Towards spatially-organized organs-on-chip: Photopatterning cell-laden thiol-ene and methacryloyl hydrogels in a microfluidic device.
Towards spatially-organized organs-on-chip: Photopatterning cell-laden thiol-ene and methacryloyl hydrogels in a microfluidic device.
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DOI：
10.1016/j.ooc.2022.100018
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发表时间：
2022-12-01
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影响因子：
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通讯作者：
Pompano, Rebecca R
中科院分区：
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作者：
Ortiz-Cardenas, Jennifer E;Zatorski, Jonathan M;Pompano, Rebecca R
用于3D细胞培养的微图案化技术能够重建组织水平的结构，但是图案化水凝胶与芯片上器官的组合以在微流体灌注下产生有组织的3D培养物仍然具有挑战性。为了解决这一技术差距，我们开发了一种用户友好的原位微图案化方案，该方案将可交联的、载有细胞的水凝胶的光刻与简单的微流体外壳集成在一起，并测试了交联化学对稳定性和空间分辨率的影响。使用用可光交联部分官能化的明胶工作，我们发现在高密度（≥ 107/mL）下包含细胞并不妨碍硫醇-异戊二烯凝胶化，但降低了甲基丙烯酰基水凝胶的储能模量。选择水凝胶组成和光剂量以匹配软组织的储能模量。为了在芯片上产生所需的图案，将载有细胞的前体溶液流入微流体室并通过光掩模暴露于405 nm光。芯片上的3D培养物是独立的，设计可以通过简单地更换光掩模来互换。硫醇-烯水凝胶产生高度精确的特征尺寸从100 - 900 μ m的直径，而甲基丙烯酰水凝胶产生轻微放大的功能。此外，只有硫醇-烯水凝胶在灌注过夜下机械稳定。重复的图案化容易产生多区域培养物，无论是单独的还是相邻的，包括在芯片上获得具有挑战性的非线性边界。作为原理证明，原代人T细胞以高区域特异性在芯片上图案化。在恒定灌注下培养12小时后，存活率保持较高（> 85%）。我们设想，这项技术将使研究人员能够对3D共培养物进行图案化，以模仿以前难以获得的器官样结构。
Micropatterning techniques for 3D cell cultures enable the recreation of tissue-level structures, but the combination of patterned hydrogels with organs-on-chip to generate organized 3D cultures under microfluidic perfusion remains challenging. To address this technological gap, we developed a user-friendly in-situ micropatterning protocol that integrates photolithography of crosslinkable, cell-laden hydrogels with a simple microfluidic housing, and tested the impact of crosslinking chemistry on stability and spatial resolution. Working with gelatin functionalized with photo-crosslinkable moieties, we found that inclusion of cells at high densities (≥ 107/mL) did not impede thiol-norbornene gelation, but decreased the storage moduli of methacryloyl hydrogels. Hydrogel composition and light dose were selected to match the storage moduli of soft tissues. To generate the desired pattern on-chip, the cell-laden precursor solution was flowed into a microfluidic chamber and exposed to 405 nm light through a photomask. The on-chip 3D cultures were self-standing and the designs were interchangeable by simply swapping out the photomask. Thiol-ene hydrogels yielded highly accurate feature sizes from 100 - 900 mum in diameter, whereas methacryloyl hydrogels yielded slightly enlarged features. Furthermore, only thiol-ene hydrogels were mechanically stable under perfusion overnight. Repeated patterning readily generated multi-region cultures, either separately or adjacent, including non-linear boundaries that are challenging to obtain on-chip. As a proof-of-principle, primary human T cells were patterned on-chip with high regional specificity. Viability remained high (> 85%) after 12-hr culture with constant perfusion. We envision that this technology will enable researchers to pattern 3D co-cultures to mimic organ-like structures that were previously difficult to obtain.