High-throughput screening for modulators of cellular contractile force

Park, Chan Young; Zhou, Enhua H.; Tambe, Dhananjay; Chen, Bo‐Hao; Lavoie, Tera; Dowell, Maria; Simeonov, Anton; Maloney, David J.; Marinković, Аleksandar; Tschumperlin, Daniel J.; Burger, Stephanie; Frykenberg, Matthew; Butler, James P.; Stamer, W. Daniel; Johnson, Mark; Solway, Julian; Fredberg, Jeffrey J.; Krishnan, Ramaswamy

doi:10.1039/c5ib00054h

Cited by 63 publications

(62 citation statements)

References 58 publications

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“…Such studies are difficult to perform on TCPS, as it significantly perturbs baseline expression levels of key myofibroblast markers and might lead to false negatives when screening for useful therapeutics. Additionally, signaling cascades initiated by therapeutics may have important interactions with mechanotransduction pathways [54]. While 3D hydrogel culture led to transcriptional profiles more similar to freshly isolated VICs, there are still many differences.…”

Section: Discussionmentioning

confidence: 99%

Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype

2016

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Valvular interstitial cells (VICs) actively maintain and repair heart valve tissue; however, persistent activation of VICs to a myofibroblast phenotype can lead to aortic stenosis. To better understand and quantify how microenvironmental cues influence VIC phenotype and myofibroblast activation, we compared expression profiles of VICs cultured on poly(ethylene glycol) (PEG) gels to those cultured on tissue culture polystyrene (TCPS), as well as fresh isolates. In general, VICs cultured in hydrogel matrices had lower levels of activation (<10%), similar to levels seen in healthy valve tissue, while VICs cultured on TCPS were ~75% activated myofibroblasts. VICs cultured on TCPS also exhibited a higher magnitude of perturbations in gene expression than soft hydrogel cultures when compared to the native phenotype. Using peptide-modified PEG gels, VICs were seeded on (2D), as well as encapsulated in (3D), matrices of the same composition and modulus. Despite similar levels of activation, VICs cultured in 2D had distinct variations in transcriptional profiles compared to those in 3D hydrogels. Genes related to cell structure and motility were particularly affected by the dimensionality of the culture platform, with higher expression levels in 2D than in 3D. These results indicate that dimensionality may play a significant role in dictating cell phenotype (e.g., through differences in polarity, diffusion of soluble signals), and emphasize the importance of using multiple metrics when characterizing cell phenotype.

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Section: Discussionmentioning

confidence: 99%

Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype

2016

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“…This approach is advantageous over CFS using PAA [13] as the material itself is more robust and uniform, and the fabrication of multiwell-silicone substrates eliminates time-consuming production steps, utilizes standard micro-fabrication procedures, and obviates the need for surface-bound fluorescent beads by embedding them as a monolayer by spin-coating. Moreover, silicone elastomers possess many material-favorable properties over PAA.…”

Section: Discussionmentioning

confidence: 99%

“…Critically, the monolithic impermeable nature of these silicone substrates makes them easy and ideal to subdivide spatially into biochemically independent wells, creating a uniform multi-well platform for traction force microscopy. Taken together, our compliant PDMS presents numerous advantages in becoming a new standard in soft substrates for TFM; these advantages are particularly important for a standardized higher-throughput technology and will enable widespread adoption of CFS from our previous approach using PAA [13] .…”

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confidence: 99%

Traction force screening enabled by compliant PDMS elastomers

Yoshie

Koushki

Kaviani

et al. 2017

Preprint

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Abstract:Acto-myosin contractility is an essential element of many aspects of cellular biology, and manifests as traction forces that cells exert on their surroundings. The central role of these forces makes them a novel principal therapeutic target in diverse diseases. This requires accurate and higher capacity measurements of traction forces; however, existing methods are largely low throughput, limiting their utility in broader applications. To address this need, we employ Fourier-transform traction force microscopy in a parallelized 96-well format, which we refer to as contractile force screening (CFS). Critically, rather than the frequently employed hydrogel polyacrylamide (PAA), we fabricate these plates using polydimethylsiloxane (PDMS) rubber. Key to this approach is that the PDMS used is very compliant, with a lower-bound Young's modulus of approximately 0.7 kPa. We subdivide these monolithic substrates spatially into biochemically independent wells, creating a uniform multiwell platform for traction force screening. We demonstrate the utility and versatility of this platform by quantifying the compound and dose-dependent contractility responses of human airway smooth muscle cells and retinal pigment epithelial cells.

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“…As a comparative storage strategy, corneas were immersed in sterile PBS and stored at 2–8°C. This approach was investigated as it is routinely used to maintain ex vivo corneal integrity over short periods prior to use in surgery [15, 16]. …”

Section: Methodsmentioning

confidence: 99%

Evaluation of 2 ex vivo Bovine Cornea Storage Protocols for Drug Delivery Applications

Thakur

Shrestha

Rupenthal³

2018

Ophthalmic Res

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The use of corneal tissue for ex vivo therapeutic evaluations is limited due to its rapid loss of viability after excision. Optimization of storage conditions may allow prolonged retention of physical tissue properties. In this study, we evaluated how storage in optimized organ culture (OC) medium at 37°C or phosphate-buffered saline (PBS) at 2–8°C impacted physical properties of bovine corneas. Tissue hydration, permeability and histology were monitored at baseline and following 1, 4 and 7 days of storage. Corneas stored in OC demonstrated significantly higher hydration and permeability when compared to those stored in PBS. Histology revealed that storage in OC consistently caused detachment of the epithelial layer by day 4 of storage, whereas both storage conditions caused a significant increase in stromal thickness and tissue vacuolation. This study highlights the limitations of currently available corneal tissue storage approaches for ex vivo drug permeation studies.

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High-throughput screening for modulators of cellular contractile force

Cited by 63 publications

References 58 publications

Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype

Microarray analyses to quantify advantages of 2D and 3D hydrogel culture systems in maintaining the native valvular interstitial cell phenotype

Traction force screening enabled by compliant PDMS elastomers

Evaluation of 2 ex vivo Bovine Cornea Storage Protocols for Drug Delivery Applications

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