Publications by authors named "Alexander Revzin"

113 Publications

Core-shell hydrogel microcapsules enable formation of human pluripotent stem cell spheroids and their cultivation in a stirred bioreactor.

Sci Rep 2021 Mar 30;11(1):7177. Epub 2021 Mar 30.

Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, 55902, USA.

Cellular therapies based on human pluripotent stem cells (hPSCs) offer considerable promise for treating numerous diseases including diabetes and end stage liver failure. Stem cell spheroids may be cultured in stirred bioreactors to scale up cell production to cell numbers relevant for use in humans. Despite significant progress in bioreactor culture of stem cells, areas for improvement remain. In this study, we demonstrate that microfluidic encapsulation of hPSCs and formation of spheroids. A co-axial droplet microfluidic device was used to fabricate 400 μm diameter capsules with a poly(ethylene glycol) hydrogel shell and an aqueous core. Spheroid formation was demonstrated for three hPSC lines to highlight broad utility of this encapsulation technology. In-capsule differentiation of stem cell spheroids into pancreatic β-cells in suspension culture was also demonstrated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-85786-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010084PMC
March 2021

Prospects and Opportunities for Microsystems and Microfluidic Devices in the Field of Otorhinolaryngology.

Clin Exp Otorhinolaryngol 2021 Feb 11;14(1):29-42. Epub 2020 Aug 11.

Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA.

Microfluidic systems can be used to control picoliter to microliter volumes in ways not possible with other methods of fluid handling. In recent years, the field of microfluidics has grown rapidly, with microfluidic devices offering possibilities to impact biology and medicine. Microfluidic devices populated with human cells have the potential to mimic the physiological functions of tissues and organs in a three-dimensional microenvironment and enable the study of mechanisms of human diseases, drug discovery and the practice of personalized medicine. In the field of otorhinolaryngology, various types of microfluidic systems have already been introduced to study organ physiology, diagnose diseases, and evaluate therapeutic efficacy. Therefore, microfluidic technologies can be implemented at all levels of otorhinolaryngology. This review is intended to promote understanding of microfluidic properties and introduce the recent literature on application of microfluidic-related devices in the field of otorhinolaryngology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.21053/ceo.2020.00626DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904428PMC
February 2021

Microfluidic confinement enhances phenotype and function of hepatocyte spheroids.

Am J Physiol Cell Physiol 2020 09 22;319(3):C552-C560. Epub 2020 Jul 22.

Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota.

A number of cell culture approaches have been described for maintenance of primary hepatocytes. Forming hepatocytes into three-dimensional (3-D) spheroids is one well-accepted method for extending epithelial phenotype of these cells. Our laboratory has previously observed enhanced function of two-dimensional (2-D, monolayer) hepatocyte cultures in microfluidic devices due to increased production of several hepato-inductive growth factors, including hepatocyte growth factor (HGF). In the present study, we wanted to test a hypothesis that culturing hepatocyte spheroids (3-D) in microfluidic devices will also result in enhanced phenotype and function. To test this hypothesis, we fabricated devices with small and large volumes. Both types of devices included a microstructured floor containing arrays of pyramidal wells to promote assembly of hepatocytes into spheroids with individual diameters of ~100 µm. The hepatocyte spheroids were found to be more functional, as evidenced by higher level of albumin synthesis, bile acid production, and hepatic enzyme expression, in low-volume compared with large-volume devices. Importantly, high functionality of spheroid cultures correlated with elevated levels of HGF secretion. Although decay of hepatic function (albumin secretion) was observed over the course 3 wk, this behavior could be abrogated by inhibiting TGF-β1 signaling. With TGF-β1 inhibitor, microfluidic hepatocyte spheroid cultures maintained high and stable levels of albumin synthesis over the course of 4 wk. To further highlight utility of this culture platform for liver disease modeling, we carried out alcohol injury experiments in microfluidic devices and tested protective effects of interleukin-22: a potential therapy for alcoholic hepatitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajpcell.00094.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509267PMC
September 2020

IRE1A Stimulates Hepatocyte-Derived Extracellular Vesicles That Promote Inflammation in Mice With Steatohepatitis.

Gastroenterology 2020 10 20;159(4):1487-1503.e17. Epub 2020 Jun 20.

Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota. Electronic address:

Background & Aims: Endoplasmic reticulum to nucleus signaling 1 (ERN1, also called IRE1A) is a sensor of the unfolded protein response that is activated in the livers of patients with nonalcoholic steatohepatitis (NASH). Hepatocytes release ceramide-enriched inflammatory extracellular vesicles (EVs) after activation of IRE1A. We studied the effects of inhibiting IRE1A on release of inflammatory EVs in mice with diet-induced steatohepatitis.

Methods: C57BL/6J mice and mice with hepatocyte-specific disruption of Ire1a (IRE1α) were fed a diet high in fat, fructose, and cholesterol to induce development of steatohepatitis or a standard chow diet (controls). Some mice were given intraperitoneal injections of the IRE1A inhibitor 4μ8C. Mouse liver and primary hepatocytes were transduced with adenovirus or adeno-associated virus that expressed IRE1A. Livers were collected from mice and analyzed by quantitative polymerase chain reaction and chromatin immunoprecipitation assays; plasma samples were analyzed by enzyme-linked immunosorbent assay. EVs were derived from hepatocytes and injected intravenously into mice. Plasma EVs were characterized by nanoparticle-tracking analysis, electron microscopy, immunoblots, and nanoscale flow cytometry; we used a membrane-tagged reporter mouse to detect hepatocyte-derived EVs. Plasma and liver tissues from patients with NASH and without NASH (controls) were analyzed for EV concentration and by RNAscope and gene expression analyses.

Results: Disruption of Ire1a in hepatocytes or inhibition of IRE1A reduced the release of EVs and liver injury, inflammation, and accumulation of macrophages in mice on the diet high in fat, fructose, and cholesterol. Activation of IRE1A, in the livers of mice, stimulated release of hepatocyte-derived EVs, and also from cultured primary hepatocytes. Mice given intravenous injections of IRE1A-stimulated, hepatocyte-derived EVs accumulated monocyte-derived macrophages in the liver. IRE1A-stimulated EVs were enriched in ceramides. Chromatin immunoprecipitation showed that IRE1A activated X-box binding protein 1 (XBP1) to increase transcription of serine palmitoyltransferase genes, which encode the rate-limiting enzyme for ceramide biosynthesis. Administration of a pharmacologic inhibitor of serine palmitoyltransferase to mice reduced the release of EVs. Levels of XBP1 and serine palmitoyltransferase were increased in liver tissues, and numbers of EVs were increased in plasma, from patients with NASH compared with control samples and correlated with the histologic features of inflammation.

Conclusions: In mouse hepatocytes, activated IRE1A promotes transcription of serine palmitoyltransferase genes via XBP1, resulting in ceramide biosynthesis and release of EVs. The EVs recruit monocyte-derived macrophages to the liver, resulting in inflammation and injury in mice with diet-induced steatohepatitis. Levels of XBP1, serine palmitoyltransferase, and EVs are all increased in liver tissues from patients with NASH. Strategies to block this pathway might be developed to reduce liver inflammation in patients with NASH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2020.06.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666601PMC
October 2020

Microfluidic devices, accumulation of endogenous signals and stem cell fate selection.

Differentiation 2020 Mar - Apr;112:39-46. Epub 2019 Dec 10.

Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA. Electronic address:

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.diff.2019.10.005DOI Listing
December 2019

Integrin β-enriched extracellular vesicles mediate monocyte adhesion and promote liver inflammation in murine NASH.

J Hepatol 2019 12 6;71(6):1193-1205. Epub 2019 Aug 6.

Division of Gastroenterology & Hepatology, Mayo Clinic, Rochester, Minnesota, United States; Division of Pediatric Gastroenterology, Mayo Clinic, Rochester, Minnesota, United States. Electronic address:

Background & Aims: Hepatic recruitment of monocyte-derived macrophages (MoMFs) contributes to the inflammatory response in non-alcoholic steatohepatitis (NASH). However, how hepatocyte lipotoxicity promotes MoMF inflammation is unclear. Here we demonstrate that lipotoxic hepatocyte-derived extracellular vesicles (LPC-EVs) are enriched with active integrin β (ITGβ), which promotes monocyte adhesion and liver inflammation in murine NASH.

Methods: Hepatocytes were treated with either vehicle or the toxic lipid mediator lysophosphatidylcholine (LPC); EVs were isolated from the conditioned media and subjected to proteomic analysis. C57BL/6J mice were fed a diet rich in fat, fructose, and cholesterol (FFC) to induce NASH. Mice were treated with anti-ITGβ neutralizing antibody (ITGβAb) or control IgG isotype.

Results: Ingenuity® Pathway Analysis of the LPC-EV proteome indicated that ITG signaling is an overrepresented canonical pathway. Immunogold electron microscopy and nanoscale flow cytometry confirmed that LPC-EVs were enriched with activated ITGβ. Furthermore, we showed that LPC treatment in hepatocytes activates ITGβ and mediates its endocytic trafficking and sorting into EVs. LPC-EVs enhanced monocyte adhesion to liver sinusoidal cells, as observed by shear stress adhesion assay. This adhesion was attenuated in the presence of ITGβAb. FFC-fed, ITGβAb-treated mice displayed reduced inflammation, defined by decreased hepatic infiltration and activation of proinflammatory MoMFs, as assessed by immunohistochemistry, mRNA expression, and flow cytometry. Likewise, mass cytometry by time-of-flight on intrahepatic leukocytes showed that ITGβAb reduced levels of infiltrating proinflammatory monocytes. Furthermore, ITGβAb treatment significantly ameliorated liver injury and fibrosis.

Conclusions: Lipotoxic EVs mediate monocyte adhesion to LSECs mainly through an ITGβ-dependent mechanism. ITGβAb ameliorates diet-induced NASH in mice by reducing MoMF-driven inflammation, suggesting that blocking ITGβ is a potential anti-inflammatory therapeutic strategy in human NASH.

Lay Summary: Herein, we report that a cell adhesion molecule termed integrin β (ITGβ) plays a key role in the progression of non-alcoholic steatohepatitis (NASH). ITGβ is released from hepatocytes under lipotoxic stress as a cargo of extracellular vesicles, and mediates monocyte adhesion to liver sinusoidal endothelial cells, which is an essential step in hepatic inflammation. In a mouse model of NASH, blocking ITGβ reduces liver inflammation, injury and fibrosis. Hence, ITGβ inhibition may serve as a new therapeutic strategy for NASH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jhep.2019.07.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6864271PMC
December 2019

Mechanical Stretch Increases Expression of CXCL1 in Liver Sinusoidal Endothelial Cells to Recruit Neutrophils, Generate Sinusoidal Microthombi, and Promote Portal Hypertension.

Gastroenterology 2019 07 11;157(1):193-209.e9. Epub 2019 Mar 11.

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota. Electronic address:

Background & Aims: Mechanical forces contribute to portal hypertension (PHTN) and fibrogenesis. We investigated the mechanisms by which forces are transduced by liver sinusoidal endothelial cells (LSECs) into pressure and matrix changes.

Methods: We isolated primary LSECs from mice and induced mechanical stretch with a Flexcell device, to recapitulate the pulsatile forces induced by congestion, and performed microarray and RNA-sequencing analyses to identify gene expression patterns associated with stretch. We also performed studies with C57BL/6 mice (controls), mice with deletion of neutrophil elastase (NE) or peptidyl arginine deiminase type IV (Pad4) (enzymes that formation of neutrophil extracellular traps [NETs]), and mice with LSEC-specific deletion of Notch1 (Notch1). We performed partial ligation of the suprahepatic inferior vena cava (pIVCL) to simulate congestive hepatopathy-induced portal hypertension in mice; some mice were given subcutaneous injections of sivelestat or underwent bile-duct ligation. Portal pressure was measured using a digital blood pressure analyzer and we performed intravital imaging of livers of mice.

Results: Expression of the neutrophil chemoattractant CXCL1 was up-regulated in primary LSECs exposed to mechanical stretch, compared with unexposed cells. Intravital imaging of livers in control mice revealed sinusoidal complexes of neutrophils and platelets and formation of NETs after pIVCL. NE and Pad4 mice had lower portal pressure and livers had less fibrin compared with control mice after pIVCL and bile-duct ligation; neutrophil recruitment into sinusoidal lumen of liver might increase portal pressure by promoting sinusoid microthrombi. RNA-sequencing of LSECs identified proteins in mechanosensitive signaling pathways that are altered in response to mechanical stretch, including integrins, Notch1, and calcium signaling pathways. Mechanical stretch of LSECs increased expression of CXCL1 via integrin-dependent activation of transcription factors regulated by Notch and its interaction with the mechanosensitive piezo calcium channel.

Conclusions: In studies of LSECs and knockout mice, we identified mechanosensitive angiocrine signals released by LSECs which promote PHTN by recruiting sinusoidal neutrophils and promoting formation of NETs and microthrombi. Strategies to target these pathways might be developed for treatment of PHTN. RNA-sequencing accession number: GSE119547.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1053/j.gastro.2019.03.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581607PMC
July 2019

Automated Droplet-Based Microfluidic Platform for Multiplexed Analysis of Biochemical Markers in Small Volumes.

Anal Chem 2019 04 27;91(8):5133-5141. Epub 2019 Mar 27.

Mayo Clinic , Rochester , Minnesota 55905 , United States.

The ability to detect multiple analytes in a small sample volume has significance for numerous areas of research, including organs-on-chip, small animal experiments, and neonatology. The objective of this study was to develop an automated microfluidics platform for multiplexed detection of analytes in microliter sample volumes. This platform employed computer-controlled microvalves to create laminar co-flows of sample and assay reagent solutions. It also contained valve-regulated cross-junction for discretizing sample/reagent mixtures into water-in-oil droplets. Microfluidic automation allowed us to control parameters related to frequency of droplet generation and the number of droplets of the same composition, as well as the size of droplets. Each droplet represented an individual enzymatic assay carried out in a sub-nanoliter (0.8 nL) volume reactor. An enzymatic reaction involving target analyte and assay reagents produced colorimetric or fluorescent signals in droplets. Importantly, intensity of optical signal was proportional to the concentration of analyte in question. This microfluidic bioanalysis platform was used in conjunction with commercial "mix-detect" assays for glucose, total bile acids, and lactate dehydrogenase (LDH). After characterizing these assays individually, we demonstrated sensitive multiplexed detection of three analytes from as little as 3 μL. In fact, this volume was sufficient to generate multiple repeat droplets for each of the three biochemical assays as well as positive control droplets, confirming the quality of assay reagents and negative control droplets to help with background subtraction. One potential application for this microfluidic bioanalysis platform involves sampling cell-conditioned media in organ-on-chip devices. To highlight this application, hepatocyte spheroids were established in microfluidic devices, injured on-chip by exposure to lipotoxic agent (palmitate), and then connected to the bioanalysis module for daily monitoring of changes in cytotoxicity (LDH), energy metabolism (glucose), and liver function (total bile acids). Microfluidic in-droplet assays revealed increased levels of LDH as well as reduction in bile acid synthesis-results that were consistent with hepatic injury. Importantly, these experiments highlighted the fact that in-droplet assays were sufficiently sensitive to detect changes in functional output of a relatively small (∼100) number of hepatocyte spheroids cultured in a microfluidic device. Moving forward, we foresee increasing the multiplexing capability of this technology and applying this platform to other biological/medical scenarios where detection of multiple analytes from a small sample volume is desired.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.analchem.8b05689DOI Listing
April 2019

Microencapsulated Immunoassays for Detection of Cytokines in Human Blood.

ACS Sens 2019 03 18;4(3):578-585. Epub 2019 Feb 18.

Department of Physiology and Biomedical Engineering , Mayo Clinic , Rochester Campus, Rochester , Minnesota 55905 , United States.

Cytokines are produced by leukocytes in blood and may be used as indicators of malignancies or infections. The objective of this study was to develop a strategy for immunosensing cytokines in whole, unprocessed human blood. Microfluidic droplet generation was employed to fabricate ∼400 μm diameter microcapsules with a hydrogel shell and an aqueous core containing sensing microbeads. The hydrogel shell was composed of poly(ethylene glycol) forming a thin (∼10 μm) immunoisolation layer protecting antibody-modified microbeads inside the capsule from immune cells on the outside. The microbeads were functionalized with antibodies against cytokines of interest: interferon (IFN)-γ and tumor necrosis factor (TNF)-α. While nonfouling, a hydrogel shell was permeable to cytokine molecules; these molecules were captured on microbeads and were detected with fluorescently labeled secondary antibodies. Calibration of encapsulated immunoassays with known concentrations of cytokines revealed a limit of detection of 14.8 and 14.4 pM for IFN-γ and TNF-α, respectively. We also demonstrated the concept of multi-cytokine detection by fabricating distinct populations of capsules carrying either anti-IFN-γ or anti-TNF-α microbeads and dispensing these capsules into a solution containing both cytokine types. Importantly, when placed into whole blood for 16 h, microcapsules were free of leukocytes, effectively protecting sensing beads from the blood components. To further demonstrate utility of this strategy, encapsulated microbeads were used for detection of IFN-γ in blood of patients with latent tuberculosis infection (LTBI) and unexposed healthy controls. When compared to gold standard technology (interferon gamma release assay or IGRA), our encapsulated immunoassay accurately predicted LTBI diagnosis in 11 out of 14 patients. Overall, encapsulation of immunoassays represents a promising strategy for keeping sensing elements operational in a highly fouling complex environment such as blood.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acssensors.8b01033DOI Listing
March 2019

Hepatocyte-Derived Lipotoxic Extracellular Vesicle Sphingosine 1-Phosphate Induces Macrophage Chemotaxis.

Front Immunol 2018 19;9:2980. Epub 2018 Dec 19.

Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, United States.

The pathophysiology of non-alcoholic steatohepatitis involves hepatocyte lipotoxicity due to excess saturated free fatty acids and concomitant proinflammatory macrophage effector responses. These include the infiltration of macrophages into hepatic cords in response to incompletely understood stimuli. Stressed hepatocytes release an increased number of extracellular vesicles (EVs), which are known to participate in intercellular signaling and coordination of the behavior of immune cell populations via their cargo. We hypothesized that hepatocyte-derived lipotoxic EVs that are enriched in sphingosine 1-phosphate (S1P) are effectors of macrophage infiltration in the hepatic microenvironment. Lipotoxic EVs were isolated from palmitate treated immortalized mouse hepatocytes and characterized by nanoparticle tracking analysis. Lipotoxic EV sphingolipids were quantified using tandem mass spectrometry. Wildtype and S1P receptor knockout bone marrow-derived macrophages were exposed to lipotoxic EV gradients in a microfluidic gradient generator. Macrophage migration toward EV gradients was captured by time-lapse microscopy and analyzed to determine directional migration. Fluorescence-activated cell sorting along with quantitative PCR and immunohistochemistry were utilized to characterize the cell surface expression of S1P receptor on intrahepatic leukocytes and hepatic expression of S1P receptor, respectively. Palmitate treatment induced the release of EVs. These EVs were enriched in S1P. Palmitate-induced S1P enriched EVs were chemoattractive to macrophages. EV S1P enrichment depended on the activity of sphingosine kinases 1 and 2, such that, pharmacological inhibition of sphingosine kinases 1 and 2 resulted in a significant reduction in EV S1P cargo without affecting the number of EVs released. When exposed to EVs derived from cells treated with palmitate in the presence of a pharmacologic inhibitor of sphingosine kinases 1 and 2, macrophages displayed diminished chemotactic behavior. To determine receptor-ligand specificity, we tested the migration responses of macrophages genetically deleted in the S1P receptor toward lipotoxic EVs. S1P receptor knockout macrophages displayed a marked reduction in their chemotactic responses toward lipotoxic palmitate-induced EVs. Palmitate-induced lipotoxic EVs are enriched in S1P through sphingosine kinases 1 and 2. S1P-enriched EVs activate persistent and directional macrophage chemotaxis mediated by the S1P receptor, a potential signaling axis for macrophage infiltration during hepatic lipotoxicity, and a potential therapeutic target for non-alcoholic steatohepatitis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2018.02980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6305739PMC
October 2019

Fabrication of composite microfluidic devices for local control of oxygen tension in cell cultures.

Lab Chip 2019 01;19(2):306-315

Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905, USA.

Oxygen tension is a central component of the cellular microenvironment and can serve as a trigger for changes in cell phenotype and function. There is a strong need to precisely control and modulate oxygen tension in cell culture systems in order to more accurately model the physiology and pathophysiology observed in vivo. The objective of this paper was to develop a simple, yet effective strategy for local control of oxygen tension in microfluidic cell cultures. Our strategy relied on fabrication of microfluidic devices using oxygen-permeable and impermeable materials. This composite device was designed so as to incorporate regions of gas permeability into the roof of the cell culture chamber and was outfitted with a reservoir for the oxygen-consuming chemical pyrogallol. When assembled and filled with pyrogallol, this device allowed oxygen depletion to occur within a specific region of the microfluidic culture chamber. The geometry and dimensions of the hypoxic region inside a microfluidic chamber were controlled by features fabricated into the oxygen-impermeable layer. Oxygen tension as low as 0.5% could be achieved using this strategy. To prove the utility of this device, we demonstrated that hypoxia induced anaerobic metabolism in a group of liver cancer cells, and that neighboring cancer cells residing under normoxic conditions upregulated the expression of transporters for taking up lactate - a product of anaerobic respiration. The microfluidic devices described here may be broadly applicable for mimicking multiple physiological scenarios where oxygen tension varies on the length scale of tens of micrometers including the cancer microenvironment, liver zonation, and luminal microenvironment of the gut.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c8lc00825fDOI Listing
January 2019

Macrophages contribute to the pathogenesis of sclerosing cholangitis in mice.

J Hepatol 2018 09 24;69(3):676-686. Epub 2018 May 24.

Division of Gastroenterology and Hepatology and the Mayo Clinic Center for Cell Signaling in Gastroenterology, Rochester, MN, USA. Electronic address:

Background & Aims: Macrophages contribute to liver disease, but their role in cholestatic liver injury, including primary sclerosing cholangitis (PSC), is unclear. We tested the hypothesis that macrophages contribute to the pathogenesis of, and are therapeutic targets for, PSC.

Methods: Immune cell profile, hepatic macrophage number, localization and polarization, fibrosis, and serum markers of liver injury and cholestasis were measured in an acute (intrabiliary injection of the inhibitor of apoptosis antagonist BV6) and chronic (Mdr2 mice) mouse model of sclerosing cholangitis (SC). Selected observations were confirmed in liver specimens from patients with PSC. Because of the known role of the CCR2/CCL2 axis in monocyte/macrophage chemotaxis, therapeutic effects of the CCR2/5 antagonist cenicriviroc (CVC), or genetic deletion of CCR2 (Ccr2 mice) were determined in BV6-injected mice.

Results: We found increased peribiliary pro-inflammatory (M1-like) and alternatively-activated (M2-like) monocyte-derived macrophages in PSC compared to normal livers. In both SC models, genetic profiling of liver immune cells identified a predominance of monocytes/macrophages; immunohistochemistry confirmed peribiliary monocyte-derived macrophage recruitment (M1>M2-polarized), which paralleled injury onset and was reversed upon resolution in acute SC mice. PSC, senescent and BV6-treated human cholangiocytes released monocyte chemoattractants (CCL2, IL-8) and macrophage-activating factors in vitro. Pharmacological inhibition of monocyte recruitment by CVC treatment or CCR2 genetic deletion attenuated macrophage accumulation, liver injury and fibrosis in acute SC.

Conclusions: Peribiliary recruited macrophages are a feature of both PSC and acute and chronic murine SC models. Pharmacologic and genetic inhibition of peribiliary macrophage recruitment decreases liver injury and fibrosis in mouse SC. These observations suggest monocyte-derived macrophages contribute to the development of SC in mice and in PSC pathogenesis, and support their potential as a therapeutic target.

Lay Summary: Primary sclerosing cholangitis (PSC) is an inflammatory liver disease which often progresses to liver failure. The cause of the disease is unclear and therapeutic options are limited. Therefore, we explored the role of white blood cells termed macrophages in PSC given their frequent contribution to other human inflammatory diseases. Our results implicate macrophages in PSC and PSC-like diseases in mice. More importantly, we found that pharmacologic inhibition of macrophage recruitment to the liver reduces PSC-like liver injury in the mouse. These exciting observations highlight potential new strategies to treat PSC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jhep.2018.05.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6098983PMC
September 2018

Synectin promotes fibrogenesis by regulating PDGFR isoforms through distinct mechanisms.

JCI Insight 2017 12 21;2(24). Epub 2017 Dec 21.

Gastroenterology Research Unit, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota, USA.

The scaffold protein synectin plays a critical role in the trafficking and regulation of membrane receptor pathways. As platelet-derived growth factor receptor (PDGFR) is essential for hepatic stellate cell (HSC) activation and liver fibrosis, we sought to determine the role of synectin on the PDGFR pathway and development of liver fibrosis. Mice with deletion of synectin from HSC were found to be protected from liver fibrosis. mRNA sequencing revealed that knockdown of synectin in HSC demonstrated reductions in the fibrosis pathway of genes, including PDGFR-β. Chromatin IP assay of the PDGFR-β promoter upon synectin knockdown revealed a pattern of histone marks associated with decreased transcription, dependent on p300 histone acetyltransferase. Synectin knockdown was found to downregulate PDGFR-α protein levels, as well, but through an alternative mechanism: protection from autophagic degradation. Site-directed mutagenesis revealed that ubiquitination of specific PDGFR-α lysine residues was responsible for its autophagic degradation. Furthermore, functional studies showed decreased PDGF-dependent migration and proliferation of HSC after synectin knockdown. Finally, human cirrhotic livers demonstrated increased synectin protein levels. This work provides insight into differential transcriptional and posttranslational mechanisms of synectin regulation of PDGFRs, which are critical to fibrogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.92821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752303PMC
December 2017

Nanowire Aptasensors for Electrochemical Detection of Cell-Secreted Cytokines.

ACS Sens 2017 11 9;2(11):1644-1652. Epub 2017 Oct 9.

Department of Biomedical Engineering, University of California, Davis , Davis, California 95616, United States.

Cytokines are small proteins secreted by immune cells in response to pathogens/infections; therefore, these proteins can be used in diagnosing infectious diseases. For example, release of a cytokine interferon (IFN)-γ from T-cells is used for blood-based diagnosis of tuberculosis (TB). Our lab has previously developed an atpamer-based electrochemical biosensor for rapid and sensitive detection of IFN-γ. In this study, we explored the use of silicon nanowires (NWs) as a way to create nanostructured electrodes with enhanced sensitivity for IFN-γ. Si NWs were covered with gold and were further functionalized with thiolated aptamers specific for IFN-γ. Aptamer molecules were designed to form a hairpin and in addition to terminal thiol groups contained redox reporter molecules methylene blue. Binding of analyte to aptamer-modified NWs (termed here nanowire aptasensors) inhibited electron transfer from redox reporters to the electrode and caused electrochemical redox signal to decrease. In a series of experiments we demonstrate that NW aptasensors responded 3× faster and were 2× more sensitive to IFN-γ compared to standard flat electrodes. Most significantly, NW aptasensors allowed detection of IFN-γ from as few as 150 T-cells/mL while ELISA did not pick up signal from the same number of cells. One of the challenges faced by ELISA-based TB diagnostics is poor performance in patients whose T-cell numbers are low, typically HIV patients. Therefore, NW aptasensors developed here may be used in the future for more sensitive monitoring of IFN-γ responses in patients coinfected with HIV/TB.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acssensors.7b00486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311073PMC
November 2017

Microchamber Cultures of Bladder Cancer: A Platform for Characterizing Drug Responsiveness and Resistance in PDX and Primary Cancer Cells.

Sci Rep 2017 09 25;7(1):12277. Epub 2017 Sep 25.

Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA.

Precision cancer medicine seeks to target the underlying genetic alterations of cancer; however, it has been challenging to use genetic profiles of individual patients in identifying the most appropriate anti-cancer drugs. This spurred the development of patient avatars; for example, patient-derived xenografts (PDXs) established in mice and used for drug exposure studies. However, PDXs are associated with high cost, long development time and low efficiency of engraftment. Herein we explored the use of microfluidic devices or microchambers as simple and low-cost means of maintaining bladder cancer cells over extended periods of times in order to study patterns of drug responsiveness and resistance. When placed into 75 µm tall microfluidic chambers, cancer cells grew as ellipsoids reaching millimeter-scale dimeters over the course of 30 days in culture. We cultured three PDX and three clinical patient specimens with 100% success rate. The turn-around time for a typical efficacy study using microchambers was less than 10 days. Importantly, PDX-derived ellipsoids in microchambers retained patterns of drug responsiveness and resistance observed in PDX mice and also exhibited in vivo-like heterogeneity of tumor responses. Overall, this study establishes microfluidic cultures of difficult-to-maintain primary cancer cells as a useful tool for precision cancer medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-12543-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612935PMC
September 2017

Microbiota-activated PPAR-γ signaling inhibits dysbiotic Enterobacteriaceae expansion.

Science 2017 08;357(6351):570-575

Department of Medical Microbiology and Immunology, School of Medicine, University of California at Davis, One Shields Avenue, Davis, CA 95616, USA.

Perturbation of the gut-associated microbial community may underlie many human illnesses, but the mechanisms that maintain homeostasis are poorly understood. We found that the depletion of butyrate-producing microbes by antibiotic treatment reduced epithelial signaling through the intracellular butyrate sensor peroxisome proliferator-activated receptor γ (PPAR-γ). Nitrate levels increased in the colonic lumen because epithelial expression of , the gene encoding inducible nitric oxide synthase, was elevated in the absence of PPAR-γ signaling. Microbiota-induced PPAR-γ signaling also limits the luminal bioavailability of oxygen by driving the energy metabolism of colonic epithelial cells (colonocytes) toward β-oxidation. Therefore, microbiota-activated PPAR-γ signaling is a homeostatic pathway that prevents a dysbiotic expansion of potentially pathogenic and by reducing the bioavailability of respiratory electron acceptors to Enterobacteriaceae in the lumen of the colon.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aam9949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5642957PMC
August 2017

Harnessing endogenous signals from hepatocytes using a low volume multi-well plate.

Integr Biol (Camb) 2017 05;9(5):427-435

Department of Biomedical Engineering, University of California, Davis, CA 95616, USA.

Hepatocytes are highly differentiated epithelial cells that lose their phenotype and function when removed from the in vivo environment. Given the importance of hepatic cultures for drug toxicity, bioartificial liver assist devices and basic biology studies, considerable efforts have been focused on the maintenance of hepatic function in vitro. The methods used to date include co-cultivation of hepatocytes with stromal cells, organizing these cells into spheroids and imbedding them into bioactive gels. Our team has recently demonstrated that primary rat hepatocytes confined to microfluidic channels in the absence of convection maintained the epithelial phenotype through upregulation of endogenous signals including hepatocyte growth factor (HGF). The objective of the present study was to transition from microfluidic devices, which are somewhat specialized and challenging to use, towards low volume multiwell plates ubiquitous in biology laboratories. Using a combination of 3D printing and micromolding we have constructed inserts that can be placed into standard 12-well plates and can be used to create low volume culture conditions under which primary hepatocytes maintained a differentiated phenotype. This phenotype enhancement was confirmed by hepatic function assays including albumin synthesis and expression. Importantly we confirmed upregulation of HGF inside the low volume culture plates and demonstrated that inhibition of HGF signaling degraded the hepatic phenotype in our cell culture platform. Overall, this study outlines a new cell culture system that leverages the low volume effects of microfluidic channels in a multiwell plate format. Beyond hepatocytes, such a system may be of use in the maintenance of other difficult-to-culture cells including stem cells and primary cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c7ib00010cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5800425PMC
May 2017

One step fabrication of hydrogel microcapsules with hollow core for assembly and cultivation of hepatocyte spheroids.

Acta Biomater 2017 03 6;50:428-436. Epub 2017 Jan 6.

Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA. Electronic address:

3D hepatic microtissues can serve as valuable liver analogues for cell-based therapies and for hepatotoxicity screening during preclinical drug development. However, hepatocytes rapidly dedifferentiate in vitro, and typically require 3D culture systems or co-cultures for phenotype rescue. In this work we present a novel microencapsulation strategy, utilizing coaxial flow-focusing droplet microfluidics to fabricate microcapsules with liquid core and poly(ethylene glycol) (PEG) gel shell. When entrapped inside these capsules, primary hepatocytes rapidly formed cell-cell contacts and assembled into compact spheroids. High levels of hepatic function were maintained inside the capsules for over ten days. The microencapsulation approach described here is compatible with difficult-to-culture primary epithelial cells, allows for tuning gel mechanical properties and diffusivity, and may be used in the future for high density suspension cell cultures.

Statement Of Significance: Our paper combines an interesting new way for making capsules with cultivation of difficult-to-maintain primary epithelial cells (hepatocytes). The microcapsules described here will enable high density suspension culture of hepatocytes or other cells and may be used as building blocks for engineering tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2017.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809154PMC
March 2017

Detecting cell-secreted growth factors in microfluidic devices using bead-based biosensors.

Microsyst Nanoeng 2017 3;3. Epub 2017 Jul 3.

Department of Biomedical Engineering, University of California, Davis, California 95616, USA.

Microfluidic systems provide an interesting alternative to standard macroscale cell cultures due to the decrease in the number of cells and reagents as well as the improved physiology of cells confined to small volumes. However, the tools available for cell-secreted molecules inside microfluidic devices remain limited. In this paper, we describe an integrated microsystem composed of a microfluidic device and a fluorescent microbead-based assay for the detection of the hepatocyte growth factor (HGF) and the transforming growth factor (TGF)-β1 secreted by primary hepatocytes. This microfluidic system is designed to separate a cell culture chamber from sensing chambers using a permeable hydrogel barrier. Cell-secreted HGF and TGF-β1 diffuse through the hydrogel barrier into adjacent sensing channels and are detected using fluorescent microbead-based sensors. The specificity of sensing microbeads is defined by the choice of antibodies; therefore, our microfluidic culture system and sensing microbeads may be applied to a variety of cells and cell-secreted factors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/micronano.2017.25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023413PMC
July 2017

Microfluidic generation of alginate microgels for the controlled delivery of lentivectors.

J Mater Chem B 2016 Nov 17;4(43):6989-6999. Epub 2016 Oct 17.

Department of Biomedical Engineering, University of California, 1 Shields Ave., Davis, CA 95616, USA.

Lentivectors are widely used for gene delivery and have been increasingly tested in clinical trials. However, achieving safe, localized, and sufficient gene expression remain key challenges for effective lentivectoral therapy. Localized and efficient gene expression can be promoted by developing material systems to deliver lentivectors. Here, we address the utility of microgel encapsulation as a strategy for the controlled release of lentivectors. Three distinct routes for ionotropic gelation of alginate were incorporated into microfluidic templating to create lentivector-loaded microgels. Comparisons of the three microgels revealed marked differences in mechanical properties, crosslinking environment, and ultimately lentivector release and functional gene expression in vitro. Gelation with chelated calcium demonstrated low utility for gene delivery due to a loss of lentivector function with acidic gelation conditions. Both calcium carbonate gelation, and calcium chloride gelation, preserved lentivector function with a more sustained transduction and gene expression over 4 days observed with calcium chloride gelated microgels. The validation of these two strategies for lentivector microencapsulation may provide a platform for controlled gene delivery.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c6tb02150fDOI Listing
November 2016

Ductular reaction-on-a-chip: Microfluidic co-cultures to study stem cell fate selection during liver injury.

Sci Rep 2016 10 31;6:36077. Epub 2016 Oct 31.

Department of Biomedical Engineering, University of California Davis, CA 95616, USA.

Liver injury modulates local microenvironment, triggering production of signals that instruct stem cell fate choices. In this study, we employed a microfluidic co-culture system to recreate important interactions in the liver stem cell niche, those between adult hepatocytes and liver progenitor cells (LPCs). We demonstrate that pluripotent stem cell-derived LPCs choose hepatic fate when cultured next to healthy hepatocytes but begin biliary differentiation program when co-cultured with injured hepatocytes. We connect this fate selection to skewing in production of hepatocyte growth factor (HGF) and transforming growth factor (TGF)-β1 caused by injury. Significantly, biliary fate selection of LPCs was not observed in the absence of hepatocytes nor did it happen in the presence of TGF-β inhibitors. Our study demonstrates that microfluidic culture systems may offer an interesting new tool for dissecting cellular interactions leading to aberrant stem cell differentiation during injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep36077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5086854PMC
October 2016

Cell biology is different in small volumes: endogenous signals shape phenotype of primary hepatocytes cultured in microfluidic channels.

Sci Rep 2016 Sep 29;6:33980. Epub 2016 Sep 29.

Department of Biomedical Engineering, University of California Davis, CA 95616, USA.

The approaches for maintaining hepatocytes in vitro are aimed at recapitulating aspects of the native liver microenvironment through the use of co-cultures, surface coatings and 3D spheroids. This study highlights the effects of spatial confinement-a less studied component of the in vivo microenvironment. We demonstrate that hepatocytes cultured in low-volume microfluidic channels (microchambers) retain differentiated hepatic phenotype for 21 days whereas cells cultured in regular culture plates under identical conditions de-differentiate after 7 days. Careful consideration of nutrient delivery and oxygen tension suggested that these factors could not solely account for enhanced cell function in microchambers. Through a series of experiments involving microfluidic chambers of various heights and inhibition of key molecular pathways, we confirmed that phenotype of hepatocytes in small volumes was shaped by endogenous signals, both hepato-inductive growth factors (GFs) such as hepatocyte growth factor (HGF) and hepato-disruptive GFs such as transforming growth factor (TGF)-β1. Hepatocytes are not generally thought of as significant producers of GFs-this role is typically assigned to nonparenchymal cells of the liver. Our study demonstrates that, in an appropriate microenvironment, hepatocytes produce hepato-inductive and pro-fibrogenic signals at the levels sufficient to shape their phenotype and function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041105PMC
http://dx.doi.org/10.1038/srep33980DOI Listing
September 2016

Functional imaging of neuron-astrocyte interactions in a compartmentalized microfluidic device.

Microsyst Nanoeng 2016 29;2:15045. Epub 2016 Feb 29.

Department of Biochemistry and Molecular Medicine, University of California, Davis, CA 95616, USA.

Traditional approaches in cultivating neural cells in a dish without orienting their interactions have had only limited success in revealing neural network properties. To enhance the experimental capabilities of studying neural circuitry , we designed an experimental system combining concepts of micropatterned surfaces, microfluidic devices and genetically encoded biosensors. Micropatterning was used to position neurons and astrocytes in defined locations and guide interactions between the two cell types. Microfluidic chambers were placed atop micropatterned surfaces to allow delivery of different pharmacological agents or viral vectors to the desired cell types. In this device, astrocytes and neurons communicated through grooves molded into the floor of the microfluidic device. By combining microfluidics with genetically encoded calcium indicators as functional readouts, we further demonstrated the utility of this device for analyzing neuron-neuron and neuron-astrocyte interactions under both healthy and pathophysiological conditions. We found that both spontaneous and evoked calcium dynamics in astrocytes can be modulated by interactions with neurons. In the future, we foresee employing the microdevices described here for studying mechanisms of neurological disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/micronano.2015.45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6444726PMC
February 2016

Embryonic Stem Cells Cultured in Microfluidic Chambers Take Control of Their Fate by Producing Endogenous Signals Including LIF.

Stem Cells 2016 06 25;34(6):1501-12. Epub 2016 Feb 25.

Department of Biomedical Engineering, University of California, Davis, Davis, California, USA.

It is important to understand the role played by endogenous signals in shaping stem cell fate decisions to develop better culture systems and to improve understanding of development processes. In this study, we describe the behavior of mouse embryonic stem cells (mESCs) inside microfluidic chambers (microchambers) operated under conditions of minimal perfusion. mESCs inside microchambers formed colonies and expressed markers of pluripotency in the absence of feeders or pluripotency-inducing signals such as leukemia inhibitory factor (LIF), while mESCs in standard cultureware differentiated rapidly. In a series of experiments, we demonstrate that remarkable differences in stem cell phenotype are due to endogenous production of LIF and other growth factors brought upon by cultivation in confines of a microchamber in the absence of perfusion (dilution). At the protein level, mESCs produced ∼140 times more LIF inside microchambers than under standard culture conditions. In addition, we demonstrate that pluripotent phenotype of stem cells could be degraded by increasing the height (volume) of the microchamber. Furthermore, we show that inhibition of LIF in microchambers, via the JAK/STAT3 pathway, leads to preferential differentiation into mesoderm that is driven by bone morphogenetic protein (BMP)-4. Collectively, we demonstrate for the first time that it is possible to design a cell culture system where stem cell fate is controlled solely by the endogenous signals. Our study may help shift the paradigm of stem cell cultivation away from relying on expensive exogenous molecules such as growth factors and toward designing culture chambers for harnessing endogenous signals. Stem Cells 2016;34:1501-1512.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.2324DOI Listing
June 2016

Sensing Conductive Hydrogels for Rapid Detection of Cytokines in Blood.

Adv Healthc Mater 2016 Mar 22;5(6):659-64, 627. Epub 2016 Jan 22.

Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA.

Conducting polymer hydrogel is fabricated atop gold or ITO electrodes and is functionalized with monoclonal antibodies. Binding of interferon-γ molecules causes redox properties of conductive hydrogel to change in a concentration-dependent fashion without the need for washing or sample handling steps. This conductive hydrogel remains functional in a fouling media such as whole blood.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adhm.201500571DOI Listing
March 2016

Microfluidic fabrication of bioactive microgels for rapid formation and enhanced differentiation of stem cell spheroids.

Acta Biomater 2016 Apr 13;34:125-132. Epub 2016 Jan 13.

University of California, Davis, 451 Health Sciences Dr., Davis, CA, USA. Electronic address:

Unlabelled: A major challenge in tissue engineering is to develop robust protocols for differentiating ES and iPS cells to functional adult tissues at a clinically relevant scale. The goal of this study is to develop a high throughput platform for generating bioactive, stem cell-laden microgels to direct differentiation in a well-defined microenvironment. We describe a droplet microfluidics system for fabricating microgels composed of polyethylene glycol and heparin, with tunable geometric, mechanical, and chemical properties, at kHz rates. Heparin-containing hydrogel particles sequestered growth factors Nodal and FGF-2, which are implicated in specifying pluripotent cells to definitive endoderm. Mouse ESCs were encapsulated into heparin microgels with a single dose of Nodal and FGF-2, and expressed high levels of endoderm markers Sox17 and FoxA2 after 5 days. These results highlight the use of microencapsulation for tailoring the stem cell microenvironment to promote directed differentiation, and may provide a straightforward path to large scale bioprocessing in the future.

Statement Of Significance: Multicellular spheroids and microtissues are valuable for tissue engineering, but fabrication approaches typically sacrifice either precision or throughput. Microfluidic encapsulation in polymeric biomaterials is a promising technique for rapidly generating cell aggregates with excellent control of microenvironmental parameters. Here we describe the microfluidic fabrication of bioactive, heparin-based microgels, and demonstrate the adsorption of heparin-binding growth factors for enhancing directed differentiation of embryonic stem cells toward endoderm. This approach also facilitated a ∼90-fold decrease in consumption of exogenous growth factors compared to conventional differentiation protocols.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.actbio.2016.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811722PMC
April 2016

Microfluidic co-cultures with hydrogel-based ligand trap to study paracrine signals giving rise to cancer drug resistance.

Lab Chip 2015 Dec 6;15(24):4614-24. Epub 2015 Nov 6.

Department of Biomedical Engineering, One Shields Ave, University of California, Davis, CA 95618, USA.

Targeted cancer therapies are designed to deactivate signaling pathways used by cancer cells for survival. However, cancer cells are often able to adapt by activating alternative survival pathways, thereby acquiring drug resistance. An emerging theory is that autocrine or paracrine growth factor signaling in the cancer microenvironment represent an important mechanism of drug resistance. In the present study we wanted to examine whether paracrine interactions between groups of melanoma cells result in resistance to vemurafenib - an FDA approved drug targeting the BRAF mutation in metastatic melanoma. We used a vemurafenib-resistant melanoma model which secretes fibroblast growth factor (FGF)-2 to test our hypothesis that this is a key paracrine mediator of resistance to vemurafenib. Sensitive cells treated with media conditioned by resistant cells did not protect from the effects of vemurafenib. To query paracrine interactions further we fabricated a microfluidic co-culture device with two parallel compartments, separated by a 100 μm wide hydrogel barrier. The gel barrier prevented resorting/contact of cells while permitting paracrine cross-talk. In this microfluidic system, sensitive cells did become refractive to the effects of vemurafenib when cultured adjacent to resistant cells. Importantly, incorporation of FGF-2 capture probes into the gel barrier separating the two cell types prevented onset of resistance to vemurafenib. Microfluidic tools described here allow for more sensitive analysis of paracrine signals, may help better understand signaling in the cancer microenvironment and may enable development of more effective cancer therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c5lc00948kDOI Listing
December 2015

Microfluidic compartments with sensing microbeads for dynamic monitoring of cytokine and exosome release from single cells.

Analyst 2016 Jan;141(2):679-88

Department of Biomedical Engineering, University of California, Davis, California 95616, USA.

Monitoring activity of single cells has high significance for basic science and diagnostic applications. Here we describe a reconfigurable microfluidic device for confining single cells along with antibody-modified sensing beads inside 20 picoliter (pL) microcompartments for monitoring cellular secretory activity. An array of ∼7000 microchambers fabricated in the roof of the reconfigurable microfluidic device could be raised or lowered by applying negative pressure. The floor of the device was micropatterned to contain cell attachment sites in registration with the microcompartments. Using this set-up, we demonstrated the detection of inflammatory cytokine IFN-γ and exosomes from single immune cells and cancer cells respectively. The detection scheme was similar in both cases: cells were first captured on the surface inside the microfluidic device, then sensing microbeads were introduced into the device so that, once the microcompartments were lowered, single cells and microbeads became confined together. The liquid bathing the beads and the cells inside the compartments also contained fluorescently-labeled secondary antibodies (Abs). The capture of cell-secreted molecules onto microbeads was followed by binding of secondary antibodies - this caused microbeads to become fluorescent. The fluorescence intensity of the microbeads changed over time, providing dynamics of single cell secretory activity. The microdevice described here may be particularly useful in the cases where panning upstream of sensing is required or to analyze secretory activity of anchorage-dependent cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c5an01648gDOI Listing
January 2016