry out in culture dish differentiation-inducing.
9. cultural method according to claim 8, wherein, described digestion meets the following conditions: digestion time is 3-6min;
Preferably, with the area of the upper surface of described culture dish for benchmark, the footprint area of described monolayer cell is 0.1-0.2.
10. cultural method according to claim 8, wherein, in step 1) before, described cultural method also comprises Matrigel matrigel or vitronectin bag by culture vessel;
Preferably, described multipotential stem cell is human pluripotent stem cell.
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| CN105969720A (en) * | 2016-07-29 | 2016-09-28 | 上海瑞鹿生物技术有限公司 | Human vascular endothelial cell culture solution and culture method |
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| 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 |
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| 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 |
| CN112662627A (en) * | 2021-01-19 | 2021-04-16 | 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 |
| 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 |
| CN114540278A (en) * | 2022-03-25 | 2022-05-27 | 青岛大学 | Microvessel in-vitro culture method and culture solution |
| CN114540278B (en) * | 2022-03-25 | 2023-11-21 | 青岛大学 | Microvascular in-vitro culture method and culture solution |
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