CN104046587A - Method for adjusting and controlling in vitro three-dimensional directed differentiation of stem cells - Google Patents
Method for adjusting and controlling in vitro three-dimensional directed differentiation of stem cells Download PDFInfo
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- CN104046587A CN104046587A CN201310076323.4A CN201310076323A CN104046587A CN 104046587 A CN104046587 A CN 104046587A CN 201310076323 A CN201310076323 A CN 201310076323A CN 104046587 A CN104046587 A CN 104046587A
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- chondroitin sulfate
- sodium alginate
- differentiation
- stem cell
- micro gel
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Abstract
The invention relates to the technical field of regenerative medicine and discloses a method for adjusting and controlling in vitro three-dimensional directed differentiation of stem cells. The method includes following steps: embedding Stem cells into calcium alginate micro gel beads containing chondroitin sulfate, adding an inductive differentiation culture liquid, adjusting and controlling the in vitro directed differentiation of the stem cells by changing a mass ratio between the sodium alginate and the chondroitin sulfate during the preparation of the micro gel beads, dissolving the calcium alginate micro gel beads in a sodium citrate solution after the differentiation process being finished, and then obtaining pure differentiation cells. The method is simple in operation and low in cost, the calcium alginate micro gel beads containing chondroitin sulfate can simulate an in vivo three-dimensional extracellular matrix so that a three-dimensional directed differentiating microenvironment which approximates an in vivo microenvironment is supplied for the stem cells, thereby improving directed differentiation efficiency and a biological function of the stem cells. The method plays an important role in applications of regenerative medicine.
Description
Technical field
The present invention relates to regenerative medicine field, be a kind of regulate and control the method for stem cell in vitro three-dimensional orientation differentiation.
Background technology
Stem cell, owing to having the ability of self and differentiation, is the desirable seed cell that damaged tissue is repaired.But when stem cell implants after damaged part, stem cell, except being divided into needed histocyte, participates in tissue repair, is also divided into the cell of other type simultaneously, even has higher tumorigenicity.Stem cell is carried out to external pre-differentiation, make it to be divided in advance ripe histocyte or precursor cell before transplanting, can improve directed differentiation efficiency, lower the ability that stem cell breaks up to other cell type in vivo, thereby improve tissue repair effect, and reduce tumorigenesis risk.
In the three-dimensional network that Growth of Cells forms at extracellular matrix in vivo, research shows that the cell under external two-dimentional culture condition lost its phenotype and function gradually, and being conducive to the function of cell, external dimensional culture maintains, specific tissue's cell micro-environment in can analogue body, promotes the directed differentiation of stem cell in vitro better.At present, the three-dimensional system of regulation and control stem cell in vitro directed differentiation, de-cytoskeleton, collagen scaffold or polylactic acid bracket etc. are adopted, what these supports had has high immunogenicity, the special cells epimatrix composition of the short differentiation of some shortages, some degraded products are unfavorable for cell cultures and tissue repair, and this has caused stem cell in vitro directed differentiation efficiency low, obtain cell and do not have the problems such as the interior normal physiological function of body.
Because existing induction differentiated system exists above-mentioned defect, seriously limit further developing and clinical application of stem cell in vitro directed differentiation technology, therefore be badly in need of the novel method of the three-dimensional differentiation of induced dry-cell of the convenient practicality of research and development, by utilize dimensional culture and add matrix components build can analogue body inner tissue microenvironment, to realize the external regulation and control to the pre-differentiation of stem cell directional, further promote the application of stem cell in regenerative medicine field.
Summary of the invention
The object of the present invention is to provide a kind of regulate and control the method for stem cell in vitro three-dimensional orientation differentiation, utilize the alginate calcium micro gel bead that contains chondroitin sulfate to realize the regulation and control to stem cell in vitro directed differentiation.
For achieving the above object, the technical solution used in the present invention is:
The present invention is embedded in stem cell in the alginate calcium micro gel bead that contains chondroitin sulfate, add induction differentiation culture liquid, the mass ratio of sodium alginate and chondroitin sulfate regulation and control stem cell in vitro directed differentiation while preparing micro gel bead by change, and after differentiation finishes, can utilize sodium citrate solution to dissolve alginate calcium micro gel bead, and then obtain pure noble cells.
Described stem cell comprises embryonic stem cell, iPS cell, mescenchymal stem cell, neural stem cell, iris pigment epithelial cells, amniotic epithelial cells or hemopoietic stem cell.
The described alginate calcium micro gel bead that contains chondroitin sulfate is served as reasons prepared through calcium liquid calciumization containing the solution of sodium alginate and chondroitin sulfate, this solution solvent is physiological saline, sodium alginate concentration is 0.3-4% (W/V), chondroitin sulfate strength of solution is 0.5-10% (W/V), and the mass ratio of sodium alginate and chondroitin sulfate is 1-20;
The alginate calcium micro gel bead diameter that contains chondroitin sulfate is 200-2000 μ m;
The molecular weight of sodium alginate is 100-1000kDa, guluronic acid and mannuronic acid ratio (GM ratio) scope is 0.2-3, and sodium alginate is that unmodified sodium alginate, arginyl-glycyl-aspartic acid (RGD) are modified sodium alginate, Isoleucine-Methionin-α-amino-isovaleric acid-L-Ala-α-amino-isovaleric acid (IKVAV) and modified sodium alginate, tyrosine-Isoleucine-glycine-Vitro By Serine/arginine (YIGSR) and modify in sodium alginate the sodium alginate of one or two or more kinds;
Chondroitin sulfate molecular-weight average is 2-40kDa;
Described directed differentiation comprises stem cell neuralward, cardiac muscle, liver, pancreas islet, skeletonization, cartilage or adipocyte directed differentiation.
The culture vessel that described directed differentiation is used is static cultivation system or bio-reactor;
Static cultivation system refers to use culturing bottle, culture plate, culture dish or culture bag to cultivate;
Bio-reactor comprises rotary reactor, filling type reactor, stirring reactor or airlift reactor.
Tool of the present invention has the following advantages:
1. simple to operate, cost is low.The alginate calcium micro gel bead that utilization of the present invention contains chondroitin sulfate regulates and controls stem cell in vitro directed differentiation, and method is simple, avoids using expensive material and technique;
2. specific tissue's differentiation microenvironment in analogue body, improves differentiation of stem cells efficiency and function.Three-dimensional alginate calcium micro gel bead provides three dimensional growth environment for stem cell, and the extracellular matrix components of in-vivo tissue has further been simulated in the interpolation of chondroitin sulfate, therefore the present invention can break up by induced dry-cell under the condition that is similar to Ti Nei specific tissue microenvironment, and its induction efficiency and cell function further improve;
4. high reactivity is collected noble cells.After differentiation finishes, utilize sodium citrate solution to dissolve micro gel bead, by the centrifugal cell that can collect differentiation under physiological condition, cytoactive is high, and function is unaffected;
5. applied range.The inventive method can promote stem cell neuralward, cardiac muscle, liver, pancreas islet, skeletonization, cartilage or adipocyte directed differentiation, and can amplification culture scale, has potential applicability in clinical practice.
Brief description of the drawings
Fig. 1 is schematic diagram of the present invention: culture vessel 1, the alginate calcium micro gel bead 2 that contains chondroitin sulfate, stem cell 3;
The cartilage specificity dyeing that Fig. 2 is cell in the alginate calcium micro gel bead that contains 20% (W/V) chondroitin sulfate;
Fig. 3 is cell II Collagen Type VI genetic expression in the alginate calcium micro gel bead of different chondroitin sulfate mass percents;
Fig. 4 is that alginate calcium micro gel bead regulation and control mescenchymal stem cell directed differentiation is neural precursor: Fig. 4 A is Nestin gene relative expression; Fig. 4 B is Nestin positive cell percentage.
Embodiment
Embodiment 1: three-dimensional induction system regulation and control human mesenchymal stem cell is external to chondrocyte's directed differentiation
Sodium alginate (molecular weight 430kDa, guluronic acid and mannuronic acid are than 1.5) powder dissolution, in physiological saline, is obtained to the solution of 3% (W/V, g/ml).Chondroitin sulfate (molecular-weight average 20kDa) powder dissolution, in physiological saline, is obtained to the solution of 5% (W/V, g/ml).By 3% (W/V, g/ml) sodium alginate, 5% (W/V, g/ml) chondroitin sulfate cellulose solution and physiological saline mix, obtain sodium alginate final concentration and be 2% (W/V, g/ml), sodium alginate/chondroitin sulfate mass ratio is respectively 1.5,4 and 9 mixing solutions.By the 5th generation human umbilical cord mesenchymal stem cells mix with this mixing solutions, adjust cell density be 6 × 10
6cellsmL
-1, this cell suspension is splashed into 100mmolL through syringe pump
-1caCl
2calcification 30min in solution, obtains being embedded with the alginate calcium micro gel bead of stem cell, then adds and contain 10
-7molL
-1dexamethasone, 1% foetal calf serum, 1%ITS
+, 40mgmL
-1l-PROLINE, 50mgmL
-1vitamins C and 100mgmL
-1the DMEM in high glucose nutrient solution of Sodium.alpha.-ketopropionate is (containing 4500mlL
-1glucose) carry out chondrocyte induction 3 weeks.Afterwards, use 55mmolL
-1sodium citrate solution soaks micro gel bead 10min, dissolve micro gel bead, centrifugal results cartilage differentiation cell, utilizes toluidine blue to carry out chondrocyte's specific stain, and analyzes the gene expression dose of the chondrocyte's II Collagen Type VI obtaining from different chondroitin sulfate content micro gel beads.Experimental result is shown in Fig. 2 and 3, and result shows, is divided into chondrocyte through three-dimensional induced dry-cell, and the genetic expression of II Collagen Type VI improves with the increase of chondroitin sulfate cellulose content, illustrates that this three-dimensional induction system can regulate and control the cartilage differentiation of stem cell.
Embodiment 2: the external neuralward precursor cell of three-dimensional induction system regulation and control human mesenchymal stem cell directed differentiation
First, prepare the sodium alginate that arginyl-glycyl-aspartic acid (RGD) is modified.By sodium alginate (molecular weight 500kDa, guluronic acid and mannuronic acid are than 2) be dissolved in 0.1M2-(N-morpholino) ethyl sulfonic acid (MES) damping fluid that contains 0.5M NaCl (pH value 6.5), obtain 1% (W/V, g/ml) sodium alginate soln.Add again 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), N-hydroxy thiosuccinimide (sulfo-NHS) and rgd peptide, stirring at room temperature reaction 24 hours.EDC and sodium alginate mol ratio are 1:20, and EDC and sulfo-NHS mol ratio are 2:1, and rgd peptide and sodium alginate mass ratio are 1:1000.Then dialyse and lyophilize, thereby obtain the sodium alginate that RGD modifies.
RGD is modified to sodium alginate powder and be dissolved in physiological saline, obtain the solution of 3% (W/V, g/ml).Chondroitin sulfate (molecular-weight average 20kDa) powder dissolution, in physiological saline, is obtained to the solution of 5% (W/V, g/ml).By 3% (W/V, g/ml) RGD modifies sodium alginate, 5% (W/V, g/ml) chondroitin sulfate cellulose solution and physiological saline mix, obtain sodium alginate final concentration and be 1.5% (W/V, g/ml), sodium alginate/chondroitin sulfate mass ratio is respectively 1.5,4 and 9 mixing solutions.By the 4th generation human umbilical cord mesenchymal stem cells mix with this mixing solutions, adjust cell density be 4 × 10
6cellsmL
-1, this cell suspension is splashed into 100mmolL through syringe pump
-1caCl
2calcification 30min in solution, obtains being embedded with the alginate calcium micro gel bead of stem cell, then adds and contain 10ngL
-1epidermal growth factor (EGF) and 10ngL
-1the RPMI1640 nutrient solution of Basic Fibroblast Growth Factor (bFGF) carries out nerve-inducing 1 week.Afterwards, use 55mmolL
-1sodium citrate solution soaks micro gel bead 10min, dissolve micro gel bead, centrifugal results Neural Differentiation cell, utilize the relative genetic expression of Nestin of the neurocyte that real-time pcr analysis obtains from different chondroitin sulfate content micro gel beads, and utilize fluorescent dye to obtain the per-cent of Nestin positive cell under each condition.Experimental result is shown in Fig. 4, and result shows, induces through three-dimensional, and Nestin gene and positive cell percentage improve with the increase of chondroitin sulfate cellulose content, illustrates that this three-dimensional induction system can regulate and control the differentiation of stem cell neuralward precursor cell.
Claims (5)
1. one kind regulates and controls the method for stem cell in vitro three-dimensional orientation differentiation, it is characterized in that: stem cell is embedded in the alginate calcium micro gel bead that contains chondroitin sulfate, add induction differentiation culture liquid, the mass ratio of sodium alginate and chondroitin sulfate regulation and control stem cell in vitro directed differentiation while preparing micro gel bead by change, and after differentiation finishes, can utilize sodium citrate solution to dissolve alginate calcium micro gel bead, and then obtain pure noble cells.
2. it is characterized in that in accordance with the method for claim 1:
Described stem cell comprises embryonic stem cell, iPS cell, mescenchymal stem cell, neural stem cell, iris pigment epithelial cells, amniotic epithelial cells or hemopoietic stem cell.
3. it is characterized in that in accordance with the method for claim 1:
The described alginate calcium micro gel bead that contains chondroitin sulfate is served as reasons prepared through calcium liquid calciumization containing the solution of sodium alginate and chondroitin sulfate, and this solution solvent is physiological saline; Employing sodium alginate concentration is 0.3-4% (W/V), and adopting chondroitin sulfate strength of solution is 0.5-10% (W/V), and the mass ratio of sodium alginate and chondroitin sulfate is 1-20;
The alginate calcium micro gel bead diameter that contains chondroitin sulfate is 200-2000 μ m;
The molecular weight of sodium alginate is 100-1000kDa, guluronic acid and mannuronic acid ratio (GM ratio) scope is 0.2-3, and sodium alginate is that unmodified sodium alginate, arginyl-glycyl-aspartic acid (RGD) are modified sodium alginate, Isoleucine-Methionin-α-amino-isovaleric acid-L-Ala-α-amino-isovaleric acid (IKVAV) and modified sodium alginate, tyrosine-Isoleucine-glycine-Vitro By Serine/arginine (YIGSR) and modify in sodium alginate the sodium alginate of one or two or more kinds;
Chondroitin sulfate molecular-weight average is 2-40kDa.
4. it is characterized in that in accordance with the method for claim 1:
Described directed differentiation comprises stem cell neuralward, cardiac muscle, liver, pancreas islet, skeletonization, cartilage or adipocyte directed differentiation.
5. it is characterized in that in accordance with the method for claim 1:
The culture vessel that described directed differentiation is used is static cultivation system or bio-reactor;
Static cultivation system refers to use culturing bottle, culture plate, culture dish or culture bag to cultivate;
Bio-reactor comprises rotary reactor, filling type reactor, stirring reactor or airlift reactor.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105734017A (en) * | 2014-12-09 | 2016-07-06 | 中国科学院大连化学物理研究所 | Method for promoting directional differentiation and proliferation of mesenchymal stem cells towards neural precursor cells |
| CN105734006A (en) * | 2014-12-09 | 2016-07-06 | 中国科学院大连化学物理研究所 | Preparation method of acellular sodium alginate bionic hydrogel |
| CN106190961A (en) * | 2016-07-27 | 2016-12-07 | 深圳爱生再生医学科技有限公司 | The method of induction stem cell in vitro directed differentiation |
| CN106589161A (en) * | 2016-12-21 | 2017-04-26 | 深圳先进技术研究院 | Modified alginic acid or alginate, preparation method thereof, biological repair material and stent |
| CN107224613A (en) * | 2016-03-23 | 2017-10-03 | 北京泰盛生物科技有限公司 | The method that Odontogenic cysts mescenchymal stem cell realizes regenerating bone or cartilage |
| CN107224607A (en) * | 2016-03-23 | 2017-10-03 | 北京泰盛生物科技有限公司 | Parodontium and gum stem cell realize tendon tissue regeneration method |
| CN115029295A (en) * | 2022-05-12 | 2022-09-09 | 汕头大学医学院 | Novel stem cell 3D differentiation method |
| CN115067321A (en) * | 2022-06-30 | 2022-09-20 | 上海市伤骨科研究所 | Nutritive capsule for medium-and long-term three-dimensional preservation of corneal tissue and preparation method thereof |
| CN115029295B (en) * | 2022-05-12 | 2024-04-30 | 汕头大学医学院 | Novel stem cell 3D differentiation method |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105734017A (en) * | 2014-12-09 | 2016-07-06 | 中国科学院大连化学物理研究所 | Method for promoting directional differentiation and proliferation of mesenchymal stem cells towards neural precursor cells |
| CN105734006A (en) * | 2014-12-09 | 2016-07-06 | 中国科学院大连化学物理研究所 | Preparation method of acellular sodium alginate bionic hydrogel |
| CN105734017B (en) * | 2014-12-09 | 2019-11-12 | 中国科学院大连化学物理研究所 | A method of promoting mesenchymal stem cells into nerve precursor directed differentiation, proliferation |
| CN105734006B (en) * | 2014-12-09 | 2020-02-14 | 中国科学院大连化学物理研究所 | Preparation method of acellular sodium alginate bionic hydrogel |
| CN107224613A (en) * | 2016-03-23 | 2017-10-03 | 北京泰盛生物科技有限公司 | The method that Odontogenic cysts mescenchymal stem cell realizes regenerating bone or cartilage |
| CN107224607A (en) * | 2016-03-23 | 2017-10-03 | 北京泰盛生物科技有限公司 | Parodontium and gum stem cell realize tendon tissue regeneration method |
| CN106190961A (en) * | 2016-07-27 | 2016-12-07 | 深圳爱生再生医学科技有限公司 | The method of induction stem cell in vitro directed differentiation |
| CN106589161A (en) * | 2016-12-21 | 2017-04-26 | 深圳先进技术研究院 | Modified alginic acid or alginate, preparation method thereof, biological repair material and stent |
| CN115029295A (en) * | 2022-05-12 | 2022-09-09 | 汕头大学医学院 | Novel stem cell 3D differentiation method |
| CN115029295B (en) * | 2022-05-12 | 2024-04-30 | 汕头大学医学院 | Novel stem cell 3D differentiation method |
| CN115067321A (en) * | 2022-06-30 | 2022-09-20 | 上海市伤骨科研究所 | Nutritive capsule for medium-and long-term three-dimensional preservation of corneal tissue and preparation method thereof |
| CN115067321B (en) * | 2022-06-30 | 2023-08-08 | 上海市伤骨科研究所 | Nutritional capsule for medium-long-term three-dimensional preservation of cornea tissue and preparation method thereof |
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