CN101649304A - Method for inducing and dividing embryonic stem cells into hepatic cells - Google Patents
Method for inducing and dividing embryonic stem cells into hepatic cells Download PDFInfo
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- CN101649304A CN101649304A CN200910190861A CN200910190861A CN101649304A CN 101649304 A CN101649304 A CN 101649304A CN 200910190861 A CN200910190861 A CN 200910190861A CN 200910190861 A CN200910190861 A CN 200910190861A CN 101649304 A CN101649304 A CN 101649304A
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Abstract
The invention relates to a method for inducing and dividing embryonic stem cells into hepatic cells, belonging to the field of biomedicine. In the invention, the trypsinized embryonic stem cells are transferred to a bioreactor containing a culture solution, the bioreactor rotates at the speed of 15-50 revolutions/minute, and the trypsinized embryonic stem cells are directionally divided into the mature hepatic cells with full functions in a weightless state under the support of the culture solution in the bioreactor. The whole technique and the method for inducing and dividing the embryonic stem cells into the hepatic cells are not only reasonable and efficient, but also simple, convenient and practical and obviously enhance the inducing and dividing efficiency of the hepatic cells; and inaddition, the number of mature hepatic cells with excellent functions is increased, thus the requirement on the experiment research of the transplant and the replacement therapy of the hepatic cellsis met.
Description
Technical field
The present invention relates to biomedical sector, specifically relating to a kind of inducing and dividing embryonic stem cells becomes hepatocellular method.
Background technology
Embryonic stem cell has and is differentiated to form any histiocytic potential, therefore can be used as Transplanted cells and alternate ideal cell source, is used for the treatment of body tissue organ damage and nonfunction.Present laboratory level can be induced to differentiate into embryonic stem cell " similar " property liver cell with certain function and feature.Used technology and method mainly are that embryonic stem cell is seeded on the common culture plate, with containing the nutrient solution inducing embryo stem cell of cytokine to the liver cell directed differentiation sophisticated, that function is arranged.Though such structure helps the gaseous interchange of cell and the picked-up of nutritive substance, but under this two-dimensional condition, because nutritive substance, gas and meta-bolites density unevenness one in the culture vessel, thereby cell can only be monolayer growth, cell density is low, and differentiation effect is bad.The more important thing is that numerous gravity that studies show that can influence the growth and the differentiation of mammalian cell, easily those cellular constituents that should combine and structure are separated.Because the effect of gravity, isolated cells is natural subsidence in nutrient solution, thereby the three-dimensional random combination that has limited between cell and cell, cell and the matrix is located with common, cell can't be realized the three-dimensional contact in the similar embryo development procedure, can not form three-dimensional arrangement, become one of difficult problem of cultivating differentiating embryonic stem cells.This influences the normal physiological function of culturing cell greatly, make the cell of differentiation only have the function of part of hepatocytes, can not satisfy with Transplanted cells and replacement therapy is the fundamental research and the experimentation on animals of purpose, more can't satisfy the application requiring of clinical Transplanted cells experiment and treatment hepatic diseases
Summary of the invention
Purpose of the present invention becomes hepatocellular method with regard to providing a kind of inducing and dividing embryonic stem cells efficient, simple, practical, that differentiation effect is good, and it can be effectively farthest be induced to differentiate into abundant amount, multiple functional liver cell with the stem cell of embryonic stem cell and other type.
The objective of the invention is to realize by such technical scheme:
With the embryonic stem cell transferred species after the trysinization in the bio-reactor that fills nutrient solution, this bio-reactor is with the rotation of 15 rev/mins-50 rev/mins rotating speed, embryonic stem cell in this bio-reactor with weightlessness, induce down to sophisticated, multiple functional liver cell directed differentiation in the support of nutrient solution.When bio-reactor during with the rotation of 15 rev/mins-50 rev/mins rotating speed, because between nutrient solution and the bio-reactor inwall, have surface tension between nutrient solution and embryonic stem cell, under surface tension effects, bio-reactor brings to bio-reactor top with the embryonic stem cell in the nutrient solution, when embryonic stem cell arrives somewhere, bio-reactor top, under action of gravity, free-falling is in weightlessness.Bio-reactor whenever rotates a circle, and all can have at least for some time to make the embryonic stem cell in the nutrient solution be in weightlessness, and this moment, embryonic stem cell was suspended in the nutrient solution with weightlessness, and suffered shearing force is very little, thereby reduces the physical abuse of pair cell; Simultaneously, because the effect of rotation, nutritive substance can fully contact with each embryonic stem cell, compare common culture condition, quicken cellular metabolism, increased replenishing of cytotrophy, thereby improved the culture condition of isolated cells, helped cell and tissue growth.Simultaneously under weightlessness, cell has three-dimensional space freedom to a certain degree in bio-reactor, can be in contact with one another by histological characteristic between cell-cell, the cell-matrix, be gathered into the comparatively cell mass of uniformity, quicken growth, propagation and the differentiation of embryonic stem cell.When embryonic stem cell was in non-weightlessness, its culture environment was suitable with common culture environment, also can not influence the propagation and the differentiation of embryonic stem cell.
It is emphasized that through testing the speed of rotation that draws bio-reactor of the present invention repeatedly and be controlled at 15 rev/mins-50 rev/mins.If the bio-reactor rotating speed is slow excessively, bio-reactor can't bring to the embryonic stem cell in the nutrient solution its top, can only rest on the bottom of bio-reactor, and embryonic stem cell can't be realized free-falling, can not be in weightlessness; If the bio-reactor rotating speed is too fast, because action of inertia, the embryonic stem cell in the nutrient solution can be close to the biological respinse wall and rotate with bio-reactor, also can not realize free-falling, can not make embryonic stem cell be in weightlessness; Draw through testing repeatedly, have only when the speed of rotation of bio-reactor is controlled at 15 rev/mins-50 rev/mins, embryonic stem cell in the nutrient solution could rely on capillary effect to be brought to the top of bio-reactor by bio-reactor, when arriving somewhere, bio-reactor top, free-falling is in weightlessness.
Owing to adopted technique scheme, the present invention to have following advantage:
(1) inducing and dividing embryonic stem cells is that hepatocellular overall technology and method were both rationally efficient, simple and practical again;
(2) it is very little to be in the suffered shearing force of the embryonic stem cell of weightlessness, helps the differentiation of the propagation of cell;
(3) embryonic stem cell is cultivation of many cells three-dimensional ball shape and differentiation, three-dimensional space freedom is to a certain degree arranged, help between cell-cell, the cell-matrix being in contact with one another and at external realization tissue reconstruction, thereby can realize the differentiation of inducing of embryo-stem cell into hepatocyte to greatest extent by histological characteristic;
(4) according to the liver differentiation characteristics of embryonic stem cell, adopt the stimulating growth factor of different periods interpolation various combinations, inducing embryo stem cell breaks up to liver cell effectively;
(5) inducing the ripe liver cell that is differentiated to form is the three-dimensional ball body of uniformity, close with hepatocellular normal physiological state, has comparatively complete cellularstructure and function;
(6) adopt container to induce differentiated hepatocellular, obviously improved the efficient that liver cell is induced differentiation, ripe hepatocellular quantity increases, and has satisfied the experimental study of hepatocyte transplantation and replacement therapy.
Embodiment
The present invention will be further described below in conjunction with embodiment:
The present invention is in the bio-reactor that fills nutrient solution with the embryonic stem cell transferred species after the trysinization, this bio-reactor rotates with 15 rev/mins-50 rev/mins rotating speed, embryonic stem cell is in weightlessness in described bio-reactor, inducing embryo stem cell is to sophisticated, multiple functional liver cell directed differentiation under the support of nutrient solution.
The present invention with the embryonic stem cell of trysinization with 0.75 * 10
6/ ml-1.5 * 10
6The density of/ml by cell implantable port transferred species in the bio-reactor that fills nutrient solution.This bio-reactor is with the rotation of 15 rev/mins-50 rev/mins rotating speed, embryonic stem cell in this bio-reactor with weightlessness, induce down to sophisticated, multiple functional liver cell directed differentiation in the support of nutrient solution.When bio-reactor during with the rotation of 15 rev/mins-50 rev/mins rotating speed, because between nutrient solution and the bio-reactor inwall, have surface tension between nutrient solution and embryonic stem cell, under surface tension effects, bio-reactor brings to bio-reactor top with the embryonic stem cell in the nutrient solution, when embryonic stem cell arrives somewhere, bio-reactor top, under action of gravity, free-falling is in weightlessness.Bio-reactor whenever rotates a circle, and all can have at least for some time to make embryonic stem cell be in weightlessness, and this moment, embryonic stem cell was suspended in the nutrient solution with weightlessness, and suffered shearing force is very little, thereby reduces the physical abuse of pair cell; Simultaneously, because the effect of rotation, nutritive substance can fully contact with each embryonic stem cell, compare common culture condition, quicken cellular metabolism, increased replenishing of cytotrophy, thereby improved the culture condition of isolated cells, helped cell and tissue growth.Simultaneously under weightlessness, cell has three-dimensional space freedom to a certain degree in bio-reactor, can be in contact with one another by histological characteristic between cell-cell, the cell-matrix, be gathered into the comparatively cell mass of uniformity, quicken growth, propagation and the differentiation of embryonic stem cell.When embryonic stem cell was in non-weightlessness, its culture environment was suitable with common culture environment, also can not influence the propagation and the differentiation of embryonic stem cell.
It is emphasized that through testing the speed of rotation that draws bio-reactor of the present invention repeatedly and be controlled at 15 rev/mins-50 rev/mins.If the bio-reactor rotating speed is slow excessively, bio-reactor can't bring to the embryonic stem cell in the nutrient solution its top, can only rest on the bottom of bio-reactor, and embryonic stem cell can't be realized free-falling, can not be in weightlessness; If the bio-reactor rotating speed is too fast, because action of inertia, the embryonic stem cell in the nutrient solution can be close to the biological respinse wall and rotate with bio-reactor, also can not realize free-falling, can not make embryonic stem cell be in weightlessness; Draw through testing repeatedly, have only when the speed of rotation of bio-reactor is controlled at 15 rev/mins-50 rev/mins, embryonic stem cell in the nutrient solution could rely on capillary effect to be brought to the top of bio-reactor by bio-reactor, when arriving somewhere, bio-reactor top, free-falling is in weightlessness.
Bio-reactor of the present invention is to be fixed on the horizontal rotating shaft, only to allow the free disperse of air and encloses container that liquid can not exosmose, is provided with the inlet and the discharge outlet that are used for implanting the implantable port of cell and are used for changing nutrient solution on this bio-reactor; This bio-reactor is done uniform circular motion around transverse axis in perpendicular.
The rotating speed of bio-reactor of the present invention can be 20 rev/mins.
The rotating speed of bio-reactor of the present invention also can be 30 rev/mins, is optimum value.
The rotating speed of bio-reactor of the present invention can also be 40 rev/mins.
In order to make embryonic stem cell well to the liver cell differentiation, the present invention adopts the different periods to add the nutrient solution of various combination composition according to the liver differentiation characteristics of embryonic stem cell, and effectively inducing embryo stem cell breaks up to liver cell, wherein
It is 10 that first all nutrient solutions contain concentration
-7M-10
-6The fibroblast growth factor of the dexamethasone of M, the methyl-sulphoxide of 0.5%-2%, 5ng/ml-20ng/ml, dexamethasone and methyl-sulphoxide all have the effect that promotes the stem cell into hepatocyte differentiation, fibroblast growth factor has the promotion cell fission, the effect of differentiation.
Second all nutrient solutions contain the pHGF that concentration is 10ng/ml-30ng/ml, the fibroblast growth factor, 10 of 5ng/ml-20ng/ml
-7M-10
-6The dexamethasone of M, pHGF have the effect that the promotion embryonic stem cell is divided into mature liver cells.
The 3rd all nutrient solutions contain OSM, the 5ng/ml-10ng/ml ITS, 10 that concentration is 10ng/ml-30ng/ml
-7M-10
-6The dexamethasone of M, OSM have the hepatocyte growth of promotion and sophisticated effect; ITS has the sophisticated effect of the liver cell of promotion.
Be to realize specific embodiments of the present invention below:
(1) under aseptic condition, cultivates embryonic stem cell, trysinization is standby: the fetal tissues stem cell is inoculated in the culturing bottle of several linings 0.1%gelatin 75cm2, cultivated with the stem cell nutrient solution that contains LIF (leukemiainhibitory factor), non-essential amino acid, glutamine, 15% foetal calf serum, microbiotic etc., guaranteed embryonic stem cell proliferate under undifferentiated state.After embryonic stem cell proliferation arrived required quantity, trysinization was standby.
(2) the required nutrient solution of the different cultivation stages of preparation embryonic stem cell; Embryonic stem cell needs to cultivate for three weeks in bio-reactor altogether, for embryonic stem cell is broken up to liver cell well, the present invention adopts the different periods to add the nutrient solution of various combination composition according to the liver differentiation characteristics of embryonic stem cell, and effectively inducing embryo stem cell breaks up to liver cell.Wherein: it is 10 that first all used nutrient solutions contain concentration
-7M-10
-6The dexamethasone of M, 0.5%-2% methyl-sulphoxide, 5ng/ml-20ng/ml fibroblast growth factor; Second all used nutrient solutions contain pHGF, the 5ng/ml-20ng/ml fibroblast growth factor, 10 that concentration is 10ng/ml-30ng/ml
-7M-10
-6The M dexamethasone; The 3rd all used nutrient solutions contain OSM, the 5ng/ml-10ng/ml ITS, 10 that concentration is 10ng/ml-30ng/ml
-7M-10
-6The M dexamethasone.
(3) inject first week of embryonic stem cell by the nutrient solution inlet that is provided with on the described bio-reactor to bio-reactor and cultivate required nutrient solution;
(4) by the cell implantable port on the bio-reactor, with 0.75 * 10
6Individual/ml-1.5 * 10
6The density of individual/ml is implanted the embryonic stem cell of trysinization and is filled in the bio-reactor of nutrient solution;
(5) three weeks of rotating and culturing in described bio-reactor of the embryonic stem cell after the described trysinization; In this process, inlet and the relief outlet of changing nutrient solution by being used for of being provided with on the described bio-reactor in every 3-4 days are changed a nutrient solution, change the amount of nutrient solution 60%-85% in the reactor at every turn.
Claims (8)
1. an inducing and dividing embryonic stem cells becomes hepatocellular method, it is characterized in that: with the embryonic stem cell transferred species after the trysinization in the bio-reactor that fills nutrient solution, described bio-reactor is with the rotation of 15 rev/mins-50 rev/mins rotating speed, embryonic stem cell in described bio-reactor with weightlessness, induce down to sophisticated, multiple functional liver cell directed differentiation in the support of nutrient solution.
2. inducing and dividing embryonic stem cells as claimed in claim 1 becomes hepatocellular method, it is characterized in that: described bio-reactor is one and is fixed on the encloses container that only allows the free disperse of air, liquid not to exosmose on the horizontal rotating shaft, is provided with the implantable port that is used for implanting cell, inlet and the discharge outlet that is used for changing nutrient solution on this encloses container; Described bio-reactor is done uniform circular motion around transverse axis in perpendicular.
3. inducing and dividing embryonic stem cells as claimed in claim 1 or 2 becomes hepatocellular method, it is characterized in that: the rotating speed of described bio-reactor is 20 rev/mins.
4. inducing and dividing embryonic stem cells as claimed in claim 1 or 2 becomes hepatocellular method, it is characterized in that: the rotating speed of described bio-reactor is 30 rev/mins.
5. inducing and dividing embryonic stem cells as claimed in claim 1 or 2 becomes hepatocellular method, it is characterized in that: the rotating speed of described bio-reactor is 40 rev/mins.
6. inducing and dividing embryonic stem cells as claimed in claim 1 becomes hepatocellular method, it is characterized in that: the embryonic stem cell after the described trysinization is with 0.75 * 10
6Individual/ml-1.5 * 10
6The density transferred species of individual/ml is in the bio-reactor that fills nutrient solution.
7. become hepatocellular method as claim 1,2 or 6 described inducing and dividing embryonic stem cells, it is characterized in that: the embryonic stem cell after the described trysinization was cultivated for three weeks in filling the bio-reactor of nutrient solution, wherein:
It is 10 that first all nutrient solutions contain concentration
-7M-10
-6The dexamethasone of M, the methyl-sulphoxide of 0.5%-2%, 5ng/ml-20ng/ml fibroblast growth factor;
Second all nutrient solutions contain the pHGF that concentration is 10ng/ml-30ng/ml, the fibroblast growth factor, 10 of 5ng/ml-20ng/ml
-7M-10
-6The dexamethasone of M;
The 3rd all nutrient solutions contain the tumour inhibitor that concentration is 10ng/ml-30ng/ml (Oncostatin M, OSM), Regular Insulin-Transferrins,iron complexes-Sodium Selenite composition (ITS), 10 of 5ng/ml-10ng/ml
-7M-10
-6The dexamethasone of M.
8. inducing and dividing embryonic stem cells as claimed in claim 7 becomes hepatocellular method, it is characterized in that comprising the steps:
(1) cultivate embryonic stem cell under aseptic condition, trysinization is standby;
(2) the required nutrient solution of the different cultivation stages of preparation embryonic stem cell; Wherein: it is 10 that described first week of nutrient solution contains concentration
-7M-10
-6The fibroblast growth factor of the dexamethasone of M, the methyl-sulphoxide of 0.5%-2%, 5ng/ml-20ng/ml; Second all nutrient solutions contain the pHGF that concentration is 10ng/ml-30ng/ml, the fibroblast growth factor, 10 of 5ng/ml-20ng/ml
-7M-10
-6The dexamethasone of M; The 3rd all nutrient solutions contain the OSM that concentration is 10ng/ml-30ng/ml, the ITS, 10 of 5ng/ml-10ng/ml
-7M-10
-6The dexamethasone of M;
(3) inject first week of embryonic stem cell by the nutrient solution inlet that is provided with on the described bio-reactor to bio-reactor and cultivate required nutrient solution;
(4) by the cell implantable port on the bio-reactor, with 0.75 * 10
6Individual/ml-1.5 * 10
6The density of individual/ml is implanted the embryonic stem cell of trysinization and is filled in the bio-reactor of nutrient solution;
(5) three weeks of rotating and culturing in described bio-reactor of the embryonic stem cell after the described trysinization; In this process, inlet and the relief outlet of changing nutrient solution by being used for of being provided with on the described bio-reactor in every 3-4 days are changed a nutrient solution, change the amount of nutrient solution 60%-85% in the reactor at every turn.
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| CN200910190861A CN101649304A (en) | 2009-09-16 | 2009-09-16 | Method for inducing and dividing embryonic stem cells into hepatic cells |
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|---|---|---|---|
| CN200910190861A CN101649304A (en) | 2009-09-16 | 2009-09-16 | Method for inducing and dividing embryonic stem cells into hepatic cells |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694462A (en) * | 2015-03-17 | 2015-06-10 | 奥思达干细胞有限公司 | Method for directionally inducing to differentiate embryonic stem cell into hepatocyte |
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2009
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104694462A (en) * | 2015-03-17 | 2015-06-10 | 奥思达干细胞有限公司 | Method for directionally inducing to differentiate embryonic stem cell into hepatocyte |
| CN104694462B (en) * | 2015-03-17 | 2018-02-13 | 奥思达干细胞有限公司 | A kind of method that embryo stem cell for directional is induced to differentiate into liver cell |
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Open date: 20100217 |