The effects of xeno-free cryopreservation on the contractile properties of human iPSC derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have advanced our ability to study the basic function of the heart and model cardiac diseases. Due to the complexities in stem cell culture and differentiation protocols, many researchers source their hiPSC-CMs from collaborators or commercial biobanks. Generally, the field has assumed the health of frozen cardiomyocytes is unchanged if the cells adhere to the substrate and commence beating. However, very few have investigated the effects of cryopreservation on hiPSC-CM's functional and transcriptional health at the cellular and molecular level. Here we review methods and challenges associated with cryopreservation, and examine the effects of cryopreservation on the functionality (contractility and calcium handling) and transcriptome of hiPSC-CMs from six healthy stem cell lines. Utilizing protein patterning methods to template physiological cell aspect ratios (7:1, length:width) in conjunction with polyacrylamide (PA) hydrogels, we measured changes in force generation and calcium handling of single hiPSC-CMs. We observed that cryopreservation altered the functionality and transcriptome of hiPSC-CMs towards larger sizes and contractile force as assessed by increased spread area and volume, single cell traction force microscopy and delayed calcium dynamics. hiPSC-CMs are broadly used for basic science research, regenerative medicine, and testing biological therapeutics. This study informs the design of experiments utilizing hiPSC-CMs to avoid confounding functional changes due to cryopreservation with other treatments.

Wafer-Scale Patterning of Protein Templates for Hydrogel Fabrication.

Human-induced pluripotent stem cell-derived cardiomyocytes are a potentially unlimited cell source and promising patient-specific in vitro model of cardiac diseases. Yet, these cells are limited by immaturity and population heterogeneity. Current in vitro studies aiming at better understanding of the mechanical and chemical cues in the microenvironment that drive cellular maturation involve deformable materials and precise manipulation of the microenvironment with, for example, micropatterns. Such microenvironment manipulation most often involves microfabrication protocols which are time-consuming, require cleanroom facilities and photolithography expertise. Here, we present a method to increase the scale of the fabrication pipeline, thereby enabling large-batch generation of shelf-stable microenvironment protein templates on glass chips. This decreases fabrication time and allows for more flexibility in the subsequent steps, for example, in tuning the material properties and the selection of extracellular matrix or cell proteins. Further, the fabrication of deformable hydrogels has been optimized for compatibility with these templates, in addition to the templates being able to be used to acquire protein patterns directly on the glass chips. With our approach, we have successfully controlled the shapes of cardiomyocytes seeded on Matrigel-patterned hydrogels.

Nano-plastics induce aquatic particulate organic matter (microgels) formation.

The pervasive presence of plastic waste in the aquatic environment is widely viewed as one of the most serious environmental challenges for current and future generations. Microplastics ultimately degrade into nano and smaller-sizes. In turn, their biological and ecological impacts become more complicated and ambiguous. Nano-plastic particles travel from freshwater systems to estuarine and oceanic regions, during which they can interact with dissolved organic matter (DOM) to form microgels. Microgel formation is ubiquitous in aquatic systems, serving as a shunt between DOM and particulate organic matter (POM), as well as playing key roles in particle aggregation/sedimentation and pollutant transport. Currently the influences and mechanisms of the aggregation behavior and environmental fate of nano-plastics in different aquatic environments is poorly understood. Here, we report that 25 nm polystyrene nano-particles in lake and river water can promote POM (microgel) formation and accelerate the DOM-POM transition. We also adjusted various salinities of water samples to simulate scenarios based on plastic transport in waters flowing from rivers to seas. The results indicate polystyrene nanoparticles can interact with organic matter to form large organic particles, which may undergo further settling in response to specific salinity levels. Polystyrene-induced microgel formation appears to involve the hydrophobic interactions between plastics and DOM. Our data provides much needed information for modeling and understanding the retention and sedimentation of nano-plastics. We show that nano-plastics alter the DOM-POM shunt to cause unanticipated perturbations in the functionality of aquatic ecosystems.