Sumey Jenna L, Harrell Abigail M, Johnston Peyton C, Caliari Steven R
Department of Chemical Engineering, University of Virginia, Charlottesville, Virginia 22903.
Department of Chemistry, University of Virginia, Charlottesville, Virginia 22903.
bioRxiv. 2023 Mar 20:2023.03.16.532853. doi: 10.1101/2023.03.16.532853.
There is tremendous interest in developing hydrogels as tunable cell culture platforms to study cell response to mechanical cues in a controlled manner. However, little is known about how common cell culture techniques, such as serial expansion on tissue culture plastic, affect subsequent cell behavior when cultured on hydrogels. In this work we leverage a methacrylated hyaluronic acid hydrogel platform to study stromal cell mechanotransduction. Hydrogels are first formed through thiol-Michael addition to model normal soft tissue (e.g., lung) stiffness ( ~ 1 kPa). Secondary crosslinking via radical photopolymerization of unconsumed methacrylates allows matching of early- ( ~ 6 kPa) and late-stage fibrotic tissue ( ~ 50 kPa). Early passage (P1) primary human mesenchymal stromal cells (hMSCs) display increased spreading, myocardin-related transcription factor-A (MRTF-A) nuclear localization, and focal adhesion size with increasing hydrogel stiffness. However, late passage (P5) hMSCs show reduced sensitivity to substrate mechanics with lower MRTF-A nuclear translocation and smaller focal adhesions on stiffer hydrogels compared to early passage hMSCs. Similar trends are observed in an immortalized human lung fibroblast line. Overall, this work highlights the implications of standard cell culture practices on investigating cell response to mechanical signals using hydrogel models.
人们对开发水凝胶作为可调谐细胞培养平台以可控方式研究细胞对机械信号的反应有着极大的兴趣。然而，对于诸如在组织培养塑料上进行传代扩增等常见细胞培养技术如何影响后续在水凝胶上培养时的细胞行为，人们了解甚少。在这项工作中，我们利用甲基丙烯酸化透明质酸水凝胶平台来研究基质细胞的机械转导。首先通过硫醇 - 迈克尔加成反应形成水凝胶，以模拟正常软组织（如肺）的硬度（约1千帕）。通过未消耗的甲基丙烯酸酯的自由基光聚合进行二次交联，可实现早期（约6千帕）和晚期纤维化组织（约50千帕）硬度的匹配。早期传代（P1）的原代人骨髓间充质基质细胞（hMSCs）随着水凝胶硬度的增加，其铺展、心肌相关转录因子 - A（MRTF - A）核定位以及粘着斑大小均增加。然而，晚期传代（P5）的hMSCs与早期传代的hMSCs相比，对底物力学的敏感性降低，在较硬水凝胶上MRTF - A核转位减少且粘着斑较小。在永生化人肺成纤维细胞系中也观察到类似趋势。总体而言，这项工作突出了标准细胞培养实践对使用水凝胶模型研究细胞对机械信号反应的影响。
