Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Third International Conference on 3Rs Research and Progress, Vishakhapatnam, 2022.

Animal experimentation has been integral to drug discovery and development and safety assessment for many years, since it provides insights into the mechanisms of drug efficacy and toxicity (e.g. pharmacology, pharmacokinetics and pharmacodynamics). However, due to species differences in physiology, metabolism and sensitivity to drugs, the animal models can often fail to replicate the effects of drugs and chemicals in human patients, workers and consumers. Researchers across the globe are increasingly applying the Three Rs principles by employing innovative methods in research and testing. The Three Rs concept focuses on: the replacement of animal models (e.g. with in vitro and in silico models or human studies), on the reduction of the number of animals required to achieve research objectives, and on the refinement of existing experimental practices (e.g. eliminating distress and enhancing animal wellbeing). For the last two years, Oncoseek Bio-Acasta Health, a 3-D cell culture-based cutting-edge translational biotechnology company, has organised an annual International Conference on 3Rs Research and Progress. This series of global conferences aims to bring together researchers with diverse expertise and interests, and provides a platform where they can share and discuss their research to promote practices according to the Three Rs principles. In November 2022, the 3rd international conference, Advances in Animal Models and Cutting-Edge Research in Alternatives, took place at the GITAM University in Vishakhapatnam (AP, India) in a hybrid format (i.e. online and in-person). These conference proceedings provide details of the presentations, which were categorised under five different topic sessions. It also describes a special interactive session on in silico strategies for preclinical research in oncology, which was held at the end of the first day.

A human kidney and liver organoid-based multi-organ-on-a-chip model to study the therapeutic effects and biodistribution of mesenchymal stromal cell-derived extracellular vesicles.

Mesenchymal stromal cell (MSC)-derived small extracellular vesicles (sEVs) show therapeutic potential in multiple disease models, including kidney injury. Clinical translation of sEVs requires further preclinical and regulatory developments, including elucidation of the biodistribution and mode of action (MoA). Biodistribution can be determined using labelled sEVs in animal models which come with ethical concerns, are time-consuming and expensive, and may not well represent human physiology. We hypothesised that, based on developments in microfluidics and human organoid technology, in vitro multi-organ-on-a-chip (MOC) models allow us to study effects of sEVs in modelled human organs like kidney and liver in a semi-systemic manner. Human kidney- and liver organoids combined by microfluidic channels maintained physiological functions, and a kidney injury model was established using hydrogenperoxide. MSC-sEVs were isolated, and their size, density and potential contamination were analysed. These sEVs stimulated recovery of the renal epithelium after injury. Microscopic analysis shows increased accumulation of PKH67-labelled sEVs not only in injured kidney cells, but also in the unharmed liver organoids, compared to healthy control conditions. In conclusion, this new MOC model recapitulates therapeutic efficacy and biodistribution of MSC-sEVs as observed in animal models. Its human background allows for in-depth analysis of the MoA and identification of potential side effects.

Organ-on-a-chip: Determine feasibility of a human liver microphysiological model to assess long-term steroid metabolites in sports drug testing.

A fundamental challenge in preventive doping research is the study of metabolic pathways of substances banned in sport. However, the pharmacological predictions obtained by conventional in vitro or in vivo animal studies are occasionally of limited transferability to humans according to an inability of in vitro models to mimic higher order system physiology or due to various species-specific differences using animal models. A more recently established technology for simulating human physiology is the "organ-on-a-chip" principle. In a multichannel microfluidic cell culture chip, 3-dimensional tissue spheroids, which can constitute artificial and interconnected microscale organs, imitate principles of the human physiology. The objective of this study was to determine if the technology is suitable to adequately predict metabolic profiles of prohibited substances in sport. As model compounds, the frequently misused anabolic steroids, stanozolol and dehydrochloromethyltestosterone (DHCMT) were subjected to human liver spheroids in microfluidic cell culture chips. The metabolite patterns produced and circulating in the chip media were then assessed by LC-HRMS/(MS) at different time points of up to 14 days of incubation at 37°C. The overall profile of observed glucurono-conjugated stanozolol metabolites excellently matched the commonly found urinary pattern of metabolites, including 3'OH-stanozolol-glucuronide and stanozolol-N-glucuronides. Similarly, but to a lower extent, the DHCMT metabolic profile was in agreement with phase-I and phase-II biotransformation products regularly seen in postadministration urine specimens. In conclusion, this pilot study indicates that the "organ-on-a-chip" technology provides a high degree of conformity with traditional human oral administration studies, providing a promising approach for metabolic profiling in sports drug testing.

Validation of Bioreactor and Human-on-a-Chip Devices for Chemical Safety Assessment.

Equipment and device qualification and test assay validation in the field of tissue engineered human organs for substance assessment remain formidable tasks with only a few successful examples so far. The hurdles seem to increase with the growing complexity of the biological systems, emulated by the respective models. Controlled single tissue or organ culture in bioreactors improves the organ-specific functions and maintains their phenotypic stability for longer periods of time. The reproducibility attained with bioreactor operations is, per se, an advantage for the validation of safety assessment. Regulatory agencies have gradually altered the validation concept from exhaustive "product" to rigorous and detailed process characterization, valuing reproducibility as a standard for validation. "Human-on-a-chip" technologies applying micro-physiological systems to the in vitro combination of miniaturized human organ equivalents into functional human micro-organisms are nowadays thought to be the most elaborate solution created to date. They target the replacement of the current most complex models-laboratory animals. Therefore, we provide here a road map towards the validation of such "human-on-a-chip" models and qualification of their respective bioreactor and microchip equipment along a path currently used for the respective animal models.

The multi-organ chip--a microfluidic platform for long-term multi-tissue coculture.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments, as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier, more quantifiable extracellular relay of signals to the cells. For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Integrating biological vasculature into a multi-organ-chip microsystem.

A chip-based system mimicking the transport function of the human cardiovascular system has been established at minute but standardized microsystem scale. A peristaltic on-chip micropump generates pulsatile shear stress in a widely adjustable physiological range within a microchannel circuit entirely covered on all fluid contact surfaces with human dermal microvascular endothelial cells. This microvascular transport system can be reproducibly established within four days, independently of the individual endothelial cell donor background. It interconnects two standard tissue culture compartments, each of 5 mm diameter, through microfluidic channels of 500 µm width. Further vessel branching and vessel diameter reduction down to a microvessel scale of approximately 40 µm width was realised by a two-photon laser ablation technique applied to inserts, designed for the convenient establishment of individual organ equivalents in the tissue culture compartments at a later time. The chip layout ensures physiological fluid-to-tissue ratios. Moreover, an in-depth microscopic analysis revealed the fine-tuned adjustment of endothelial cell behaviour to local shear stresses along the microvasculature of the system. Time-lapse and 3D imaging two-photon microscopy were used to visualise details of spatiotemporal adherence of the endothelial cells to the channel system and to each other. The first indicative long-term experiments revealed stable performance over two and four weeks. The potential application of this system for the future establishment of human-on-a-chip systems and basic human endothelial cell research is discussed.

Skin and hair on-a-chip: in vitro skin models versus ex vivo tissue maintenance with dynamic perfusion.

Substantial progress has been achieved over the last few decades in the development of skin equivalents to model the skin as an organ. However, their static culture still limits the emulation of essential physiological properties crucial for toxicity testing and compound screening. Here, we describe a dynamically perfused chip-based bioreactor platform capable of applying variable mechanical shear stress and extending culture periods. This leads to improvements of culture conditions for integrated in vitro skin models, ex vivo skin organ cultures and biopsies of single hair follicular units.

'Human-on-a-chip' developments: a translational cutting-edge alternative to systemic safety assessment and efficiency evaluation of substances in laboratory animals and man?

Various factors, including the phylogenetic distance between laboratory animals and humans, the discrepancy between current in vitro systems and the human body, and the restrictions of in silico modelling, have generated the need for new solutions to the ever-increasing worldwide dilemma of substance testing. This review provides a historical sketch on the accentuation of this dilemma, and highlights fundamental limitations to the countermeasures taken so far. It describes the potential of recently-introduced microsystems to emulate human organs in 'organ-on-a-chip' devices. Finally, it focuses on an in-depth analysis of the first devices that aimed to mimic human systemic organ interactions in 'human-on-a-chip' systems. Their potential to replace acute systemic toxicity testing in animals, and their inability to provide alternatives to repeated dose long-term testing, are discussed. Inspired by the latest discoveries in human biology, tissue engineering and micro-systems technology, this review proposes a paradigm shift to overcome the apparent challenges. A roadmap is outlined to create a new homeostatic level of biology in 'human-on-a-chip' systems in order to, in the long run, replace systemic repeated dose safety evaluation and disease modelling in animals.

Cartilage oligomeric matrix protein (COMP) forms part of the connective tissue of normal human hair follicles.

Hair follicle cycling is driven by epithelial-mesenchymal interactions (EMI), which require extracellular matrix (ECM) modifications to control the crosstalk between key epithelial- and mesenchymal-derived growth factors and cytokines. The exact roles of these ECM modifications in hair cycle-associated EMI are still unknown. Here, we used differential microarray analysis of laser capture-microdissected human scalp hair follicles (HF) to identify new ECM components that distinguish fibroblasts from the connective tissue sheath (CTS) from those of the follicular dermal papilla (DP). These analyses provide the first evidence that normal human CTS fibroblasts are characterized by the selective in situ-transcription of cartilage oligomeric matrix protein (COMP). Following this up on the protein level, COMP was found to be hair cycle-dependent, suggesting critical role in this process: COMP is expressed during telogen and early anagen at regions of EMI and is degraded during catagen (only the CTS adjacent to the bulge remains COMP+ during catagen). Notably, COMP gene expression in vitro suggests direct correlation with the expression of TGFß2 in CTS fibroblasts. This raises the question whether COMP expression undergoes regulation by transforming growth factor, beta (TGFß) signalling. The intrafollicular COMP expression suggests to be functionally important and deserves further scrutiny in hair biology as indicated by the fact that altered COMP expression might be associated with scant fine hair in the case of some chondrodysplasia and scleroderma patients. Together these results reveal for the first time that COMP is part of the ECM and suggests its important role in normal human HF biology.

De novo formation and ultra-structural characterization of a fiber-producing human hair follicle equivalent in vitro.

Across many tissues and organs, the ability to create an organoid, the smallest functional unit of an organ, in vitro is the key both to tissue engineering and preclinical testing regimes. The hair follicle is an organoid that has been much studied based on its ability to grow quickly and to regenerate after trauma. But hair follicle formation in vitro has been elusive. Replacing hair lost due to pattern baldness or more severe alopecia, including that induced by chemotherapy, remains a significant unmet medical need. By carefully analyzing and recapitulating the growth conditions of hair follicle formation, we recreated human hair follicles in tissue culture that were capable of producing hair. Our microfollicles contained all relevant cell types and their structure and orientation resembled in some ways excised hair follicle specimens from human skin. This finding offers a new window onto hair follicle development. Having a robust culture system for hair follicles is an important step towards improved hair regeneration as well as to an understanding of how marketed drugs or drug candidates, including cancer chemotherapy, will affect this important organ.

Design and prototyping of a chip-based multi-micro-organoid culture system for substance testing, predictive to human (substance) exposure.

Dynamic miniaturized human multi-micro-organ bioreactor systems are envisaged as a possible solution for the embarrassing gap of predictive substance testing prior to human exposure. A rational approach was applied to simulate and design dynamic long-term cultures of the smallest possible functional human organ units, human "micro-organoids", on a chip the shape of a microscope slide. Each chip contains six identical dynamic micro-bioreactors with three different micro-organoid culture segments each, a feed supply and waste reservoirs. A liver, a brain cortex and a bone marrow micro-organoid segment were designed into each bioreactor. This design was translated into a multi-layer chip prototype and a routine manufacturing procedure was established. The first series of microscopable, chemically resistant and sterilizable chip prototypes was tested for matrix compatibility and primary cell culture suitability. Sterility and long-term human cell survival could be shown. Optimizing the applied design approach and prototyping tools resulted in a time period of only 3 months for a single design and prototyping cycle. This rapid prototyping scheme now allows for fast adjustment or redesign of inaccurate architectures. The designed chip platform is thus ready to be evaluated for the establishment and maintenance of the human liver, brain cortex and bone marrow micro-organoids in a systemic microenvironment.