Microphysiological System Research Articles

Algae-based flexible localized oxygen control around Cells: An approach leading to more Biomimetic microphysiological systems

Oxygen levels are crucial in the cellular microenvironment, affecting cellular function. Controlling pericellular oxygen is essential for understanding cell-oxygen interactions, disease mechanisms, and developing therapies. In this study, an algae strain was engineered as a biological oxygen-control material, integrated with a light-controlled microphysiological system to regulate pericellular oxygen. This technique could provide different oxygen levels and varying profiles for different locations within one microphysiological system. Additionally, the pericellular oxygen dynamics were faster than those achieved by other methods. By utilizing this algae-integrated microfluidic chip, mesenchymal stem cells differentiated into adipocytes with improved phenotypic characteristics, including increased numbers and sizes of lipid droplets, under 21-day stage-specific oxygen concentrations. Furthermore, this technique effectively mitigated the need to vent significant amounts of oxygen or nitrogen into the laboratory during long-term or virus-infested experiments, thereby enhancing both safety and practicality. The algae-integrated microfluidic chip also served as a platform for constructing a stroke-related vascular-on-chip model, enabling the evaluation of the protective effects of oxygen therapy on endothelial injury induced by oxygen-glucose deprivation/reperfusion (OGD/R). The results indicated that waving hyperoxic dynamics were most effective in safeguarding vascular tissue against OGD/R compared to other hyperoxic dynamics. This finding can guide the clinical oxygen inhalation mode during stroke management. All the aforementioned results demonstrated that the algae-light microphysiological system offered a technical platform capable of precise, dynamic pericellular oxygen control across the disciplines of biology and pharmaceutics. Moreover, The cost-effective cultivation of algae on a large scale made this method suitable for wider use.

Predicting Clinical Outcomes of SARS‐CoV‐2 Drug Efficacy with a High‐Throughput Human Airway Microphysiological System (Adv. Biology 11/2024)

Predicting Clinical Outcomes of SARS‐CoV‐2 Drug Efficacy with a High‐Throughput Human Airway Microphysiological System (Adv. Biology 11/2024)

RCAN1.4 regulates tumor cell engraftment and invasion in a thyroid cancer to lung metastasis-on-a-chip microphysiological system

Progressive metastasis is the primary cause of cancer-related deaths. It has been recognized that many cancers are characterized by long periods of stability followed by subsequent progression. Genes termed metastasis progression suppressors (MPS) are functional gatekeepers of this process, and their loss leads to late-stage progression. Previously, we identified regulator of calcineurin 1, isoform 4 (RCAN1.4) as a functional MPS for several cancers, including thyroid cancer, a tumor type prone to metastatic dormancy. RCAN1.4 knockdown increases expression of the cancer-promoting transcription factor NFE2-like bZIP transcription factor (NFE2L3), and through this mechanism increases cancer cell proliferation and invasion inin vitroandin vivoand promotes metastatic potential to lungs in tail vein models. However, the mechanisms by which RCAN 1.4 regulates specific metastatic steps is incompletely characterized. Studies of the metastatic cascade are limited in mouse systems due to high cost and long duration. Here, we have shown the creation of a thyroid-to-lung metastasis-on-a-chip (MOC) model to address these limitations, allowing invasion analysis and quantification on a single cell level. We then deployed the platform to investigate RCAN1.4 knockdown in fluorescently tagged hTh74 and FTC236 thyroid cancer cell lines. Cells were circulated through microfluidic channels, running parallel to lung hydrogel constructs allowing tumor cell-lung tissue interactions. Similar to studies in mouse models, RCAN1.4 knockdown increased NFE2L3 expression, globally increased invasion distance into lung constructs and had cell line and clonally dependent variations on bulk metastatic burden. In line with previousin vivoobservations, RCAN1.4 knockdown had a greater impact on hTh74 metastatic propensity than FTC236. In summary, we have developed and validated a novel MOC system evaluate and quantify RCAN1.4-regulated thyroid cancer cell lung adherence and invasion. This system creates opportunities for more detailed and rapid mechanistic studies the metastatic cascade and creates opportunities for translational assay development.

A microvascularized in vitro liver model for disease modeling and drug discovery

Drug discovery for complex liver diseases faces alarming attrition rates. The lack of non-clinical models that recapitulate key aspects of liver (patho)-physiology is likely contributing to the inefficiency of developing effective treatments. Of particular notice is the common omission of an organized microvascular component despite its importance in maintaining liver function and its involvement in the development of several pathologies. Increasing the complexity ofin vitromodels is usually associated with a lack of scalability and robustness which hinders their implementation in drug development pipelines. Here, we describe a comprehensive liver microphysiological system comprising stellates, liver-derived endothelial cells and hepatocytes conceived within a scalable and automated platform. We show that endothelial cells self-organize in a microvascular network when co-cultured with stellates in a hydrogel. In a tri-culture, hepatocytes polarize accordingly, with a basolateral side facing blood vessels and an apical side facing bile-canaliculi-like structures. Stellates interact and surround the hollow microvessels. Steatosis was induced by exogenous administration of fatty acids which could be prevented by co-administration of firsocostat. Administration of TGF-βresulted in an activated stellate cells phenotype which could be prevented by the co-administration of SB-431542. The model was implemented on a microtiter plate format comprising 64 chips which enabled the development of a fully automated, multiplexed fibrosis assay with a robust Z' factor suitable for high-throughput applications.

A single-cell 3D dynamic volume control system for chondrocytes.

In articular cartilage, zone-specific cellular morphology is a typical characteristic of cartilage tissue, which is related with chondrocyte function, inflammation and osteoarthritis (OA). Chondrocyte hypertrophic phenotype is a criticle physiological process which indicates a hallmark of chondrocyte terminal differentiation and bone formation. Thus, developing a in vitro cell culture system for dynamic regulation of single chondrocyte volume at a three-dimensional (3D) level is particularly necessary for understanding how physical cues of matrix microenvironment regulate chondrocyte fate and the degeneration of articular cartilage. Here, based on the soft lithography techniques, we have constructed well-defined single-cell 3D dynamic volume control system to recapitulate the physiological matrix microenvironment of single chondrocyte niche. The results of finite element analysis indicated that the stress and strain distribution in the cell culture region is homogeneous during the stretching process. Additionally, 3D dynamic volume expansion and compression of single cells in physiological or hyperphysiological can be realized in this cell culture system. Our device for single-cell 3D dynamic culture provides a microphysiological culture system for chondrocytes to explore the mechanisms of cartilage hypertrophy, as well as develops a new paradigm for functional cartilage tissue engineering and regenerative medicine.

A metabolic dysfunction-associated steatotic liver acinus biomimetic induces pancreatic islet dysfunction in a coupled microphysiology system

Preclinical and clinical studies suggest that lipid-induced hepatic insulin resistance is a primary defect that predisposes to dysfunction in islets, implicating a perturbed liver-pancreas axis underlying the comorbidity of T2DM and MASLD. To investigate this hypothesis, we developed a human biomimetic microphysiological system (MPS) coupling our vascularized liver acinus MPS (vLAMPS) with pancreatic islet MPS (PANIS) enabling MASLD progression and islet dysfunction to be assessed. The modular design of this system (vLAMPS-PANIS) allows intra-organ and inter-organ dysregulation to be deconvoluted. When compared to normal fasting (NF) conditions, under early metabolic syndrome (EMS) conditions, the standalone vLAMPS exhibited characteristics of early stage MASLD, while no significant differences were observed in the standalone PANIS. In contrast, with EMS, the coupled vLAMPS-PANIS exhibited a perturbed islet-specific secretome and a significantly dysregulated glucose stimulated insulin secretion response implicating direct signaling from the dysregulated liver acinus to the islets. Correlations between several pairs of a vLAMPS-derived and a PANIS-derived factors were significantly altered under EMS, as compared to NF conditions, mechanistically connecting MASLD and T2DM associated hepatic-factors with islet-derived GLP-1 synthesis and regulation. Since vLAMPS-PANIS is compatible with patient-specific iPSCs, this platform represents an important step towards addressing patient heterogeneity, identifying disease mechanisms, and advancing precision medicine.

A microphysiological system for studying barrier health of live tissues in real time

Epithelial cells create barriers that protect many different components in the body from their external environment. Increased gut barrier permeability (leaky gut) has been linked to several chronic inflammatory diseases. Understanding the cause of leaky gut and effective interventions are elusive due to the lack of tools that maintain tissue’s physiological environment while elucidating cellular functions under various stimuli ex vivo. Here we present a microphysiological system that records real-time barrier permeability of mouse colon in a physiological environment over extended durations. The system includes a microfluidic chamber; media composition that preserves microbiome and creates necessary oxygen gradients across the barrier; and integrated sensor electrodes for acquiring transepithelial electrical resistance (TEER). Our results demonstrate that the system can maintain tissue viability for up to 72 h. The TEER sensors can distinguish levels of barrier permeability when treated with collagenase and low pH media and detect different thickness in the tissue explant.

Adipo-on-chip: a microphysiological system to culture human mesenchymal stem cells with improved adipogenic differentiation

Adipo-on-chip: a microphysiological system to culture human mesenchymal stem cells with improved adipogenic differentiation

Hepatotoxic assessment in a microphysiological system: Simulation of the drug absorption and toxic process after an overdosed acetaminophen on intestinal-liver-on-chip

To compensate the limitation of animal models, new models were proposed for drug safety evaluation to refine and reduce existing models. To mimic drug absorption and metabolism and predict toxicokinetic and toxic effects in an in vitro intestinal-liver microphysiological system (MPS), we constructed an intestinal-liver-on-chip and detected the acute liver injury process after an overdose of acetaminophen (APAP). Caco-2 and HT29-MTX-E12 cell lines were utilized to establish intestinal equivalents, along with HepG2, HUVEC-T1, and THP-1 induced by PMA and human hepatic stellate cell to establish liver equivalents. The APAP concentration was determined using high-performance liquid chromatography, and the toxicokinetic parameters were fitted using the non-compartmental analysis method by Phoenix. Changes in liver injury biomarkers aspartate aminotransferase and alanine aminotransferase, and liver function marker albumin indicated that the short-term culture of the two organs-on-chip model was stable for 4 days. Reactive oxygen species signaling was enhanced after APAP administration, along with decreased mitochondrial membrane potential, activated caspase-3, and enhanced p53 signaling, indicating a toxic response induced by APAP overdose. In the gut-liver MPS model, we fitted the toxicokinetic parameters and simulated the hepatotoxicity procedure following an APAP overdose, which will facilitate the organ-on-chips application in drug toxicity assays.

Improved functionality of hepatic spheroids cultured in acoustic levitation compared to existing 2D and 3D models

Hepatic spheroids are of high interest in basic research, drug discovery and cell therapy. Existing methods for spheroid culture present advantages and drawbacks. An alternative technology is explored: the hepatic spheroid formation and culture in an acoustofluidic chip, using HepaRG cell line. Spheroid formation and morphology, cell viability, genetic stability, and hepatic functions are analyzed after 6 days of culture in acoustic levitation. They are compared to 2D culture and non-levitated 3D cultures. Sizes of the 25 spheroids created in a single acoustofluidic microphysiological system are homogeneous. The acoustic parameters in our system do not induce cell mortality nor DNA damage. Spheroids are cohesive and dense. From a functional point of view, hepatic spheroids obtained by acoustic levitation exhibit polarity markers, secrete albumin and express hepatic genes at higher levels compared to 2D and low attachment 3D cultures. In conclusion, this microphysiological system proves not only to be suitable for long-term culture of hepatic spheroids, but also to favor differentiation and functionality within 6 days of culture.

Facile photopatterning of perfusable microchannels in hydrogels for microphysiological systems.

Perfusable hydrogels have garnered substantial attention in recent years for the fabrication of microphysiological systems. However, current methodologies to fabricate microchannels in hydrogel platforms involve sophisticated equipment and techniques, which hinder progress of the field. In this protocol, we present a cost-effective, simple, versatile and ultrafast method to create perfusable microchannels of complex shapes in photopolymerizable hydrogels. Our method uses one-step UV photocross-linking and a photomask printed on inexpensive transparent films, to photopattern both synthetic (PEG-norbornene) and natural (hyaluronic acid-norbornene) hydrogels in just 0.8 s. Moreover, these perfusable hydrogels are fully integrated into a custom-made microfluidic device that allows continuous fluid perfusion when connected to an external pump system. This methodology can be easily reproduced by professionals with basic laboratory skills and a fundamental knowledge of polymers and materials science. In this protocol, we demonstrate the functionality of our photopatterned hydrogels by seeding human endothelial cells into the microchannels, culturing them under dynamic conditions for 7 d, and exposing them to inflammatory stimuli to elicit cellular responses. This highlights the versatility of our platform in fabricating microphysiological systems and different microenvironments. The fabrication of perfusable channels within the hydrogels, including the fabrication of the microfluidic devices, requires ~3 d. The development of the cell-seeded microphysiological system, including the stimulation of cells, takes ~7 d. In conclusion, our approach provides a straightforward and widely applicable solution to simplify and reduce the cost of biofabrication techniques for developing functional in vitro models using perfusable three-dimensional hydrogels.

Adipocyte inflammation is the primary driver of hepatic insulin resistance in a human iPSC-based microphysiological system

Interactions between adipose tissue, liver and immune system are at the center of metabolic dysfunction-associated steatotic liver disease and type 2 diabetes. To address the need for an accurate in vitro model, we establish an interconnected microphysiological system (MPS) containing white adipocytes, hepatocytes and proinflammatory macrophages derived from isogenic human induced pluripotent stem cells. Using this MPS, we find that increasing the adipocyte-to-hepatocyte ratio moderately affects hepatocyte function, whereas macrophage-induced adipocyte inflammation causes lipid accumulation in hepatocytes and MPS-wide insulin resistance, corresponding to initiation of metabolic dysfunction-associated steatotic liver disease. We also use our MPS to identify and characterize pharmacological intervention strategies for hepatic steatosis and systemic insulin resistance and find that the glucagon-like peptide-1 receptor agonist semaglutide improves hepatocyte function by acting specifically on adipocytes. These results establish our MPS modeling the adipose tissue-liver axis as an alternative to animal models for mechanistic studies or drug discovery in metabolic diseases.

Construction of Microphysiological System by Integration of Cells and Microdevices

Construction of Microphysiological System by Integration of Cells and Microdevices

A Microphysiological System with an Anaerobic Air-Liquid Interface and Functional Mucus Layer for Coculture of Intestinal Bacteria and Primary Human Colonic Epithelium.

Coculture of intestinal bacteria with primary human intestinal epithelium provides a valuable tool for investigating host-colon bacterial interactions and for testing and screening therapeutics. However, most current intestinal model systems lack key physiological features of the in vivo colon, such as both a proper oxygen microenvironment and a mucus layer. In this work, a new in vitro colonic microphysiological system is demonstrated with a cell-derived, functional mucus that closely resembles the in vivo colonic mucosa and apical microenvironment by employing an anaerobic air-liquid interface culture. The human primary colon epithelial cells in this new in vitro system exhibit high cell viability (>98%) with ≈100 μm thick functional mucus layer on average. Successful coculture of model anaerobic gut bacterial strains Lactobacillus rhamnosus GG and Anaerobutyricum hallii without loss in human cell viability demonstrates that this new model can be an invaluable tool for future studies of the impact of commensal and pathogenic bacteria on the colon.

A Pumpless, High‐Throughput Microphysiological System to Mimic Enteric Innervation of Duodenal Epithelium and the Impact on Barrier Function

AbstractEnteric neurons are critical in maintaining organ homeostasis within the small intestine, and their dysregulation are implicated in gastrointestinal disorders and neurodegenerative diseases. Most in vitro models lack enteric innervation, limiting basic discovery and disease modeling research. Here, a high‐throughput 3D microphysiological system (MPS), or organ chip is presented that supports a primary epithelial monolayer interfacing directly with encapsulated primary enteric neurons. The device features twelve 3D MPSs per device and gravity‐driven flow via a laboratory rocker to induce biomimetic shear stress on the epithelium culture and provide continuous nutrient presentation. Intestinal and neural tissue exhibited expected morphologies. Neural gene upregulation in the epithelium suggests RNA contamination from proximal enteric neurons extending neurites toward the epithelial monolayer. With the enteric nervous system (ENS), barrier integrity significantly increased for both TEER and permeability assays, a 1.25‐fold greater resistance and 10% lower permeability as compared to epithelium cultured alone. The presence of the ENS resulted in a significant (1.4‐fold) reduction in epidermal growth factor (EGF). Additionally, several key epithelial genes are compared between duodenal tissue and epithelial monolayers with and without neurons present. Results demonstrated changes in cytokine gene expression and WNT pathways, highlighting innervation is essential to create more biomimetic and physiologically relevant in vitro models.

Evaluating nephrotoxicity reduction in a novel polymyxin B formulation: insights from a 3D kidney-on-a-chip model.

This study aimed to assess the nephrotoxicity associated with VRP-034 (novel formulation of polymyxin B [PMB]) compared to marketed PMB in a three-dimensional (3D) kidney-on-a-chip model. To model the human kidney proximal tubule for analysis, tubular structures were established using 23 triple-channel chips seeded with RPTEC/hTERT1 cells. These cells were exposed to VRP-034 or PMB at seven concentrations (1-200 µM) over 12, 24, and 48 h. A suite of novel kidney injury biomarkers, cell health, and inflammatory markers were quantitatively assessed in the effluent. Additionally, caspase and cytochrome C levels were measured, and cell viability was evaluated using calcein AM and ethidium homodimer-1 (EthD-1). Exposure to marketed PMB resulted in significantly elevated levels (P < 0.05) of four key biomarkers (KIM-1, cystatin C, clusterin, and OPN) compared to VRP-034, particularly at clinically relevant concentrations of ≥10 µM. At 25 µM, all biomarkers demonstrated a significant increase (P < 0.05) with marketed PMB exposure compared to VRP-034. Inflammatory markers (interleukin-6 and interleukin-8) increased significantly (P < 0.05) with marketed PMB at concentrations of ≥5 µM, relative to VRP-034. VRP-034 displayed superior cell health outcomes, exhibiting lower lactate dehydrogenase release, while ATP levels remained comparable. Morphological analysis revealed that marketed PMB induced more severe damage, disrupting tubular integrity. Both treatments activated cytochrome C, caspase-3, caspase-8, caspase-9, and caspase-12 in a concentration-dependent manner; however, caspase activation was significantly reduced (P < 0.05) with VRP-034. This study demonstrates that VRP-034 significantly reduces nephrotoxicity compared to marketed PMB within a 3D microphysiological system, suggesting its potential to enable the use of full therapeutic doses of PMB with an improved safety profile, addressing the need for less nephrotoxic polymyxin antibiotics.

P04-09 Development of a microphysiological system to quantify the liver fibrosis AOP in vitro

P04-09 Development of a microphysiological system to quantify the liver fibrosis AOP in vitro

S21-04 A microphysiological system of the liver that recapitulates the cellular composition and organization of the hepatic lobule

S21-04 A microphysiological system of the liver that recapitulates the cellular composition and organization of the hepatic lobule

Paracrine Activation of STAT3 Drives GM-CSF Expression in Breast Carcinoma Cells, Generating a Symbiotic Signaling Network with Breast Carcinoma-Associated Fibroblasts.

This study evaluated the paracrine signaling between breast carcinoma-associated fibroblasts (CAFs) and breast cancer (BCa) cells. Resolving cell-cell communication in the BCa tumor microenvironment (TME) will aid the development of new therapeutics. Here, we utilized our patented TAME (tissue architecture and microenvironment engineering) 3D culture microphysiological system, which is a suitable pathomimetic avatar for the study of the BCa TME. We cultured in 3D BCa cells and CAFs either alone or together in cocultures and found that when cocultured, CAFs enhanced the invasive characteristics of tumor cells, as shown by increased proliferation and spread of tumor cells into the surrounding matrix. Secretome analysis from 3D cultures revealed a relatively high secretion of IL-6 by CAFs. A marked increase in the secretion of granulocyte macrophage-colony stimulating factor (GM-CSF) when carcinoma cells and CAFs were in coculture was also observed. We theorized that the CAF-secreted IL-6 functions in a paracrine manner to induce GM-CSF expression and secretion from carcinoma cells. This was confirmed by evaluating the activation of STAT3 and gene expression of GM-CSF in carcinoma cells exposed to CAF-conditioned media (CAF-CM). In addition, the treatment of CAFs with BCa cell-CM yielded a brief upregulation of GM-CSF followed by a marked decrease, indicating a tightly regulated control of GM-CSF in CAFs. Secretion of IL-6 from CAFs drives the activation of STAT3 in BCa cells, which in turn drives the expression and secretion of GM-CSF. As a result, CAFs exposed to BCa cell-secreted GM-CSF upregulate inflammation-associated genes such as IL-6, IL-6R and IL-8, thereby forming a positive feedback loop. We propose that the tight regulation of GM-CSF in CAFs may be a novel regulatory pathway to target for disrupting the CAF:BCa cell symbiotic relationship. These data provide yet another piece of the cell-cell communication network governing the BCa TME.

Fast formation and maturation enhancement of human liver organoids using a liver-organoid-on-a-chip.

Background: Spatial and functional hepatic zonation, established by the heterogeneous tissue along the portal-central axis of the liver, is important for ensuring optimal liver function. Researchers have attempted to develop reliable hepatic models to mimic the liver microenvironment and analyze liver function using hepatocytes cultured in the developed systems. However, mimicking the liver microenvironment in vitro remains a great challenge owing to the lack of perfusable vascular networks in the model systems and the limitation in maintaining hepatocyte function over time. Methods: In this study, we established a microphysiological system that operated under continuous flush medium flow, thereby allowing the supply of nutrients and oxygen to liver organoids and the removal of waste and release of cytokines therefrom, similar to the function of blood vessels. Results: The application of microphysiological system to organoid culture was advantageous for reducing the differentiation time and enhancing the functional maturity of human liver organoid. Conclusion: Hence, our microphysiological culture system might open the possibility of the miniaturized liver model system into a single device to enable more rational in vitro assays of liver response.