A microgroove patterned multiwell cell culture plate for high-throughput studies of cell alignment.

Grooved substrates are commonly used to guide cell alignment and produce in vitro tissues that mimic certain aspects of in vivo cellular organization. These more sophisticated tissues provide valuable in vitro models for testing drugs and for dissecting out molecular mechanisms that direct tissue organization. To increase the accessibility of these tissue models we describe a simple and yet reproducible strategy to produce 1 µm-spaced grooved well plates suitable for conducting automated analysis of cellular responses. We characterize the alignment of four human cell types: retinal epithelial cells, umbilical vein endothelial cells, foreskin fibroblasts, and human pluripotent stem-cell-derived cardiac cells on grooves. We find all cells align along the grooves to differing extents at both sparse and confluent densities. To increase the sophistication of in vitro tissue organization possible, we also created hybrid substrates with controlled patterns of microgrooved and flat regions that can be identified in real-time using optical microscopy. Using our hybrid patterned surfaces we explore: (i) the ability of neighboring cells to provide a template to organize surrounding cells that are not directly exposed to grooved topographic cues, and (ii) the distance over which this template effect can operate in confluent cell sheets. We find that in fibroblast sheets, but not epithelial sheets, cells aligned on grooves can direct alignment of neighboring cells in flat regions over a limited distance of approximately 200 µm. Our hybrid surface plate provides a novel tool for studying the collective response of groups of cells exposed to differential topographical cues.

Multiwell plate tools for controlling cellular alignment with grooved topography.

In many tissues, cells must be aligned for proper function. This alignment can occur at the cellular and/or subcellular (protein/molecular) level. The alignment of cytoskeletal components, in fact, precedes whole cell alignment. A variety of methods exist to manipulate cytoskeletal and whole cell alignment; one of the simplest and most predictable involves seeding adherent cells onto defined substrate topography. We present here two methods to create grooved multiwell plates: one involving microfabrication, which allows for custom design of substrate topography, and a simpler, inexpensive method using commercially available diffraction gratings. We also include methods for manual and automatic quantification of cell alignment.

Nonautonomous contact guidance signaling during collective cell migration.

Directed migration of groups of cells is a critical aspect of tissue morphogenesis that ensures proper tissue organization and, consequently, function. Cells moving in groups, unlike single cells, must coordinate their migratory behavior to maintain tissue integrity. During directed migration, cells are guided by a combination of mechanical and chemical cues presented by neighboring cells and the surrounding extracellular matrix. One important class of signals that guide cell migration includes topographic cues. Although the contact guidance response of individual cells to topographic cues has been extensively characterized, little is known about the response of groups of cells to topographic cues, the impact of such cues on cell-cell coordination within groups, and the transmission of nonautonomous contact guidance information between neighboring cells. Here, we explore these phenomena by quantifying the migratory response of confluent monolayers of epithelial and fibroblast cells to contact guidance cues provided by grooved topography. We show that, in both sparse clusters and confluent sheets, individual cells are contact-guided by grooves and show more coordinated behavior on grooved versus flat substrates. Furthermore, we demonstrate both in vitro and in silico that the guidance signal provided by a groove can propagate between neighboring cells in a confluent monolayer, and that the distance over which signal propagation occurs is not significantly influenced by the strength of cell-cell junctions but is an emergent property, similar to cellular streaming, triggered by mechanical exclusion interactions within the collective system.

High-throughput fingerprinting of human pluripotent stem cell fate responses and lineage bias.

Populations of cells create local environments that lead to emergent heterogeneity. This is particularly evident with human pluripotent stem cells (hPSCs): microenvironmental heterogeneity limits hPSC cell fate control. We developed a high-throughput platform to screen hPSCs in configurable microenvironments in which we optimized colony size, cell density and other parameters to achieve rapid and robust cell fate responses to exogenous cues. We used this platform to perform single-cell protein expression profiling, revealing that Oct4 and Sox2 costaining discriminates pluripotent, neuroectoderm, primitive streak and extraembryonic cell fates. We applied this Oct4-Sox2 code to analyze dose responses of 27 developmental factors to obtain lineage-specific concentration optima and to quantify cell line-specific endogenous signaling pathway activation and differentiation bias. We demonstrated that short-term responses predict definitive endoderm induction efficiency and can be used to rescue differentiation of cell lines reticent to cardiac induction. This platform will facilitate high-throughput hPSC-based screening and quantification of lineage-induction bias.

HER2-targeted liposomal doxorubicin displays enhanced anti-tumorigenic effects without associated cardiotoxicity.

Anthracycline-based regimens are a mainstay of early breast cancer therapy, however their use is limited by cardiac toxicity. The potential for cardiotoxicity is a major consideration in the design and development of combinatorial therapies incorporating anthracyclines and agents that target the HER2-mediated signaling pathway, such as trastuzumab. In this regard, HER2-targeted liposomal doxorubicin was developed to provide clinical benefit by both reducing the cardiotoxicity observed with anthracyclines and enhancing the therapeutic potential of HER2-based therapies that are currently available for HER2-overexpressing cancers. While documenting the enhanced therapeutic potential of HER2-targeted liposomal doxorubicin can be done with existing models, there has been no validated human cardiac cell-based assay system to rigorously assess the cardiotoxicity of anthracyclines. To understand if HER2-targeting of liposomal doxorubicin is possible with a favorable cardiac safety profile, we applied a human stem cell-derived cardiomyocyte platform to evaluate the doxorubicin exposure of human cardiac cells to HER2-targeted liposomal doxorubicin. To the best of our knowledge, this is the first known application of a stem cell-derived system for evaluating preclinical cardiotoxicity of an investigational agent. We demonstrate that HER2-targeted liposomal doxorubicin has little or no uptake into human cardiomyocytes, does not inhibit HER2-mediated signaling, results in little or no evidence of cardiomyocyte cell death or dysfunction, and retains the low penetration into heart tissue of liposomal doxorubicin. Taken together, this data ultimately led to the clinical decision to advance this drug to Phase I clinical testing, which is now ongoing as a single agent in HER2-expressing cancers.