Journal: Advanced Biosystems

Abbreviation

Adv Biosyst

Publisher

Wiley-VCH

Journal Volumes

ISSN

2366-7478

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2018 - 202010
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Publications 1 - 10 of 10
  • Calcium-Mediated Liposome Fusion to Engineer Giant Lipid Vesicles with Cytosolic Proteins and Reconstituted Mammalian Proteins
    Item type: Journal Article
    Schmid, Yannick R.F.; Scheller, Leo; Buchmann, Sebastian; et al. (2020)
    Advanced Biosystems
    Giant unilamellar lipid vesicles (GUVs) are widely used as model membrane systems and provide an excellent basis to construct artificial cells. To construct more sophisticated artificial cells, proteins-in particular membrane proteins-need to be incorporated in GUVs. However, current methods for protein reconstitution have limited throughput or are not generally applicable for all proteins because they depend on detergent solubilization. This limitation is addressed here by introducing calcium-mediated membrane fusion to transfer proteins between negatively charged GUVs and cell-derived plasma membrane vesicles (CDVs), derived from HEK293T cells overexpressing a membrane receptor protein. Fusion conditions are optimized using large unilamellar vesicles and GUVs containing phosphatidylserines and fusogenic lipids. The approach is then applied to induce lipid mixing and subsequent transfer of the overexpressed membrane receptor from CDVs into GUVs. The membrane receptor is detected by immunofluorescence on GUVs that underwent lipid mixing with CDVs. Those GUVs also exhibit esterase activity because cytosolic esterases entrapped in the CDVs are transferred during membrane fusion. Thus, content mixing is demonstrated. Using CDVs circumvents the need to purify or solubilize proteins. Moreover, calcium-mediated fusion allows transfer of lipids, water-soluble and membrane bound proteins in one step, resulting in a semi-synthetic cell.
  • Cell-Derived Vesicles as TRPC1 Channel Delivery Systems for the Recovery of Cellular Respiratory and Proliferative Capacities
    Item type: Journal Article
    Kurth, Felix; Tai, Yee K.; Parate, Dinesh; et al. (2020)
    Advanced Biosystems
    Pulsed electromagnetic fields (PEMFs) are capable of specifically activating a TRPC1‐mitochondrial axis underlying cell expansion and mitohormetic survival adaptations. This study characterizes cell‐derived vesicles (CDVs) generated from C2C12 murine myoblasts and shows that they are equipped with the sufficient molecular machinery to confer mitochondrial respiratory capacity and associated proliferative responses upon their fusion with recipient cells. CDVs derived from wild type C2C12 myoblasts include the cation‐permeable transient receptor potential (TRP) channels, TRPC1 and TRPA1, and directly respond to PEMF exposure with TRPC1‐mediated calcium entry. By contrast, CDVs derived from C2C12 muscle cells in which TRPC1 has been genetically knocked‐down using CRISPR/Cas9 genome editing, do not. Wild type C2C12‐derived CDVs are also capable of restoring PEMF‐induced proliferative and mitochondrial activation in two C2C12‐derived TRPC1 knockdown clonal cell lines in accordance to their endogenous degree of TRPC1 suppression. C2C12 wild type CDVs respond to menthol with calcium entry and accumulation, likewise verifying TRPA1 functional gating and further corroborating compartmental integrity. Proteomic and lipidomic analyses confirm the surface membrane origin of the CDVs providing an initial indication of the minimal cellular machinery required to recover mitochondrial function. CDVs hence possess the potential of restoring respiratory and proliferative capacities to senescent cells and tissues.
  • Integrated Microphysiological Systems: Transferable Organ Models and Recirculating Flow
    Item type: Review Article
    Renggli, Kasper; Rousset, Nassim; Lohasz, Christian; et al. (2019)
    Advanced Biosystems
    Studying and understanding of tissue and disease mechanisms largely depend on the availability of suitable and representative biological model systems. These model systems should be carefully engineered and faithfully reproduce the biological system of interest to understand physiological effects, pharmacokinetics, and toxicity to better identify new drug compounds. By relying on microfluidics, microphysiological systems (MPSs) enable the precise control of culturing conditions and connections of advanced in vitro 3D organ models that better reproduce in vivo environments. This review focuses on transferable in vitro organ models and integrated MPSs that host these transferable biological units and enable interactions between different tissue types. Interchangeable and transferrable in vitro organ models allow for independent quality control of the biological model before system assembly and building MPS assays on demand. Due to the complexity and different maturation times of individual in vitro tissues, off-chip production and quality control entail improved stability and reproducibility of the systems and results, which is important for large-scale adoption of the technology. Lastly, the technical and biological challenges and open issues for realizing and implementing integrated MPSs with transferable in vitro organ models are discussed.
  • In Vitro Platform for Studying Human Insulin Release Dynamics of Single Pancreatic Islet Microtissues at High Resolution
    Item type: Journal Article
    Misun, Patrick M.; Yesildag, Burçak; Forschler, Felix; et al. (2020)
    Advanced Biosystems
    Insulin is released from pancreatic islets in a biphasic and pulsatile manner in response to elevated glucose levels. This highly dynamic insulin release can be studied in vitro with islet perifusion assays. Herein, a novel platform to perform glucose-stimulated insulin secretion (GSIS) assays with single islets is presented for studying the dynamics of insulin release at high temporal resolution. A standardized human islet model is developed and a microfluidic hanging-drop-based perifusion system is engineered, which facilitates rapid glucose switching, minimal sample dilution, low analyte dispersion, and short sampling intervals. Human islet microtissues feature robust and long-term glucose responsiveness and demonstrate reproducible dynamic GSIS with a prominent first phase and a sustained, pulsatile second phase. Perifusion of single islet microtissues produces a higher peak secretion rate, higher secretion during the first and second phases of insulin release, as well as more defined pulsations during the second phase in comparison to perifusion of pooled islets. The developed platform enables to study compound effects on both phases of insulin secretion as shown with two classes of insulin secretagogs. It provides a new tool for studying physiologically relevant dynamic insulin secretion at comparably low sample-to-sample variation and high temporal resolution.
  • Predicting Metabolism‐Related Drug–Drug Interactions Using a Microphysiological Multitissue System
    Item type: Journal Article
    Lohasz, Christian; Bonanini, Flavio; Hoelting, Lisa; et al. (2020)
    Advanced Biosystems
    Drug–drug interactions (DDIs) occur when the pharmacological activity of one drug is altered by a second drug. As multimorbidity and polypharmacotherapy are becoming more common due to the increasing age of the population, the risk of DDIs is massively increasing. Therefore, in vitro testing methods are needed to capture such multiorgan events. Here, a scalable, gravity‐driven microfluidic system featuring 3D microtissues (MTs) that represent different organs for the prediction of drug–drug interactions is used. Human liver microtissues (hLiMTs) are combined with tumor microtissues (TuMTs) and treated with drug combinations that are known to cause DDIs in vivo. The testing system is able to capture and quantify DDIs upon co‐administration of the anticancer prodrugs cyclophosphamide or ifosfamide with the antiretroviral drug ritonavir. Dosage of ritonavir inhibits hepatic metabolization of the two prodrugs to different extents and decreases their efficacy in acting on TuMTs. The flexible MT compartment design of the system, the use of polystyrene as chip material, and the assembly of several chips in stackable plates offer the potential to significantly advance preclinical substance testing. The possibility of testing a broad variety of drug combinations to identify possible DDIs will improve the drug development process and increase patient safety. © 2020 Wiley‐VCH GmbH.
  • Microfluidic Hydrogel Hanging-Drop Network for Long-Term Culturing of 3D Microtissues and Simultaneous High-Resolution Imaging
    Item type: Journal Article
    Aeby, Elise A.; Misun, Patrick M.; Hierlemann, Andreas; et al. (2018)
    Advanced Biosystems
    hree-dimensional (3D) microtissues, cultured in microfluidic platforms, enable to study complex biological mechanisms that cannot be replicated in two-dimensional cell cultures. Deeper insights can be obtained if these 3D culture systems are rendered compatible with high-resolution time-lapse imaging systems, which requires precise placement and immobilization of the specimen while ensuring high viability and functionality of the 3D cell constructs. This article presents a versatile microfluidic platform for long-term culturing and analysis of 3D microtissues. The platform is compatible with time-lapse high-resolution confocal microscopy. Hanging hydrogel drops enable the precise placement and stable immobilization of the microtissues in the microfluidic chip. The chip includes perfusion capability to apply drugs, staining and clearing solutions. The features of the chip are demonstrated by studying (i) colon cancer microtissues to monitor tissue growth and cell death; on-chip clearing was used to augment the penetration depth for endpoint imaging; (ii) primary human liver microtissues were exposed to cytochalasin D to observe its effect on the bile canaliculi. The results obtained with both sample types demonstrate the suitability of the system for investigating complex processes in organotypic 3D microtissues, down to single-cell level, and for observation of physiologically relevant processes at subcellular scale.
  • The Axon Initial Segment is the Dominant Contributor to the Neuron's Extracellular Electrical Potential Landscape
    Item type: Journal Article
    Bakkum, Douglas J.; Obien, Marie E.J.; Radivojevic, Milos; et al. (2019)
    Advanced Biosystems
    Extracellular voltage fields, produced by a neuron's action potentials, provide a widely used means for studying neuronal and neuronal-network function. The neuron's soma and dendrites are thought to drive the extracellular action potential (EAP) landscape, while the axon's contribution is usually considered less important. However, by recording voltages of single neurons in dissociated rat cortical cultures and Purkinje cells in acute mouse cerebellar slices through hundreds of densely packed electrodes, it is found, instead, that the axon initial segment dominates the measured EAP landscape, and, surprisingly, the soma only contributes to a minor extent. As expected, the recorded dominant signal has negative polarity (charge entering the cell) and initiates at the distal end. Interestingly, signals with positive polarity (charge exiting the cell) occur near some but not all dendritic branches and occur after a delay. Such basic knowledge about which neuronal compartments contribute to the extracellular voltage landscape is important for interpreting results from all electrical readout schemes. Finally, initiation of the electrical activity at the distal end of the axon initial segment (AIS) and subsequent spreading into the axon proper and backward through the proximal AIS toward the soma are confirmed. The corresponding extracellular waveforms across different neuronal compartments could be tracked.
  • Parallelized Impedance-Based Platform for Continuous Dose-Response Characterization of Antischistosomal Drugs
    Item type: Journal Article
    Ravaynia, Paolo S.; Lombardo, Flavio C.; Biendl, Stefan; et al. (2020)
    Advanced Biosystems
    Schistosomiasis is an acute and chronic disease caused by tropical parasitic worms of the genus Schistosoma , which parasitizes annually over 200 million people worldwide. Screening of antischistosomal compounds is hampered by the low throughput and potential subjectivity of the visual evaluation of the parasite phenotypes, which affects the current drug assays. Here, an impedance‐based platform, capable of assessing the viability of Schistosoma mansoni schistosomula exposed to drugs, is presented. This automated and parallelized platform enables unbiased and continuous measurements of dose–response relationships for more than 48 h. The platform performance is established by exposure of schistosomula to three test compounds, praziquantel, oxethazaine, and mefloquine, which are known to affect the larvae phenotypes. The system is thereafter used to investigate the response of schistosomula to methiothepine, an antipsychotic compound, which causes complex drug‐induced effects. Continuous monitoring of the parasites reveals transient behavioral phenotypes and allows for extracting temporal characteristics of dose–response curves, which are essential for selecting drugs that feature high activity and fast kinetics of action. These measurements demonstrate that impedance‐based detection provides a wealth of information for the in vitro characterization of candidate antischistosomals and, represents a promising tool for the identification of new lead compounds.
  • Generation of 3D Soluble Signal Gradients in Cell-Laden Hydrogels Using Passive Diffusion
    Item type: Journal Article
    Ahrens, Lucas; Tanaka, Simon; Vonwil, Daniel; et al. (2019)
    Advanced Biosystems
    Soluble signal gradients play an important role in organ patterning, cell migration, and differentiation. Currently, signal gradients in 2D cell culture are realized using microfluidics and here cells are exposed to high and nonphysiological shear stress. Tissue morphogenesis (organogenesis) however occurs in 3D and therefore there is a need for simple and practical systems to impose gradients to cells dispersed in 3D matrix. Herein, a 3D gradient generator based on passive diffusion elements that recapitulates interstitial flow and is capable of imposing predictable gradients over long length scales (6 mm) lasting up to 48 h to cells dispersed in a hydrogel environment is reported. Using recombinant human WNT3A (rhWNT3A), the spatiotemporal activation of the canonical WNT pathway in human epithelial kidney cells and human mesenchymal stems cells expressing a green fluorescence protein reporter on a transcription factor/lymphoid enhancer-binding factor (TCF/LEF) promoter is demonstrated. By refining computation models based on experimental findings, the diffusion coefficient of rhWNT3A in presence of human cells in 3D is determined. Furthermore, the formation of rhBMP4 gradients is visualized using immunohistochemistry by staining for phospho-SMAD1/5, the downstream targets of the bone morphogenetic protein (BMP) pathway. The simplicity of the gradient generator is expected to spur its adoption in studying developmental biology paradigms in vitro.
  • Alginate Sulfate Substrates Control Growth Factor Binding and Growth of Primary Neurons: Toward Engineered 3D Neural Networks
    Item type: Journal Article
    Kfoury, Georges; El Habbaki, Vanessa; Malaeb, Waddah; et al. (2020)
    Advanced Biosystems
    Sulfated glycosaminoglycans (sGAGs) are vital molecules of the extracellular matrix (ECM) of the nervous system known to regulate proliferation, migration, and differentiation of neurons mainly through binding relevant growth factors. Alginate sulfate (AlgSulf) mimics sGAGs and binds growth factors such as basic fibroblast growth factor (FGF-2). Here, thin films of biotinylated AlgSulf (b-AlgSulfn) are engineered with sulfation degrees (DS = 0.0 and 2.7) and the effect of polysaccharide concentration on FGF-2 and nerve growth factor (β-NGF) binding and subsequent primary neural viability and neurite outgrowth is assessed. An increase in b-AlgSulfn concentration results in higher FGF-2 and β-NGF binding as demonstrated by greater frequency and dissipation shifts measured with quartz crystal microbalance with dissipation monitoring (QCM-D). Primary neurons seeded on the 2D b-AlgSulfn films maintain high viability comparable to positive controls grown on poly-d-lysine. Neurons grown in 3D AlgSulf hydrogels (DS = 0.8) exhibit a significantly higher viability, neurite numbers and mean branch length compared to neurons grown in nonsulfated controls. Finally, a first step is made toward constructing 3D neuronal networks by controllably patterning neurons encapsulated in AlgSulf into an alginate carrier. The substrates and neural networks developed in the current study can be used in basic and applied neural applications.
Publications 1 - 10 of 10