海藻酸钠微胶囊作为一个人类胚胎干细胞（胚胎干细胞）的繁殖和分化的3D平台，以不同的谱系
Summary
我们已经优化了微胶囊技术作为一种有效的繁殖和胚胎干细胞分化为内胚层和多巴胺（DA）神经元的3D平台1。它还提供了一个机会，从宿主细胞移植过程中的免疫隔离。这个平台可以适应其他类型的细胞。
Abstract
人类胚胎干细胞（胚胎干细胞）正在成为一个有吸引力的替代细胞替代治疗源，扩大在文化，因为它们可以无限期地在身体的任何类型的细胞分化。不同类型的生物材料也被用于干细胞培养提供了一个微环境，模仿的干细胞小生境1-3。后者是重要的促进提供一个三维（3D）环境中，如封装4细胞与细胞间的相互作用，细胞增殖，分化成特定的谱系，以及组织机构。 2三维文化的半渗透膜的范围内对细胞封装的原则，涉及诱捕的活细胞。这些膜允许交换横跨膜的营养物质，氧气和刺激，而从主机比胶囊孔径大的抗体和免疫细胞被排除。在这里，我们预文化派的做法和三维使用海藻酸钠微囊微环境中的人类胚胎干细胞多巴胺神经元分化。我们已经修改了培养条件，以加强封装的人类胚胎干细胞的可行性。我们先前已经表明，除了显着增强的3D平台盘绕线圈P160-Rho相关激酶（ROCK）抑制剂Y-27632和人类的胎儿成纤维细胞调节血清更换介质（HFF-CM）封装的人类胚胎干细胞的可行性在细胞中表达明确的内胚层标记基因1。我们现在已经传播到人类胚胎干细胞高效分化的多巴胺神经元用于这个3D平台。多巴胺神经细胞分化的最后阶段后，蛋白和基因表达分析表明酪氨酸羟化酶（TH）的表达增加，多巴胺神经元的标志，> 2周后的100倍。我们假设，我们的3D平台，使用海藻酸钠微囊可能是有用的，以研究细胞增殖和定向分化人类胚胎干细胞，以不同的谱系。这个3D系统还允许从人类胚胎干细胞饲养层细胞的分离，并在分化过程中，也有可能在将来移植免疫隔离。
Protocol
下面所有的程序进行了II级生物安全柜内部使用无菌技术。在下表中列出所用的试剂和设备。 1。 1.1％的海藻酸钠的制备（W / V） 加入纯化的海藻酸钠0.275克（高葡萄糖醛酸含量≥60％，粘度> 200 MPa级小号，内毒素≤100 EU / G）在无菌的50毫升管，并添加25毫升无菌0.1％的明胶溶液，提前准备（0.5 gelatin/500毫升的Milli-Q H 2 O和由蒸压溶解）。 涡管约30秒，?…
Discussion
利用小鼠胚胎干细胞和胚胎干细胞的一些研究已经证明，生物材料和组织工程2,3三维培养系统的好处。我们作为一个合适的3D平台研究钡海藻酸钠相比，人类胚胎干细胞的增殖和分化，自人类胚胎干细胞表现出显着高于可行性封装比钡海藻酸钠海藻酸钙海藻酸钙微胶囊。这种文化系统还允许一个高密度细胞培养和交流的营养和氧气穿过细胞膜7。胶囊内自发分化先前已观察到的，它…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作是由NHMRC项目资助＃568969（PSS）和医学院，新南威尔士大学，干细胞的倡议。（KSS）支持。
Materials
| Name of the reagent | Company | Catalogue number | Notes |
| Alginate (Pronova UP MVG) | NovoMatrix | 4200101 | high glucuronic acid content ≥60%, viscosity >200 mPa s, and endotoxin <100 EU/g |
| Gelatin | Sigma-Aldrich | G1890-100G | |
| 0.9% NaCl | Baxter healthcare | AHF7123 | |
| Type J1 bead generator | Nisco engineering Inc | SPA-0447 | |
| Multi-Phaser syringe pump | New Era Pump Systems Inc | Model NE-1000 | |
| Ezi-Flow Medical Flowmeter | Gascon Systems | G0149 | |
| Y-27632 | Merck | 688000 | |
| Human Serum Albumin | Sigma-Aldrich | A4327-1G | |
| Accutase | Millipore | SCR005 | |
| 14G x 2” I.V. catheter | Terumo | SR-OX1451C | |
| Knockout-DMEM | Invitrogen | 10829-018 | For SR medium (basal) |
| GlutaMAX -I | Invitrogen | 35050-061 | For SR medium (2 mM) |
| Knockout Serum Replacement | Invitrogen | 10828-028 | For SR medium (20%) |
| Penicillin-Streptomycin | Invitrogen | 15070063 | For SR medium (2.5 U/ml) |
| Insulin-Transferrin-Selenium (ITS) | Invitrogen | 41400045 | For SR medium (1x) |
| β-Mercaptoethanol | Invitrogen | 21985-023 | For SR medium (0.1 mM) |
| MEM NEAA Solution | Invitrogen | 11140-050 | For SR medium (5 mM) |
| Glasgow Minimum Essential Medium | Invitrogen | 11710035 | For DA neural differentiation medium (basal) |
| Knockout Serum Replacement | Invitrogen | 10828-028 | For DA neural differentiation medium (10%) |
| Sodium pyruvate | Invitrogen | 11360070 | For DA neural differentiation medium (1 mM) |
| MEM NEAA Solution | Invitrogen | 11140-050 | For DA neural differentiation medium (0.1 mM) |
| β-Mercaptoethanol | Invitrogen | 21985-023 | For DA neural differentiation medium (0.1 mM) |
| Sonic hedgehog (SHH) | R & D Systems | 1314-SH-025/CF | For DA neural differentiation (100 ng/ml) |
| Fibroblast growth factor 8a (FGF8a) | R & D Systems | 4745-F8-050 | For DA neural differentiation (100 ng/ml) |
References
- Chayosumrit, M., Tuch, B., Sidhu, K. Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm. Biomaterials. 31, 505-514 (2010).
- Dean, S. K., Yulyana, Y., Williams, G., Sidhu, K. S., Tuch, B. E. Differentiation of encapsulated embryonic stem cells after transplantation. Transplantation. 82, 1175-1184 (2006).
- Siti-Ismail, N., Bishop, A. E., Polak, J. M., Mantalaris, A. The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance. Biomaterials. 29, 3946-3952 (2008).
- Dawson, E., Mapili, G., Erickson, K., Taqvi, S., Roy, K. Biomaterials for stem cell differentiation. Adv. Drug Deliv. Rev. 60, 215-228 (2008).
- Orive, G. Cell encapsulation: promise and progress. Nat. Med. 9, 104-107 (2003).
- Watanabe, K. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681-686 (2007).
- Dang, S. M., Gerecht-Nir, S., Chen, J., Itskovitz-Eldor, J., Zandstra, P. W. Controlled, scalable embryonic stem cell differentiation culture. Stem Cells. 22, 275-282 (2004).
- Drukker, M. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc. Natl. Acad. Sci. U S A. 99, 9864-9869 (2002).
- Calafiore, R. Standard Technical Procedures for Microencapsulation of Human Islets for Graft into Nonimmunosuppressed Patients With Type 1 Diabetes Mellitus. Transplantation Proceedings. 38, 1156-1157 (2006).
- Cho, M. S. Highly efficient and large-scale generation of functional dopamine neurons from human embryonic stem cells. Proc. Natl. Acad. Sci. U.S.A. 105, 3392-3397 (2008).
- Vazin, T., Chen, J., Lee, C. T., Amable, R., Freed, W. J. Assessment of stromal-derived inducing activity in the generation of dopaminergic neurons from human embryonic stem cells. Stem Cells. 26, 1517-1525 (2008).
Tags
Alginate Microcapsule3D PlatformPropagationDifferentiationHuman Embryonic Stem CellsHESCBiomaterialsStem Cell CulturesMicroenvironmentCell-to-cell InteractionCell ProliferationSpecific LineagesEncapsulationSemi-permeable Membranes3D CulturesNutrients ExchangeOxygen ExchangeStimuli ExchangeAntibodies ExclusionImmune Cells ExclusionAlginate MicrocapsulesCulture Conditions EnhancementP160-Rho-associated Coiled-coil Kinase InhibitorROCK InhibitorY-27632Human Fetal Fibroblast-conditioned Serum Replacement Medium
Citer Cet Article
Sidhu, K., Kim, J., Chayosumrit, M., Dean, S., Sachdev, P. Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages. J. Vis. Exp. (61), e3608, doi:10.3791/3608 (2012).