bims-orenst Biomed News
on Organs-on-chips and engineered stem cell models
Issue of 2021–05–16
seven papers selected by
Joram Mooiweer, University of Groningen



  1. Nat Commun. 2021 May 14. 12(1): 2805
      Engineered bacteria (synthetic biotics) represent a new class of therapeutics that leverage the tools of synthetic biology. Translational testing strategies are required to predict synthetic biotic function in the human body. Gut-on-a-chip microfluidics technology presents an opportunity to characterize strain function within a simulated human gastrointestinal tract. Here, we apply a human gut-chip model and a synthetic biotic designed for the treatment of phenylketonuria to demonstrate dose-dependent production of a strain-specific biomarker, to describe human tissue responses to the engineered strain, and to show reduced blood phenylalanine accumulation after administration of the engineered strain. Lastly, we show how in vitro gut-chip models can be used to construct mechanistic models of strain activity and recapitulate the behavior of the engineered strain in a non-human primate model. These data demonstrate that gut-chip models, together with mechanistic models, provide a framework to predict the function of candidate strains in vivo.
    DOI:  https://doi.org/10.1038/s41467-021-23072-5
  2. Sci Rep. 2021 May 14. 11(1): 10327
      Flow conditions have been shown to be important in improving longevity and functionality of primary hepatocytes, but the impact of flow on HepaRG cells is largely unknown. We studied the expression of genes encoding CYP enzymes and transporter proteins and CYP1 and CYP3A4 activity during 8 weeks of culture in HepaRG cells cultured under static conditions (conventional 24-/96-well plate culture with common bicarbonate/CO2 buffering) and under flow conditions in an organ-on-chip (OOC) device. Since the OOC-device is a closed system, bicarbonate/CO2 buffering was not possible, requiring application of another buffering agent, such as HEPES. In order to disentangle the effects of HEPES from the effects of flow, we also applied HEPES-supplemented medium in static cultures and studied gene expression and CYP activity. We found that cells cultured under flow conditions in the OOC-device, as well as cells cultured under static conditions with HEPES-supplemented medium, showed more stable gene expression levels. Furthermore, only cells cultured in the OOC-device showed relatively high baseline CYP1 activity, and their gene expression levels of selected CYPs and transporters were most similar to gene expression levels in human primary hepatocytes. However, there was a decrease in baseline CYP3A4 activity under flow conditions compared to HepaRG cells cultured under static conditions. Altogether, the present study shows that HepaRG cells cultured in the OOC-device were more stable than in static cultures, being a promising in vitro model to study hepatoxicity of chemicals upon chronic exposure.
    DOI:  https://doi.org/10.1038/s41598-021-89710-6
  3. Lab Chip. 2021 May 10.
      Glioblastoma multiforme (GBM) is the most common and the most aggressive type of primary brain malignancy. Glioblastoma stem-like cells (GSCs) can migrate in vascular niches within or away from the tumour mass, increasing tumour resistance to treatments and contributing to relapses. To study individual GSC migration and their interactions with the perivasculature of the tumour microenvironment, there is a need to develop a human organotypic in vitro model. Herein, we demonstrated a perivascular niche-on-a-chip, in a serum-free condition with gravity-driven flow, that supported the stemness of patient-derived GSCs and foetal neural stem cells grown in a three-dimensional environment (3D). Endothelial cells from three organ origins, (i) human brain microvascular endothelial cells (hCMEC/D3), (ii) human umbilical vein endothelial cells (HUVECs) and, (iii) human lung microvascular endothelial cells (HMVEC-L) formed rounded microvessels within the extracellular-matrix integrated microfluidic chip. By optimising cell extraction protocols, systematic studies were performed to evaluate the effects of serum-free media, 3D cell cultures, and the application of gravity-driven flow on the characteristics of endothelial cells and their co-culture with GSCs. Our results showed the maintenance of adherent and tight junction markers of hCMEC/D3 in the serum-free culture and that gravity-driven flow was essential to support adequate viability of both the microvessel and the GSCs in co-culture (>80% viability at day 3). Endpoint biological assays showed upregulation of neovascularization-related genes (e.g., angiopoietins, vascular endothelial growth factor receptors) in endothelial cells co-cultured with GSCs in contrast to the neural stem cell reference that showed insignificant changes. The on-chip platform further permitted live-cell imaging of GSC - microvessel interaction, enabling quantitative analysis of GSC polarization and migration. Overall, our comparative genotypic (i.e. qPCR) and phenotypic (i.e. vessel permeability and GSC migration) studies showed that organotypic (brain cancer cells-brain endothelial microvessel) interactions differed from those within non-tissue specific vascular niches of human origin. The development and optimization of this on-chip perivascular niche, in a serum-free flowable culture, could provide the next level of complexity of an in vitro system to study the influence of glioma stem cells on brain endothelium.
    DOI:  https://doi.org/10.1039/d1lc00271f
  4. Lab Chip. 2021 May 12.
      Mechanistic understanding of atherosclerosis is largely hampered by the lack of a suitable in vitro human arterial model that recapitulates the arterial wall structure, and the interplay between different cell types and the surrounding extracellular matrix (ECM). This work introduces a novel microfluidic endothelial cell (EC)-smooth muscle cell (SMC) 3D co-culture platform that replicates the structural and biological aspects of the human arterial wall for modeling early atherosclerosis. Using a modified surface tension-based ECM patterning method, we established a well-defined intima-media-like structure, and identified an ECM composition (collagen I and Matrigel mixture) that retains the SMCs in a quiescent and aligned state, characteristic of a healthy artery. Endothelial stimulation with cytokines (IL-1β and TNFα) and oxidized low-density lipoprotein (oxLDL) was performed on-chip to study various early atherogenic events including endothelial inflammation (ICAM-1 expression), EC/SMC oxLDL uptake, SMC migration, and monocyte-EC adhesion. As a proof-of-concept for drug screening applications, we demonstrated the atheroprotective effects of vitamin D (1,25(OH)2D3) and metformin in mitigating cytokine-induced monocyte-EC adhesion and SMC migration. Overall, the developed arterial wall model facilitates quantitative and multi-factorial studies of EC and SMC phenotype in an atherogenic environment, and can be readily used as a platform technology to reconstitute multi-layered ECM tissue biointerfaces.
    DOI:  https://doi.org/10.1039/d1lc00131k
  5. Biotechnol J. 2021 May 13. e2000355
      Cultured neuronal networks (CNNs) are a robust model to closely investigate neuronal circuits' formation and monitor their structural properties evolution. Typically, neurons are cultured in plastic plates or, more recently, in microfluidic platforms with potentially a wide variety of neuroscience applications. As a biological protocol, cell culture integration with a microfluidic system provides benefits such as accurate control of cell seeding area, culture medium renewal, or lower exposure to contamination. The objective of this report is to present a novel neuronal network on a chip device, including a chamber, fabricated from PDMS, vinyl and glass connected to a microfluidic platform to perfuse the continuous flow of culture medium. Network growth is compared in chips and traditional Petri dishes to validate the microfluidic chip performance. The network assessment is performed by computing relevant topological measures like the number of connected neurons, the clustering coefficient, and the shortest path between any pair of neurons throughout the culture's life. The results demonstrate that neuronal circuits on a chip have a more stable network structure and lifespan than developing in conventional settings, and therefore this setup is an advantageous alternative to current culture methods. This technology could lead to challenging applications such as batch drug testing of in vitro cell culture models. From the engineering perspective, a device's advantage is the chance to develop custom designs more efficiently than other microfluidic systems. This article is protected by copyright. All rights reserved.
    Keywords:  biochips; biological circuits; biomaterials; neuronal culture; neurons on a chip
    DOI:  https://doi.org/10.1002/biot.202000355
  6. FASEB J. 2021 Jun;35(6): e21643
      The endothelial glycocalyx is a dynamic structure integral to blood vessel hemodynamics and capable of tightly regulating a range of biological processes (ie, innate immunity, inflammation, and coagulation) through dynamic changes in its composition of the brush structure. Evaluating the specific roles of the endothelial glycocalyx under a range of pathophysiologic conditions has been a challenge in vitro as it is difficult to generate functional glycocalyces using commonly employed 2D cell culture models. We present a new multi-height microfluidic platform that promotes the growth of functional glycocalyces by eliciting unique shear stress forces over a continuous human umbilical vein endothelial cell monolayer at magnitudes that recapitulate the physical environment in arterial, capillary and venous regions of the vasculature. Following 72 hours of shear stress, unique glycocalyx structures formed within each region that were distinct from that observed in short (3 days) and long-term (21 days) static cell culture. The model demonstrated glycocalyx-specific properties that match the characteristics of the endothelium in arteries, capillaries and veins, with respect to surface protein expression, platelet adhesion, lymphocyte binding and nanoparticle uptake. With artery-to-capillary-to-vein transition on a continuous endothelial monolayer, this in vitro platform is an improved system over static cell culture for more effectively studying the role of the glycocalyx in endothelial biology and disease.
    Keywords:  blood; endothelial biology; mechanosensing; microfluidic; shear stress
    DOI:  https://doi.org/10.1096/fj.201802376RRRR
  7. Adv Sci (Weinh). 2021 05;8(9): 2003332
      Epithelial, stem-cell derived organoids are ideal building blocks for tissue engineering, however, scalable and shape-controlled bio-assembly of epithelial organoids into larger and anatomical structures is yet to be achieved. Here, a robust organoid engineering approach, Multi-Organoid Patterning and Fusion (MOrPF), is presented to assemble individual airway organoids of different sizes into upscaled, scaffold-free airway tubes with predefined shapes. Multi-Organoid Aggregates (MOAs) undergo accelerated fusion in a matrix-depleted, free-floating environment, possess a continuous lumen, and maintain prescribed shapes without an exogenous scaffold interface. MOAs in the floating culture exhibit a well-defined three-stage process of inter-organoid surface integration, luminal material clearance, and lumina connection. The observed shape stability of patterned MOAs is confirmed by theoretical modelling based on organoid morphology and the physical forces involved in organoid fusion. Immunofluorescent characterization shows that fused MOA tubes possess an unstratified epithelium consisting mainly of tracheal basal stem cells. By generating large, shape-controllable organ tubes, MOrPF enables upscaled organoid engineering towards integrated organoid devices and structurally complex organ tubes.
    Keywords:  bio‐assembly; organoids; respiratory system; tissue engineering; tissue morphogenesis
    DOI:  https://doi.org/10.1002/advs.202003332