The FDA modernisation act 2.0: Bringing non-animal technologies to the regulatory table.

The FDA modernisation Act 2.0 marks a game-changing legislation enabling drug registration without the absolute requirement for the use of animals in safety toxicology assessment. We discuss landmark developments in the legislation under which the FDA operates and consider the implications of this most recent chapter in the evolution of the drug regulation pathway, focussing on new opportunities to embed microphysiological systems.

ACE2 Expression in Organotypic Human Airway Epithelial Cultures and Airway Biopsies.

Coronavirus disease 2019 (COVID-19) caused by infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an acute respiratory disease with systemic complications. Therapeutic strategies for COVID-19, including repurposing (partially) developed drugs are urgently needed, regardless of the increasingly successful vaccination outcomes. We characterized two-dimensional (2D) and three-dimensional models (3D) to establish a physiologically relevant airway epithelial model with potential for investigating SARS-CoV-2 therapeutics. Human airway basal epithelial cells maintained in submerged 2D culture were used at low passage to retain the capacity to differentiate into ciliated, club, and goblet cells in both air-liquid interface culture (ALI) and airway organoid cultures, which were then analyzed for cell phenotype makers. Airway biopsies from non-asthmatic and asthmatic donors enabled comparative evaluation of the level and distribution of immunoreactive angiotensin-converting enzyme 2 (ACE2). ACE2 and transmembrane serine proteinase 2 (TMPRSS2) mRNA were expressed in ALI and airway organoids at levels similar to those of native (i.e., non-cultured) human bronchial epithelial cells, whereas furin expression was more faithfully represented in ALI. ACE2 was mainly localized to ciliated and basal epithelial cells in human airway biopsies, ALI, and airway organoids. Cystic fibrosis appeared to have no influence on ACE2 gene expression. Neither asthma nor smoking status had consistent marked influence on the expression or distribution of ACE2 in airway biopsies. SARS-CoV-2 infection of ALI cultures did not increase the levels of selected cytokines. Organotypic, and particularly ALI airway cultures are useful and practical tools for investigation of SARS-CoV-2 infection and evaluating the clinical potential of therapeutics for COVID-19.

Comprehensive multiplexed superfusion system enables physiological emulation in cell culture: exemplification by persistent circadian entrainment.

Cells and tissues are routinely cultured in vitro for biological research with findings being extrapolated to their host organ and tissue function. However, most samples are cultured and studied in unphysiological environments, without temporal variation in the biochemical cues that are ubiquitous in vivo. The artificiality of these conditions undermines the predictive value of cell culture studies. We ascribe the prevalence of this suboptimal culture methodology to the lack of practical continuous flow systems that are economical and robust. Here, we design and implement an expandable multiplexed flow system for cell culture superfusion. By expanding on the concept of the planar peristaltic pump, we fabricated a highly compact and multiplexed pump head with up to 48 active pump lines. The pump is incorporated into a custom, open-top superfusion system configured for conventional multi-well culture plates. We then demonstrated the utility of the system for in vitro circadian entrainment using a daily cortisol pulse, generating a sustained circadian amplitude that is essential for physiological emulation and chrono-pharmacological studies. The multiplexed pump is complemented by a package of fluidic interconnection and management methods enabling user-friendly and scalable operation. Collectively, the suite of technologies provides a much-needed improvement in physiological emulation to support the predictive value of in vitro biomedical and biological research.

Gaming Motivation and Negative Psychosocial Outcomes in Male Adolescents: An Individual-Centered 1-Year Longitudinal Study.

"Gaming motivation" is a useful concept to draw upon when considering inconsistencies in the effects of online gaming on psychosocial wellbeing. However, most prior studies that utilize it are cross-sectional and do not allow that individuals can be driven by multiple motives. The present study uses an individual-centered method to classify gaming motivation styles of male adolescents and longitudinally observes the relationship between gaming motivations and psychosocial outcomes. A total of 929 healthy, male, adolescent gamers were recruited in October 2019 and classified into "recreational" "achiever," and "escaper" categories according to their baseline gaming motivations and self-esteem levels. Then, 1-year incidence rates of players and relative risks (RRs) of social withdrawal problems, anxiety/depression syndrome, and self-destructive/identity problems were assessed. Recreational players were found to have the lowest incidence of all the three psychosocial problems among the three categories, achievers only had a moderate risk of social withdrawal, compared to recreational players, while escapers showed a strong risk for social withdrawal, anxiety/depression, and self-destructive/identity problems, relative to recreational gamers. Overall, the different motivation subgroups were associated with different psychosocial problems. Both achievers and escapers were found to be maladaptive, but their psychosocial outcomes were different, a finding that provides further insight into the psychological mechanisms underlying these subgroups.

Artificial Microniche Array with Spatially Structured Biochemical Cues.

We present here a method to create arrays of microcavities that can be differentially coated on their bottom, side, and top with different proteins. These cavities range in size from single cell to multicellular aggregate. We provide detailed protocols to create such arrays with some variations using different materials and different coating proteins. The use of such cavities as bona fide artificial microniches to mimic cellular microenvironments has been already established and is referenced.

Probing compression versus stretch activated recruitment of cortical actin and apical junction proteins using mechanical stimulations of suspended doublets.

We report an experimental approach to study the mechanosensitivity of cell-cell contact upon mechanical stimulation in suspended cell-doublets. The doublet is placed astride an hourglass aperture, and a hydrodynamic force is selectively exerted on only one of the cells. The geometry of the device concentrates the mechanical shear over the junction area. Together with mechanical shear, the system also allows confocal quantitative live imaging of the recruitment of junction proteins (e.g., E-cadherin, ZO-1, occludin, and actin). We observed the time sequence over which proteins were recruited to the stretched region of the contact. The compressed side of the contact showed no response. We demonstrated how this mechanism polarizes the stress-induced recruitment of junctional components within one single junction. Finally, we demonstrated that stabilizing the actin cortex dynamics abolishes the mechanosensitive response of the junction. Our experimental design provides an original approach to study the role of mechanical force at a cell-cell contact with unprecedented control over stress application and quantitative optical analysis.