Cyclo olefin polymer-based solvent-free mass-productive microphysiological systems

Dynamic microphysiological system chip platform for high-throughput, customizable, and multi-dimensional drug screening

To increase success rates of clinical studies, preclinical evaluation systems have been expected to improve human predictability. In addition, future preclinical studies need to become more sophisticated and efficient on the back ground of the adoption of FDA Modernization Act 2.0 and the 3R principle promotion of animal tests. In this review, we will discuss about the efficiency of in vivo imaging in preclinical studies taking 'an attempt to establishment of in vitro in vivo extraporation (IVIVE) model for seizure risk assessment using microphysiological system (MPS) and magnetic resonance imaging (MRI)', and 'an attempt to predict drug delivery to the alveoli' as examples. In the seizure risk assessment of new drugs so far, primary cultures of rodent neurons and in vivo behavioral observation have been mainly used, however, since the human induced pluripotent stem cell (iPSC) technology was reported, the need for IVIVE model is more and more increasing to improve human predictability. As an MPS, we here introduce microelectrode array (MEA) system recording of primary culture of rodent neurons, while as in vivo experiments, we here introduce the measurement of cerebrospinal fluid (CSF) concentrations and MRI imaging of forebrains of the rats i.p. injected with seizurogenic compounds. In case of inhalation drugs, it has been difficult to confirm whether or not the drugs surely reach alveoli. We visualized two-dimensional spatial localization of inhaled ciclesonide (CIC) in rat lungs after administration of a single dose of a CIC aerosol using by desorption electrospray ionization-time of flight mass spectrometry imaging (DESI-MSI).

In recent years, many researchers have proposed biohybrid systems that integrate biological materials, especially cells or cultured tissues formed by in vitro cell culture, and microdevices to utilize the functions of cultured cellular tissues in an engineering way. In this paper, we focus on microphysiological systems composed of microfluidic channels or microdevices and cells or cultured tissues and introduce the functional characteristics of these microphysiological systems. In the microphysiological system, we can adjust cell culture conditions under mechanical stress control and bring out the dynamic performance of the cultured tissue. The systems can be applied not only to drug development to replace laboratory animals, but also to the development of advanced robots with cellular functions. Therefore, we believe that the biohybrid systems are a promising technology to apply cells and cultured tissues in various fields.

Microphysiological systems (MPS) are drawing increasing interest from academia and from biomedical industry due to their improved capability to capture human physiology. MPS offer an advanced in vitro platform that can be used to study human organ and tissue level functions in health and in diseased states more accurately than traditional single cell cultures or even animal models. Key features in MPS include microenvironmental control and monitoring as well as high biological complexity of the target tissue. To reach these qualities, cross-disciplinary collaboration from multiple fields of science is required to build MPS. Here, we review different areas of expertise and describe essential building blocks of heart MPS including relevant cardiac cell types, supporting matrix, mechanical stimulation, functional measurements, and computational modelling. The review presents current methods in cardiac MPS and provides insights for future MPS development with improved recapitulation of human physiology.

Cyclic Olefin Copolymer (COC) is an amorphous thermoplastic polymer synthesized through the catalytic copolymerization of α-olefin and cyclic olefin. When used in pre-filled syringes and pharmaceutical packaging, COCs require radiation sterilization. The radiation sterilization alters the microstructure of COC, which ultimately affects its performance and biosafety. In this study, to investigate the effects of γ-radiation on COC microstructures, ethylene-norbornene copolymers with various compositions, representative of COC, are studied by nuclear magnetic resonance (NMR) and small angle X-ray scattering (SAXS) techniques. During irradiation, the COC containing 35 mol% norbornene produced free radicals that triggered migration and reaction processes, leading to the formation of entanglements within flexible chain segments. This, in turn, affected nearby ring structures with high steric hindrance, resulting in a 9.2% decrease in internal particle size and an increase in particle spacing. Conversely, when the norbornene content in COC was increased to 57 mol%, the internal particle size increased by 17.9%, while the particle spacing decreased.

Oral bioavailability is a crucial pharmacokinetic (PK) parameter optimized by drug developers working on new therapeutic compounds. The goal is to obtain adequate systemic exposure and to establish safe and effective therapeutic dosages. Critical to optimizing oral bioavailability is understanding a compound’s susceptibility to first-pass metabolism, determined by the intricate relationship between gut absorption, metabolism, and hepatic metabolism. Efforts to model this relationship in vitro have led to the emergence of microphysiological systems (MPSs) that consist of multiple, fluidically linked organs. Here, we describe an MPS that links the gut and liver, capable of simulating both oral and intravenous dosing routes and is made up of entirely primary human cells: a gut barrier tissue comprised of an intestinal epithelial monolayer derived from the human jejunum, and a liver microtissue, derived from primary human hepatocytes. Functionality of gut and liver tissues is maintained in coculture enabling the PK investigation of oral compounds. We combine the primary Gut/Liver MPS with a mechanistic mathematical model to generate organ-specific PK parameters and estimate human oral bioavailability and its components: the fraction absorbed (Fa), the fraction escaping gut wall elimination (Fg), and the fraction escaping hepatic elimination (Fh). We used the CYP3A-mediated compound midazolam, which is subjected to both intestinal and hepatic extraction, to demonstrate the transformative potential of the primary Gut/Liver MPS to mechanistically model the PKs of oral compounds in vitro.Significance StatementBioavailability underpins the success or when insufficient, can stall the development of oral therapeutics. The drug discovery process lacks in vitro assays that can profile the contribution to bioavailability by the gut and liver. The primary Gut/Liver MPS outlined in this study bridges this gap.

Innervated adrenomedullary microphysiological system to model nicotine and opioid exposure

Microphysiological systems (MPSs) reconstitute tissue interfaces and organ functions, presenting a promising alternative to animal models in drug development. However, traditional materials like polydimethylsiloxane (PDMS) often interfere by absorbing hydrophobic molecules, affecting drug testing accuracy. Additive manufacturing, including 3D bioprinting, offers viable solutions. GlioFlow3D, a novel microfluidic platform combining extrusion bioprinting and stereolithography (SLA) is introduced. GlioFlow3D integrates primary human cells and glioblastoma (GBM) lines in hydrogel‐based microchannels mimicking vasculature, within an SLA resin framework using cost‐effective materials. The study introduces a robust protocol to mitigate SLA resin cytotoxicity. Compared to PDMS, GlioFlow3D demonstrated lower small molecule absorption, which is relevant for accurate testing of small molecules like Temozolomide (TMZ). Computational modeling is used to optimize a pumpless setup simulating interstitial fluid flow dynamics in tissues. Co‐culturing GBM with brain endothelial cells in GlioFlow3D showed enhanced CD133 expression and TMZ resistance near vascular interfaces, highlighting spatial drug resistance mechanisms. This PDMS‐free platform promises advanced drug testing, improving preclinical research and personalized therapy by elucidating complex GBM drug resistance mechanisms influenced by the tissue microenvironment.

Drugs may undergo costly preclinical studies but still fail to demonstrate their efficacy in clinical trials, which makes it challenging to discover new drugs. Both in vitro and in vivo models are essential for disease research and therapeutic development. However, these models cannot simulate the physiological and pathological environment in the human body, resulting in limited drug detection and inaccurate disease modelling, failing to provide valid guidance for clinical application. Organs-on-chips (OCs) are devices that serve as a micro-physiological system or a tissue-on-a-chip; they provide accurate insights into certain functions and the pathophysiology of organs to precisely predict the safety and efficiency of drugs in the body. OCs are faster, more economical, and more precise. Thus, they are projected to become a crucial addition to, and a long-term replacement for, traditional preclinical cell cultures, animal studies, and even human clinical trials. This paper first outlines the nature of OCs and their significance, and then details their manufacturing-related materials and methodology. It also discusses applications of OCs in drug screening and disease modelling and treatment, and presents the future perspective of OCs.

Chinese hamster ovary (CHO) cells are frequently used for recombinant protein production (RPP) as a host. While the RPP has been proven successful, there is still a compelling need for further improvement. Cyclo olefin polymer (COP) is a plastic material widely utilized due to its properties including its low protein absorption. We applied this as a raw material for RPP cell culture to see if the COP is suitable. A recombinant CHO cell line expressing the human erythropoietin (hEPO) gene under the control of the Moloney murine leukemia virus-long terminal repeat (MMLV-LTR) was established. When the cells were cultured in a dish made from COP, the cells attached to the bottom, and then started to float and form spheroids. RNASeq data analysis suggested the epithelial-mesenchymal transition (EMT) was triggered with receptor tyrosine kinase activation shortly after cultivation. It coincided with the hEPO transcription increase. After the cell floating, though EMT marker gene expression subsided, a hEPO expression increase sustained. When fibronectin was applied to COP dish surface, the cell floating was suppressed and hEPO expression decreased. We then treated cells with MβCD, a drug that destroys the lipid raft, eliminating molecules in the raft. This facilitated cell floating and spheroid formation coincided with hEPO expression enhancement. These results suggest interactions between a cell and COP surface might trigger the EMT and the subsequent event, both of which activated the MMLV-LTR promoter. Thus, employing COP for culturing cells, a potent RPP system could be established with its advantage for efficient protein purification.

This study introduces a novel surface modification process involving vacuum ultraviolet (VUV) irradiation followed by oxygen plasma treatment to enhance the adhesion between a cycloolefin polymer (COP) film, known for its excellent dielectric properties, and a copper plating layer with a direct sputtered copper seed layer, intended for use in antenna circuits for 6 G communications. Various surface analyses revealed that oxygen plasma treatment after VUV irradiation increased the quantity of functional groups in the extreme surface layer (∼10 nm) of the COP film more than oxygen plasma treatment alone and removed the brittle modified layer formed by VUV irradiation. This dual effect resulted in a peel strength of 1 kN m−1, exceeding the 0.8 kN m−1 achieved with oxygen plasma treatment alone, confirming the synergistic effect of VUV irradiation and oxygen plasma treatment. Based on the surface analysis results, a process model was constructed explaining how oxygen plasma treatment etched away part of the modified layer containing the functional groups formed by VUV irradiation while simultaneously introducing additional functional groups to the newly exposed surface layer.

We have developed a novel approach for the fabrication of 6G communication antenna substrates by directly sputtering a copper seed layer onto a resin with low dielectric properties and low adhesiveness, without relying on a metal adhesion layer such as titanium. This study presents a detailed analysis of the effects of two surface modification methods—vacuum ultraviolet (VUV) irradiation and oxygen plasma treatment—performed before sputtering the copper seed layer on the interfacial structure between a cycloolefin polymer (COP) film and copper plating layer. Using scanning transmission electron microscopy, energy-dispersive x-ray spectroscopy, and selected area electron diffraction, we confirmed that both surface modification methods promote the formation of polycrystalline Cu2O at the interface between the COP film and the copper plating layer. VUV irradiation enhances the smoothness of the COP surface, resulting in the uniform distribution of hydrophilic functional groups. These functional groups stably interact with the sputtered copper, leading to the oxidation of copper during annealing and the formation of a consistent Cu2O layer. In contrast, oxygen plasma treatment selectively etches regions of the COP surface with high oxygen affinity, creating fine structures and concentrating the hydrophilic functional groups in the recessed areas. After sputtering, copper interacts with these localized functional groups, and during annealing, limited diffusion causes the copper to oxidize and aggregates into a Cu2O particle structure. Thus, this study demonstrates that the structures of polycrystalline Cu2O formed at the interface vary depending on the surface modification methods and elucidates the mechanisms behind these differences through analysis of surface morphology and the distribution of functional groups induced by each method.

Cancer microenvironment has a significant impact on the progression of the disease. In particular, hypoxia is the key driver of cancer survival, invasion, and chemoresistance. Although several in vitro models have been developed to study hypoxia-related cancer pathology, the complex interplay of the cancer microenvironment observedin vivo has not been reproduced yet owing to the lack of precise spatial control. Instead, 3D biofabrication approaches have been proposed to create microphysiological systems for better emulation of cancer ecology and accurate anticancer treatment evaluation. Herein, we propose a 3D cell-printing approach to fabricate a hypoxic cancer-on-a-chip. The hypoxia-inducing components in the chip were determined based on a computer simulation of the oxygen distribution. Cancer-stroma concentric rings were printed using bioinks containing glioblastoma cells and endothelial cells to recapitulate a type of solid cancer. The resulting chip realized central hypoxia and aggravated malignancy in cancer with the formation of representative pathophysiological markers. Overall, the proposed approach for creating a solid-cancer-mimetic microphysiological system is expected to bridge the gap between in vivo and in vitro models for cancer research.

Physiological supporting systems, such as the vascular network and excretion system, are crucial for the effective functioning of organs. This study demonstrates that when a body-on-a-chip microdevice is coupled with miniaturized physiological support systems, it can create a multi-organ microphysiological system capable of more accurately mimicking the physiological complexity of a body, thereby offering potential for preclinical drug testing. To exemplify this concept, we have developed a model system comprising 18 types of microtissues interconnected by a vascular network that replicates the in vivo blood distribution among the organs. Furthermore, this system includes an excretory system with a micro-stirrer that ensures elimination efficiency akin to in vivo conditions. Our findings indicate that this system can: (1) survive and function for almost two months; (2) achieve two-compartment pharmacokinetics of a drug; (3) investigate the dynamic relationship between the tissue distribution and toxicity of a drug; (4) establish the multimorbidity model and evaluate the effectiveness of polypharmacy, challenging tasks with traditional animal models; (5) reduce animal usage in drug evaluations. Notably, features from points (2) to (4) are capabilities not achievable by other in vitro models. The strategy proposed in this study can also be applied to the development of multi-organ microphysiological systems that mimic the physiological complexity of human organs or the entire body.

Microphysiological systems (MPS) are powerful predictive tools for assessing drug-induced kidney injuries. Previous MPS have examined single regions of the nephron, but lack simultaneous filtration, reabsorption, and secretion functionality. Here, we developed a partially open MPS that structurally and functionally recapitulated the glomerular filtration barrier, proximal tubular reabsorption, and secretion for seven days. The system introduced a recirculation circuit and an open filtrate output as a source of functional testing. As a proof-of-concept, a tri-culture of immortalized podocytes, umbilical vein endothelial cells, and proximal tubule (PCT) cells were housed in a single MPS: T-junction, glomerulus housing unit, and PCT chip. The MPS successfully retained blood serum protein, reabsorbed glucose, secreted creatinine, and expressed cell-type specific proteins (VE-cadherin, nephrin, and ZO-1). To simulate drug-induced kidney injuries, the system was perfused with cisplatin and adriamycin, and then tested using serum albumin filtration, glucose clearance, and lactate dehydrogenase release. The glomerulus and PCT MPS demonstrated a complex, dynamic microenvironment and recreated some in vivo-like functions in basal and drug-induced conditions, offering a novel prototype for preclinical testing.