A sense of proximity: Cell packing modulates oxygen consumption.

Accurately modeling oxygen transport and consumption is crucial to predict metabolic dynamics in cell cultures and optimize the design of tissue and organ models. We present a methodology to characterize the Michaelis-Menten oxygen consumption parameters in vitro, integrating novel experimental techniques and computational tools. The parameters were derived for hepatic cell cultures with different dimensionality (i.e., 2D and 3D) and with different surface and volumetric densities. To quantify cell packing regardless of the dimensionality of cultures, we devised an image-based metric, referred to as the proximity index. The Michaelis-Menten parameters were related to the proximity index through an uptake coefficient, analogous to a diffusion constant, enabling the quantitative analysis of oxygen dynamics across dimensions. Our results show that Michaelis-Menten parameters are not constant for a given cell type but change with dimensionality and cell density. The maximum consumption rate per cell decreases significantly with cell surface and volumetric density, while the Michaelis-Menten constant tends to increase. In addition, the dependency of the uptake coefficient on the proximity index suggests that the oxygen consumption rate of hepatic cells is superadaptive, as they modulate their oxygen utilization according to its local availability and to the proximity of other cells. We describe, for the first time, how cells consume oxygen as a function of cell proximity, through a quantitative index, which combines cell density and dimensionality. This study enhances our understanding of how cell-cell interaction affects oxygen dynamics and enables better prediction of aerobic metabolism in tissue models, improving their translational value.

Multi-disciplinary Insights from the First European Forum on Visceral Myopathy 2022 Meeting.

Visceral myopathy is a rare, life-threatening disease linked to identified genetic mutations in 60% of cases. Mostly due to the dearth of knowledge regarding its pathogenesis, effective treatments are lacking. The disease is most commonly diagnosed in children with recurrent or persistent disabling episodes of functional intestinal obstruction, which can be life threatening, often requiring long-term parenteral or specialized enteral nutritional support. Although these interventions are undisputedly life-saving as they allow affected individuals to avoid malnutrition and related complications, they also seriously compromise their quality of life and can carry the risk of sepsis and thrombosis. Animal models for visceral myopathy, which could be crucial for advancing the scientific knowledge of this condition, are scarce. Clearly, a collaborative network is needed to develop research plans to clarify genotype-phenotype correlations and unravel molecular mechanisms to provide targeted therapeutic strategies. This paper represents a summary report of the first 'European Forum on Visceral Myopathy'. This forum was attended by an international interdisciplinary working group that met to better understand visceral myopathy and foster interaction among scientists actively involved in the field and clinicians who specialize in care of people with visceral myopathy.

An integrated in silico-in vitro approach for bioprinting core-shell bioarchitectures.

Biological tissues possess a high degree of structural complexity characterized by curvature and stratification of different tissue layers. Despite recent advances in in vitro technology, current engineering solutions do not comprise both of these features. In this paper, we present an integrated in silico-in vitro strategy for the design and fabrication of biological barriers with controlled curvature and architecture. Analytical and computational tools combined with advanced bioprinting methods are employed to optimize living inks for bioprinting-structured core-shell constructs based on alginate. A finite element model is used to compute the hindered diffusion and crosslinking phenomena involved in the formation of core-shell structures and to predict the width of the shell as a function of material parameters. Constructs with a solid alginate-based shell and a solid, liquid, or air core can be reproducibly printed using the workflow. As a proof of concept, epithelial cells and fibroblasts were bioprinted respectively in a liquid core (10 mg/mL Pluronic) and in a solid shell (20 mg/mL alginate plus 20 mg/mL gelatin, used for providing the cells with adhesive moieties). These constructs had a roundness of 97.6% and an average diameter of 1500 ±136 µm. Moreover, their viability was close to monolayer controls (74.12% ± 22.07%) after a week in culture, and the paracellular transport was twice that of cell-free constructs, indicating cell polarization.

Advanced 3D Models of Human Brain Tissue Using Neural Cell Lines: State-of-the-Art and Future Prospects.

Human-relevant three-dimensional (3D) models of cerebral tissue can be invaluable tools to boost our understanding of the cellular mechanisms underlying brain pathophysiology. Nowadays, the accessibility, isolation and harvesting of human neural cells represents a bottleneck for obtaining reproducible and accurate models and gaining insights in the fields of oncology, neurodegenerative diseases and toxicology. In this scenario, given their low cost, ease of culture and reproducibility, neural cell lines constitute a key tool for developing usable and reliable models of the human brain. Here, we review the most recent advances in 3D constructs laden with neural cell lines, highlighting their advantages and limitations and their possible future applications.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

Biomedical Research on Substances of Abuse: The Italian Case Study.

Substances of abuse have the potential to cause addiction, habituation or altered consciousness. Most of the research on these substances focuses on addiction, and is carried out through observational and clinical studies on humans, or experimental studies on animals. The transposition of the EU Directive 2010/63 into Italian law in 2014 (IT Law 2014/26) includes a ban on the use of animals for research on substances of abuse. Since then, in Italy, public debate has continued on the topic, while the application of the Article prohibiting animal research in this area has been postponed every couple of years. In the light of this debate, we briefly review a range of methodologies - including animal and non-animal, as well as patient or population-based studies - that have been employed to address the biochemical, neurobiological, toxicological, clinical and behavioural effects of substances of abuse and their dependency. We then discuss the implications of the Italian ban on the use of animals for such research, proposing concrete and evidence-based solutions to allow scientists to pursue high-quality basic and translational studies within the boundaries of the regulatory and legislative framework.

Biomedical engineering in low- and middle-income settings: analysis of current state, challenges and best practices.

Supporting the expansion of best practices in Biomedical Engineering (BME) can facilitate pathway toward the providing universal health coverage and more equitable and accessible healthcare technologies, especially in low- and middle-income (LMI) settings. These best practices can act as drivers of change and may involve scientific-technological issues, human intervention during technology development, educational aspects, social performance management for improved interactions along the medical technology life cycle, methods for managing resources and approaches for the establishment of regulatory frameworks. The aim of our study was to identify weaknesses and strengths of the scientific, technological, socio-political, regulatory and educational landscape in BME in LMI resource settings. We thus analysed the current state-of-the-art through six dimensions considered fundamental for advancing quality and equity in healthcare: 1) relevant and 2) emergent technologies, 3) new paradigms in medical technology development, 4) innovative BME education, 5) regulation and standardization for novel approaches, and 6) policy making. In order to evaluate and compare their relevance, maturity and implementation challenges, they were assessed through a questionnaire to which 100 professionals from 35 countries with recognized experience in the field of BME and its application to LMI settings responded. The results are presented and discussed, highlighting the main challenges and pinpointing relevant areas where intervention, including local lobbying and international promotion of best practices is necessary. We were also able to identify areas where minimal effort is required to make big changes in global health. Supplementary information: The online version contains supplementary material available at 10.1007/s12553-022-00657-8.

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Advances in Animal Models and Cutting-Edge Research in Alternatives: Proceedings of the Second International Conference on 3Rs Research and Progress, Hyderabad, 2021.

The fact that animal models fail to replicate human disease faithfully is now being widely accepted by researchers across the globe. As a result, they are exploring the use of alternatives to animal models. The time has come to refine our experimental practices, reduce the numbers and eventually replace the animals used in research with human-derived and human-relevant 3-D disease models. Oncoseek Bio-Acasta Health, which is an innovative biotechnology start-up company based in Hyderabad and Vishakhapatnam, India, organises an annual International Conference on 3Rs Research and Progress. In 2021, this conference was on 'Advances in Research Animal Models and Cutting-Edge Research in Alternatives'. This annual conference is a platform that brings together eminent scientists and researchers from various parts of the world, to share recent advances from their research in the field of alternatives to animals including new approach methodologies, and to promote practices to help refine animal experiments where alternatives are not available. This report presents the proceedings of the conference, which was held in hybrid mode (i.e. virtual and in-person) in November 2021.

Characterizing and Engineering Biomimetic Materials for Viscoelastic Mechanotransduction Studies.

The mechanical behavior of soft tissue extracellular matrix is time dependent. Moreover, it evolves over time due to physiological processes as well as aging and disease. Measuring and quantifying the time-dependent mechanical behavior of soft tissues and materials pose a challenge, not only because of their labile and hydrated nature but also because of the lack of a common definition of terms and understanding of models for characterizing viscoelasticity. Here, we review the most important measurement techniques and models used to determine the viscoelastic properties of soft hydrated materials-or hydrogels-underlining the difference between viscoelastic behavior and the properties and descriptors used to quantify viscoelasticity. We then discuss the principal factors, which determine tissue viscoelasticity in vivo and summarize what we currently know about cell response to time-dependent materials, outlining fundamental factors that have to be considered when interpreting results. Particular attention is given to the relationship between the different time scales involved (mechanical, cellular and observation time scales), as well as scaling principles, all of which must be considered when designing viscoelastic materials and performing experiments for biomechanics or mechanobiology applications. From this overview, key considerations and directions for furthering insights and applications in the emergent field of cell viscoelastic mechanotransduction are provided. Impact statement Our tissues are viscoelastic: they respond to mechanical stresses and strains in a time-dependent manner. Their mechanical behavior also evolves over time due to growth, aging, remodeling and disease. Understanding cell response to time-dependent and time-evolving mechanical cues is important for a better comprehension of a wide number of pathophysiological processes and for the design of biomimetic substrates, which can be used as physiologically relevant in vitro models and in regenerative medicine applications. This review highlights the importance of a more rigorous approach toward viscoelastic material design and testing for cell mechanobiology studies, which embrace the entire spectrum of elasto- and viscotransduction.

Scaling of joint mass and metabolism fluctuations in in silico cell-laden spheroids.

Variations and fluctuations are characteristic features of biological systems and are also manifested in cell cultures. Here, we describe a computational pipeline for identifying the range of three-dimensional (3D) cell-aggregate sizes in which nonisometric scaling emerges in the presence of joint mass and metabolic rate fluctuations. The 3D cell-laden spheroids with size and single-cell metabolic rates described by probability density functions were randomly generated in silico. The distributions of the resulting metabolic rates of the spheroids were computed by modeling oxygen diffusion and reaction. Then, a method for estimating scaling exponents of correlated variables through statistically significant data collapse of joint probability distributions was developed. The method was used to identify a physiologically relevant range of spheroid sizes, where both nonisometric scaling and a minimum oxygen concentration (0.04 mol⋅m-3) is maintained. The in silico pipeline described enables the prediction of the number of experiments needed for an acceptable collapse and, thus, a consistent estimate of scaling parameters. Using the pipeline, we also show that scaling exponents may be significantly different in the presence of joint mass and metabolic-rate variations typically found in cells. Our study highlights the importance of incorporating fluctuations and variability in size and metabolic rates when estimating scaling exponents. It also suggests the need for taking into account their covariations for better understanding and interpreting experimental observations both in vitro and in vivo and brings insights for the design of more predictive and physiologically relevant in vitro models.

Engineering and Monitoring 3D Cell Constructs with Time-Evolving Viscoelasticity for the Study of Liver Fibrosis In Vitro.

Liver fibrosis is generally associated with an over-production and crosslinking of extracellular matrix proteins, causing a progressive increase in both the elastic and viscous properties of the hepatic tissue. We describe a strategy for mimicking and monitoring the mechano-dynamics of the 3D microenvironment associated with liver fibrosis. Cell-laden gelatin hydrogels were crosslinked with microbial transglutaminase using a purpose-designed cytocompatible two-step protocol, which allows for the exposure of cells to a mechanically changing environment during culturing. A bioreactor was re-engineered to monitor the mechanical properties of cell constructs over time. The results showed a shift towards a more elastic (i.e., solid-like) behaviour, which is likely related to an increase in cell stress. The method effectively mimics the time-evolving mechanical microenvironment associated with liver fibrosis and could provide novel insights into pathophysiological processes in which both elastic and viscous properties of tissues change over time.

The Role of the 3Rs for Understanding and Modeling the Human Placenta.

Modeling the physiology of the human placenta is still a challenge, despite the great number of scientific advancements made in the field. Animal models cannot fully replicate the structure and function of the human placenta and pose ethical and financial hurdles. In addition, increasingly stricter animal welfare legislation worldwide is incentivizing the use of 3R (reduction, refinement, replacement) practices. What efforts have been made to develop alternative models for the placenta so far? How effective are they? How can we improve them to make them more predictive of human pathophysiology? To address these questions, this review aims at presenting and discussing the current models used to study phenomena at the placenta level: in vivo, ex vivo, in vitro and in silico. We describe the main achievements and opportunities for improvement of each type of model and critically assess their individual and collective impact on the pursuit of predictive studies of the placenta in line with the 3Rs and European legislation.

Breathing in vitro: Designs and applications of engineered lung models.

The aim of this review is to provide a systematic design guideline to users, particularly engineers interested in developing and deploying lung models, and biologists seeking to identify a suitable platform for conducting in vitro experiments involving pulmonary cells or tissues. We first discuss the state of the art on lung in vitro models, describing the most simplistic and traditional ones. Then, we analyze in further detail the more complex dynamic engineered systems that either provide mechanical cues, or allow for more predictive exposure studies, or in some cases even both. This is followed by a dedicated section on microchips of the lung. Lastly, we present a critical discussion of the different characteristics of each type of system and the criteria which may help researchers select the most appropriate technology according to their specific requirements. Readers are encouraged to refer to the tables accompanying the different sections where comprehensive and quantitative information on the operating parameters and performance of the different systems reported in the literature is provided.

Tools and approaches for analyzing the role of mitochondria in health, development and disease using human cerebral organoids.

Mitochondria are cellular organelles involved in generating energy to power various processes in the cell. Although the pivotal role of mitochondria in neurogenesis was demonstrated (first in animal models), very little is known about their role in human embryonic neurodevelopment and its pathology. In this respect human-induced pluripotent stem cells (hiPSC)-derived cerebral organoids provide a tractable, alternative model system of the early neural development and disease that is responsive to pharmacological and genetic manipulations, not possible to apply in humans. Although the involvement of mitochondria in the pathogenesis and progression of neurodegenerative diseases and brain dysfunction has been demonstrated, the precise role they play in cell life and death remains unknown, compromising the development of new mitochondria-targeted approaches to treat human diseases. The cerebral organoid model of neurogenesis and disease in vitro provides an unprecedented opportunity to answer some of the most fundamental questions about mitochondrial function in early human neurodevelopment and neural pathology. Largely an unexplored territory due to the lack of tools and approaches, this review focuses on recent technological advancements in fluorescent and molecular tools, imaging systems, and computational approaches for quantitative and qualitative analyses of mitochondrial structure and function in three-dimensional cellular assemblies-cerebral organoids. Future developments in this direction will further facilitate our understanding of the important role or mitochondrial dynamics and energy requirements during early embryonic development. This in turn will provide a further understanding of how dysfunctional mitochondria contribute to disease processes.

A Bioinspired in vitro Lung Model to Study Particokinetics of Nano-/Microparticles Under Cyclic Stretch and Air-Liquid Interface Conditions.

Evolution has endowed the lung with exceptional design providing a large surface area for gas exchange area (ca. 100 m2) in a relatively small tissue volume (ca. 6 L). This is possible due to a complex tissue architecture that has resulted in one of the most challenging organs to be recreated in the lab. The need for realistic and robust in vitro lung models becomes even more evident as causal therapies, especially for chronic respiratory diseases, are lacking. Here, we describe the Cyclic I n VI tro Cell-stretch (CIVIC) "breathing" lung bioreactor for pulmonary epithelial cells at the air-liquid interface (ALI) experiencing cyclic stretch while monitoring stretch-related parameters (amplitude, frequency, and membrane elastic modulus) under real-time conditions. The previously described biomimetic copolymeric BETA membrane (5 µm thick, bioactive, porous, and elastic) was attempted to be improved for even more biomimetic permeability, elasticity (elastic modulus and stretchability), and bioactivity by changing its chemical composition. This biphasic membrane supports both the initial formation of a tight monolayer of pulmonary epithelial cells (A549 and 16HBE14o-) under submerged conditions and the subsequent cell-stretch experiments at the ALI without preconditioning of the membrane. The newly manufactured versions of the BETA membrane did not improve the characteristics of the previously determined optimum BETA membrane (9.35% PCL and 6.34% gelatin [w/v solvent]). Hence, the optimum BETA membrane was used to investigate quantitatively the role of physiologic cyclic mechanical stretch (10% linear stretch; 0.33 Hz: light exercise conditions) on size-dependent cellular uptake and transepithelial transport of nanoparticles (100 nm) and microparticles (1,000 nm) for alveolar epithelial cells (A549) under ALI conditions. Our results show that physiologic stretch enhances cellular uptake of 100 nm nanoparticles across the epithelial cell barrier, but the barrier becomes permeable for both nano- and micron-sized particles (100 and 1,000 nm). This suggests that currently used static in vitro assays may underestimate cellular uptake and transbarrier transport of nanoparticles in the lung.

Development of a dynamic in vitro stretch model of the alveolar interface with aerosol delivery.

We describe the engineering design, computational modeling, and empirical performance of a moving air-liquid interface (MALI) bioreactor for the study of aerosol deposition on cells cultured on an elastic, porous membrane which mimics both air-liquid interface exposure conditions and mechanoelastic motion of lung tissue during breathing. The device consists of two chambers separated by a cell layer cultured on a porous, flexible membrane. The lower (basolateral) chamber is perfused with cell culture medium simulating blood circulation. The upper (apical) chamber representing the air compartment of the lung is interfaced to an aerosol generator and a pressure actuation system. By cycling the pressure in the apical chamber between 0 and 7 kPa, the membrane can mimic the periodic mechanical strain of the alveolar wall. Focusing on the engineering aspects of the system, we show that membrane strain can be monitored by measuring changes in pressure resulting from the movement of media in the basolateral chamber. Moreover, liquid aerosol deposition at a high dose delivery rate (>1 µl cm-2 min-1 ) is highly efficient (ca. 51.5%) and can be accurately modeled using finite element methods. Finally, we show that lung epithelial cells can be mechanically stimulated under air-liquid interface and stretch-conditions without loss of viability. The MALI bioreactor could be used to study the effects of aerosol on alveolar cells cultured at the air-liquid interface in a biodynamic environment or for toxicological or therapeutic applications.

One-step automated bioprinting-based method for cumulus-oocyte complex microencapsulation for 3D in vitro maturation.

Three-dimensional in vitro maturation (3D IVM) is a promising approach to improve IVM efficiency as it could prevent cumulus-oocyte complex (COC) flattening and preserve its structural and functional integrity. Methods reported to date have low reproducibility and validation studies are limited. In this study, a bioprinting based production process for generating microbeads containing a COC (COC-microbeads) was optimized and its validity tested in a large animal model (sheep). Alginate microbeads were produced and characterized for size, shape and stability under culture conditions. COC encapsulation had high efficiency and reproducibility and cumulus integrity was preserved. COC-microbeads underwent IVM, with COCs cultured in standard 2D IVM as controls. After IVM, oocytes were analyzed for nuclear chromatin configuration, bioenergetic/oxidative status and transcriptional activity of genes biomarker of mitochondrial activity (TFAM, ATP6, ATP8) and oocyte developmental competence (KHDC3, NLRP5, OOEP and TLE6). The 3D system supported oocyte nuclear maturation more efficiently than the 2D control (P<0.05). Ooplasmic mitochondrial activity and reactive oxygen species (ROS) generation ability were increased (P<0.05). Up-regulation of TFAM, ATP6 and ATP8 and down-regulation of KHDC3, NLRP5 expression were observed in 3D IVM. In conclusion, the new bioprinting method for producing COC-microbeads has high reproducibility and efficiency. Moreover, 3D IVM improves oocyte nuclear maturation and relevant parameters of oocyte cytoplasmic maturation and could be used for clinical and toxicological applications.

Investigating Curcumin/Intestinal Epithelium Interaction in a Millifluidic Bioreactor.

Multidrug resistance is still an obstacle for chemotherapeutic treatments. One of the proteins involved in this phenomenon is the P-glycoprotein, P-gp, which is known to be responsible for the efflux of therapeutic substances from the cell cytoplasm. To date, the identification of a drug that can efficiently inhibit P-gp activity remains a challenge, nevertheless some studies have identified natural compounds suitable for that purpose. Amongst them, curcumin has shown an inhibitory effect on the protein in in vitro studies using Caco-2 cells. To understand if flow can modulate the influence of curcumin on the protein's activity, we studied the uptake of a P-gp substrate under static and dynamic conditions. Caco-2 cells were cultured in bioreactors and in Transwells and the basolateral transport of rhodamine-123 was assessed in the two systems as a function of the P-gp activity. Experiments were performed with and without pre-treatment of the cells with an extract of curcumin or an arylmethyloxy-phenyl derivative to evaluate the inhibitory effect of the natural substance with respect to a synthetic compound. The results indicated that the P-gp activity of the cells cultured in the bioreactors was intrinsically lower, and that the effect of both natural and synthetic inhibitors was up modulated by the presence of flow. Our study underlies the fact that the use of more sophisticated and physiologically relevant in vitro models can bring new insights on the therapeutic effects of natural substances such as curcumin.