Publications by authors named "Gulnaz Stybayeva"

27 Publications

  • Page 1 of 1

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

Predictive value of olfactory and taste symptoms in the diagnosis of COVID-19: A systematic review and meta-analysis.

Clin Exp Otorhinolaryngol 2021 Jan 25. Epub 2021 Jan 25.

Department of Otolaryngology-Head and Neck Surgery, Bucheon St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Korea.

Objectives: This study evaluated the diagnostic value of the various symptoms of COVID-19 in the screening of this disease.

Methods: Two authors (working independently) comprehensively reviewed six databases (PubMed, Cochrane database, Embase, Web of Science, SCOPUS, and Google Scholar) from their dates of inception until November 2020. Patient-reported symptoms, including otolaryngologic and general symptoms, were evaluated for their predictive values in adults who underwent testing for COVID-19. True-positive, true-negative, false-positive, and false-negative data were extracted from each study. The methodological quality of included studies was evaluated using the Quality Assessment of Diagnostic Accuracy Studies tool (ver. 2).

Results: Twenty-eight prospective and retrospective studies were included in the meta-analysis. The diagnostic odds ratio (DOR) of a change in olfaction and/or taste was 10.20 (95% confidence interval [CI], 8.43; 12.34). The area under the summary receiver operating characteristic curve was 0.8. Olfactory and/or taste changes had a low sensitivity (0.57, 95%CI: 0.47; 0.66) but moderate negative (0.78, 95%CI: 0.69; 0.85] and positive (0.78, 95%CI: 0.66; 0.87) predictive values and a high specificity (0.91, (95%CI: 0.83; 0.96). Olfactory and/or taste changes had a higher diagnostic value than the other otolaryngologic symptoms, a higher DOR and specificity, and a similar or higher diagnostic value than the other general symptoms.

Conclusion: Among otolaryngologic symptoms, olfactory and/or taste dysfunction was the most highly associated with COVID-19 and its general symptoms and should be considered when screening for the disease.
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http://dx.doi.org/10.21053/ceo.2020.02369DOI Listing
January 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.
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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.
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http://dx.doi.org/10.1152/ajpcell.00094.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509267PMC
September 2020

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

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.
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http://dx.doi.org/10.1021/acssensors.7b00486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311073PMC
November 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.
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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.
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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.
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http://dx.doi.org/10.1038/micronano.2017.25DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023413PMC
July 2017

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.
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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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041105PMC
http://dx.doi.org/10.1038/srep33980DOI Listing
September 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.
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http://dx.doi.org/10.1002/adhm.201500571DOI Listing
March 2016

Liver injury-on-a-chip: microfluidic co-cultures with integrated biosensors for monitoring liver cell signaling during injury.

Lab Chip 2015 Dec;15(23):4467-78

Department of Biomedical Engineering, University of California, Davis, 451 Health Sciences, Davis, CA, USA.

Tissue injury triggers complex communication between cells via secreted signaling molecules such as cytokines and growth factors. Discerning when and where these signals begin and how they propagate over time is very challenging with existing cell culture and analysis tools. The goal of this study was to develop new tools in the form of microfluidic co-cultures with integrated biosensors for local and continuous monitoring of secreted signals. Specifically, we focused on how alcohol injury affects TGF-β signaling between two liver cell types, hepatocytes and stellate cells. Activation of stellate cells happens early during liver injury and is at the center of liver fibrosis. We demonstrated that alcohol injury to microfluidic co-cultures caused significantly higher levels of stellate cell activation compared to conditioned media and transwell injury experiments. This highlighted the advantage of the microfluidic co-culture: placement of two cell types in close proximity to ensure high local concentrations of injury-promoting secreted signals. Next, we developed a microsystem consisting of five chambers, two for co-culturing hepatocytes with stellate cells and three additional chambers containing miniature aptamer-modified electrodes for monitoring secreted TGF-β. Importantly, the walls separating microfluidic chambers were actuatable; they could be raised or lowered to create different configurations of the device. The use of reconfigurable microfluidics and miniature biosensors revealed that alcohol injury causes hepatocytes to secrete TGF-β molecules, which diffuse over to neighboring stellate cells and trigger production of additional TGF-β from stellate cells. Our results lend credence to the emerging view of hepatocytes as active participants of liver injury. Broadly speaking, our microsystem makes it possible to monitor paracrine crosstalk between two cell types communicating via the same signaling molecule (e.g. TGF-β).
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http://dx.doi.org/10.1039/c5lc00874cDOI Listing
December 2015

Reconfigurable microfluidic device with integrated antibody arrays for capture, multiplexed stimulation, and cytokine profiling of human monocytes.

Biomicrofluidics 2015 Jul 6;9(4):044115. Epub 2015 Aug 6.

Department of Biomedical Engineering, University of California , 451 Health Sciences Drive, Suite 2619, Davis, California 95616, USA.

Monocytes represent a class of immune cells that play a key role in the innate and adaptive immune response against infections. One mechanism employed by monocytes for sensing foreign antigens is via toll-like receptors (TLRs)-transmembrane proteins that distinguish classes of foreign pathogens, for example, bacteria (TLR4, 5, and 9) vs. fungi (TLR2) vs. viruses (TLR3, 7, and 8). Binding of antigens activates a signaling cascade through TLR receptors that culminate in secretion of inflammatory cytokines. Detection of these cytokines can provide valuable clinical data for drug developers and disease investigations, but this usually requires a large sample volume and can be technically inefficient with traditional techniques such as flow cytometry, enzyme-linked immunosorbent assay, or luminex. This paper describes an approach whereby antibody arrays for capturing cells and secreted cytokines are encapsulated within a microfluidic device that can be reconfigured to operate in serial or parallel mode. In serial mode, the device represents one long channel that may be perfused with a small volume of minimally processed blood. Once monocytes are captured onto antibody spots imprinted into the floor of the device, the straight channel is reconfigured to form nine individually perfusable chambers. To prove this concept, the microfluidic platform was used to capture monocytes from minimally processed human blood in serial mode and then to stimulate monocytes with different TLR agonists in parallel mode. Three cytokines, tumor necrosis factor-α, interleukin (IL)-6, and IL-10, were detected using anti-cytokine antibody arrays integrated into each of the six chambers. We foresee further use of this device in applications such as pediatric immunology or drug/vaccine testing where it is important to balance small sample volume with the need for high information content.
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http://dx.doi.org/10.1063/1.4928128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529433PMC
July 2015

HLA Class I Depleted hESC as a Source of Hypoimmunogenic Cells for Tissue Engineering Applications.

Tissue Eng Part A 2015 Oct 10;21(19-20):2559-71. Epub 2015 Sep 10.

1 Pharmacology and Physiology Department, School of Medicine and Health Sciences, The George Washington University , Washington, District of Columbia.

Background: Rapidly improving protocols for the derivation of autologous cells from stem cell sources is a welcome development. However, there are many circumstances when off-the-shelf universally immunocompatible cells may be needed. Embryonic stem cells (ESCs) provide a unique opportunity to modify the original source of differentiated cells to minimize their rejection by nonautologous hosts.

Hypothesis: Immune rejection of nonautologous human embryonic stem cell (hESC) derivatives can be reduced by downregulating human leukocyte antigen (HLA) class I molecules, without affecting the ability of these cells to differentiate into specific lineages.

Methods And Results: Beta-2-microglobulin (B2M) expression was decreased by lentiviral transduction using human anti-HLA class I light-chain B2M short hairpin RNA. mRNA levels of B2M were decreased by 90% in a RUES2-modified hESC line, as determined by quantitative real time-polymerase chain reaction analysis. The transduced cells were selected under puromycin pressure and maintained in an undifferentiated state. The latter was confirmed by Oct4 and Nanog expression, and by the formation of characteristic round-shaped colonies. B2M downregulation led to diminished HLA-I expression on the cell surface, as determined by flow cytometry. When used as target cells in a mixed lymphocyte reaction assay, transduced hESCs and their differentiated derivatives did not stimulate allogeneic T-cell proliferation. Using a cardiac differentiation protocol, transduced hESCs formed a confluent layer of cardiac myocytes and maintained a low level of B2M expression. Transduced hESCs were also successfully differentiated into a hepatic lineage, validating their capacity to differentiate into multiple lineages.

Conclusions: HLA-I depletion does not preclude hESC differentiation into cardiac or hepatic lineages. This methodology can be used to engineer tissue from nonautologous hESC sources with improved immunocompatibility.
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http://dx.doi.org/10.1089/ten.TEA.2015.0105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605353PMC
October 2015

Exposure to phthalates affects calcium handling and intercellular connectivity of human stem cell-derived cardiomyocytes.

PLoS One 2015 23;10(3):e0121927. Epub 2015 Mar 23.

Department of Pharmacology & Physiology, The George Washington University, Washington, DC, United States of America.

Background: The pervasive nature of plastics has raised concerns about the impact of continuous exposure to plastic additives on human health. Of particular concern is the use of phthalates in the production of flexible polyvinyl chloride (PVC) products. Di-2-ethylhexyl-phthalate (DEHP) is a commonly used phthalate ester plasticizer that imparts flexibility and elasticity to PVC products. Recent epidemiological studies have reported correlations between urinary phthalate concentrations and cardiovascular disease, including an increased risk of high blood pressure and coronary risk. Yet, there is little direct evidence linking phthalate exposure to adverse effects in human cells, including cardiomyocytes.

Methods And Results: The effect of DEHP on calcium handling was examined using monolayers of gCAMP3 human embryonic stem cell-derived cardiomyocytes, which contain an endogenous calcium sensor. Cardiomyocytes were exposed to DEHP (5 - 50 μg/mL), and calcium transients were recorded using a Zeiss confocal imaging system. DEHP exposure (24 - 72 hr) had a negative chronotropic and inotropic effect on cardiomyocytes, increased the minimum threshold voltage required for external pacing, and modified connexin-43 expression. Application of Wy-14,643 (100 μM), an agonist for the peroxisome proliferator-activated receptor alpha, did not replicate DEHP's effects on calcium transient morphology or spontaneous beating rate.

Conclusions: Phthalates can affect the normal physiology of human cardiomyocytes, including DEHP elicited perturbations in cardiac calcium handling and intercellular connectivity. Our findings call for additional studies to clarify the extent by which phthalate exposure can alter cardiac function, particularly in vulnerable patient populations who are at risk for high phthalate exposure.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0121927PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370601PMC
February 2016

Photodegradable hydrogels for capture, detection, and release of live cells.

Angew Chem Int Ed Engl 2014 Jul 16;53(31):8221-4. Epub 2014 Jun 16.

Department of Biomedical Engineering, University of California Davis, One Shields Ave, Davis, CA 95616 (USA).

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T-cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV-induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95% purity of CD4 and CD8 T-cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti-CD4 and anti-TNF-α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence "halo" after immunofluorescent staining and could be retrieved by site-specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.
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http://dx.doi.org/10.1002/anie.201404323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380505PMC
July 2014

Reconfigurable microfluidics combined with antibody microarrays for enhanced detection of T-cell secreted cytokines.

Biomicrofluidics 2013 14;7(2):24105. Epub 2013 Mar 14.

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

Cytokines are small proteins secreted by leukocytes in blood in response to infections, thus offering valuable diagnostic information. Given that the same cytokines may be produced by different leukocyte subsets in blood, it is beneficial to connect production of cytokines to specific cell types. In this paper, we describe integration of antibody (Ab) microarrays into a microfluidic device to enable enhanced cytokine detection. The Ab arrays contain spots specific to cell-surface antigens as well as anti-cytokine detection spots. Infusion of blood into a microfluidic device results in the capture of specific leukocytes (CD4 T-cells) and is followed by detection of secreted cytokines on the neighboring Ab spots using sandwich immunoassay. The enhancement of cytokine signal comes from leveraging the concept of reconfigurable microfluidics. A three layer polydimethylsiloxane microfluidic device is fabricated so as to contain six microchambers (1 mm × 1 mm × 30 μm) in the ceiling of the device. Once the T-cell capture is complete, the device is reconfigured by withdrawing liquid from the channel, causing the chambers to collapse onto Ab arrays and enclose cell/anti-cytokine spots within a 30 nl volume. In a set of proof-of-concept experiments, we demonstrate that ∼90% pure CD4 T-cells can be captured inside the device and that signals for three important T-cell secreted cytokines, tissue necrosis factor-alpha, interferon-gamma, and interleukin-2, may be enhanced by 2 to 3 folds through the use of reconfigurable microfluidics.
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http://dx.doi.org/10.1063/1.4795423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612111PMC
January 2014

A zinc finger protein array for the visual detection of specific DNA sequences for diagnostic applications.

Nucleic Acids Res 2011 Mar 5;39(5):e29. Epub 2010 Dec 5.

Genome Center, Department of Pharmacology, University of California, Davis, CA 95616, USA.

The visual detection of specific double-stranded DNA sequences possesses great potential for the development of diagnostics. Zinc finger domains provide a powerful scaffold for creating custom DNA-binding proteins that recognize specific DNA sequences. We previously demonstrated sequence-enabled reassembly of TEM-1 β-lactamase (SEER-LAC), a system consisting of two inactive fragments of β-lactamase each linked to engineered zinc finger proteins (ZFPs). Here the SEER-LAC system was applied to develop ZFP arrays that function as simple devices to identify bacterial double-stranded DNA sequences. The ZFP arrays provided a quantitative assay with a detection limit of 50 fmol of target DNA. The method could distinguish target DNA from non-target DNA within 5 min. The ZFP arrays provided sufficient sensitivity and high specificity to recognize specific DNA sequences. These results suggest that ZFP arrays have the potential to be developed into a simple and rapid point-of-care (POC) diagnostic for the multiplexed detection of pathogens.
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http://dx.doi.org/10.1093/nar/gkq1214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3061069PMC
March 2011

Detecting interferon-gamma release from human CD4 T-cells using surface plasmon resonance.

Colloids Surf B Biointerfaces 2010 Oct 25;80(2):251-5. Epub 2010 Jun 25.

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

Cytokine secretion by leukocytes is an important indicator of immune response to pathogens and therefore has significant implications in disease diagnostics. Given heterogeneity of leukocyte subsets and the ability of multiple cell subsets to secrete the same cytokines, connecting cytokine production to a specific leukocyte subset is a distinct challenge. In the present paper we describe a strategy combining antibody (Ab)-based affinity cell separation and surface plasmon resonance (SPR) for capturing human CD4 T-cells and for label-free detection of cell-secreted interferon (IFN)-gamma--an important inflammatory cytokine. Human blood was introduced into a flow chamber modified with anti-CD4 Abs resulting in capture of CD4(+) T-cells. After mitogenic activation of cells inside the flow chamber, culture medium was routed onto an SPR chip modified with monoclonal IFN-gamma Abs. SPR signal observed in this experiment correlated with cytokine production by T-cells. The strategy of combining SPR detection with cell purification may be used in the future for label-free, sensitive detection of multiple cytokines or proteins secreted by the desired cell subset.
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http://dx.doi.org/10.1016/j.colsurfb.2010.06.015DOI Listing
October 2010

Lensfree holographic imaging of antibody microarrays for high-throughput detection of leukocyte numbers and function.

Anal Chem 2010 May;82(9):3736-44

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

Characterization of leukocytes is an integral part of blood analysis and blood-based diagnostics. In the present paper, we combine lensless holographic imaging with antibody microarrays for rapid and multiparametric analysis of leukocytes from human blood. Monoclonal antibodies (Abs) specific for leukocyte surface antigens (CD4 and CD8) and cytokines (TNF-alpha, IFN-gamma, IL-2) were printed in an array so as to juxtapose cell capture and cytokine detection antibody (Ab) spots. Integration of Ab microarrays into a microfluidic flow chamber (4 muL volume) followed by incubation with human blood resulted in capture of CD4 and CD8 T-cells on specific Ab spots. On-chip mitogenic activation of these cells induced release of cytokine molecules that were subsequently captured on neighboring anticytokine Ab spots. The binding of IL-2, TNF-alpha, and IFN-gamma molecules on their respective Ab spots was detected using horseradish peroxidase (HRP)-labeled anticytokine Abs and a visible color reagent. Lensfree holographic imaging was then used to rapidly ( approximately 4 s) enumerate CD4 and CD8 T-lymphocytes captured on Ab spots and to quantify the cytokine signal emanating from IL-2, TNF-alpha, and IFN-gamma spots on the same chip. To demonstrate the utility of our approach for infectious disease monitoring, blood samples of healthy volunteers and human immunodeficiency virus (HIV)-infected patients were analyzed to determine the CD4/CD8 ratio, an important HIV/AIDS diagnostic marker. The ratio obtained by lensfree on-chip imaging of CD4 and CD8 T-cells captured on Ab spots was in close agreement with conventional microscopy-based cell counting. The present paper, describing tandem use of Ab microarrays and lensfree holographic imaging, paves the way for future development of miniature cytometry devices for multiparametric blood analysis at the point of care or in a resource-limited setting.
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http://dx.doi.org/10.1021/ac100142aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2864520PMC
May 2010

Detecting cytokine release from single T-cells.

Anal Chem 2009 Oct;81(19):8150-6

Biomedical Engineering, University of California, Davis, USA.

The cytokine production by leukocytes correlates with body's ability to mount an immune response and therefore has high diagnostic value. In the present study we employed microfabricated surfaces to capture T-cells from minimally processed human blood, arrange these cells into a single cell array, and then detect interferon (IFN)-gamma released from individual cells. The fabrication of cell capture surfaces started with coating a silane-modified glass slide with a uniform layer of poly(ethylene glycol) (PEG) hydrogel. The hydrogel-coated slide was lyophilized and then incubated with a mixture of monoclonal anti-IFN-gamma and anti-CD4 antibodies (Abs). To define sites for single cell attachment, PEG hydrogel microwells (20 microm diameter) were photolithographically patterned on top of the Ab-containing hydrogel layer. This micropatterning process resulted in fabrication of PEG hydrogel microwells with Ab-decorated bottom and nonfouling walls. To minimize the blood volume requirement and to precisely define shear stress conditions, the engineered surface was enclosed inside a PDMS-based microfluidic device. Introduction of red blood cell (RBC) depleted whole human blood followed by controlled washing led to the isolation of individual CD4 T-cells within PEG microwells. Mitogenic activation and immunofluorescent staining performed inside the microfluidic chamber revealed IFN-gamma cytokine signal colocalized with specific T-cells. The device and process presented here will be expanded in the future to enable multiparametric functional analysis of immune cells organized into high density single cell arrays.
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http://dx.doi.org/10.1021/ac901390jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2755614PMC
October 2009

Micropatterned co-cultures of T-lymphocytes and epithelial cells as a model of mucosal immune system.

Biochem Biophys Res Commun 2009 Mar 31;380(3):575-80. Epub 2009 Jan 31.

Department of Biomedical Engineering, University of California, 451 East Health Sciences Drive, Davis, CA 95616, USA.

Gut-associated lymphoid tissue is a major target and reservoir of human immunodeficiency virus (HIV)-infected T-cells. Our studies seek to recapitulate, in vitro, interactions between HIV-infected T-lymphocytes and intestinal epithelial cells in order to investigate the mechanisms underlying the disruption of normal epithelial cell and barrier function. Here, we describe a novel approach for creating co-cultures of healthy or HIV-infected T-lymphocytes (Jurkat) and human intestinal epithelial (HT-29) cells where both cell types are positioned on the same surface in a price spatial configuration (micropattern). This co-culture method simplified observation/monitoring of the two cell types and was particularly suited for laser microdissection-based retrieval of the desired cells for downstream gene expressions studies. DNA microarray analysis of epithelial cells retrieved from co-cultures with HIV-1-infected vs. uninfected Jurkat cells revealed that epithelial cells from HIV-infected co-cultures exhibited gene expression patterns consistent with disruption of epithelial barrier formation. Overall, the micropatterned co-culture system described here is envisioned as a valuable new tool for delineating how HIV and other infections contribute to dysfunction of mucosal epithelium.
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http://dx.doi.org/10.1016/j.bbrc.2009.01.164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2659419PMC
March 2009

A microdevice for multiplexed detection of T-cell-secreted cytokines.

Lab Chip 2008 Dec 30;8(12):2197-205. Epub 2008 Sep 30.

Department of Biomedical Engineering, University of California, Davis, 451 East Health Sciences St. #2619, Davis, CA 95616, USA.

Cytokines are produced by immune cells in response to viral or bacterial pathogens and therefore have significant diagnostic value. The goal of the present study was to develop a miniature device for detection of interleukin (IL)-2 and interferon (IFN)-gamma cytokines secreted by a small population of CD4 and CD8 T-cells. Microarrays of T-cell- and cytokine-specific Ab spots were printed onto poly(ethylene glycol) (PEG) hydrogel-coated glass slides and enclosed inside a microfluidic device, creating a miniature ( approximately 3 microL) immunoreaction chamber. Introduction of the red blood cell (RBC) depleted whole human blood into the microfluidic device followed by washing at a pre-defined shear stress resulted in isolation of pure CD4 and CD8 T-cells on their respective Ab spots. Importantly, the cells became localized next to anti-IL-2 and -IFN-gamma Ab spots. Mitogenic activation of the captured T-cells was followed by immunofluorescent staining (all steps carried out inside a microfluidic device), revealing concentration gradients of surface-bound cytokine molecules. A microarray scanner was then used to quantify the concentration of IFN-gamma and IL-2 near CD4 and CD8 T-cells. This study represents one of the first demonstrations of a microdevice for capturing desired T-cell subsets from a small blood volume and determining, on-chip, cytokine profiles of the isolated cells. Such a microdevice is envisioned as an immunology tool for multi-parametric analysis of T-cell function with direct applications in diagnosis/monitoring of HIV and other infectious diseases.
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http://dx.doi.org/10.1039/b810244aDOI Listing
December 2008

Multifunctional protein microarrays for cultivation of cells and immunodetection of secreted cellular products.

Anal Chem 2008 Aug 22;80(16):6351-7. Epub 2008 Jul 22.

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

The microarray format is being used extensively for combinatorial screening of cellular interactions with proteins, small molecules, or biomaterials. The utility of microarray-based cell cultivation approaches may be enhanced further by incorporating biosensing elements alongside the cell-adhesive ligands to enable local detection of secreted cellular products. The concept of combining cells and sensing elements in the same microarray is demonstrated in the present paper with hepatocytes serving as a model cellular system. Robotic microarraying was employed to print arrays of 300-mum-diameter collagen (I) spots alongside the antibody (Ab) spots specific to liver proteins: albumin and alpha1-antitrypsin (alpha1-AT). Protein microarrays were printed onto poly(ethylene glycol) hydrogel-coated glass slides, thus eliminating nonspecific adsorption of cells or proteins. When incubated with printed microarrays, hepatocytes became localized on collagen (I) domains but did not attach on Ab spots or elsewhere on hydrogel-coated glass substrates. Liver-specific proteins secreted by hepatocytes were captured on Ab domains in the immediate vicinity of the cells, detected with a sandwich immunofluorescent assay and quantified using a microarray scanner. Importantly, hepatic albumin and alpha1-AT production detected in the microarray was comparable to enzyme-linked immunosorbent assay measurements of these proteins. In the future, the juxtaposition of sensing Ab regions with cell arrays will be particularly useful for the detection of local appearance or loss of phenotype of cells interacting with the printed components of the cellular microenvironment.
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http://dx.doi.org/10.1021/ac8007626DOI Listing
August 2008

Phenotypic and molecular characterizations of Yersinia pestis isolates from Kazakhstan and adjacent regions.

Microbiology (Reading) 2007 Jan;153(Pt 1):169-77

Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, 3500 Rampart Rd, Fort Collins, CO 80522, USA.

Recent interest in characterizing infectious agents associated with bioterrorism has resulted in the development of effective pathogen genotyping systems, but this information is rarely combined with phenotypic data. Yersinia pestis, the aetiological agent of plague, has been well defined genotypically on local and worldwide scales using multi-locus variable number tandem repeat analysis (MLVA), with emphasis on evolutionary patterns using old isolate collections from countries where Y. pestis has existed the longest. Worldwide MLVA studies are largely based on isolates that have been in long-term laboratory culture and storage, or on field material from parts of the world where Y. pestis has potentially circulated in nature for thousands of years. Diversity in these isolates suggests that they may no longer represent the wild-type organism phenotypically, including the possibility of altered pathogenicity. This study focused on the phenotypic and genotypic properties of 48 Y. pestis isolates collected from 10 plague foci in and bordering Kazakhstan. Phenotypic characterization was based on diagnostic tests typically performed in reference laboratories working with Y. pestis. MLVA was used to define the genotypic relationships between the central-Asian isolates and a group of North American isolates, and to examine Kazakh Y. pestis diversity according to predefined plague foci and on an intermediate geographical scale. Phenotypic properties revealed that a large portion of this collection lacks one or more plasmids necessary to complete the blocked flea/mammal transmission cycle, has lost Congo red binding capabilities (Pgm-), or both. MLVA analysis classified isolates into previously identified biovars, and in some cases groups of isolates collected within the same plague focus formed a clade. Overall, MLVA did not distinguish unique phylogeographical groups of Y. pestis isolates as defined by plague foci and indicated higher genetic diversity among older biovars.
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http://dx.doi.org/10.1099/mic.0.29059-0DOI Listing
January 2007