bims-orenst Biomed News
on Organs-on-chips and engineered stem cell models
Issue of 2021–04–18
ten papers selected by
Joram Mooiweer, University of Groningen
  1. Islet-on-a-chip: Biomimetic micropillar-based microfluidic system for three-dimensional pancreatic islet cell culture.
  2. High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia.
  3. Leukocyte transmigration efficiency and longitudinal forward-thrusting force in a microfluidic Transwell device.
  4. An engineered neurovascular unit for modeling neuroinflammation.
  5. Measuring barrier function in organ-on-chips with cleanroom-free integration of multiplexable electrodes.
  6. FleXert: A Soft, Actuatable Multiwell Plate Insert for Cell Culture under Stretch.
  7. Studying Tumor Angiogenesis and Cancer Invasion in a Three-Dimensional Vascularized Breast Cancer Micro-Environment.
  8. Elucidating molecular mechanisms of acquired resistance to BRAF inhibitors in melanoma using a microfluidic device and deep sequencing.
  9. Emulating Early Atherosclerosis in a Vascular Microphysiological System Using Branched Tissue-Engineered Blood Vessels.
  10. Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation.
  1. Biosens Bioelectron. 2021 Apr 02. pii: S0956-5663(21)00252-9. [Epub ahead of print]183 113215
    Islet-on-a-chip: Biomimetic micropillar-based microfluidic system for three-dimensional pancreatic islet cell culture.
    Patrycja Sokolowska, Kamil Zukowski, Justyna Janikiewicz, Elzbieta Jastrzebska, Agnieszka Dobrzyn, Zbigniew Brzozka.
      Type 2 diabetes is currently one of the most common metabolic diseases, affecting all ages worldwide. As the incidence of type 2 diabetes increases, a growing number of studies focus on islets of Langerhans. A three-dimensional research model that maps islet morphology and maintains hormonal balance in vivo is still needed. In this work, we present an Islet-on-a-chip system, specifically a micropillar-based microfluidic platform for three-dimensional pancreatic islet cell culture and analysis. The microfluidic system consisted of two culture chambers that were equipped with 15 circular microtraps each, which were built with seven round micropillars each. Micropillars in the structure of microtraps supported cell aggregation by limiting the growth surface and minimizing wall shear stress, thereby ensuring proper medium diffusion and optimal culture conditions for cell aggregates. Our system is compatible with microwell plate readers and confocal laser scanning microscopes. Because of optimization of the immunostaining method, the appropriate cell distribution and high viability and proliferation up to 72 h of culture were confirmed. Enzyme-linked immunosorbent assays were performed to measure insulin and glucagon secretion after stimulation with different glucose concentrations. To our knowledge, this is the first Lab-on-a-chip system which enables the formation and three-dimensional culture of cell aggregates composed of commercially available α and β pancreatic islet cells. The specific composition and arrangement of cells in the obtained model corresponds to the arrangement of the cells in rodent pancreatic islets in vivo. This Islet-on-a-chip system may be utilized to test pathogenic effectors and future therapeutic agents.
    Keywords:  Glucagon secretion; Insulin secretion; Islet-on-a-chip diabetes mellitus; Lab-on-a-chip system; Microfluidic system; Pancreatic islet; Three-dimensional cellular model
    DOI:  https://doi.org/10.1016/j.bios.2021.113215
  2. Lab Chip. 2021 Apr 15.
    High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia.
    A Nicolas, F Schavemaker, K Kosim, D Kurek, M Haarmans, M Bulst, K Lee, S Wegner, T Hankemeier, J Joore, K Domansky, H L Lanz, P Vulto, S J Trietsch.
      Assessment of epithelial barrier function is critically important for studying healthy and diseased biological models. Here we introduce an instrument that measures transepithelial electrical resistance (TEER) of perfused epithelial tubes in the microfluidic OrganoPlate platform. The tubules are grown in microfluidic channels directly against an extracellular matrix, obviating the need for artificial filter membranes. We present TEER measurements on Caco-2 intestinal and renal proximal tubule epithelium. Forty tubules on one single plate were interrogated in less than a minute. We show that TEER measurement is significantly more sensitive than a fluorescent reporter leakage assay in response to staurosporine. We demonstrate a 40-channel time-lapse data acquisition over a 25 hour time period under flow conditions. We furthermore observed a 50% reduction in Caco-2 TEER values following exposure to a cocktail of inflammatory cytokines. To our best knowledge, this is the first instrument of its kind that allows routine TEER studies in perfused organ-on-a-chip systems without interference by artificial filter membranes. We believe the apparatus will contribute to accelerating routine adoption of perfused organ-on-a-chip systems in academic research and in industrial drug development.
    DOI:  https://doi.org/10.1039/d0lc00770f
  3. Biophys J. 2021 Apr 07. pii: S0006-3495(21)00292-7. [Epub ahead of print]
    Leukocyte transmigration efficiency and longitudinal forward-thrusting force in a microfluidic Transwell device.
    Laurene Aoun, Paulin Nègre, Cristina Gonsales, Valentine Seveau de Noray, Sophie Brustlein, Martine Biarnes-Pelicot, Marie-Pierre Valignat, Olivier Theodoly.
      Transmigration of leukocytes across blood vessels walls is a critical step of the immune response. Transwell assays examine transmigration properties in vitro by counting cells passages through a membrane, however the difficulty of in situ imaging hampers a clear disentanglement of the roles of adhesion, chemokinesis and chemotaxis. We used here microfluidic Transwells to image the cells transition from 2D migration on a surface to 3D migration in a confining microchannels, and measure cells longitudinal forward-thrusting force in microchannels. Primary human effector T lymphocytes adhering with integrins LFA-1 (αLβ2) had a marked propensity to transmigrate in transwells without chemotactic cue. Both adhesion and contractility were important to overcome the critical step of nucleus penetration, but were remarkably dispensable for 3D migration in smooth microchannels deprived of topographic features. Transmigration in smooth channels was qualitatively consistent with a propulsion by treadmilling of cell envelope and squeezing of cell trailing edge. Stalling conditions of 3D migration were then assessed by imposing pressure drops across microchannels. Without specific adhesion, the cells slid backwards with sub-nanonewton forces, showing that 3D migration under stress is strongly limited by a lack of adhesion and friction with channels. With specific LFA-1 mediated adhesion, stalling occurred at around 3 and 6 nN in respectively 2x4 and 4x4 μm2 channels, supporting that stalling of adherent cells was under pressure control rather than force control. The stall pressure of 4 mbar is consistent with the pressure of actin filament polymerization that mediates lamellipod growth. The arrest of adherent cells under stress seems therefore controlled by the compression of the cell leading edge that perturbs cells front-rear polarization, and triggers adhesion failure or polarization reversal. While stalling assays in microfluidic Transwells do not mimic in vivo transmigration, they provide a powerful tool to scrutinize 2D/3D migration, barotaxis and chemotaxis.
    DOI:  https://doi.org/10.1016/j.bpj.2021.03.037
  4. Biofabrication. 2021 Apr 13.
    An engineered neurovascular unit for modeling neuroinflammation.
    Suyeong Seo, Chihoon Choi, Kyung Sik Lee, Seung Kim, Kangwon Lee, Nakwon Choi, Hong Jun Lee, Sang-Hoon Cha, Hong Nam Kim.
      The neurovascular unit (NVU) comprises multiple types of brain cells, including brain endothelial cells, astrocytes, pericytes, neurons, microglia, and oligodendrocytes. Each cell type contributes to the maintenance of the molecular transport barrier and brain tissue homeostasis. Several disorders and diseases of the central nervous system, including neuroinflammation, Alzheimer's disease, stroke, and multiple sclerosis, have been associated with dysfunction of the NVU. As a result, there has been increased demand for the development of NVU in vitro models. Here, we present a three-dimensional (3D) immortalized human cell-based NVU model generated by organizing the brain microvasculature in a collagen matrix embedded with six different types of cells that comprise the NVU. By surrounding a perfusable brain endothelium with six types of NVU-composing cells, we demonstrated a significant impact of the 3D co-culture on the maturation of barrier function, which is supported by cytokines secreted from NVU-composing cells. Furthermore, NVU-composing cells alleviated the inflammatory responses induced by lipopolysaccharides. Our human cell-based NVU in vitro model could enable elucidation of both physiological and pathological mechanisms in the human brain and evaluation of safety and efficacy in the context of high-content analysis during the process of drug development.
    Keywords:  blood-brain barrier; coculture; in vitro model; neuroinflammation; neurovascular unit; vascular permeability
    DOI:  https://doi.org/10.1088/1758-5090/abf741
  5. Lab Chip. 2021 Apr 15.
    Measuring barrier function in organ-on-chips with cleanroom-free integration of multiplexable electrodes.
    Elsbeth G B M Bossink, Mariia Zakharova, Douwe S de Bruijn, Mathieu Odijk, Loes I Segerink.
      Transepithelial/transendothelial electrical resistance (TEER) measurements can be applied in organ-on-chips (OoCs) to estimate the barrier properties of a tissue or cell layer in a continuous, non-invasive, and label-free manner. Assessing the barrier integrity in in vitro models is valuable for studying and developing barrier targeting drugs. Several systems for measuring the TEER have been shown, but each of them having their own drawbacks. This article presents a cleanroom-free fabrication method for the integration of platinum electrodes in a polydimethylsiloxane OoC, allowing the real-time assessment of the barrier function by employing impedance spectroscopy. The proposed method and electrode arrangement allow visual inspection of the cells cultured in the device at the site of the electrodes, and multiplexing of both the electrodes in one OoC and the number of OoCs in one device. The effectiveness of our system is demonstrated by lining the OoC with intestinal epithelial cells, creating a gut-on-chip, where we monitored the formation, as well as the disruption and recovery of the cell barrier during a 21 day culture period. The application is further expanded by creating a blood-brain-barrier, to show that the proposed fabrication method can be applied to monitor the barrier formation in the OoC for different types of biological barriers.
    DOI:  https://doi.org/10.1039/d0lc01289k
  6. ACS Biomater Sci Eng. 2021 Apr 12.
    FleXert: A Soft, Actuatable Multiwell Plate Insert for Cell Culture under Stretch.
    Sara Correia Carreira, Majid Taghavi, Elizabeth Pavez Loriè, Jonathan Rossiter.
      Porous multiwell plate inserts are widely used in biomedical research to study transport processes or to culture cells/tissues at the air-liquid interface. These inserts are made of rigid materials and used under static culture conditions, which are unrepresentative of biological microenvironments. Here, we present FleXert, a soft, actuatable cell culture insert that interfaces with six-well plates. It is made of polydimethylsiloxane (PDMS) and comprises a porous PDMS membrane as cell/tissue support. FleXerts can be pneumatically actuated using a standard syringe pump, imparting tensile strains of up to 30%. A wide range of actuation patterns can be achieved by varying the air pressure and pumping rate. Facile surface functionalization of FleXert's porous PDMS membrane with fibronectin enables adhesion of human dermal fibroblasts and strains developing on FleXert's membrane are successfully transduced to the cell layer. 3D tissue models, such as fibroblast-laden collagen gels, can also be anchored to PDMS following polydopamine coating. Furthermore, collagen-coated FleXert membranes support the establishment of a human skin model, demonstrating the material's excellent biocompatibility required for tissue engineering. In contrast to existing technologies, FleXerts do not require costly fabrication equipment or custom-built culture chambers, making them a versatile and low-cost solution for tissue engineering and biological barrier penetration studies under physiological strain. This paper is an extensive toolkit for multidisciplinary mechanobiology studies, including detailed instructions for a wide variety of methods such as device fabrication, theoretical modeling, cell culture, and image analysis techniques.
    Keywords:  biointerface; in vitro model; mechanical stimulation; soft actuator; tissue engineering
    DOI:  https://doi.org/10.1021/acsbiomaterials.0c01448
  7. Adv Biol (Weinh). 2021 Apr 15. e2100090
    Studying Tumor Angiogenesis and Cancer Invasion in a Three-Dimensional Vascularized Breast Cancer Micro-Environment.
    Madhuri Dey, Bugra Ayan, Marina Yurieva, Derya Unutmaz, Ibrahim T Ozbolat.
      Metastatic breast cancer is one of the deadliest forms of malignancy, primarily driven by its characteristic micro-environment comprising cancer cells interacting with stromal components. These interactions induce genetic and metabolic alterations creating a conducive environment for tumor growth. In this study, a physiologically relevant 3D vascularized breast cancer micro-environment is developed comprising of metastatic MDA-MB-231 cells and human umbilical vein endothelial cells loaded in human dermal fibroblasts laden fibrin, representing the tumor stroma. The matrix, as well as stromal cell density, impacts the transcriptional profile of genes involved in tumor angiogenesis and cancer invasion, which are hallmarks of cancer. Cancer-specific canonical pathways and activated upstream regulators are also identified by the differential gene expression signatures of these composite cultures. Additionally, a tumor-associated vascular bed of capillaries is established exhibiting dilated vessel diameters, representative of in vivo tumor physiology. Further, employing aspiration-assisted bioprinting, cancer-endothelial crosstalk, in the form of collective angiogenesis of tumor spheroids bioprinted at close proximity, is identified. Overall, this bottom-up approach of tumor micro-environment fabrication provides an insight into the potential of in vitro tumor models and enables the identification of novel therapeutic targets as a preclinical drug screening platform.
    Keywords:  aspiration-assisted bioprinting; in vitro tumor models; metastatic breast cancer; tumor angiogenesis; tumor micro-environments
    DOI:  https://doi.org/10.1002/adbi.202100090
  8. Genomics Inform. 2021 Mar;19(1): e2
    Elucidating molecular mechanisms of acquired resistance to BRAF inhibitors in melanoma using a microfluidic device and deep sequencing.
    Jiyeon Han, Yeonjoo Jung, Yukyung Jun, Sungsu Park, Sanghyuk Lee.
      BRAF inhibitors (e.g., vemurafenib) are widely used to treat metastatic melanoma with the BRAF V600E mutation. The initial response is often dramatic, but treatment resistance leads to disease progression in the majority of cases. Although secondary mutations in the mitogen-activated protein kinase signaling pathway are known to be responsible for this phenomenon, the molecular mechanisms governing acquired resistance are not known in more than half of patients. Here we report a genome- and transcriptome-wide study investigating the molecular mechanisms of acquired resistance to BRAF inhibitors. A microfluidic chip with a concentration gradient of vemurafenib was utilized to rapidly obtain therapy-resistant clones from two melanoma cell lines with the BRAF V600E mutation (A375 and SK-MEL-28). Exome and transcriptome data were produced from 13 resistant clones and analyzed to identify secondary mutations and gene expression changes. Various mechanisms, including phenotype switching and metabolic reprogramming, have been determined to contribute to resistance development differently for each clone. The roles of microphthalmia-associated transcription factor, the master transcription factor in melanocyte differentiation/dedifferentiation, were highlighted in terms of phenotype switching. Our study provides an omics-based comprehensive overview of the molecular mechanisms governing acquired resistance to BRAF inhibitor therapy.
    Keywords:  RNA sequencing; cancer drug resistance; melanoma; microfluidic device; targeted therapy; whole exome sequencing
    DOI:  https://doi.org/10.5808/gi.20074
  9. Adv Biol (Weinh). 2021 Apr;5(4): e2000428
    Emulating Early Atherosclerosis in a Vascular Microphysiological System Using Branched Tissue-Engineered Blood Vessels.
    Jounghyun H Lee, Zaozao Chen, Siyu He, JoyceK Zhou, Alexander Tsai, GeorgeA Truskey, Kam W Leong.
      Atherosclerosis begins with the accumulation of cholesterol-carrying lipoproteins on blood vessel walls and progresses to endothelial cell dysfunction, monocyte adhesion, and foam cell formation. Endothelialized tissue-engineered blood vessels (TEBVs) have previously been fabricated to recapitulate artery functionalities, including vasoconstriction, vasodilation, and endothelium activation. Here, the initiation of atherosclerosis is emulated by designing branched TEBVs (brTEBVs) of various geometries treated with enzyme-modified low-density-lipoprotein (eLDL) and TNF-α to induce endothelial cell dysfunction and adhesion of perfused human monocytes. Locations of monocyte adhesion under pulsatile flow are identified, and the hemodynamics in the brTEBVs are characterized using particle image velocimetry (PIV) and computational fluid dynamics (CFD). Monocyte adhesion is greater at the side outlets than at the main outlets or inlets, and is greatest at larger side outlet branching angles (60° or 80° vs 45°). In PIV experiments, the branched side outlets are identified as atherosclerosis-prone areas where fluorescent particles show a transient swirling motion following flow pulses; in CFD simulations, side outlets with larger branching angles show higher vorticity magnitude and greater flow disturbance than other areas. These results suggest that the branched TEBVs with eLDL/TNF-α treatment provide a physiologically relevant model of early atherosclerosis for preclinical studies.
    Keywords:  atherosclerosis; bifurcated arteries; microphysiological systems; pulsatile flows; tissue-engineered blood vessels
    DOI:  https://doi.org/10.1002/adbi.202000428
  10. Sci Rep. 2021 Apr 16. 11(1): 8355
    Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation.
    Nathan Jeger-Madiot, Lousineh Arakelian, Niclas Setterblad, Patrick Bruneval, Mauricio Hoyos, Jérôme Larghero, Jean-Luc Aider.
      In recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.
    DOI:  https://doi.org/10.1038/s41598-021-87459-6
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