A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression

Lee, Jason; Ochoa, Miguel Armenta; Maceda, Pablo; Yoon, Euisik; Samarneh, Lara; Wong, Mitchell; Baker, Aaron

doi:10.1038/s41598-020-72158-5

Cited by 8 publications

(6 citation statements)

References 45 publications

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“…The stretch plate, with ANSI standard plate contour dimensions, is automation compatible for easy integration into screening systems to facilitate high throughput endpoints such as target ID and drug discovery. While other stretch devices have been developed previously for high throughput and screening, [103][104][105][106][107] the current 192w stretch plate is of higher well capacity than other reported 96-well format systems and provides this screening advantage. Moreover, the stretch plate silicone elastomer resin design could facilitate large scale injection-molding manufacturing for rapid production and bolster high throughput research.…”

Section: Discussionmentioning

confidence: 99%

“…The chondrocyte stretch gene signature, characterized by L1000 gene expression profiling and RASL-seq, and with adequate intra-plate consistency relative to static gene expression, illustrates feasibility of utilizing transcriptional readouts with this system. Alternative stretch readouts, such as protein expression, 103,107 cell proliferation, 17,108 morphological alignment with stretch, 104,106,109 or calcium signaling, 110 may also be employed.…”

Section: Discussionmentioning

confidence: 99%

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A high throughput cell stretch device for investigating mechanobiology in vitro

Pratt,

Plunkett,

Kuzu

et al. 2024

APL Bioengineering

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Mechanobiology is a rapidly advancing field, with growing evidence that mechanical signaling plays key roles in health and disease. To accelerate mechanobiology-based drug discovery, novel in vitro systems are needed that enable mechanical perturbation of cells in a format amenable to high throughput screening. Here, both a mechanical stretch device and 192-well silicone flexible linear stretch plate were designed and fabricated to meet high throughput technology needs for cell stretch-based applications. To demonstrate the utility of the stretch plate in automation and screening, cell dispensing, liquid handling, high content imaging, and high throughput sequencing platforms were employed. Using this system, an assay was developed as a biological validation and proof-of-concept readout for screening. A mechano-transcriptional stretch response was characterized using focused gene expression profiling measured by RNA-mediated oligonucleotide Annealing, Selection, and Ligation with Next-Gen sequencing. Using articular chondrocytes, a gene expression signature containing stretch responsive genes relevant to cartilage homeostasis and disease was identified. The possibility for integration of other stretch sensitive cell types (e.g., cardiovascular, airway, bladder, gut, and musculoskeletal), in combination with alternative phenotypic readouts (e.g., protein expression, proliferation, or spatial alignment), broadens the scope of high throughput stretch and allows for wider adoption by the research community. This high throughput mechanical stress device fills an unmet need in phenotypic screening technology to support drug discovery in mechanobiology-based disease areas.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A high throughput cell stretch device for investigating mechanobiology in vitro

Pratt,

Plunkett,

Kuzu

et al. 2024

APL Bioengineering

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“…Our group has previously created a high throughput device that applies mechanical stretch to cancer cells in a 6 × 96 well format 18 – 21 . Cells were cultured on custom 96-well culture plates with a flexible silicone membrane culture surface.…”

Section: Materials and Methods (Online)mentioning

confidence: 99%

Biomechanical regulation of breast cancer metastasis and progression

Spencer

Sligar

Chavarria

et al. 2021

Sci Rep

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Physical activity has been consistently linked to decreased incidence of breast cancer and a substantial increase in the length of survival of patients with breast cancer. However, the understanding of how applied physical forces directly regulate breast cancer remains limited. We investigated the role of mechanical forces in altering the chemoresistance, proliferation and metastasis of breast cancer cells. We found that applied mechanical tension can dramatically alter gene expression in breast cancer cells, leading to decreased proliferation, increased resistance to chemotherapeutic treatment and enhanced adhesion to inflamed endothelial cells and collagen I under fluidic shear stress. A mechanistic analysis of the pathways involved in these effects supported a complex signaling network that included Abl1, Lck, Jak2 and PI3K to regulate pro-survival signaling and enhancement of adhesion under flow. Studies using mouse xenograft models demonstrated reduced proliferation of breast cancer cells with orthotopic implantation and increased metastasis to the skull when the cancer cells were treated with mechanical load. Using high throughput mechanobiological screens we identified pathways that could be targeted to reduce the effects of load on metastasis and found that the effects of mechanical load on bone colonization could be reduced through treatment with a PI3Kγ inhibitor.

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“…20 It is, however, less clear how mechanical stretch of the flexible substrate leads to forces on the nucleus. In recent studies from our laboratory, we have found that physiological waveforms of stretch can lead to differential responsiveness of many cell types, 21,22 and in mesenchymal stem cells (MSCs), lead to increased vascular phenotypes and regenerative properties. 23 In the these studies, we demonstrated that a very specific types of mechanical stretch induce the MSCs into the vascular phenotypes.…”

Section: Introductionmentioning

confidence: 99%

“…This study provides a computational model for understanding the forces that different regions of the cells are experiencing when they are stretched at various levels of culture membrane strain. As our previous work has highlighted the importance of physiological waveforms, [21][22][23] we examined the differences between the standard sine waveform and a brachial waveform of mechanical stretch that mimics the stretch of the brachial artery during the cardiac cycle. Our previous study demonstrated that brachial waveforms delivered at 0.1 Hz frequency of loading and 7.5%-12.5% maximal strain resulted in MSCs with enhanced vascular regenerative properties and maximal activation of Yap/Taz (Yes-associated protein/transcriptional coactivator with PDZ-binding motif) signaling.…”

Section: Introductionmentioning

confidence: 99%

A Computational Model of Mechanical Stretching of Cultured Cells on a Flexible Membrane

Massidda,

Ashirov,

Demkov

et al. 2024

Preprint

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Mechanical forces applied to cells are known to regulate a wide variety of biological processes. Recent studies have supported that mechanical forces can cause nuclear deformation, leading to significant alterations in the gene expression and chromatin landscape of the cell. While the stresses and strains applied to cells is it is often known or controlled experimentally on a macroscopic length scale, it is often unclear what the actual forces and displacements are at the microscopic level of the cell. In this work, we created a model of cell deformation during application of mechanical stretch to cultured cells growth on a flexible membrane. This configuration is commonly used is in experimental studies as a means to apply controlled mechanical strains to adherent cultured cells. The parameters used in the study were used for application of strain to a mesenchymal stem cell stretched on a membrane. computational model was created to simulate the stresses and strains within the cell under a variety of stain amplitudes, waveforms and frequencies of mechanical loading with the range of commonly used experimental systems. The results demonstrate the connection between mechanical loading parameters applied through the flexible membrane and the resulting stresses and strains within the cell and nucleus. Using a viscoelastic model of chromatin, we connected the results provide to a rough model of resulting deformation within chromatin from the forces applied to the nucleus. Overall, the model is useful in providing insight between experimentally applied mechanical forces and the actual forces within the cell to better interpret the results of experimental studies.Statement of SignificanceIn this work, we created a computational model of the mechanical stretching of cell on a flexible membrane under cyclic mechanical loading. This model provides insight into the forces and displacements inside of cell that result from that application of stretch. As many experiments use this set up, our work is relevant to interpreting many studies that use mechanical stretch to stimulate mechanotransduction.

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A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression

Cited by 8 publications

References 45 publications

A high throughput cell stretch device for investigating mechanobiology in vitro

A high throughput cell stretch device for investigating mechanobiology in vitro

Biomechanical regulation of breast cancer metastasis and progression

A Computational Model of Mechanical Stretching of Cultured Cells on a Flexible Membrane

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