bims-orenst Biomed News
on Organs-on-chips and engineered stem cell models
Issue of 2021‒02‒28
sixteen papers selected by
Joram Mooiweer
University of Groningen

  1. ACS Biomater Sci Eng. 2021 Feb 24.
      Organs-on-chips are microphysiological in vitro models of human organs and tissues that rely on culturing cells in a well-controlled microenvironment that has been engineered to include key physical and biochemical parameters. Some systems contain a single perfused microfluidic channel or a patterned hydrogel, whereas more complex devices typically employ two or more microchannels that are separated by a porous membrane, simulating the tissue interface found in many organ subunits. The membranes are typically made of synthetic and biologically inert materials that are then coated with extracellular matrix (ECM) molecules to enhance cell attachment. However, the majority of the material remains foreign and fails to recapitulate the native microenvironment of the barrier tissue. Here, we study microfluidic devices that integrate a vitrified membrane made of collagen-I hydrogel (VC). The biocompatibility of this membrane was confirmed by growing a healthy population of stem cell derived endothelial cells (iPSC-EC) and immortalized retinal pigment epithelium (ARPE-19) on it and assessing morphology by fluorescence microscopy. Moreover, VC membranes were subjected to biochemical degradation using collagenase II. The effects of this biochemical degradation were characterized by the permeability changes to fluorescein. Topographical changes on the VC membrane after enzymatic degradation were also analyzed using scanning electron microscopy. Altogether, we present a dynamically bioresponsive membrane integrated in an organ-on-chip device with which disease-related ECM remodeling can be studied.
    Keywords:  collagenase; organ-on-a-chip; permeability; vitrified collagen membrane
  2. Int J Biochem Cell Biol. 2021 Feb 23. pii: S1357-2725(21)00044-3. [Epub ahead of print] 105960
      Netrin-4, recognized in neural and vascular development, is highly expressed by mature endothelial cells. The function of this netrin-4 in vascular biology after development has remained unclear. We found that the expression of netrin-4 is highly regulated in endothelial cells and is important for quiescent healthy endothelium. Netrin-4 expression is upregulated in endothelial cells cultured under laminar flow conditions, while endothelial cells stimulated with tumor necrosis factor alpha resulted in decreased netrin-4 expression. Targeted reduction of netrin-4 in endothelial cells resulted in increased expression of vascular cell adhesion molecule 1 and intercellular adhesion molecule 1. Besides, these endothelial cells were more prone to monocyte adhesion and showed impaired barrier function, measured in electric cell-substrate impedance sensing system, as well as in an 'organ-on-a-chip' microfluidic system. Importantly, endothelial cells with reduced levels of netrin-4 showed increased expression of the senescence-associated markers cyclin-dependent kinase inhibitor-1 and -2A, an increased cell size and decreased ability to proliferate. Consistent with the gene expression profile, netrin-4 reduction was accompanied with more senescent associated β-galactosidase activity, which could be rescued by adding netrin-4 protein. Finally, using human decellularized kidney extracellular matrix scaffolds, we found that pre-treatment of the scaffolds with netrin-4 increased numbers of endothelial cells adhering to the matrix, showing a pro-survival effect of netrin-4. Taken together, netrin-4 acts as an anti-senescence and anti-inflammation factor in endothelial cell function and our results provide insights as to maintain endothelial homeostasis and supporting vascular health.
    Keywords:  barrier function; endothelial cells; inflammation; netrin-4; senescence
  3. Lab Chip. 2021 Feb 22.
      A microfluidics-based three-dimensional skin-on-chip (SoC) model is developed in this study to enable quantitative studies of transendothelial and transepithelial migration of human T lymphocytes in mimicked skin inflammatory microenvironments and to test new drug candidates. The keys results include 1) CCL20-dependent T cell transmigration is significantly inhibited by an engineered CCL20 locked dimer (CCL20LD), supporting the potential immunotherapeutic use of CCL20LD for treating skin diseases such as psoriasis; 2) transepithelial migration of T cells in response to a CXCL12 gradient mimicking T cell egress from the skin is significantly reduced by a sphingosine-1-phosphate (S1P) background, suggesting the role of S1P for T cell retention in inflamed skin tissues; and 3) T cell transmigration is induced by inflammatory cytokine stimulated epithelial cells in the SoC model. Collectively, the developed SoC model recreates a dynamic multi-cellular micro-environment that enables quantitative studies of T cell transmigration at a single cell level in response to physiological cutaneous inflammatory mediators and potential drugs.
  4. Macromol Biosci. 2021 Feb 24. e2100016
      Tissue models mimic the complex 3D structure of human tissues, which allows the study of pathologies and the development of new therapeutic strategies. The introduction of perfusion overcomes the diffusion limitation and enables the formation of larger tissue constructs. Furthermore, it provides the possibility to investigate the effects of hematogenously administered medications. In this study, the applicability of hydrophilic polytetrafluoroethylene (PTFE) membranes as vessel-like constructs for further use in perfused tissue models is evaluated. The presented approach allows the formation of stable and leakproof tubes with a mean diameter of 654.7 µm and a wall thickness of 84.2 µm. A polydimethylsiloxane (PDMS) chip acts as a perfusion bioreactor and provides sterile conditions. As proof of concept, endothelial cells adhere to the tube's wall, express vascular endothelial cadherin (VE-cadherin) between neighboring cells, and resist perfusion at a shear rate of 0.036 N m-2 for 48 h. Furthermore, the endothelial cell layer delays significantly the diffusion of fluorescently labeled molecules into the surrounding collagen matrix and leads to a twofold reduced diffusion velocity. This approach represents a cost-effective alternative to introduce stable vessel-like constructs into tissue models, which allows adapting the surrounding matrix to the tissue properties in vivo.
    Keywords:  PTFE; endothelial cells; perfusion bioreactor
  5. J Biotechnol. 2021 Feb 19. pii: S0168-1656(21)00057-2. [Epub ahead of print]
      The limited availability of primary human β-cells/islets and their quality (due to donor diversity) restrict the development of in vitro models for diabetes research. Human induced pluripotent stem cells (hiPSCs) may be a promising cell-source for diabetes studies, anti-diabetic drug screening and personalized therapies. However, achieving levels of maturity/functionality that are comparable to the in vivo situation and islets rebuilt from iPSCs is still challenging. Here, we compare and discuss two strategies for culturing human pancreatic β-cells derived from hiPSCs in microfluidic biochips. First, we confirmed that the protocol in conventional Petri 2D monolayer led to insulin, PDX1 and MAFA positive staining, to C-Peptide productive cells, and to tissue responsive to high/low glucose and GLP1 stimulation. This protocol and its subsequent modifications (including extracellular matrix coating, cell adhesion time, cell inoculation density, flow rate) was not successful in the 2D biochip culture. We proposed a second strategy using 3D spheroids created from honeycomb static cultures. Spheroids in static experiments carried out over 14 days demonstrated that they expressed high levels of β-cell markers (INS mRNA) and higher α-cell markers (GCG mRNA and glucagon positive staining), when compared to Petri 2D cultures. Furthermore, the 3D spheroids were specifically able to secrete insulin in response to both high/low glucose stimulation and GLP1 exposure. The spheroids were successfully inoculated into biochips and maintained for 10 days in perfusion. The 3D biochip cultures increased mRNA levels of GCG and maintained high levels of β-cell markers and responsiveness to both high/low glucose and GLP1 stimulation. Finally, C-peptide and insulin secretion were higher in biochips when compared to static spheroids. These results illustrate the promising potential for hiPSCs derived β-cells and their spheroid-based pancreas-on-chip model for pancreatic disease/diabetes modeling and anti-diabetic drug screening.
    Keywords:  3D spheroids; human induced pluripotent stem cells; microfluidic culture; β-pancreatic cells
  6. Biotechnol Bioeng. 2021 Feb 21.
      Previous in vitro studies interrogating the endothelial response to physiologically-relevant flow regimes require specialized pumps to deliver time-dependent waveforms that imitate in vivo blood flow. The aim of this study is to create a low-cost and broadly adaptable approach to mimic physiological flow, and then use this system to characterize the effect of flow separation on velocity and shear stress profiles in a 3D topology. The flow apparatus incorporates a programmable linear actuator that superposes oscillations on a constant mean flow driven by a peristaltic pump to emulate flow in the carotid artery. The flow is perfused through a three-dimensional in vitro model of the blood-brain barrier designed to induce separated flow. Experimental flow patterns measured by microparticle image velocimetry and modeled by computational fluid dynamics reveal periodic changes in the instantaneous shear stress along the channel wall. Moreover, the time-dependent flow causes periodic flow separation zones, resulting in variable reattachment points during the cycle. The effects of these complex flow regimes are assessed by evaluating the integrity of the in vitro blood-brain barrier model. Permeability assays and immunostaining for proteins associated with tight junctions reveal barrier breakdown in the region of disturbed flow. In conclusion, the flow system described here creates complex, physiologically-relevant flow profiles that provide deeper insight into the fluid dynamics of separated flow and pave the way for future studies interrogating the cellular response to complex flow regimes. This article is protected by copyright. All rights reserved.
    Keywords:  microfluidics; physiological flow; shear stress
  7. J Virol. 2021 Feb 26. pii: JVI.00110-21. [Epub ahead of print]
      Infections with SARS-CoV-2 can be asymptomatic, but they can also be accompanied by a variety of symptoms that result in mild to severe coronavirus disease-19 (COVID-19) and are sometimes associated with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including interferon expression. By contrast, the adjacent endothelial layer was neither infected nor did it show productive virus replication or interferon release. With prolonged infection, both cell types were damaged, and the barrier function was deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokines or components induced during infection, which further results in the damage of the epithelial/endothelial barrier function and viral dissemination.IMPORTANCESARS-CoV-2 challenges healthcare systems and societies worldwide in unprecedented ways. Although numerous new studies have been conducted, research to better understand the molecular pathogen-host interactions are urgently needed. For this, experimental models have to be developed and adapted. In the present study we used mono cell-culture systems and we established a complex chip model, where epithelial and endothelial cells are cultured in close proximity. We demonstrate that epithelial cells can be infected with SARS-CoV-2, while the endothelium did not show any infection signs. Since SARS-CoV-2 is able to establish viremia, the link to thromboembolic events in severe COVID-19 courses is evident. However, whether the endothelial layer is damaged by the viral pathogens or whether other endothelial-independent homeostatic factors are induced by the virus is essential for understanding the disease development. Therefore, our study is important as it demonstrates that the endothelial layer could not be infected by SARS-CoV-2 in our in vitro experiments, but we were able to show the destruction of the epithelial-endothelial barrier in our chip model. From our experiments we can assume that virus-induced host factors disturbed the epithelial-endothelial barrier function and thereby promote viral spread.
  8. Int J Pharm. 2021 Feb 20. pii: S0378-5173(21)00195-2. [Epub ahead of print] 120391
      The mucus covering of epithelial tissues presents one significant biological barrier to the uptake and absorption of particulate carriers. Improved understanding of the mechanisms mediating the transport of nanoparticles across such mucus layers would accelerate their development as optimised mucosal drug delivery formulations (e.g. via oral and rectal routes). Herein, an in vitro mucus model ("Mucus-on-Chip") was developed to enable the interaction and transport of functionalised nanoparticles and reconstituted mucus to be quantitatively investigated in real-time. We verified that the diffusion of nanoparticles into mucus is highly dependent on their biointerfacial properties. Muco-inert modification (PEGylation) significantly enhanced the mucopenetration of 50 nm and 200 nm nanoparticles, whereas limited mucopenetration was observed for pectin coated mucoadhesive nanoparticles. Furthermore, this model can be easily adapted to mimic specific physiological mucus environments. Mucus pre-treated with a mucolytic agent displayed reduced barrier function and therefore significantly accelerated mucopenetration of nanoparticles, which was independent of their size and biointerfacial properties. This new "Mucus-on-Chip" methodology provides detailed insight into the dynamics of nanoparticle-mucus interaction, which can be applied to refine the design of particulate formulations for more efficient mucosal drug delivery.
    Keywords:  Dynamic nanoparticle-mucus interaction; Microfluidic methodology; Mucosal drug delivery; Physicochemical properties; Versatility
  9. J Biomech. 2021 Feb 14. pii: S0021-9290(21)00110-X. [Epub ahead of print]119 110330
      Advances in microphysiological systems have prompted the need for long-term cell culture under physiological flow conditions. Conventional laboratory pumps typically lack the ability to deliver cell culture media at the low flow rates required to meet the physiological ranges of fluid flow, and are often pulsatile or require flow reversal. Here, a microfluidic-based pump is presented, which allows for the controlled delivery of media for vascular microphysiological applications. The performance of the pump was characterized in a range of microfluidic systems, including straight channels of varying dimensions and self-assembled microvascular networks. A theoretical framework was developed based on lumped element analysis to predict the performance of the pump for different fluidic configurations and a finite element model of the included check-valves. The use of the pump for microvascular physiological studies demonstrated the utility of this system to recapitulate vascular fluid transport phenomena in microphysiological systems, which may find applications in disease models and drug screening.
    Keywords:  Endothelial culture; Immune cell circulation; Lumped element modelling; Microvascular networks; Pulsatile flow
  10. Sci Transl Med. 2021 Feb 24. pii: eabd6299. [Epub ahead of print]13(582):
      The kidney plays a critical role in fluid homeostasis, glucose control, and drug excretion. Loss of kidney function due to drug-induced nephrotoxicity affects over 20% of the adult population. The kidney proximal tubule is a complex vascularized structure that is particularly vulnerable to drug-induced nephrotoxicity. Here, we introduce a model of vascularized human kidney spheroids with integrated tissue-embedded microsensors for oxygen, glucose, lactate, and glutamine, providing real-time assessment of cellular metabolism. Our model shows that both the immunosuppressive drug cyclosporine and the anticancer drug cisplatin disrupt proximal tubule polarity at subtoxic concentrations, leading to glucose accumulation and lipotoxicity. Impeding glucose reabsorption using glucose transport inhibitors blocked cyclosporine and cisplatin toxicity by 1000- to 3-fold, respectively. Retrospective study of 247 patients who were diagnosed with kidney damage receiving cyclosporine or cisplatin in combination with the sodium-glucose cotransporter-2 (SGLT2) inhibitor empagliflozin showed significant (P < 0.001) improvement of kidney function, as well as reduction in creatinine and uric acid, markers of kidney damage. These results demonstrate the potential of sensor-integrated kidney-on-chip platforms to elucidate mechanisms of action and rapidly reformulate effective therapeutic solutions, increasing drug safety and reducing the cost of clinical and commercial failures.
  11. Sci Rep. 2021 Feb 26. 11(1): 4747
      Atherosclerosis is a complex process involving progressive pathological events, including monocyte adhesion to the luminal endothelial surface. We have developed a functional in vitro adhesion assay using BioFlux microfluidic technology to investigate THP-1 (human acute monocytic leukaemia cell) monocyte adhesion to human aortic endothelial cells (HAECs). The effect of whole smoke conditioned media (WSCM) generated from University of Kentucky reference cigarette 3R4F, electronic cigarette vapour conditioned media (eVCM) from an electronic nicotine delivery system (ENDS) product (Vype ePen) and nicotine on monocyte adhesion to HAECs was evaluated. Endothelial monolayers were grown in microfluidic channels and exposed to 0-1500 ng/mL nicotine or nicotine equivalence of WSCM or eVCM for 24 h. Activated THP-1 cells were perfused through the channels and a perfusion, adhesion period and wash cycle performed four times with increasing adhesion period lengths (10, 20, 30 and 40 min). THP-1 cell adhesion was quantified by counting adherent cells. WSCM induced dose-dependent increases in monocyte adhesion compared to vehicle control. No such increases were observed for eVCM or nicotine. Adhesion regulation was linked to increased ICAM-1 protein expression. Staining of ICAM-1 in HAECs and CD11b (MAC-1) in THP-1 cells demonstrated adhesion molecule co-localisation in BioFlux plates. The ICAM-1 adhesion response to WSCM was downregulated by transfecting HAECs with ICAM-1 siRNA. We conclude that the BioFlux system is able to model human monocyte adhesion to primary human endothelial cells in vitro and WSCM drives the greatest increase in monocyte adhesion via a mechanism involving endothelial ICAM-1 expression.
  12. Front Bioeng Biotechnol. 2021 ;9 616830
      Evolution has endowed the lung with exceptional design providing a large surface area for gas exchange area (ca. 100 m2) in a relatively small tissue volume (ca. 6 L). This is possible due to a complex tissue architecture that has resulted in one of the most challenging organs to be recreated in the lab. The need for realistic and robust in vitro lung models becomes even more evident as causal therapies, especially for chronic respiratory diseases, are lacking. Here, we describe the Cyclic I n VI tro Cell-stretch (CIVIC) "breathing" lung bioreactor for pulmonary epithelial cells at the air-liquid interface (ALI) experiencing cyclic stretch while monitoring stretch-related parameters (amplitude, frequency, and membrane elastic modulus) under real-time conditions. The previously described biomimetic copolymeric BETA membrane (5 μm thick, bioactive, porous, and elastic) was attempted to be improved for even more biomimetic permeability, elasticity (elastic modulus and stretchability), and bioactivity by changing its chemical composition. This biphasic membrane supports both the initial formation of a tight monolayer of pulmonary epithelial cells (A549 and 16HBE14o-) under submerged conditions and the subsequent cell-stretch experiments at the ALI without preconditioning of the membrane. The newly manufactured versions of the BETA membrane did not improve the characteristics of the previously determined optimum BETA membrane (9.35% PCL and 6.34% gelatin [w/v solvent]). Hence, the optimum BETA membrane was used to investigate quantitatively the role of physiologic cyclic mechanical stretch (10% linear stretch; 0.33 Hz: light exercise conditions) on size-dependent cellular uptake and transepithelial transport of nanoparticles (100 nm) and microparticles (1,000 nm) for alveolar epithelial cells (A549) under ALI conditions. Our results show that physiologic stretch enhances cellular uptake of 100 nm nanoparticles across the epithelial cell barrier, but the barrier becomes permeable for both nano- and micron-sized particles (100 and 1,000 nm). This suggests that currently used static in vitro assays may underestimate cellular uptake and transbarrier transport of nanoparticles in the lung.
    Keywords:  ALI culture; bioinspired membrane; cyclic stretch; lung cell model; particle study
  13. Nature. 2021 Feb 24.
      The small intestine is the main organ for nutrient absorption, and its extensive resection leads to malabsorption and wasting conditions referred to as short bowel syndrome (SBS). Organoid technology enables an efficient expansion of intestinal epithelium tissue in vitro1, but reconstruction of the whole small intestine, including the complex lymphovascular system, has remained challenging2. Here we generate a functional small intestinalized colon (SIC) by replacing the native colonic epithelium with ileum-derived organoids. We first find that xenotransplanted human ileum organoids maintain their regional identity and form nascent villus structures in the mouse colon. In vitro culture of an organoid monolayer further reveals an essential role for luminal mechanistic flow in the formation of villi. We then develop a rat SIC model by repositioning the SIC at the ileocaecal junction, where the epithelium is exposed to a constant luminal stream of intestinal juice. This anatomical relocation provides the SIC with organ structures of the small intestine, including intact vasculature and innervation, villous structures, and the lacteal (a fat-absorbing lymphatic structure specific to the small intestine). The SIC has absorptive functions and markedly ameliorates intestinal failure in a rat model of SBS, whereas transplantation of colon organoids instead of ileum organoids invariably leads to mortality. These data provide a proof of principle for the use of intestinal organoids for regenerative purposes, and offer a feasible strategy for SBS treatment.
  14. Front Cell Dev Biol. 2021 ;9 621442
      Paracrine signaling in the tissue microenvironment is a central mediator of morphogenesis, and modeling this dynamic intercellular activity in vitro is critical to understanding normal and abnormal development. For example, Sonic Hedgehog (Shh) signaling is a conserved mechanism involved in multiple developmental processes and strongly linked to human birth defects including orofacial clefts of the lip and palate. SHH ligand produced, processed, and secreted from the epithelial ectoderm is shuttled through the extracellular matrix where it binds mesenchymal receptors, establishing a gradient of transcriptional response that drives orofacial morphogenesis. In humans, complex interactions of genetic predispositions and environmental insults acting on diverse molecular targets are thought to underlie orofacial cleft etiology. Consequently, there is a need for tractable in vitro approaches that model this complex cellular and environmental interplay and are sensitive to disruption across the multistep signaling cascade. We developed a microplate-based device that supports an epithelium directly overlaid onto an extracellular matrix-embedded mesenchyme, mimicking the basic tissue architecture of developing orofacial tissues. SHH ligand produced from the epithelium generated a gradient of SHH-driven transcription in the adjacent mesenchyme, recapitulating the gradient of pathway activity observed in vivo. Shh pathway activation was antagonized by small molecule inhibitors of epithelial secretory, extracellular matrix transport, and mesenchymal sensing targets, supporting the use of this approach in high-content chemical screening of the complete Shh pathway. Together, these findings demonstrate a novel and practical microphysiological model with broad utility for investigating epithelial-mesenchymal interactions and environmental signaling disruptions in development.
    Keywords:  3D extracellular matrix; chemical screening; cleft lip and palate; embryonic morphogenesis; epithelial mesenchymal cross-talk; gene environment interaction; paracrine signaling; signaling gradient
  15. Nat Rev Mater. 2021 Feb 19. 1-19
      Organoids are in vitro miniaturized and simplified model systems of organs that have gained enormous interest for modelling tissue development and disease, and for personalized medicine, drug screening and cell therapy. Despite considerable success in culturing physiologically relevant organoids, challenges remain to achieve real-life applications. In particular, the high variability of self-organizing growth and restricted experimental and analytical access hamper the translatability of organoid systems. In this Review, we argue that many limitations of traditional organoid culture can be addressed by engineering approaches at all levels of organoid systems. We investigate cell surface and genetic engineering approaches, and discuss stem cell niche engineering based on the design of matrices that allow spatiotemporal control of organoid growth and shape-guided morphogenesis. We examine how microfluidic approaches and lessons learnt from organs-on-a-chip enable the integration of mechano-physiological parameters and increase accessibility of organoids to improve functional readouts. Applying engineering principles to organoids increases reproducibility and provides experimental control, which will, ultimately, be required to enable clinical translation.
    Keywords:  Morphogenesis; Organogenesis; Stem cells; Tissue engineering
  16. Biotechnol Bioeng. 2021 Feb 25.
      Microfluidic-based technologies enable the development of cell culture systems that provide tailored microenvironmental inputs to mammalian cells. Primary myoblasts can be induced to differentiate into multinucleated skeletal muscle cells, myotubes, which are a relevant model system for investigating skeletal muscle metabolism and physiology in vitro. However, it remains challenging to differentiate primary myoblasts into mature myotubes in microfluidics devices. Here we investigated the effects of integrating continuous (solid) and intermittent (dashed) walls in microfluidic channels as topological constraints in devices designed to promote the alignment and maturation of primary myoblast-derived myotubes. The topological constraints caused alignment of the differentiated myotubes, mimicking the native anisotropic organization of skeletal muscle cells. Interestingly, dashed walls facilitated the maturation of skeletal muscle cells, as measured by quantifying myotube cell area and number of nuclei per myotube. Together, our results suggest that integrating dashed walls as topographic constraints in microfluidic devices supports the alignment and maturation of primary myoblast-derived myotubes. This article is protected by copyright. All rights reserved.
    Keywords:  alignment; maturation; multinucleated myotubes; primary myoblasts; topological constraints