This work is supported by NHMRC Program Grant # 568969 (PSS) and Faculty of Medicine, University of New South Wales, Stem Cell Initiative (KSS).

All of the procedures below are carried out using aseptic techniques inside a Class II Biosafety Cabinet. Reagents and equipments used are listed in the tables below.
1. Preparation of 1.1% Alginate (w/v)
<ol>
	<li>Add 0.275 g of purified sodium alginate (high glucuronic acid content &ge;60%, viscosity &gt;200 mPa s, and endotoxin &le;100 EU/g) in a sterile 50 ml tube and add 25 ml sterile 0.1% gelatin solution prepared earlier (0.5g gelatin/500 ml milli-Q H2O and dissolved by aut...

<ol>
	<li>Chayosumrit, M., Tuch, B., Sidhu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+microcapsule+for+propagation+and+directed+differentiation+of+hESCs+to+definitive+endoderm.">Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm.</a> Biomaterials. 31, 505-514 (2010).</li><li>Dean, S. K., Yulyana, Y., Williams, G., Sidhu, K. S., Tuch, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+encapsulated+embryonic+stem+cells+after+transplantation.">Differentiation of encapsulated embryonic stem cells after transplantation.</a> Transplantation. 82, 1175-1184 (2006).</li><li>Siti-Ismail, N., Bishop, A. E., Polak, J. M., Mantalaris, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+benefit+of+human+embryonic+stem+cell+encapsulation+for+prolonged+feeder-free+maintenance.">The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance.</a> Biomaterials. 29, 3946-3952 (2008).</li><li>Dawson, E., Mapili, G., Erickson, K., Taqvi, S., Roy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+for+stem+cell+differentiation.">Biomaterials for stem cell differentiation.</a> Adv. Drug Deliv. Rev. 60, 215-228 (2008).</li><li>Orive, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+encapsulation:+promise+and+progress.">Cell encapsulation: promise and progress.</a> Nat. Med. 9, 104-107 (2003).</li><li>Watanabe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ROCK+inhibitor+permits+survival+of+dissociated+human+embryonic+stem+cells.">A ROCK inhibitor permits survival of dissociated human embryonic stem cells.</a> Nat. Biotechnol. 25, 681-686 (2007).</li><li>Dang, S. M., Gerecht-Nir, S., Chen, J., Itskovitz-Eldor, J., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled,+scalable+embryonic+stem+cell+differentiation+culture.">Controlled, scalable embryonic stem cell differentiation culture.</a> Stem Cells. 22, 275-282 (2004).</li><li>Drukker, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+expression+of+MHC+proteins+in+human+embryonic+stem+cells.">Characterization of the expression of MHC proteins in human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U S A. 99, 9864-9869 (2002).</li><li>Calafiore, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+Technical+Procedures+for+Microencapsulation+of+Human+Islets+for+Graft+into+Nonimmunosuppressed+Patients+With+Type+1+Diabetes+Mellitus.">Standard Technical Procedures for Microencapsulation of Human Islets for Graft into Nonimmunosuppressed Patients With Type 1 Diabetes Mellitus.</a> Transplantation Proceedings. 38, 1156-1157 (2006).</li><li>Cho, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+and+large-scale+generation+of+functional+dopamine+neurons+from+human+embryonic+stem+cells.">Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 3392-3397 (2008).</li><li>Vazin, T., Chen, J., Lee, C. T., Amable, R., Freed, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+stromal-derived+inducing+activity+in+the+generation+of+dopaminergic+neurons+from+human+embryonic+stem+cells.">Assessment of stromal-derived inducing activity in the generation of dopaminergic neurons from human embryonic stem cells.</a> Stem Cells. 26, 1517-1525 (2008).</li></ol>

We have nothing to disclose.

Several studies using mouse embryonic stem cells and hESC have demonstrated the benefits of 3D culture system in biomaterials and tissue engineering2,3. We used calcium alginate microcapsules as a suitable 3D platform to study hESC propagation and differentiation in comparison to barium alginate since hESC showed significant higher viability when encapsulated in calcium alginate than barium alginate. This culture system also allows a high-density cell culture and exchange of nutrients and oxygen across the mem...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Notes</td>
 </tr>
 <tr>
 <td>Alginate (Pronova UP MVG) </td>
 <td>NovoMatrix</td>
 <td>4200101</td>
 <td>high glucuronic acid content &ge;60%, viscosity &gt;200 mPa s, and endotoxin &lt;100 EU/g</td>
 </tr>
 <tr>
 <td>Gelatin</td>
 <td>Sigma-Aldrich</td>
 <td>G1890-100G </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.9% NaCl</td>
 <td>Baxter healthcare</td>
 <td>AHF7123</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Type J1 bead generator </td>
 <td>Nisco engineering Inc</td>
 <td>SPA-0447</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Multi-Phaser syringe pump</td>
 <td>New Era Pump Systems Inc</td>
 <td>Model NE-1000 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ezi-Flow Medical Flowmeter</td>
 <td>Gascon Systems</td>
 <td>G0149</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Y-27632</td>
 <td>Merck</td>
 <td>688000 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Human Serum Albumin</td>
 <td>Sigma-Aldrich</td>
 <td>A4327-1G </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Accutase</td>
 <td>Millipore</td>
 <td>SCR005 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>14G x 2&rdquo; I.V. catheter</td>
 <td>Terumo</td>
 <td>SR-OX1451C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Knockout-DMEM</td>
 <td>Invitrogen</td>
 <td>10829-018</td>
 <td>For SR medium (basal)</td>
 </tr>
 <tr>
 <td>GlutaMAX -I </td>
 <td>Invitrogen</td>
 <td>35050-061</td>
 <td>For SR medium (2 mM)</td>
 </tr>
 <tr>
 <td>Knockout Serum Replacement </td>
 <td>Invitrogen</td>
 <td>10828-028</td>
 <td>For SR medium (20%)</td>
 </tr>
 <tr>
 <td>Penicillin-Streptomycin</td>
 <td>Invitrogen</td>
 <td>15070063</td>
 <td>For SR medium (2.5 U/ml)</td>
 </tr>
 <tr>
 <td>Insulin-Transferrin-Selenium (ITS)</td>
 <td>Invitrogen</td>
 <td>41400045</td>
 <td>For SR medium (1x)</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>Invitrogen</td>
 <td>21985-023</td>
 <td>For SR medium (0.1 mM)</td>
 </tr>
 <tr>
 <td>MEM NEAA Solution</td>
 <td>Invitrogen</td>
 <td>11140-050</td>
 <td>For SR medium (5 mM)</td>
 </tr>
 <tr>
 <td>Glasgow Minimum Essential Medium</td>
 <td>Invitrogen</td>
 <td>11710035 </td>
 <td>For DA neural differentiation medium (basal)</td>
 </tr>
 <tr>
 <td>Knockout Serum Replacement </td>
 <td>Invitrogen</td>
 <td>10828-028</td>
 <td>For DA neural differentiation medium (10%)</td>
 </tr>
 <tr>
 <td>Sodium pyruvate</td>
 <td>Invitrogen</td>
 <td>11360070</td>
 <td>For DA neural differentiation medium (1 mM)</td>
 </tr>
 <tr>
 <td>MEM NEAA Solution</td>
 <td>Invitrogen</td>
 <td>11140-050</td>
 <td>For DA neural differentiation medium (0.1 mM)</td>
 </tr>
 <tr>
 <td>&beta;-Mercaptoethanol</td>
 <td>Invitrogen</td>
 <td>21985-023</td>
 <td>For DA neural differentiation medium (0.1 mM)</td>
 </tr>
 <tr>
 <td>Sonic hedgehog (SHH)</td>
 <td>R &amp; D Systems</td>
 <td>1314-SH-025/CF</td>
 <td>For DA neural differentiation
 (100 ng/ml)</td>
 </tr>
 <tr>
 <td>Fibroblast growth factor 8a (FGF8a) </td>
 <td>R &amp; D Systems</td>
 <td>4745-F8-050</td>
 <td>For DA neural differentiation
 (100 ng/ml)</td>
 </tr>
 </tbody>
</table>

alginate microcapsule as a 3d platform for propagation and differentiation of human embryonic stem cells (hesc) to different lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Watch this Scientific Journal Video about Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages at JoVE.com

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

alginate-microcapsule-as-3d-platform-for-propagation-differentiation

Research

JoVE Journal

Bioengineering

16.2K Görüntüleme.  The University of New South Wales. Biz endoderm ve dopaminerjik (DA) nöronlara yayılması ve embriyonik kök hücrelerin farklılaşması için etkili bir 3D platform olarak bir mikroenkapsülasyon tekniği optimize etmiş. Ayrıca nakli sırasında ana hücreleri bağışıklık izolasyonu için bir fırsat sağlar. Bu platform, diğer hücre türleri için adapte edilebilir.