Development of a Probability-Based In Vitro Eye Irritation Screening Platform.

Traditional eye irritation assessments, which rely on animal models or ex vivo tissues, face limitations due to ethical concerns, costs, and low throughput. Although numerous in vitro tests have been developed, none have successfully reconciled the need for high experimental throughput with the accurate prediction of irritation potential, attributable to the complexity of irritation mechanisms. Simple cell models, while suitable for high-throughput screening, offer limited mechanistic insights, contrasting with more physiologically relevant but less scalable complex organotypic corneal tissue constructs. This study presents a novel strategy to enhance the predictive accuracy of screening-compatible simple cell models in eye irritation testing. Our method combines the results of two in vitro assays-cell apoptosis and nociceptor (TRPV1) activation-using micropatterned chips to partition human corneal epithelial cells into numerous discrete small populations. Following exposure to test compounds, we measure apoptosis and nociceptor activation responses. The large datasets collected from the cell micropatterns facilitate binarization and statistical fitting to calculate a mathematical probability, which assesses the compound's potential to cause eye irritation. This method potentially enables the amalgamation of multiple mechanistic readouts into a singular index, providing a more accurate and reliable prediction of eye irritation potential in a format amenable to high-throughput screening.

Bio-mimicking Shear Stress Environments for Enhancing Mesenchymal Stem Cell Differentiation.

Mesenchymal stem cells (MSCs) are multipotent stromal cells, with the ability to differentiate into mesodermal (e.g., adipocyte, chondrocyte, hematopoietic, myocyte, osteoblast), ectodermal (e.g., epithelial, neural) and endodermal (e.g., hepatocyte, islet cell) lineages based on the type of induction cues provided. As compared to embryonic stem cells, MSCs hold a multitude of advantages from a clinical translation perspective, including ease of isolation, low immunogenicity and limited ethical concerns. Therefore, MSCs are a promising stem cell source for different regenerative medicine applications. The in vitro differentiation of MSCs into different lineages relies on effective mimicking of the in vivo milieu, including both biochemical and mechanical stimuli. As compared to other biophysical cues, such as substrate stiffness and topography, the role of fluid shear stress (SS) in regulating MSC differentiation has been investigated to a lesser extent although the role of interstitial fluid and vascular flow in regulating the normal physiology of bone, muscle and cardiovascular tissues is well-known. This review aims to summarise the current state-of-the-art regarding the role of SS in the differentiation of MSCs into osteogenic, cardiovascular, chondrogenic, adipogenic and neurogenic lineages. We will also highlight and discuss the potential of employing SS to augment the differentiation of MSCs to other lineages, where SS is known to play a role physiologically but has not yet been successfully harnessed for in vitro differentiation, including liver, kidney and corneal tissue lineage cells. The incorporation of SS, in combination with biochemical and biophysical cues during MSC differentiation, may provide a promising avenue to improve the functionality of the differentiated cells by more closely mimicking the in vivo milieu.

Topography elicits distinct phenotypes and functions in human primary and stem cell derived endothelial cells.

The effective deployment of arterial (AECs), venous (VECs) and stem cell-derived endothelial cells (PSC-ECs) in clinical applications requires understanding of their distinctive phenotypic and functional characteristics, including their responses to microenvironmental cues. Efforts to mimic the in-vivo vascular basement membrane milieu have led to the design and fabrication of nano- and micro-topographical substrates. Although the basement membrane architectures of arteries and veins are different, investigations into the effects of substrate topographies have so far focused on generic EC characteristics. Thus, topographical modulation of arterial- or venous-specific EC phenotype and function remains unknown. Here, we comprehensively evaluated the effects of 16 unique topographies on primary AECs, VECs and human PSC-ECs using a Multi Architectural (MARC) Chip. Gratings and micro-lenses augmented venous-specific phenotypes and depressed arterial functions in VECs; while AECs did not respond consistently to topography. PSC-ECs exhibited phenotypic and functional maturation towards an arterial subtype with increased angiogenic potential, NOTCH1 and Ac-LDL expression on gratings. Specific topographies could elicit different phenotypic and functional changes, despite similar morphological response in different ECs, demonstrating no direct correlation between the two responses.

Environmental Specification of Pluripotent Stem Cell Derived Endothelial Cells Toward Arterial and Venous Subtypes.

Endothelial cells (ECs) are required for a multitude of cardiovascular clinical applications, such as revascularization of ischemic tissues or endothelialization of tissue engineered grafts. Patient derived primary ECs are limited in number, have donor variabilities and their in vitro phenotypes and functions can deteriorate over time. This necessitates the exploration of alternative EC sources. Although there has been a recent surge in the use of pluripotent stem cell derived endothelial cells (PSC-ECs) for various cardiovascular clinical applications, current differentiation protocols yield a heterogeneous EC population, where their specification into arterial or venous subtypes is undefined. Since arterial and venous ECs are phenotypically and functionally different, inappropriate matching of exogenous ECs to host sites can potentially affect clinical efficacy, as exemplified by venous graft mismatch when placed into an arterial environment. Therefore, there is a need to design and employ environmental cues that can effectively modulate PSC-ECs into a more homogeneous arterial or venous phenotype for better adaptation to the host environment, which will in turn contribute to better application efficacy. In this review, we will first give an overview of the developmental and functional differences between arterial and venous ECs. This provides the foundation for our subsequent discussion on the different bioengineering strategies that have been investigated to varying extent in providing biochemical and biophysical environmental cues to mature PSC-ECs into arterial or venous subtypes. The ability to efficiently leverage on a combination of biochemical and biophysical environmental cues to modulate intrinsic arterio-venous specification programs in ECs will greatly facilitate future translational applications of PSC-ECs. Since the development and maintenance of arterial and venous ECs in vivo occur in disparate physio-chemical microenvironments, it is conceivable that the application of these environmental factors in customized combinations or magnitudes can be used to selectively mature PSC-ECs into an arterial or venous subtype.

Self-aligning Tetris-Like (TILE) modular microfluidic platform for mimicking multi-organ interactions.

Multi-organ perfusion systems offer the unique opportunity to mimic different physiological systemic interactions. However, existing multi-organ culture platforms have limited flexibility in specifying the culture conditions, device architectures, and fluidic connectivity simultaneously. Here, we report a modular microfluidic platform that addresses this limitation by enabling easy conversion of existing microfluidic devices into tissue and fluid control modules with self-aligning magnetic interconnects. This enables a 'stick-n-play' approach to assemble planar perfusion circuits that are amenable to both bioimaging-based and analytical measurements. A myriad of tissue culture and flow control TILE modules were successfully constructed with backward compatibility. Finally, we demonstrate applications in constructing recirculating multi-organ systems to emulate liver-mediated bioactivation of nutraceuticals and prodrugs to modulate their therapeutic efficacies in the context of atherosclerosis and cancer. This platform greatly facilitates the integration of existing organs-on-chip models to provide an intuitive and flexible way for users to configure different multi-organ perfusion systems.

Determination of critical shear stress for maturation of human pluripotent stem cell-derived endothelial cells towards an arterial subtype.

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm 2 . We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm 2 , which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm 2 , whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm 2 was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.

Investigation of benzoyloximes as benzoylating reagents: benzoyl-Oxyma as a selective benzoylating reagent.

Hydroxybenzotriazole (HOBt) and HOBt-derived reagents have been classified as Class I explosives, with restrictions on their transportation and storage. We explored a range of benzoylated oxime-based reagents as alternatives to benzoyloxybenzotriazole (BBTZ) for the selective benzoylation of carbohydrate polyols. Benzoylated oximes derived from 2-hydroximino-malononitrile, ethyl 2-hydroximino-2-cyanoacetate (Oxyma), and tert-butyl 2-hydroximino-2-cyanoacetate were most effective for benzoylation of a simple primary alcohol, with yields approaching that obtained for BBTZ. When applied to carbohydrate diols, the most effective reagent was identified as benzoyl-Oxyma. Benzoyl-Oxyma is a highly crystalline, readily prepared alternative to BBTZ, useful in the selective benzoylation of carbohydrate polyols.