From animal testing to in vitro systems: advancing standardization in microphysiological systems.

Limitations with cell cultures and experimental animal-based studies have had the scientific and industrial communities searching for new approaches that can provide reliable human models for applications such as drug development, toxicological assessment, and in vitro pre-clinical evaluation. This has resulted in the development of microfluidic-based cultures that may better represent organs and organ systems in vivo than conventional monolayer cell cultures. Although there is considerable interest from industry and regulatory bodies in this technology, several challenges need to be addressed for it to reach its full potential. Among those is a lack of guidelines and standards. Therefore, a multidisciplinary team of stakeholders was formed, with members from the US Food and Drug Administration (FDA), the National Institute of Standards and Technology (NIST), European Union, academia, and industry, to provide a framework for future development of guidelines/standards governing engineering concepts of organ-on-a-chip models. The result of this work is presented here for interested parties, stakeholders, and other standards development organizations (SDOs) to foster further discussion and enhance the impact and benefits of these efforts.

Validation of a 3D perfused cell culture platform as a tool for humanised preclinical drug testing in breast cancer using established cell lines and patient-derived tissues.

3D cell culture models of cancer are currently being developed to recapitulate in vivo physiological conditions and to assess therapeutic responses. However, most models failed to incorporate the biochemical and biophysical stimuli from fluid flow. In this study, a three-dimensional scaffold, SeedEZ was applied within the PerfusionPal perfused culture system to investigate how perfusion, and blood-like oxygen delivery influenced breast cancer cell growth and their responses to a commonly used breast cancer drug tamoxifen. Our results showed that breast cancer cells could be maintained over 3 weeks in PerfusionPal with increased cell viability compared to static 3D culture in fully humanised conditions. This platform also supported examining the effect of tamoxifen on breast cancer cell lines and in primary patient-derived breast cancer samples. Future work is warranted to further the adaption for fully humanised assessment of drug effectiveness in a patient personalized approach with the aim to reduce the burden of animal use in cancer research and increase the degree of human pre-clinical data translation to clinic.

A 3D Cell Culture Organ-on-a-Chip Platform With a Breathable Hemoglobin Analogue Augments and Extends Primary Human Hepatocyte Functions in vitro.

Remarkable advances in three-dimensional (3D) cell cultures and organ-on-a-chip technologies have opened the door to recapitulate complex aspects of human physiology, pathology, and drug responses in vitro. The challenges regarding oxygen delivery, throughput, assay multiplexing, and experimental complexity are addressed to ensure that perfused 3D cell culture organ-on-a-chip models become a routine research tool adopted by academic and industrial stakeholders. To move the field forward, we present a throughput-scalable organ-on-a-chip insert system that requires a single tube to operate 48 statistically independent 3D cell culture organ models. Then, we introduce in-well perfusion to circumvent the loss of cell signaling and drug metabolites in otherwise one-way flow of perfusate. Further, to augment the relevancy of 3D cell culture models in vitro, we tackle the problem of oxygen transport by blood using, for the first time, a breathable hemoglobin analog to improve delivery of respiratory gases to cells, because in vivo approximately 98% of oxygen delivery to cells takes place via reversible binding to hemoglobin. Next, we show that improved oxygenation shifts cellular metabolic pathways toward oxidative phosphorylation that contributes to the maintenance of differentiated liver phenotypes in vitro. Lastly, we demonstrate that the activity of cytochrome P450 family of drug metabolizing enzymes is increased and prolonged in primary human hepatocytes cultured in 3D compared to two-dimensional (2D) cell culture gold standard with important ramifications for drug metabolism, drug-drug interactions and pharmacokinetic studies in vitro.

SeedEZ™ Interdigitated Electrodes and Multifunctional Layered Biosensor Composites (MLBCs): A Paradigm Shift in the Development of In Vitro BioMicrosystems.

We have developed a new technology for the realization of composite biosensor systems, capable of measuring electrical and electrophysiological signals from electrogenic cells, using SeedEZ™ 3D cell culture-scaffold material. This represents a paradigm-shift for BioMEMS processing; 'Biology-Microfabrication' versus the standard 'Microfabrication-Biology' approach. An Interdigitated Electrode (IDE) developed on the 3D cell-scaffold was used to successfully monitor acute cardiomyocyte growth and controlled population decline. We have further characterized processability of the 3D scaffold, demonstrated long-term biocompatibility of the scaffold with various cell lines and developed a multifunctional layered biosensor composites (MLBCs) using SeedEZ™ and other biocompatible substrates for future multilayer sensor integration.

Protease-degradable PEG-maleimide coating with on-demand release of IL-1Ra to improve tissue response to neural electrodes.

Neural electrodes are an important part of brain-machine interface devices that can restore functionality to patients with sensory and movement disorders. Chronically implanted neural electrodes induce an unfavorable tissue response which includes inflammation, scar formation, and neuronal cell death, eventually causing loss of electrode function. We developed a poly(ethylene glycol) hydrogel coating for neural electrodes with non-fouling characteristics, incorporated an anti-inflammatory agent, and engineered a stimulus-responsive degradable portion for on-demand release of the anti-inflammatory agent in response to inflammatory stimuli. This coating reduces in vitro glial cell adhesion, cell spreading, and cytokine release compared to uncoated controls. We also analyzed the in vivo tissue response using immunohistochemistry and microarray qRT-PCR. Although no differences were observed among coated and uncoated electrodes for inflammatory cell markers, lower IgG penetration into the tissue around PEG+IL-1Ra coated electrodes indicates an improvement in blood-brain barrier integrity. Gene expression analysis showed higher expression of IL-6 and MMP-2 around PEG+IL-1Ra samples, as well as an increase in CNTF expression, an important marker for neuronal survival. Importantly, increased neuronal survival around coated electrodes compared to uncoated controls was observed. Collectively, these results indicate promising findings for an engineered coating to increase neuronal survival and improve tissue response around implanted neural electrodes.

The effect of conditional inactivation of beta 1 integrins using twist 2 Cre, Osterix Cre and osteocalcin Cre lines on skeletal phenotype.

Skeletal development and growth are complex processes regulated by multiple microenvironmental cues, including integrin-ECM interactions. The ß1 sub-family of integrins is the largest integrin sub-family and constitutes the main integrin binding partners of collagen I, the major ECM component of bone. As complete ß1 integrin knockout results in embryonic lethality, studies of ß1 integrin function in vivo rely on tissue-specific gene deletions. While multiple in vitro studies indicate that ß1 integrins are crucial regulators of osteogenesis and mineralization, in vivo osteoblast-specific perturbations of ß1 integrins have resulted in mild and sometimes contradictory skeletal phenotypes. To further investigate the role of ß1 integrins on skeletal phenotype, we used the Twist2-Cre, Osterix-Cre and osteocalcin-Cre lines to generate conditional ß1 integrin deletions, where Cre is expressed primarily in mesenchymal condensation, pre-osteoblast, and mature osteoblast lineage cells respectively within these lines. Mice with Twist2-specific ß1 integrin disruption were smaller, had impaired skeletal development, especially in the craniofacial and vertebral tissues at E19.5, and did not survive beyond birth. Osterix-specific ß1 integrin deficiency resulted in viable mice which were normal at birth but displayed early defects in calvarial ossification, incisor eruption and growth as well as femoral bone mineral density, structure, and mechanical properties. Although these defects persisted into adulthood, they became milder with age. Finally, a lack of ß1 integrins in mature osteoblasts and osteocytes resulted in minor alterations to femur structure but had no effect on mineral density, biomechanics or fracture healing. Taken together, our data indicate that ß1 integrin expression in early mesenchymal condensations play an important role in skeletal ossification, while ß1 integrin-ECM interactions in pre-osteoblast, odontoblast- and hypertrophic chondryocyte-lineage cells regulate incisor eruption and perinatal bone formation in both intramembranously and endochondrally formed bones in young, rapidly growing mice. In contrast, the osteocalcin-specific ß1 integrin deletion had only minor effects on skeletal phenotype.

Host response to microgel coatings on neural electrodes implanted in the brain.

The performance of neural electrodes implanted in the brain is often limited by host response in the surrounding brain tissue, including astrocytic scar formation, neuronal cell death, and inflammation around the implant. We applied conformal microgel coatings to silicon neural electrodes and examined host responses to microgel-coated and uncoated electrodes following implantation in the rat brain. In vitro analyses demonstrated significantly reduced astrocyte and microglia adhesion to microgel-coated electrodes compared to uncoated controls. Microgel-coated and uncoated electrodes were implanted in the rat brain cortex and the extent of activated microglia and astrocytes as well as neuron density around the implant were evaluated at 1, 4, and 24 weeks postimplantation. Microgel coatings reduced astrocytic recruitment around the implant at later time points. However, microglial response indicated persistence of inflammation in the area around the electrode. Neuronal density around the implanted electrodes was also lower for both implant groups compared to the uninjured control. These results demonstrate that microgel coatings do not significantly improve host responses to implanted neural electrodes and underscore the need for further improvements in implantable materials.

Stimulus-evoked high frequency oscillations are present in neuronal networks on microelectrode arrays.

Pathological high frequency oscillations (250-600 Hz) are present in the brains of epileptic animals and humans. The etiology of these oscillations and how they contribute to the diseased state remains unclear. This work identifies the presence of microstimulation-evoked high frequency oscillations (250-400 Hz) in dissociated neuronal networks cultured on microelectrode arrays (MEAs). Oscillations are more apparent with higher stimulus voltages. As with in vivo studies, activity is isolated to a single electrode, however, the MEA provides improved spatial resolution with no spread of the oscillation to adjacent electrodes 200 µm away. Oscillations develop across four weeks in vitro. Oscillations still occur in the presence of tetrodotoxin and synaptic blockers, and they cause no apparent disruption in the ability of oscillation-presenting electrodes to elicit directly evoked action potentials (dAPs) or promote the spread of synaptic activity throughout the culture. Chelating calcium with ethylene glycol tetraacetic acid (EGTA) causes a temporal prolongation of the oscillation. Finally, carbenoxolone significantly reduces or eliminates the high frequency oscillations. Gap junctions may play a significant role in maintaining the oscillation given the inhibitory effect of carbenoxolone, the propagating effect of reduced calcium conditions and the isolated nature of the activity as demonstrated in previous studies. This is the first demonstration of stimulus-evoked high frequency oscillations in dissociated cultures. Unlike current models that rely on complex in vivo recording conditions, this work presents a simple controllable model in neuronal cultures on MEAs to further investigate how the oscillations occur at the molecular level and how they may contribute to the pathophysiology of disease.

The lipoprotein receptor LR11 regulates amyloid beta production and amyloid precursor protein traffic in endosomal compartments.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cognitive decline and neuropathological changes, including the deposition of amyloid beta (Abeta) in senile plaques. The mechanisms causing the disease and Abeta accumulation are not well understood, but important genetic associations with apolipoprotein E genotype and involvement of lipoprotein receptors have become apparent. LR11 (also known as SorLA), a member of the low-density lipoprotein receptor family, has been identified previously as an altered transcript in microarray analyses of samples from human AD cases. Here, we show neuronal expression of the lipoprotein receptor LR11 in control brain in regions vulnerable to AD neuropathology and marked reduction of LR11 expression in these regions in AD brains before cell death. Overexpression of LR11 drastically reduces levels of extracellular Abeta and also lowers levels of total cellular amyloid precursor protein (APP). LR11 colocalizes with APP and regulates its trafficking in endocytic compartments, which are important intracellular sites for APP processing and Abeta generation. Endogenous LR11 localizes to neuronal multivesicular bodies in both rat and human brain. The robust correlation between reduced LR11 expression and AD neuropathology and its potent effects on extracellular Abeta levels suggest that this neuronal lipoprotein receptor could play an important role in AD pathogenesis.