Yoshie Haruka, Koushki Newsha, Molter Clayton, Siegel Peter M, Krishnan Ramaswamy, Ehrlicher Allen J
Department of Bioengineering, McGill University.
Goodman Cancer Research Centre, McGill University; Department of Medicine, McGill University.
J Vis Exp. 2019 Jun 1(148). doi: 10.3791/59364.
Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications, however, require direct quantification of these forces. We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems. In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-β, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-β exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.
细胞收缩性在生物学的多个方面都至关重要，驱动着从运动和分裂到组织收缩及机械稳定性等一系列过程，是多细胞动物生命的核心要素。在贴壁细胞中，肌动蛋白-肌球蛋白收缩表现为细胞对其底物施加的牵引力。细胞收缩性失调出现在众多病理状况中，使得收缩性成为使用生物物理学作为指标的多种诊断方法中有前景的靶点。此外，新的治疗策略可基于纠正细胞收缩性的明显功能障碍。然而，这些应用需要直接量化这些力。我们开发了基于硅酮弹性体的并行多孔板形式的牵引力显微镜（TFM）。我们使用硅橡胶，特别是聚二甲基硅氧烷（PDMS），而非常用的水凝胶聚丙烯酰胺（PAA），这使我们能够制造出坚固且惰性的底物，其保质期无限，无需特殊储存条件。与基于柱形PDMS且模量在吉帕范围的方法不同，此处使用的PDMS非常柔顺，模量范围约为0.4千帕至100千帕。我们通过将这些大的整体底物在空间上划分为生物化学独立的孔，创建了一个用于TFM的高通量平台，从而形成了一个与现有多孔系统兼容的用于牵引力筛选的多孔板平台。在本论文中，我们使用这个多孔板牵引力系统来研究上皮-间质转化（EMT）；我们通过将NMuMG细胞暴露于转化生长因子-β（TGF-β）来诱导EMT，并量化EMT过程中的生物物理变化。我们测量收缩性作为TGF-β暴露浓度和持续时间的函数。我们在此的发现证明了并行化TFM在疾病生物物理学背景下的实用性。
