构建模块化水凝胶片的微模式化宏观缩放3D细胞结构
Summary
We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.
Abstract
水凝胶可以在使用微流体或微图案的技术，以提供在体内样的三维（3D）几何组织微观尺度进行图案化。所得三维水凝胶为基础的蜂窝结构已被引入作为替代动物实验为先进的生物研究，药理学试验和器官移植的应用程序。尽管水凝胶为基础的颗粒和纤维，可以容易地制造，这是很难操纵它们用于组织重建。在这种视频中，我们描述了一种制造方法，微图案化藻酸盐水凝胶片材，以及它们的组件，以形成一个宏观尺度三维细胞培养体系具有受控细胞微环境。 200微米，具有精确的微图案 – 采用钙胶凝剂薄雾形式，薄水凝胶片材易于与厚度在100〜生成。细胞然后可与水凝胶片材在几何指导培养独立的条件。此外，水凝胶片材可以容易地使用微量具有端切割尖端操纵，并且可以通过使用图案化的聚二甲基硅氧烷（PDMS）帧堆叠它们组装成多层结构。这些模块化水凝胶片，其可以使用一个浅显工艺制造，具有的体外药物分析和生物学研究，包括微观和宏观结构和组织重建的功能研究潜在的应用。
Introduction
水凝胶是特别有前途的生物材料，并且预计将在基础生物学，药理学试验和医学重要1的水凝胶为基础的蜂窝构造的Biofabrication已经建议减少使用的动物实验中，2,3-取代移植组织，4和提高基于细胞的测定。5,6-含有水（加氢）的粘弹性材料（凝胶）允许进行封装和保持在支架结构，以控制3D细胞微环境的大量细胞。与微流体或微图案的技术的指导组合，该水凝胶结构的几何形状可以精确地在细胞比例控制。迄今为止，多种水凝胶的形状，包括颗粒，7 – 9纤维，10 – 12，片材，13 – 15已经被用作建筑物单元在自下而上的Appro酸痛到宏观规模的多细胞结构的制造。
既基于水凝胶的颗粒和纤维一直容易且迅速地制造为应用程序作为微尺度蜂窝环境中，使用微流体装置的流体控制。然而，如工程化组织的基本单位，它将是复杂的重新排列，并扩大其体积为宏观结构。16，是更难以实现宏观规模构建体相比，以产生微米级的基本模块。可用于水凝胶基的构建体的片状单元通过简单的组装过程来增加支架的体积。必然，水凝胶片材的堆叠层不仅提供一个容积增加，而且在一个三维空间中的几何延伸。
我们以前曾报道制造微图案化的水凝胶片材，13的方法– 15连同其组装成多捻ERED蜂窝架构。该技术使得复杂微图案和蜂窝结构的模块化设计通过多层结构的层叠过程。通过堆叠模块化水凝胶片，这是微图案的制造中，可以实现一个三维细胞培养系统具有受控的宏观尺度细胞微环境。此视频协议描述了一个简单而强大的制造方法，可用于构建模块化水凝胶片材，基于人肝癌细胞系（HepG2细胞）。我们证明本文简单操作这些图案化的模块化的水凝胶片材，和它们的组装成的多层结构。
Protocol
Representative Results
Discussion
这个协议提供了一种制造模块化的水凝胶片，并把它们组装，以形成三维的细胞支架的一个简单方法。
为了构建在短时间内鲜明图案化藻酸盐的结构，我们应确定一个交联的过程，可以创造足够刚性的结构，以从模具保持复杂微图案，以及维持细胞活力和新陈代谢。我们已经开发出一种交联方法，包括溶胶 – 凝胶转变，喷使用以雾状形式的加湿器的交联试剂。如果没有这种?…
Declarações
The authors have nothing to disclose.
Acknowledgements
This research was supported by a National Leading Research Laboratory Program (Grant NRF-2013R1A2A1A05006378) through the National Research Foundation of Korea funded by the Ministry of Science, ICT and Future Planning. The authors also acknowledge a KAIST Systems Healthcare Program.
Materials
| Sylgard 184 Silicone Elastomer Kit | Dow Corning Corporation | 000000000001064291 | |
| Pluronic F-127 | Sigma-Aldrich | P2443 | Powdered nonionic surfactant |
| Alginic acid sodium salt, low viscosity | Alfa Aesar | B25266 | |
| Calcium chloride dihydrate | Sigma-Aldrich | C7902 | |
| Ultrasonic humidifier | MediHeim | MH-2800 | Modified equipment, Maximum sprayed rate: 250 mL/h |
| Nylon net filter hydrofilic, 180 μm | EMD Millipore | NY8H04700 | |
| Polycarbonate mold | Customized mold for fabrication of a PDMS frame pattern |
Referências
- Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Deliv. Rev. 64 (Supplement), 18-23 (2012).
- Lan, S., Starly, B. Alginate based 3D hydrogels as an in vitro co-culture model platform for the toxicity screening of new chemical entities. Toxicol. Appl. Pharm. 256 (1), 62-72 (2011).
- Szot, C. S., Buchanan, C. F., Freeman, J. W., Rylander, M. N. 3D in vitro bioengineered tumors based on collagen I hydrogels. Biomaterials. 32 (31), 7905-7912 (2011).
- Lim, F., Sun, A. M. Microencapsulated islets as bioartificial endocrine pancreas. Science. 210 (4472), 908-910 (1980).
- Koh, W. G., Itle, L. J., Pishko, M. V. Molding of hydrogel microstructures to create multiphenotype cell microarrays. Anal. Chem. 75 (21), 5783-5789 (2003).
- Xu, Y., et al. A Microfluidic Hydrogel Capable of Cell Preservation without Perfusion Culture under Cell-Based Assay Conditions. Adv Mater. 22 (28), 3017-3021 (2010).
- Um, E., Lee, D. S., Pyo, H. S., Park, J. K. Continuous generation of hydrogel beads and encapsulation of biological materials using a microfluidic droplet-merging channel. Microfluid. Nanofluid. 5 (4), 541-549 (2008).
- Lee, D. H., Lee, W., E, U. m., Park, J. K. Microbridge structures for uniform interval control of flowing droplets in microfluidic networks. Biomicrofluidics. 5 (3), 034117 (2011).
- Lee, D. H., Bae , C. Y., Han, J. I., Park, J. K. In situ analysis of heterogeneity in the lipid content of single green microalgae in alginate hydrogel microcapsules. Anal. Chem. 85 (18), 8749-8756 (2013).
- Yamada, M., Sugaya, S., Naganuma, Y., Seki, M. Microfluidic synthesis of chemically and physically anisotropic hydrogel microfibers for guided cell growth and networking. Soft Matter. 8 (11), 3122-3130 (2012).
- Yamada, M., et al. Controlled formation of heterotypic hepatic micro-organoids in anisotropic hydrogel microfibers for long-term preservation of liver-specific functions. Biomaterials. 33 (33), 8304-8315 (2012).
- Onoe, H., et al. Metre-long cell-laden microfibres exhibit tissue morphologies and functions. Nat. Mater. 12 (6), 584-590 (2013).
- Lee, W., Son, J., Yoo, S. S., Park, J. K. Facile and Biocompatible Fabrication of Chemically Sol−Gel Transitional Hydrogel Free-Standing Microarchitectures. 12 (1), 14-18 (2011).
- Lee, W., et al. Cellular hydrogel biopaper for patterned 3D cell culture and modular tissue reconstruction. Adv. Healthcare Mater. 1 (5), 635-639 (2012).
- Bae, C. Y., Min, M. K., Kim, H., Park, J. K. Geometric effect of the hydrogel grid structure on in vitro formation of homogeneous MIN6 cell clusters. Lab Chip. 14 (13), 2183-2190 (2014).
- Bruzewicz, D. A., McGuigan, A. P., Whitesides, G. M. Fabrication of a modular tissue construct in a microfluidic chip. Lab Chip. 8 (5), 663-671 (2008).
- Choi, S., Park, J. K. Two-step photolithography to fabricate multilevel microchannels. Biomicrofluidics. 4 (4), 046503 (2010).
- Lee, B. R., et al. In situ formation and collagen-alginate composite encapsulation of pancreatic islet spheroids. Biomaterials. 33 (3), 837-845 (2012).
- Cabodi, M., Choi, N. W., Gleghorn, J. P., Lee, C. S., Bonassar, L. J., Stroock, A. D. A microfluidic biomaterial. J. Am. Chem. Soc. 127 (40), 13788-13789 (2005).
- Choi, N. W., Cabodi, M., Held, B., Gleghorn, J. P., Bonassar, L. J., Stroock, A. D. Microfluidic scaffolds for tissue engineering. Nat. Mater. 6 (11), 908-915 (2007).
- Rowley, J. A., Madlambayan, G., Mooney, D. J. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials. 20 (1), 45-53 (1999).
