A Multiwell Platform for Studying Stiffness-Dependent Cell Biology

Abstract

Adherent cells are typically cultured on rigid substrates that are orders of magnitude stiffer than their tissue of origin. Here, we describe a method to rapidly fabricate 96 and 384 well platforms for routine screening of cells in tissue-relevant stiffness contexts. Briefly, polyacrylamide (PA) hydrogels are cast in glass-bottom plates, functionalized with collagen, and sterilized for cell culture. The Young's modulus of each substrate can be specified from 0.3 to 55 kPa, with collagen surface density held constant over the stiffness range. Using automated fluorescence microscopy, we captured the morphological variations of 7 cell types cultured across a physiological range of stiffness within a 384 well plate. We performed assays of cell number, proliferation, and apoptosis in 96 wells and resolved distinct profiles of cell growth as a function of stiffness among primary and immortalized cell lines. We found that the stiffness-dependent growth of normal human lung fibroblasts is largely invariant with collagen density, and that differences in their accumulation are amplified by increasing serum concentration. Further, we performed a screen of 18 bioactive small molecules and identified compounds with enhanced or reduced effects on soft versus rigid substrates, including blebbistatin, which abolished the suppression of lung fibroblast growth at 1 kPa. The ability to deploy PA gels in multiwell plates for high throughput analysis of cells in tissue-relevant environments opens new opportunities for the discovery of cellular responses that operate in specific stiffness regimes.

Matrix stiffness reverses the effect of actomyosin tension on cell proliferation

“…Using this system, we compared cell growth on collagen I-coated rigid (glass) versus relatively soft, physiologically relevant PA gels (1 kPa elastic [Young's] modulus) (Discher et al, 2005;Klein et al, 2009;Liu et al, 2010;Paszek et al, 2005), and confirmed that inhibition of myosin II with blebbistatin evoked divergent dose-dependent effects on cell number (Fig. 1A), enhancing proliferation on a soft matrix while inhibiting proliferation on glass (Mih et al, 2011). We documented a similar trend of increasing BrdU incorporation, indicative of ongoing DNA synthesis, when blebbistatin was applied to cells on 0.3 and 1 kPa gels, and decreasing BrdU incorporation when blebbistatin was applied on rigid surfaces (Fig.…”

“…To delineate the combined effects of matrix stiffness and modulation of actomyosin contractile function on cell proliferation, we synthesized polyacrylamide (PA) hydrogels of defined stiffness within 96-well glass-bottom plates (Mih et al, 2011). The gels were functionalized with monomeric collagen I at a constant density across all stiffness conditions, allowing for the isolation of matrix stiffness effects.…”

“…Yet, it has also been shown in some cases that actomyosin contractility is not predictive of cell proliferation (Roca-Cusachs et al, 2008) and that inhibition of Rho-associated protein kinase (ROCK) partially rescues rounded endothelial cells from G1 arrest (Mammoto et al, 2004). In fact, inhibition of nonmuscle myosin II in fibroblasts grown on a physiologically soft matrix rescues their proliferation to levels attained on rigid substrates (Mih et al, 2011). This raises a mystery as to why inhibition of myosin in such contexts should exert such a profound stimulatory effect on cell proliferation, and whether our understanding of tensional control of growth, gleaned mostly from studies on rigid substrates, is incomplete.…”

Complementary, Semiautomated Methods for Creating Multidimensional PEG-Based Biomaterials

“…This also allows users to capture stiffness when 3D geometry is unnecessary to answer the biological question at hand. Others have developed 2D hydrogel platforms, 7,20 which produce perfectly flat hydrogels, but they are complicated to fabricate 7 [35][36][37] and it was used for 2D PEG-PC hydrogels when they were first developed for coverslips. 34 Irgacure is not a very efficient photoinitiator 26 because it does not absorb light readily at 365 nm, 35 but this wavelength is used in other platforms that include cell encapsulation to limit damage to the cells.…”

Coming to terms with tissue engineering and regenerative medicine in the lung

“…Added to these passive forces are the active mechanical forces imposed on both airways and vasculature by luminal contraction and relaxation in response to changing demands, and by endogenous and exogenous factors such as local and circulating agonists and antagonists. Although in silico models (79,83,99,132,148,196), in vitro and ex vivo systems (59,92,103,108,118,176,193,202,203), and whole-organ approaches (94,143,205,206) have been developed to explore the importance of such mechanical forces, further advances in airway, lung parenchyma, and vascular mechanobiology will be critical for bioengineering a lung capable of withstanding the internal and external forces exerted on the transplanted lung within the chest cavity and will be critical to ensuring that lung function occurs without airway collapse, injury, or failure of gas exchange. The challenges to our current understanding of lung mechanobiology and their implications for a bioengineered lung are discussed in Materials, Matrix, and Mechanobiology.…”