Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography
Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography
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DOI：
10.3791/2636
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发表时间：
2011-02-01
影响因子：
1.2
通讯作者：
Moore, Michael J.
中科院分区：
文献类型：
--
作者：
Curley, J. Lowry;Jennings, Scott R.;Moore, Michael J.
越来越多的模式化细胞培养环境正在成为研究细胞特征的相关技术，许多研究人员认为需要3D环境来代表体外实验，以更好地模拟体内质量（1-3）。在癌症研究（4）、神经工程（5）、心脏生理学（6）和细胞-基质相互作用（7，8）等领域的研究表明，传统单层培养和3D构建体之间的细胞行为有很大不同。水凝胶被用作3D环境，因为它们的多样性、多功能性和通过功能化调整分子组成的能力（9-12）。存在许多用于产生作为细胞支持基质的构建体的技术，包括静电纺丝（13）、弹性体印模（14）、喷墨印刷（15）、添加剂光刻（16）、静态光掩模投影光刻（17）和动态掩模微立体光刻（18）。不幸的是，这些方法涉及多个生产步骤和/或设备，不容易适用于常规的细胞和组织培养方法。该方案中采用的技术采用后两种方法，使用数字光致变色装置（DMD）来产生用于交联几何特异性聚乙二醇（PEG）水凝胶的动态光掩模，通过UV引发的自由基聚合来诱导。由此产生的“2.5D”结构为神经生长提供了受约束的3D环境。我们采用双水凝胶方法，其中PEG作为细胞限制性区域，为由Puramatrix或琼脂糖制成的不成形但细胞允许的自组装凝胶提供结构。整个组织外植体，如胚胎背根神经节（DRG），可以掺入到双水凝胶构建体中用于实验测定，如神经突生长。另外，解离的细胞可以包封在可光交联或自聚合的水凝胶中，或者使用细胞限制性的微图案化选择性地粘附到可渗透的支撑膜上。使用DMD，我们创建了水凝胶结构，厚度接近1 mm，但薄膜（
Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities (1-3). Studies in fields such as cancer research (4), neural engineering 5, cardiac physiology (6), and cell-matrix interaction(7,8)have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization (9-12). Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning(13), elastomer stamps(14), inkjet printing(15), additive photopatterning(16), static photomask projection-lithography(17), and dynamic mask microstereolithography(18). Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to similar to 1mm thick, but thin film (