Development of a robotic-assisted handling and manipulation system for the high-scale bioproduction of 3D-bioprinted organ-on-a-chip devices.

Organs-on-a-chip (OoCs) have proven to mimic the basic physiological behavior of organs and the influence of therapeutics on them in greater detail than conventional models, resulting in enormous projected market growth rates. However, the breakthrough to profitable commercialization of that technology has not yet been achieved, partly because the production process chain is characterized by a high proportion of manual laboratory work. The present work addresses this point. Utilizing affordable components, a demonstrator was developed that can be integrated into an existing 3D-bioprinting system and enables the automated production of perfusion-ready OoC devices starting from pre-fabricated injection-molded microfluidic chips. To this end, a corresponding process chain was first defined, and an expandable, configurable algorithm was developed and validated in the form of a finite state machine (FSM). This algorithm controls a modified 4-axis robot arm that covers the steps upstream and downstream of the printing process in the manufacturing process and achieves success rates of up to 100 %. A virtual interface between the robot and printer enables mutual communication and full integration of the algorithm into the process chain. Steps that pose a challenge for the automation of the process chain and appropriate countermeasures and optimizations were identified. This lays the foundation for scaling and standardizing the automated production of OoCs.

Corrigendum: Applying new approach methodologies to assess next-generation tobacco and nicotine products.

[This corrects the article DOI: 10.3389/ftox.2024.1376118.].

Customizable Microfluidic Devices: Progress, Constraints, and Future Advances.

The field of microfluidics encompasses the study of fluid behavior within micro-channels and the development of miniature systems featuring internal compartments or passageways tailored for fluid control and manipulation. Microfluidic devices capitalize on the unique chemical and physical properties exhibited by fluids at the microscopic scale. In contrast to their larger counterparts, microfluidic systems offer a multitude of advantages. Their implementation facilitates the investigation and utilization of reduced sample, solvent, and reagent volumes, thus yielding decreased operational expenses. Owing to their compact dimensions, these devices allow for the concurrent execution of multiple procedures, leading to expedited experimental timelines. Over the past two decades, microfluidics has undergone remarkable advancements, evolving into a multifaceted discipline. Subfields such as organ-on-a-chip and paper-based microfluidics have matured into distinct fields of study. Nonetheless, while scientific progress within the microfluidics realm has been notable, its translation into autonomous end-user applications remains a frontier to be fully explored. This paper sets forth the central objective of scrutinizing the present research paradigm, prevailing limitations, and potential prospects of customizable microfluidic devices. Our inquiry revolves around the latest strides achieved, prevailing constraints, and conceivable trajectories for adaptable microfluidic technologies. We meticulously delineate existing iterations of microfluidic systems, elucidate their operational principles, deliberate upon encountered limitations, and provide a visionary outlook toward the future trajectory of microfluidic advancements. In summation, this work endeavors to shed light on the current state of microfluidic systems, underscore their operative intricacies, address incumbent challenges, and unveil promising pathways that chart the course toward the next frontier of microfluidic innovation.

An update on microfluidic multi-organ-on-a-chip systems for reproducing drug pharmacokinetics: the current state-of-the-art.

INTRODUCTION: Advances in the accessibility of manufacturing technologies and iPSC-based modeling have accelerated the overall progress of organs-on-a-chip. Notably, the progress in multi-organ systems is not progressing with equal speed, indicating that there are still major technological barriers to overcome that may include biological relevance, technological usability as well as overall accessibility. AREAS COVERED: We here review the progress in the field of multi-tissue- and body-on-a-chip pre and post- SARS-CoV-2 pandemic and review five selected studies with increasingly complex multi-organ chips aiming at pharmacological studies. EXPERT OPINION: We discuss future and necessary advances in the field of multi-organ chips including how to overcome challenges regarding cell diversity, improved culture conditions, model translatability as well as sensor integrations to enable microsystems to cover organ-organ interactions in not only toxicokinetic but more importantly pharmacodynamic and -kinetic studies.

Applying new approach methodologies to assess next-generation tobacco and nicotine products.

In vitro toxicology research has accelerated with the use of in silico, computational approaches and human in vitro tissue systems, facilitating major improvements evaluating the safety and health risks of novel consumer products. Innovation in molecular and cellular biology has shifted testing paradigms, with less reliance on low-throughput animal data and greater use of medium- and high-throughput in vitro cellular screening approaches. These new approach methodologies (NAMs) are being implemented in other industry sectors for chemical testing, screening candidate drugs and prototype consumer products, driven by the need for reliable, human-relevant approaches. Routine toxicological methods are largely unchanged since development over 50 years ago, using high-doses and often employing in vivo testing. Several disadvantages are encountered conducting or extrapolating data from animal studies due to differences in metabolism or exposure. The last decade saw considerable advancement in the development of in vitro tools and capabilities, and the challenges of the next decade will be integrating these platforms into applied product testing and acceptance by regulatory bodies. Governmental and validation agencies have launched and applied frameworks and "roadmaps" to support agile validation and acceptance of NAMs. Next-generation tobacco and nicotine products (NGPs) have the potential to offer reduced risks to smokers compared to cigarettes. These include heated tobacco products (HTPs) that heat but do not burn tobacco; vapor products also termed electronic nicotine delivery systems (ENDS), that heat an e-liquid to produce an inhalable aerosol; oral smokeless tobacco products (e.g., Swedish-style snus) and tobacco-free oral nicotine pouches. With the increased availability of NGPs and the requirement of scientific studies to support regulatory approval, NAMs approaches can supplement the assessment of NGPs. This review explores how NAMs can be applied to assess NGPs, highlighting key considerations, including the use of appropriate in vitro model systems, deploying screening approaches for hazard identification, and the importance of test article characterization. The importance and opportunity for fit-for-purpose testing and method standardization are discussed, highlighting the value of industry and cross-industry collaborations. Supporting the development of methods that are accepted by regulatory bodies could lead to the implementation of NAMs for tobacco and nicotine NGP testing.

A Comprehensive Review of Organ-on-a-Chip Technology and Its Applications.

Organ-on-a-chip (OOC) is an emerging technology that simulates an artificial organ within a microfluidic cell culture chip. Current cell biology research focuses on in vitro cell cultures due to various limitations of in vivo testing. Unfortunately, in-vitro cell culturing fails to provide an accurate microenvironment, and in vivo cell culturing is expensive and has historically been a source of ethical controversy. OOC aims to overcome these shortcomings and provide the best of both in vivo and in vitro cell culture research. The critical component of the OOC design is utilizing microfluidics to ensure a stable concentration gradient, dynamic mechanical stress modeling, and accurate reconstruction of a cellular microenvironment. OOC also has the advantage of complete observation and control of the system, which is impossible to recreate in in-vivo research. Multiple throughputs, channels, membranes, and chambers are constructed in a polydimethylsiloxane (PDMS) array to simulate various organs on a chip. Various experiments can be performed utilizing OOC technology, including drug delivery research and toxicology. Current technological expansions involve multiple organ microenvironments on a single chip, allowing for studying inter-tissue interactions. Other developments in the OOC technology include finding a more suitable material as a replacement for PDMS and minimizing artefactual error and non-translatable differences.

Revolutionizing nephrology research: expanding horizons with kidney-on-a-chip and beyond.

Organs-on-a-chip (OoC) is a microengineered three-dimensional cell culture system developed for decades. Utilizing microfluidic technology, OoC cultivates cells on perfusable channels to construct in vitro organ models, enabling the simulation of organ-level functions under physiological and pathophysiological conditions. The superior simulation capabilities compared to traditional animal experiments and two-dimensional cell cultures, making OoC a valuable tool for in vitro research. Recently, the application of OoC has extended to the field of nephrology, where it replicates various functional units, including glomerulus-on-a-chip, proximal tubule-on-a-chip, distal tubule-on-a-chip, collecting duct-on-a-chip, and even the entire nephron-on-a-chip to precisely emulate the structure and function of nephrons. Moreover, researchers have integrated kidney models into multi-organ systems, establishing human body-on-a-chip platforms. In this review, the diverse functional kidney units-on-a-chip and their versatile applications are outlined, such as drug nephrotoxicity screening, renal development studies, and investigations into the pathophysiological mechanisms of kidney diseases. The inherent advantages and current limitations of these OoC models are also examined. Finally, the synergy of kidney-on-a-chip with other emerging biomedical technologies are explored, such as bioengineered kidney and bioprinting, and a new insight for chip-based renal replacement therapy in the future are prospected.

Modeling the Progression of Placental Transport from Early- to Late-Stage Pregnancy by Tuning Trophoblast Differentiation and Vascularization.

The early-stage placental barrier is characterized by a lack of fetal circulation and by a thick trophoblastic barrier, whereas the later-stage placenta consists of vascularized chorionic villi encased in a thin, differentiated trophoblast layer, ideal for nutrient transport. In this work, predictive models of early- and late-stage placental transport are created using blastocyst-derived placental stem cells (PSCs) by modulating PSC differentiation and model vascularization. PSC differentiation results in a thinner, fused trophoblast layer, as well as an increase in human chorionic gonadotropin secretion, barrier permeability, and secretion of certain inflammatory cytokines, which are consistent with in vivo findings. Further, gene expression confirms this shift toward a differentiated trophoblast subtype. Vascularization results in a molecule type- and size-dependent change in dextran and insulin permeability. These results demonstrate that trophoblast differentiation and vascularization have critical effects on placental barrier permeability and that this model can be used as a predictive measure to assess fetal toxicity of xenobiotic substances at different stages of pregnancy.

Creating mini-pregnancy models in vitro with clinical perspectives.

During the last decade, organs-on-chips or organoids microphysiological analysis platforms (MAP) have garnered attention in the practical applications of disease models, drug discovery, and developmental biology. Research on pregnant women has firm limitations due to ethical issues; thus, remodelling human pregnancy in vitro is highly beneficial for treatment modality development via disease remodelling or drug monitoring. This review highlights current efforts in bioengineering devices to reproduce human pregnancy and emphasises the significant convergence of biology, engineering, and maternal-foetal medicine. First, we review recent achievements in culturing cells from tissues involved in pregnancy; specifically, trophoblasts from the placenta. Second, we highlight developments in the reconstitution of pregnancy-related female reproductive organs across several structural and functional interpretations. Last, we examine research on the fundamental comprehension of pregnancy-associated diseases to find bioengineering solutions. Recreating human pregnancy through an engineered model is naturally complex; nevertheless, challenges are inevitable to progress precision medicine.

Opportunities and challenges to engineer 3D models of tumor-adaptive immune interactions.

Augmenting adaptive immunity is a critical goal for developing next-generation cancer therapies. T and B cells infiltrating the tumor dramatically influence cancer progression through complex interactions with the local microenvironment. Cancer cells evade and limit these immune responses by hijacking normal immunologic pathways. Current experimental models using conventional primary cells, cell lines, or animals have limitations for studying cancer-immune interactions directly relevant to human biology and clinical translation. Therefore, engineering methods to emulate such interplay at local and systemic levels are crucial to expedite the development of better therapies and diagnostic tools. In this review, we discuss the challenges, recent advances, and future directions toward engineering the tumor-immune microenvironment (TME), including key elements of adaptive immunity. We first offer an overview of the recent research that has advanced our understanding of the role of the adaptive immune system in the tumor microenvironment. Next, we discuss recent developments in 3D in-vitro models and engineering approaches that have been used to study the interaction of cancer and stromal cells with B and T lymphocytes. We summarize recent advancement in 3D bioengineering and discuss the need for 3D tumor models that better incorporate elements of the complex interplay of adaptive immunity and the tumor microenvironment. Finally, we provide a perspective on current challenges and future directions for modeling cancer-immune interactions aimed at identifying new biological targets for diagnostics and therapeutics.

Biosensors integrated 3D organoid/organ-on-a-chip system: A real-time biomechanical, biophysical, and biochemical monitoring and characterization.

As a full-fidelity simulation of human cells, tissues, organs, and even systems at the microscopic scale, Organ-on-a-Chip (OOC) has significant ethical advantages and development potential compared to animal experiments. The need for the design of new drug high-throughput screening platforms and the mechanistic study of human tissues/organs under pathological conditions, the evolving advances in 3D cell biology and engineering, etc., have promoted the updating of technologies in this field, such as the iteration of chip materials and 3D printing, which in turn facilitate the connection of complex multi-organs-on-chips for simulation and the further development of technology-composite new drug high-throughput screening platforms. As the most critical part of organ-on-a-chip design and practical application, verifying the success of organ model modeling, i.e., evaluating various biochemical and physical parameters in OOC devices, is crucial. Therefore, this paper provides a logical and comprehensive review and discussion of the advances in organ-on-a-chip detection and evaluation technologies from a broad perspective, covering the directions of tissue engineering scaffolds, microenvironment, single/multi-organ function, and stimulus-based evaluation, and provides a more comprehensive review of the progress in the significant organ-on-a-chip research areas in the physiological state.

Elucidation of the liver pathophysiology of COVID-19 patients using liver-on-a-chips.

SARS-CoV-2 induces severe organ damage not only in the lung but also in the liver, heart, kidney, and intestine. It is known that COVID-19 severity correlates with liver dysfunction, but few studies have investigated the liver pathophysiology in COVID-19 patients. Here, we elucidated liver pathophysiology in COVID-19 patients using organs-on-a-chip technology and clinical analyses. First, we developed liver-on-a-chip (LoC) which recapitulating hepatic functions around the intrahepatic bile duct and blood vessel. We found that hepatic dysfunctions, but not hepatobiliary diseases, were strongly induced by SARS-CoV-2 infection. Next, we evaluated the therapeutic effects of COVID-19 drugs to inhibit viral replication and recover hepatic dysfunctions, and found that the combination of anti-viral and immunosuppressive drugs (Remdesivir and Baricitinib) is effective to treat hepatic dysfunctions caused by SARS-CoV-2 infection. Finally, we analyzed the sera obtained from COVID-19 patients, and revealed that COVID-19 patients, who were positive for serum viral RNA, are likely to become severe and develop hepatic dysfunctions, as compared with COVID-19 patients who were negative for serum viral RNA. We succeeded in modeling the liver pathophysiology of COVID-19 patients using LoC technology and clinical samples.

A Microfluidic Device for Long-Term Maintenance of Organotypic Liver Cultures.

Liver cultures may be used for disease modeling, testing therapies and predicting drug-induced injury. The complexity of the liver cultures has evolved from hepatocyte monocultures to co-cultures with non-parenchymal cells and finally to precision-cut liver slices. The latter culture format retains liver's native biomolecular and cellular complexity and therefore holds considerable promise for in vitro testing. However, liver slices remain functional for ~72 h in vitro and display limited utility for some disease modeling and therapy testing applications that require longer culture times. This paper describes a microfluidic device for longer-term maintenance of functional organotypic liver cultures. Our microfluidic culture system was designed to enable direct injection of liver tissue into a culture chamber through a valve-enabled side port. Liver tissue was embedded in collagen and remained functional for up to 31 days, highlighted by continued production of albumin and urea. These organotypic cultures also expressed several enzymes involved in xenobiotic metabolism. Conversely, matched liver tissue embedded in collagen in a 96-well plate lost its phenotype and function within 3-5 days. The microfluidic organotypic liver cultures described here represent a significant advance in liver cultivation and may be used for future modeling of liver diseases or for individualized liver-directed therapies.

OOCDB: A Comprehensive, Systematic, and Real-time Organs-on-a-chip Database.

Organs-on-a-chip is a microfluidic microphysiological system that uses microfluidic technology to analyze the structure and function of living human cells at the tissue and organ levels in vitro. Organs-on-a-chip technology, as opposed to traditional two-dimensional cell culture and animal models, can more closely simulate pathologic and toxicologic interactions between different organs or tissues and reflect the collaborative response of multiple organs to drugs. Despite the fact that many organs-on-a-chip-related data have been published, none of the current databases have all of the following functions: searching, downloading, as well as analyzing data and results from the literature on organs-on-a-chip. Therefore, we created an organs-on-a-chip database (OOCDB) as a platform to integrate information about organs-on-a-chip from various sources, including literature, patents, raw data from microarray and transcriptome sequencing, several open-access datasets of organs-on-a-chip and organoids, and data generated in our laboratory. OOCDB contains dozens of sub-databases and analysis tools, and each sub-database contains various data associated with organs-on-a-chip, with the goal of providing researchers with a comprehensive, systematic, and convenient search engine. Furthermore, it offers a variety of other functions, such as mathematical modeling, three-dimensional modeling, and citation mapping, to meet the needs of researchers and promote the development of organs-on-a-chip. The OOCDB is available at http://www.organchip.cn.

A rodent thyroid-liver chip to capture thyroid toxicity on organ function level.

Endocrine disruption by environmental chemicals continues to be a concern for human safety. The rat, a widely used model organism in toxicology, is very sensitive to chemical-induced thyroid perturbation, e.g., histopathological alterations in thyroid tissue. Species differences in the susceptibility to thyroid perturbation lead to uncertainty in human safety risk assessments. Hazard identification and characterization of chemically induced thyroid perturbation would therefore benefit from in vitro models addressing different mechanisms of action in a single functional assay, ideally across species. We here introduce a rat thyroid-liver chip that enables simultaneous identification of direct and indirect (liver-mediated) thyroid perturbation on organ-level functions in vitro. A second manuscript describes our work toward a human thyroid-liver chip (Kühnlenz et al., 2022). The presented microfluidic model consisting of primary rat thyroid follicles and liver 3D spheroids maintains a tissue-specific phenotype for up to 21 days. More precisely, the thyroid model exhibits a follicular architecture expressing basolateral and apical markers and secretes T4. Likewise, liver spheroids retain hepatocellular characteristics, e.g., a stable release of albumin and urea, the presence of bile canalicular networks, and the formation of T4-glucuronide. Experiments with reference chemicals demonstrated proficiency to detect direct and indirect mechanisms of thyroid perturbation through decreased thyroid hormone secretion and increased gT4 formation, respectively. Prospectively this rat thyroid-liver chip model, together with its human counterpart, may support a species-specific quantitative in vitro to in vivo extrapolation to improve a data-driven and evidence-based human safety risk assessment with significant contributions to the 3R principles.

A microfluidic thyroid-liver platform to assess chemical safety in humans.

Thyroid hormones (THs) are crucial regulators of human metabolism and early development. During the safety assessment of plant protection products, the human relevance of chemically induced TH perturbations observed in test animals remains uncertain. European regulatory authorities request follow-up in vitro studies to elucidate human-relevant interferences on thyroid gland function or TH catabolism through hepatic enzyme induction. However, human in vitro assays based on single molecular initiating events poorly reflect the complex TH biology and related liver-thyroid axis. To address this complexity, we present human three-dimensional thyroid and liver organoids with key functions of TH metabolism. The thyroid model resembles in vivo-like follicular architecture and a TSH-dependent triiodothyronine synthesis over 21 days, which is inhibited by methimazole. The HepaRG-based liver model, secreting the critical TH-binding proteins albumin and thyroxine-binding globulin, emulates an active TH catabolism via the formation of glucuronidated and sulfated thyroxine (gT4/sT4). Activation of the nuclear receptors PXR and AHR was demonstrated via the induction of specific CYP isoenzymes by rifampicin, pregnenolone-16α-carbonitrile, and ß-naphthoflavone. However, this nuclear receptor activation, assumed to regulate UDP-glucuronosyltransferases and sulfotransferases, appeared to have no effect on gT4 and sT4 formation in this human-derived hepatic cell line model. Finally, established single-tissue models were successfully co-cultured in a perfused two-organ chip for 21 days. In conclusion, this model presents a first step towards a complex multimodular human platform that will help to identify both direct and indirect thyroid disruptors that are relevant from a human safety perspective.

Modeling Crevicular Fluid Flow and Host-Oral Microbiome Interactions in a Gingival Crevice-on-Chip.

Gingival crevice and gingival crevicular fluid (GCF) flow play a crucial role at the gingiva-oral microbiome interface which contributes toward maintaining the balance between gingival health and periodontal disease. Interstitial flow of GCF strongly impacts the host-microbiome interactions and tissue responses. However, currently available in vitro preclinical models largely disregard the dynamic nature of gingival crevicular microenvironment, thus limiting the progress in the development of periodontal therapeutics. Here, a proof-of-principle "gingival crevice-on-chip" microfluidic platform to culture gingival connective tissue equivalent (CTE) under dynamic interstitial fluid flow mimicking the GCF is described. On-chip co-culture using oral symbiont (Streptococcus oralis) shows the potential to recapitulate microbial colonization, formation of biofilm-like structures at the tissue-microbiome interface, long-term co-culture, and bacterial clearance secondary to simulated GCF (s-GCF) flow. Further, on-chip exposure of the gingival CTEs to the toll-like receptor-2 (TLR-2) agonist or periodontal pathogen Fusobacterium nucleatum demonstrates the potential to mimic early gingival inflammation. In contrast to direct exposure, the induction of s-GCF flow toward the bacterial front attenuates the secretion of inflammatory mediators demonstrating the protective effect of GCF flow. This proposed in vitro platform offers the potential to study complex host-microbe interactions in periodontal disease and the development of periodontal therapeutics under near-microphysiological conditions.

Organ-On-A-Chip Database Revealed-Achieving the Human Avatar in Silicon.

Organ-on-a-chip (OOC) provides microphysiological conditions on a microfluidic chip, which makes up for the shortcomings of traditional in vitro cellular culture models and animal models. It has broad application prospects in drug development and screening, toxicological mechanism research, and precision medicine. A large amount of data could be generated through its applications, including image data, measurement data from sensors, ~omics data, etc. A database with proper architecture is required to help scholars in this field design experiments, organize inputted data, perform analysis, and promote the future development of novel OOC systems. In this review, we overview existing OOC databases that have been developed, including the BioSystics Analytics Platform (BAP) developed by the University of Pittsburgh, which supports study design as well as data uploading, storage, visualization, analysis, etc., and the organ-on-a-chip database (Ocdb) developed by Southeast University, which has collected a large amount of literature and patents as well as relevant toxicological and pharmaceutical data and provides other major functions. We used examples to overview how the BAP database has contributed to the development and applications of OOC technology in the United States for the MPS consortium and how the Ocdb has supported researchers in the Chinese Organoid and Organs-On-A-Chip society. Lastly, the characteristics, advantages, and limitations of these two databases were discussed.

Modular Microfluidics: Current Status and Future Prospects.

This review mainly studies the development status, limitations, and future directions of modular microfluidic systems. Microfluidic technology is an important tool platform for scientific research and plays an important role in various fields. With the continuous development of microfluidic applications, conventional monolithic microfluidic chips show more and more limitations. A modular microfluidic system is a system composed of interconnected, independent modular microfluidic chips, which are easy to use, highly customizable, and on-site deployable. In this paper, the current forms of modular microfluidic systems are classified and studied. The popular fabrication techniques for modular blocks, the major application scenarios of modular microfluidics, and the limitations of modular techniques are also discussed. Lastly, this review provides prospects for the future direction of modular microfluidic technologies.