Advanced biomedical applications based on emerging 3D cell culturing platforms.

It is of great value to develop reliable in vitro models for cell biology and toxicology. However, ethical issues and the decreasing number of donors restrict the further use of traditional animal models in various fields, including the emerging fields of tissue engineering and regenerative medicine. The huge gap created by the restrictions in animal models has pushed the development of the increasingly recognized three-dimensional (3D) cell culture, which enables cells to closely simulate authentic cellular behaviour such as close cell-to-cell interactions and can achieve higher functionality. Furthermore, 3D cell culturing is superior to the traditional 2D cell culture, which has obvious limitations and cannot closely mimic the structure and architecture of tissues. In this study, we review several methods used to form 3D multicellular spheroids. The extracellular microenvironment of 3D spheroids plays a role in many aspects of biological sciences, including cell signalling, cell growth, cancer cell generation, and anti-cancer drugs. More recently, they have been explored as basic construction units for tissue and organ engineering. We review this field with a focus on the previous research in different areas using spheroid models, emphasizing aqueous two-phase system (ATPS)-based techniques. Multi-cellular spheroids have great potential in the study of biological systems and can closely mimic the in vivo environment. New technologies to form and analyse spheroids such as the aqueous two-phase system and magnetic levitation are rapidly overcoming the technical limitations of spheroids and expanding their applications in tissue engineering and regenerative medicine.

Microfluidic Skin-on-a-Chip Models: Toward Biomimetic Artificial Skin.

The role of skin in the human body is indispensable, serving as a barrier, moderating homeostatic balance, and representing a pronounced endpoint for cosmetics and pharmaceuticals. Despite the extensive achievements of in vitro skin models, they do not recapitulate the complexity of human skin; thus, there remains a dependence on animal models during preclinical drug trials, resulting in expensive drug development with high failure rates. By imparting a fine control over the microenvironment and inducing relevant mechanical cues, skin-on-a-chip (SoC) models have circumvented the limitations of conventional cell studies. Enhanced barrier properties, vascularization, and improved phenotypic differentiation have been achieved by SoC models; however, the successful inclusion of appendages such as hair follicles and sweat glands and pigmentation relevance have yet to be realized. The present Review collates the progress of SoC platforms with a focus on their fabrication and the incorporation of mechanical cues, sensors, and blood vessels.

Recent Progress in Lab-On-a-Chip Systems for the Monitoring of Metabolites for Mammalian and Microbial Cell Research.

Lab-on-a-chip sensing technologies have changed how cell biology research is conducted. This review summarises the progress in the lab-on-a-chip devices implemented for the detection of cellular metabolites. The review is divided into two subsections according to the methods used for the metabolite detection. Each section includes a table which summarises the relevant literature and also elaborates the advantages of, and the challenges faced with that particular method. The review continues with a section discussing the achievements attained due to using lab-on-a-chip devices within the specific context. Finally, a concluding section summarises what is to be resolved and discusses the future perspectives.

Next-generation technologies for studying host-pathogen interactions: a focus on dual transcriptomics, CRISPR/Cas9 screening and organs-on-chips.

Pathogens constantly interact with their hosts and the environment, and therefore have evolved unique virulence mechanisms to target and breach host defense barriers and manipulate host immune response to establish an infection. Advances in technologies that allow genome mining, gene editing such as CRISPR/Cas9, genomic, epigenomic and transcriptomic studies such as dual RNA-seq, coupled with bioinformatics, have accelerated the field of host-pathogen interactions within a broad range of infection models. Underpinning of the molecular changes that accompany invasion of eukaryotic cells with pathogenic microorganisms at the intersection of host, pathogen and their local environment has provided a better understanding of infectious disease mechanisms and antimicrobial strategies. The recent evolution of physiologically relevant three-dimensional (3-D) tissue/organ models and microfluidic organ-on-chip devices also provided a window to a more predictive framework of infectious disease processes. These approaches combined hold the potential to highly impact discovery of novel drug targets and vaccine candidates of the future. Here, we review three of the available and emerging technologies-dual RNA-seq, CRISPR/Cas9 screening and organs-on-chips, applicable to the high throughput study and deciphering of interaction networks between pathogens and their hosts that are critical for the development of novel therapeutics.

Impact of organ-on-a-chip technology on pharmaceutical R&D costs.

Healthcare systems are faced with the challenge of providing innovative treatments, while shouldering high drug costs that pharmaceutical companies justify by the high costs of R&D. An emergent technology that could transform R&D efficiency is organ-on-a-chip. The technology bridges the gap between preclinical testing and human trials through better predictive models, significantly impacting R&D costs. Here, we present an expert survey on the future role of organ-on-a-chip in drug discovery and its potential quantitative impact. We find that the technology has the potential to reduce R&D costs significantly, driven by changes in direct costs, success rates and the length of the R&D process. Finally, we discuss regulatory challenges to efficiency improvements.

Challenges and Opportunities in the Design of Liver-on-Chip Microdevices.

The liver is the central hub of xenobiotic metabolism and consequently the organ most prone to cosmetic- and drug-induced toxicity. Failure to detect liver toxicity or to assess compound clearance during product development is a major cause of postmarketing product withdrawal, with disastrous clinical and financial consequences. While small animals are still the preferred model in drug development, the recent ban on animal use in the European Union created a pressing need to develop precise and efficient tools to detect human liver toxicity during cosmetic development. This article includes a brief review of liver development, organization, and function and focuses on the state of the art of long-term cell culture, including hepatocyte cell sources, heterotypic cell-cell interactions, oxygen demands, and culture medium formulation. Finally, the article reviews emerging liver-on-chip devices and discusses the advantages and pitfalls of individual designs. The goal of this review is to provide a framework to design liver-on-chip devices and criteria with which to evaluate this emerging technology.

Digital Manufacturing for Microfluidics.

The microfluidics field is at a critical crossroads. The vast majority of microfluidic devices are presently manufactured using micromolding processes that work very well for a reduced set of biocompatible materials, but the time, cost, and design constraints of micromolding hinder the commercialization of many devices. As a result, the dissemination of microfluidic technology-and its impact on society-is in jeopardy. Digital manufacturing (DM) refers to a family of computer-centered processes that integrate digital three-dimensional (3D) designs, automated (additive or subtractive) fabrication, and device testing in order to increase fabrication efficiency. Importantly, DM enables the inexpensive realization of 3D designs that are impossible or very difficult to mold. The adoption of DM by microfluidic engineers has been slow, likely due to concerns over the resolution of the printers and the biocompatibility of the resins. In this article, we review and discuss the various printer types, resolution, biocompatibility issues, DM microfluidic designs, and the bright future ahead for this promising, fertile field.

IVF-on-a-Chip: Recent Advances in Microfluidics Technology for In Vitro Fertilization.

In vitro fertilization (IVF) has been one of the most exciting modern medical technologies. It has transformed the landscape of human infertility treatment. However, current IVF procedures still provide limited accessibility and affordability to most infertile couples because of the multiple cumbersome processes and heavy dependence on technically skilled personnel. Microfluidics technology offers unique opportunities to automate IVF procedures, reduce stress imposed upon gametes and embryos, and minimize the operator-to-operator variability. This article describes the rapidly evolving state of the application of microfluidics technology in the field of IVF, summarizes the diverse angles of how microfluidics has been complementing or transforming current IVF protocols, and discusses the challenges that motivate continued innovation in this field.

Dielectrophoresis Manipulation: Versatile Lateral and Vertical Mechanisms.

Discussing the topic of the capability of dielectrophoresis (DEP) devices in terms of the selective detection and rapid manipulation of particles based on the DEP force (FDEP) via contactless methods is challenging in medical research, drug discovery and delivery. Nonetheless, the process of the selective detection and rapid manipulation of particles via contactless DEP based on dielectric particles and the surrounding medium can reduce the effects of major issues, including physical contact with the particles and medium contamination to overcome operational difficulties. In this review, DEP microelectromechanical system (MEMS) microelectrodes with a tapered profile for the selective detection and rapid manipulation of particles were studied and compared with those of conventional designs with a straight-cut profile. The main objective of this manuscript is to review the versatile mechanism of tapered DEP MEMS microelectrodes for the purpose of selective detection and rapid manipulation. Thus, this review provides a versatile filtration mechanism with the potential for a glomerular-based membrane in an artificial kidneys' development solution for implementing engineered particles and cells by lateral attraction as well as vertical repulsion in the development of lab-on-a-chip applications. For tapered DEP MEMS microelectrodes, the scope of this study methodology involved the characterisation of DEP, modelling of the polarisation factor and the dynamic dielectric changes between the particles and medium. Comprehensive discussions are presented on the capability of tapered DEP microelectrodes to drive the selected particles and the simulation, fabrication and testing of the tapered profile. This study revealed an outstanding performance with the capability of producing two regions of high electric field intensity at the bottom and top edges of the side wall of tapered microelectrodes. Observations on particle separation mainly by the lateral attraction force of particles with positive DEP on the y-axis and vertical repulsion force of particles with negative DEP on the z-axis proved an efficient and uniform FDEP produced by tapered electrodes. In conclusion, this study confirmed the reliability and efficiency of the tapered DEP microelectrodes in the process of selective detection and rapid manipulation at a higher efficiency rate than straight-cut microelectrodes, which is significant in DEP technology applications.

3D biosensors in advanced medical diagnostics of high mortality diseases.

Cardiovascular diseases, cancer, and diabetes are high mortality diseases, which account for almost two thirds of all deaths worldwide. Their early detection and continuous evaluation are fundamental for an improved patient prognosis and reduced socioeconomic impact. Current biosensor technologies are typically based on the analysis of whole blood samples from patients for the detection of disease-specific biomarkers. However, these technologies display serious shortcomings, such as reduced sensitivity and dynamic range, limited in vivo applicability, and lack of continuous monitoring. There is the urgent need for new diagnostic and treatment follow-up tools, which allow for the early detection of the pathology as well as for the continuous monitoring of the physiological responses to specific therapies. During the last years, a new generation of biosensor technologies with improved performance has emerged in the biomedical sector. The combination of advanced biomaterial methods, biochemical tools, and micro/nanotechnology approaches has resulted in the development of innovative three-dimensional (3D) biosensor platforms for advanced medical diagnosis. In this review, we report the most recent advances in the field of 3D biosensors for clinical applications, focusing on the diagnosis and monitoring of cardiovascular diseases, cancer, and diabetes. We discuss about their clinical performance compared to standard biosensor technologies, their implantable capability, and their integration into microfluidic devices to develop clinically-relevant models. Overall, we anticipate that 3D biosensors will drive us toward a new paradigm in medical diagnosis, resulting in real-time in vivo biosensors capable to significantly improve patient prognosis.

Multi-Organs-on-Chips: Towards Long-Term Biomedical Investigations.

With advantageous features such as minimizing the cost, time, and sample size requirements, organ-on-a-chip (OOC) systems have garnered enormous interest from researchers for their ability for real-time monitoring of physical parameters by mimicking the in vivo microenvironment and the precise responses of xenobiotics, i.e., drug efficacy and toxicity over conventional two-dimensional (2D) and three-dimensional (3D) cell cultures, as well as animal models. Recent advancements of OOC systems have evidenced the fabrication of 'multi-organ-on-chip' (MOC) models, which connect separated organ chambers together to resemble an ideal pharmacokinetic and pharmacodynamic (PK-PD) model for monitoring the complex interactions between multiple organs and the resultant dynamic responses of multiple organs to pharmaceutical compounds. Numerous varieties of MOC systems have been proposed, mainly focusing on the construction of these multi-organ models, while there are only few studies on how to realize continual, automated, and stable testing, which still remains a significant challenge in the development process of MOCs. Herein, this review emphasizes the recent advancements in realizing long-term testing of MOCs to promote their capability for real-time monitoring of multi-organ interactions and chronic cellular reactions more accurately and steadily over the available chip models. Efforts in this field are still ongoing for better performance in the assessment of preclinical attributes for a new chemical entity. Further, we give a brief overview on the various biomedical applications of long-term testing in MOCs, including several proposed applications and their potential utilization in the future. Finally, we summarize with perspectives.

Integrated chemiluminescence-based lab-on-chip for detection of life markers in extraterrestrial environments.

The detection of life markers is a high priority task in the exploration of the Solar System. Biochips performing in-situ multiplex immunoassays are a very promising approach alternative to gas chromatography coupled with mass spectrometry. As part of the PLEIADES project, we present the development of a chemiluminescence-based, highly integrated analytical platform for the detection of biomarkers outside of the Earth. The PLEIADES device goes beyond the current lab-on-chip approaches that still require bulky external instrumentation for their operation. It exploits an autonomous capillary force-driven microfluidic network, an array of thin-film hydrogenated amorphous silicon photosensors, and chemiluminescence bioassays to provide highly sensitive analyte detection in a very simple and compact configuration. Adenosine triphosphate was selected as the target life marker. Three bioassay formats have been developed, namely (a) a bioluminescence assay exploiting a luciferase mutant with enhanced thermal and pH stability and (b and c) binding assays exploiting antibodies or functional nucleic acids (aptamers) as biospecific recognition elements and peroxidase or DNAzymes as chemiluminescence reporters. Preliminary results, showing limits of detection in the nanomolar range, confirm the validity of the proposed approach.

Organs-on-a-chip: Current applications and consideration points for in vitro ADME-Tox studies.

Assay systems using in vitro cultured cells are increasingly applied for evaluation of the efficacy, safety, and toxicity of drug candidates. In vitro cell-based assays have two main applications in the drug discovery process: searching for a compound that is effective against the target disease (seed investigation) and confirmation of safety during use of the identified compounds (safety assessment). Currently available in vitro cell-based assays have been designed to evaluate the efficacy and toxicity in single organs, but the in vivo pharmacokinetics and pharmacodynamics of the administered drug candidates have not been considered. Thus, an evaluation system that interconnects cell culture units, one of which has appropriate drug metabolism activities and the other assesses the efficacy and toxicity of compounds, is needed. Accordingly, the in vitro ADME-Tox culture system known as organs-on-a-chip has been proposed. In this review, after introducing the organs-on-a-chip system, the evaluation of enterohepatic circulation and the gut-liver axis relationship will be presented as an example of the application of the organs-on-a-chip system for ADME studies based on inter-organ network. Additionally, the functions required for the organs-on-a-chip system and the necessity of standardization of cells mounted on the chip system will be discussed.

Organ/body-on-a-chip based on microfluidic technology for drug discovery.

Although animal experiments are indispensable for preclinical screening in the drug discovery process, various issues such as ethical considerations and species differences remain. To solve these issues, cell-based assays using human-derived cells have been actively pursued. However, it remains difficult to accurately predict drug efficacy, toxicity, and organs interactions, because cultivated cells often do not retain their original organ functions and morphologies in conventional in vitro cell culture systems. In the µTAS research field, which is a part of biochemical engineering, the technologies of organ-on-a-chip, based on microfluidic devices built using microfabrication, have been widely studied recently as a novel in vitro organ model. Since it is possible to physically and chemically mimic the in vitro environment by using microfluidic device technology, maintenance of cellular function and morphology, and replication of organ interactions can be realized using organ-on-a-chip devices. So far, functions of various organs and tissues, such as the lung, liver, kidney, and gut have been reproduced as in vitro models. Furthermore, a body-on-a-chip, integrating multi organ functions on a microfluidic device, has also been proposed for prediction of organ interactions. We herein provide a background of microfluidic systems, organ-on-a-chip, Body-on-a-chip technologies, and their challenges in the future.

Human lung epithelial cell cultures for analysis of inhaled toxicants: Lessons learned and future directions.

The epithelium that covers the conducting airways and alveoli is a primary target for inhaled toxic substances, and therefore a focus in inhalation toxicology. The increasing concern about the use of animal models has stimulated the development of in vitro cell culture models for analysis of the biological effects of inhaled toxicants. However, the validity of the current in vitro models and their acceptance by regulatory authorities as an alternative to animal models is a reason for concern, and requires a critical review. In this review, focused on human lung epithelial cell cultures as a model for inhalation toxicology, we discuss the choice of cells for these models, the cell culture system used, the method of exposure as well as the various read-outs to assess the cellular response. We argue that rapid developments in the 3D culture of primary epithelial cells, the use of induced pluripotent stem cells for generation of lung epithelial cells and the development of organ-on-a-chip technology are among the important developments that will allow significant advances in this field. Furthermore, we discuss the various routes of application of inhaled toxicants by air-liquid interface models as well as the vast array of read-outs that may provide essential information. We conclude that close collaboration between researchers from various disciplines is essential for development of valid methods that are suitable for replacement of animal studies for inhalation toxicology.

Lab-on-Chip Devices: Gaining Ground Losing Size.

Portable analytical devices are notably gaining relevance in the panorama of urgent testing. Such devices have the potential to play an important role as easy-to-handle tools in critical situations. Epidemic infectious disease agents (e.g., Ebola virus, Coronavirus, Zika virus) could be controlled more easily by testing travelers on-site at the country borders to prevent outbreaks from spreading. The increasing incidence of hospital-acquired infections caused by antibiotic resistant pathogens could be minimized by point-of-care microbial analysis as well as rapid screening tests of bacteria resistance. The threat of bioterrorism using novel unknown bioweapons has never been so high, thus, in-the-field early identification of the biological agent is crucial for triggering a coordinated response. Food allergies are a growing public health concern-allergic reactions can result in anaphylactic shock, which can prove fatal in minutes-thus, the ability to test foods for common allergens, rapidly and locally, before ingestion, would improve food safety for those with allergies. Lab-on-chip devices are becoming widely available for diverse applications and are becoming increasingly affordable. However, to shrink in price and size simultaneously, some trade-offs must be made. In this Perspective, we present considerations about product specifications, design concepts, and application scenarios.