bims-orenst 2022-02-13 papers

bims-orenst

Biomed News

on Organs-on-chips and engineered stem cell models

Issue of 2022–02–13
eight papers selected by
Joram Mooiweer, University of Groningen

Contributions of the microbiome to intestinal inflammation in a gut-on-a-chip.
Continous, non-invasive monitoring of oxygen consumption in a parallelized microfluidic in vitro system provides novel insight into the response to nutrients and drugs of primary human hepatocytes.
Design and fabrication of an integrated 3D dynamic multicellular liver-on-a-chip and its application in hepatotoxicity screening.
Vasculature-on-a-chip platform with innate immunity enables identification of angiopoietin-1 derived peptide as a therapeutic for SARS-CoV-2 induced inflammation.
Organoid-based Expansion of Patient-Derived Primary Alveolar Type-2 Cells for Establishment of Alveolus Epithelial Lung-Chip Cultures.
Prolonged culturing of iPSC-derived brain endothelial-like cells is associated with quiescence, downregulation of glycolysis, and resistance to disruption by an Alzheimer's brain milieu.
Scaffold-Free Tubular Engineered Heart Tissue From Human Induced Pluripotent Stem Cells Using Bio-3D Printing Technology in vivo.
Anti-CD47 antibody treatment attenuates liver inflammation and fibrosis in experimental non-alcoholic steatohepatitis models.

Nano Converg. 2022 Feb 08. 9(1): 8

Contributions of the microbiome to intestinal inflammation in a gut-on-a-chip.

Min Seo Jeon, Yoon Young Choi, Sung Jun Mo, Jang Ho Ha, Young Seo Lee, Hee Uk Lee, Soo Dong Park, Jae-Jung Shim, Jung-Lyoul Lee, Bong Geun Chung.

  The intestinal microbiome affects a number of biological functions of the organism. Although the animal model is a powerful tool to study the relationship between the host and microbe, a physiologically relevant in vitro human intestinal system has still unmet needs. Thus, the establishment of an in vitro living cell-based system of the intestine that can mimic the mechanical, structural, absorptive, transport and pathophysiological properties of the human intestinal environment along with its commensal bacterial strains can promote pharmaceutical development and potentially replace animal testing. In this paper, we present a microfluidic-based gut model which allows co-culture of human and microbial cells to mimic the gastrointestinal structure. The gut microenvironment is recreated by flowing fluid at a low rate (21 μL/h) over the microchannels. Under these conditions, we demonstrated the capability of gut-on-a-chip to recapitulate in vivo relevance epithelial cell differentiation including highly polarized epithelium, mucus secretion, and tight membrane integrity. Additionally, we observed that the co-culture of damaged epithelial layer with the probiotics resulted in a substantial responded recovery of barrier function without bacterial overgrowth in a gut-on-a-chip. Therefore, this gut-on-a-chip could promote explorations interaction with host between microbe and provide the insights into questions of fundamental research linking the intestinal microbiome to human health and disease.

Keywords:  Gut-on-a-chip; Inflammation bowel disease; Microbiome; Shear stress

DOI:  https://doi.org/10.1186/s40580-022-00299-6
EXCLI J. 2022 ;21 144-161

Continous, non-invasive monitoring of oxygen consumption in a parallelized microfluidic in vitro system provides novel insight into the response to nutrients and drugs of primary human hepatocytes.

Marius Busche, Dominik Rabl, Jan Fischer, Christian Schmees, Torsten Mayr, Rolf Gebhardt, Martin Stelzle.

  Oxygen plays a fundamental role in cellular energy metabolism, differentiation and cell biology in general. Consequently, in vitro oxygen sensing can be used to assess cell vitality and detect specific mechanisms of toxicity. In 2D in vitro models currently used, the oxygen supply provided by diffusion is generally too low, especially for cells having a high oxygen demand. In organ-on-chip systems, a more physiologic oxygen supply can be generated by establishing unidirectional perfusion. We established oxygen sensors in an easy-to-use and parallelized organ-on-chip system. We demonstrated the applicability of this system by analyzing the influence of fructose (40 mM, 80 mM), ammonium chloride (100 mM) and Na-diclofenac (50 µM, 150 µM, 450 µM, 1500 µM) on primary human hepatocytes (PHH). Fructose treatment for two hours showed an immediate drop of oxygen consumption (OC) with subsequent increase to nearly initial levels. Treatment with 80 mM glucose, 20 mM lactate or 20 mM glycerol did not result in any changes in OC which demonstrates a specific effect of fructose. Application of ammonium chloride for two hours did not show any immediate effects on OC, but qualitatively changed the cellular response to FCCP treatment. Na-diclofenac treatment for 24 hours led to a decrease of the maximal respiration and reserve capacity. We also demonstrated the stability of our system by repeatedly treating cells with 40 mM fructose, which led to similar cell responses on the same day as well as on subsequent days. In conclusion, our system enables in depth analysis of cellular respiration after substrate treatment in an unidirectional perfused organ-on-chip system.

Keywords:  in vitro; liver; metabolism; model; organ-on-chip; oxygen; perfusion; sensors; toxicity

DOI:  https://doi.org/10.17179/excli2021-4351
Talanta. 2022 Jan 29. pii: S0039-9140(22)00058-3. [Epub ahead of print]241 123262

Design and fabrication of an integrated 3D dynamic multicellular liver-on-a-chip and its application in hepatotoxicity screening.

Yi-Bo Zheng, Li-Dong Ma, Jian-Lin Wu, Yi-Ming Wang, Xian-Sheng Meng, Ping Hu, Qiong-Lin Liang, Yuan-Yuan Xie, Guo-An Luo.

  Nowadays, major methods of in vitro hepatotoxicity research are still based on traditional static two- or three-dimensional cell culture, although these means could investigate some toxic chemicals induced hepatotoxicity, but most of these toxicities failed to reappear in human, at least not in similar or calculable dose level. These failures may cause by the monoculture of only hepatocytes, ignored the signal communication to other non-parenchymal cells in liver tissue, also other complex microenvironment such as endothelial barrier, shear stress and other factors which were really existed in vivo but absent here, final leading to a low reliability of experimental results. In this study, a three-dimensional dynamic multi-cellular liver-on-a-chip device (3D-DMLoC) was developed to reproduce the microenvironment of in vivo liver tissue, including the simulation of hepatic sinusoid, perisinusoidal space and continuous liquid perfusion, hepatocytes could gather to some 3D cell spheroids in this chip. The perfusion could bring a real-time exchange of chemicals, nutrients, metabolites, supply suitable oxygen and a weak shear stress. The pressure and oxygen distribution inner the chip were simulated and evaluated by COMSOL Multiphysics software. HepaRG were co-cultured with HUVEC for 7 days in this chip, expression of hepatic polarization protein ZO-1 and MRP2, liver function factors ALB, UREA and CYP450s were almost all higher than in traditional static culture. Several drugs and heavy metal ions induced hepatotoxicity were then investigated, LDH released from hepatocyte spheroids in mostly 3D-DMLoC groups were higher than same-dosed 2D group, indicated the spheroids were more sensibility to the toxins. The hepatoxicity might be induced by acute hepatocytes injury according to the ratios of secreted AST/ALT contents. In conclusion, a liver-on-a-chip device was successfully developed and verified for better reproducing the in vivo physiological microenvironment of liver. It could be applied for easily, efficiently, and accurately screening the potential hepatotoxic chemicals in future.

Keywords:  Cell co-culture; Hepatocyte spheroids; Hepatotoxicity; Liver-on-a-chip; Microfluidics

DOI:  https://doi.org/10.1016/j.talanta.2022.123262
Lab Chip. 2022 Feb 10.

Vasculature-on-a-chip platform with innate immunity enables identification of angiopoietin-1 derived peptide as a therapeutic for SARS-CoV-2 induced inflammation.

Rick Xing Ze Lu, Benjamin Fook Lun Lai, Naimeh Rafatian, Dakota Gustafson, Scott B Campbell, Arinjay Banerjee, Robert Kozak, Karen Mossman, Samira Mubareka, Kathryn L Howe, Jason E Fish, Milica Radisic.

Coronavirus disease 2019 (COVID-19) was primarily identified as a novel disease causing acute respiratory syndrome. However, as the pandemic progressed various cases of secondary organ infection and damage by severe respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported, including a breakdown of the vascular barrier. As SARS-CoV-2 gains access to blood circulation through the lungs, the virus is first encountered by the layer of endothelial cells and immune cells that participate in host defense. Here, we developed an approach to study SARS-CoV-2 infection using vasculature-on-a-chip. We first modeled the interaction of virus alone with the endothelialized vasculature-on-a-chip, followed by the studies of the interaction of the virus exposed-endothelial cells with peripheral blood mononuclear cells (PBMCs). In an endothelial model grown on a permeable microfluidic bioscaffold under flow conditions, both human coronavirus (HCoV)-NL63 and SARS-CoV-2 presence diminished endothelial barrier function by disrupting VE-cadherin junctions and elevating the level of pro-inflammatory cytokines such as interleukin (IL)-6, IL-8, and angiopoietin-2. Inflammatory cytokine markers were markedly more elevated upon SARS-CoV-2 infection compared to HCoV-NL63 infection. Introduction of PBMCs with monocytes into the vasculature-on-a-chip upon SARS-CoV-2 infection further exacerbated cytokine-induced endothelial dysfunction, demonstrating the compounding effects of inter-cellular crosstalk between endothelial cells and monocytes in facilitating the hyperinflammatory state. Considering the harmful effects of SARS-CoV-2 on endothelial cells, even without active virus proliferation inside the cells, a potential therapeutic approach is critical. We identified angiopoietin-1 derived peptide, QHREDGS, as a potential therapeutic capable of profoundly attenuating the inflammatory state of the cells consistent with the levels in non-infected controls, thereby improving the barrier function and endothelial cell survival against SARS-CoV-2 infection in the presence of PBMC.

DOI: https://doi.org/10.1039/d1lc00817j
Am J Physiol Lung Cell Mol Physiol. 2022 Feb 09.

Organoid-based Expansion of Patient-Derived Primary Alveolar Type-2 Cells for Establishment of Alveolus Epithelial Lung-Chip Cultures.

Sander van Riet, Annemarie van Schadewijk, P Padmini S J Khedoe, Ronald W A L Limpens, Montserrat Bárcena, Jan Stolk, Pieter S Hiemstra, Anne M van der Does.

  Development of effective treatment strategies for lung tissue destruction as seen in emphysema would greatly benefit from representative human in vitro models of the alveolar compartment. Studying how cellular cross-talk and/or (altered) biomechanical cues affect alveolar epithelial function could provide new insight for tissue repair strategies. Preclinical models of the alveolus ideally combine human primary patient-derived lung cells with advanced cell culture applications such as breathing-related stretch, to reliably represent the alveolar microenvironment. To test the feasibility of such a model, we isolated primary alveolar type-2 cells (AEC2) from patient-derived lung tissues including those from patients with severe emphysema, using magnetic bead-based selection of cells expressing the AEC2 marker HTII-280. We obtained pure alveolar feeder-free organoid cultures using a minimally modified commercial medium. This was confirmed by known AEC2 markers as well as by detection of lamellar bodies using electron microscopy. Following (organoid-based) expansion, cells were seeded on both cell culture inserts and the Chip-S1® Organ-Chip that has a flexible PDMS membrane enabling the application of dynamic stretch. AEC2 cultured for 7 days on inserts or the chip maintained expression of HTII-280, pro-surfactant protein C (SP-C), SP-A and SP-B and zonula occludens-1 (ZO-1) also in the presence of stretch. AEC2 cultured on the chip showed lower expression levels of epithelial-mesenchymal transition-related vimentin expression compared to static cultures on inserts. The combination of a straightforward culture method of patient-derived AEC2 and their application in microfluidic chip cultures, supports successful development of more representative human preclinical models of the (diseased) alveolar compartment.

Keywords:  Alveolar type-2 cells; Cell culture; Emphysema; Organoid; Organs-on-Chips

DOI:  https://doi.org/10.1152/ajplung.00153.2021
Fluids Barriers CNS. 2022 Feb 05. 19(1): 10

Prolonged culturing of iPSC-derived brain endothelial-like cells is associated with quiescence, downregulation of glycolysis, and resistance to disruption by an Alzheimer's brain milieu.

Lindsey M Williams, Takashi Fujimoto, Riley R Weaver, Aric F Logsdon, Kira M Evitts, Jessica E Young, William A Banks, Michelle A Erickson.

   BACKGROUND: Human induced pluripotent stem cell (hiPSC)-derived brain endothelial-like cells (iBECs) are a robust, scalable, and translatable model of the human blood-brain barrier (BBB). Prior works have shown that high transendothelial electrical resistance (TEER) persists in iBECs for at least 2 weeks, emphasizing the utility of the model for longer term studies. However, most studies evaluate iBECs within the first few days of subculture, and little is known about their proliferative state, which could influence their functions. In this study, we characterized iBEC proliferative state in relation to key BBB properties at early (2 days) and late (9 days) post-subculture time points.
METHODS: hiPSCs were differentiated into iBECs using fully defined, serum-free medium. The proportion of proliferating cells was determined by BrdU assays. We evaluated TEER, expression of glycolysis enzymes and tight and adherens junction proteins (TJP and AJP), and glucose transporter-1 (GLUT1) function by immunoblotting, immunofluorescence, and quantifying radiolabeled tracer permeabilities. We also compared barrier disruption in response to TNF-α and conditioned medium (CM) from hiPSC-derived neurons harboring the Alzheimer's disease (AD)-causing Swedish mutation (APPSwe/+).
RESULTS: A significant decline in iBEC proliferation over time in culture was accompanied by adoption of a more quiescent endothelial metabolic state, indicated by downregulation of glycolysis-related proteins and upregulation GLUT1. Interestingly, upregulation of GLUT1 was associated with reduced glucose transport rates in more quiescent iBECs. We also found significant decreases in claudin-5 (CLDN5) and vascular endothelial-cadherin (VE-Cad) and a trend toward a decrease in platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas zona occludens-1 (ZO-1) increased and occludin (OCLN) remained unchanged. Despite differences in TJP and AJP expression, there was no difference in mean TEER on day 2 vs. day 9. TNF-α induced disruption irrespective of iBEC proliferative state. Conversely, APPSwe/+ CM disrupted only proliferating iBEC monolayers.
CONCLUSION: iBECs can be used to study responses to disease-relevant stimuli in proliferating vs. more quiescent endothelial cell states, which may provide insight into BBB vulnerabilities in contexts of development, brain injury, and neurodegenerative disease.

Keywords:  Alzheimer’s disease (AD); Blood–brain barrier (BBB); Glucose transporter-1 (GLUT1); Glycolysis; Human induced pluripotent stem cells (iPSCs); Quiescence; Tight junction protein (TJP)

DOI:  https://doi.org/10.1186/s12987-022-00307-1
Front Cardiovasc Med. 2021 ;8 806215

Scaffold-Free Tubular Engineered Heart Tissue From Human Induced Pluripotent Stem Cells Using Bio-3D Printing Technology in vivo.

Yujiro Kawai, Shugo Tohyama, Kenichi Arai, Tadashi Tamura, Yusuke Soma, Keiichi Fukuda, Hideyuki Shimizu, Koichi Nakayama, Eiji Kobayashi.

  Engineered heart tissues (EHTs) that are fabricated using human induced pluripotent stem cells (hiPSCs) have been considered as potential cardiac tissue substitutes in case of heart failure. In the present study, we have created hiPSC-derived cardiac organoids (hiPSC-COs) comprised of hiPSC-derived cardiomyocytes, human umbilical vein endothelial cells, and human fibroblasts. To produce a beating conduit for patients suffering from congenital heart diseases, we constructed scaffold-free tubular EHTs (T-EHTs) using hiPSC-COs and bio-3D printing with needle arrays. The bio-3D printed T-EHTs were cut open and transplanted around the abdominal aorta as well as the inferior vena cava (IVC) of NOG mice. The transplanted T-EHTs were covered with the omentum, and the abdomen was closed after completion of the procedure. Additionally, to compare the functionality of hiPSC-COs with that of T-EHTs, we transplanted the former around the aorta and IVC as well as injecting them into the subcutaneous tissue on the back of the mice. After 1 m of the transplantation procedures, we observed the beating of the T-EHTs in the mice. In histological analysis, the T-EHTs showed clear striation of the myocardium and vascularization compared to hiPSC-COs transplanted around the aorta or in subcutaneous tissue. Based on these results, bio-3D-printed T-EHTs exhibited a better maturation in vivo as compared to the hiPSC-COs. Therefore, these beating T-EHTs may form conduits for congenital heart disease patients, and T-EHT transplantation can form a treatment option in such cases.

Keywords:  bio-3D bioprinting; cardiomyocyte; engineered heart tissue; human-induced pluripotent stem cell; tubular tissue

DOI:  https://doi.org/10.3389/fcvm.2021.806215
Liver Int. 2022 Feb 07.

Anti-CD47 antibody treatment attenuates liver inflammation and fibrosis in experimental non-alcoholic steatohepatitis models.

Taesik Gwag, Eric Ma, Changcheng Zhou, Shuxia Wang.

   BACKGROUND AND AIMS: With the epidemic burden of obesity and metabolic diseases, nonalcoholic fatty liver disease (NAFLD) including steatohepatitis (NASH) has become the most common chronic liver disease in the western world. NASH may progress to cirrhosis and hepatocellular carcinoma. Currently no treatment is available for NASH. Therefore, finding a therapy for NAFLD/NASH is in urgent need. Previously we have demonstrated that mice lacking CD47 or its ligand thrombospondin1 (TSP1) are protected from obesity-associated NALFD. This suggests that CD47 blockade might be a novel treatment for obesity-associated metabolic disease. Thus, in this study, the therapeutic potential of an anti-CD47 antibody in NAFLD progression was determined.
METHODS: Both diet-induced NASH mouse model and human NASH organoid model were utilized in this study. NASH was induced in mice by feeding with diet enriched with fat, fructose and cholesterol (AMLN diet) for 20 weeks and then treated with anti-CD47 antibody or control IgG for 4 weeks. Body weight, body composition and liver phenotype were analyzed.
RESULTS: We found that anti-CD47 antibody treatment did not affect mice body weight, fat mass, or liver steatosis. However, liver immune cell infiltration, inflammation and fibrosis were significantly reduced by anti-CD47 antibody treatment. In vitro data further showed that CD47 blockade prevented hepatic stellate cell activation and NASH progression in a human NASH organoid model.
CONCLUSION: Collectively, these data suggest that anti-CD47 antibody might be a new therapeutic option for obesity-associated NASH and liver fibrosis.

Keywords:  AMLN diet; CD47; NAFLD; NASH; obesity; organoid

DOI:  https://doi.org/10.1111/liv.15182