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Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted by cells. EVs contain biological information related to parental cells and provide biomarkers for disease diagnosis. We have previously shown that the levels of podocin and nephrin expression on urinary EVs may be used to diagnose renal injury associated with preeclampsia. This paper describes a nanoparticle-enabled immunoassay integrated with an electrochemical plate for quantifying podocin and nephrin expression in urinary EVs. The strategy entailed capturing EVs on an electrode surface and then labeling EVs with gold nanoparticles that are both functionalized with antibodies for target specificity and impregnated with redox-active metal ions for electrochemical detection. These immunoprobes produced an electrochemical redox signal proportional to the expression level of EV surface markers. Electrochemical immunoassays were carried out in a novel microtiter plate that contained 16 wells with working electrodes connected to onboard counter/reference electrodes via capillary valves. Upon validation with recombinant proteins, a microtiter plate was used for analysis of urinary EVs from healthy and preeclamptic pregnant women. This analysis revealed a higher podocin to nephrin ratio for preeclamptic women compared to healthy controls (4.31 vs 1.69) suggesting that this ratio may be used for disease diagnosis.

(1) Background: To determine the optimal cut-off values of two risk stratification systems to discriminate malignant thyroid nodules and to compare the diagnostic performance; (2) Methods: True and false positive and negative data were collected, and methodological quality was assessed for forty-six studies involving 39,085 patients; (3) Results: The highest area under the receiver operating characteristic (ROC) curve (AUC) of ACR and Kwak TI-RADS were 0.875 and 0.884. Based on the optimal sensitivity and specificity, the highest accuracy values of ROC curves or diagnostic odds ratios (DOR) were taken as the cut-off values for TR4 (moderate suspicious) and 4B. The sensitivity, specificity, DOR, and AUC by ACR (TR4) and Kwak TI-RADS (4B) for malignancy risk stratification of thyroid nodules were 94.3% and 96.4%; 52.2% and 53.7%; 17.5185 and 31.8051; 0.786 and 0.884, respectively. There were no significant differences in diagnostic accuracy in any of the direction comparisons of the two systems; (4) Conclusions: ACR and Kwak TI-RADS had good diagnostic performances (AUCs > 85%). Although we determined the best cut-off values in individual risk stratification systems based on statistical assessment, clinicians can adjust the optimal cut-off value according to the clinical purpose of the ultrasonography because raising or lowering cut-points leads to reciprocal changes in sensitivity and specificity.

Objectives. This study evaluated the diagnostic value of various symptoms of coronavirus disease 2019 (COVID-19) in screening for 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. The predictive value of patient-reported symptoms, including otolaryngologic and general symptoms, was evaluated 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 the 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 closely associated with COVID-19 and its general symptoms, and should therefore be considered when screening for the disease.

Liver cultures may be used for modeling disease progression, testing therapies and predicting drug induced liver injury. The complexity of the liver cultures has evolved over the years from monocultures of hepatocytes to co-cultures with non-parenchymal cells and finally to precision cut liver slices. The latter culture format retains biomolecular and cellular complexity of the native liver and therefore holds considerable promise for in vitro testing. However, liver slices remain functional for ~72 h in vitro and hold only limited utility for some of the disease modeling and therapy testing applications that require longer culture times. This paper describes a microfluidic device for longer-term maintenance of functional organotypic liver cultures. Our microfluidic culture system was designed to enable direct injection of liver tissue into a culture chamber through a valve-enabled side port. Liver tissue was embedded in collagen and remained functional for up to 31 days as highlighted by continued production of albumin. These organotypic cultures also produced bile and urea and expressed several enzymes involved in metabolism of xenobiotics. In contrast, matched liver tissue embedded in collagen and cultured in the same media in a 96-well plate lost its phenotype and function on the timescale of 3 to 5 days. The microfluidic organotypic liver cultures described here represent a significant advance in liver cultivation and may be used in the future for modeling liver diseases or for individualized selection of liver-directed therapies.

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.

We developed a microfluidic device for the rapid analysis of biomarkers in small volumes of whole blood. This device includes an onboard plasma separation module connected to a downstream bioanalysis module in which plasma mixes with reagents and the results of a colorimetric assay are recorded. Actuation of onboard microvalves within a bioanalysis module creates active mixing conditions that allowed us to achieve solution homogeneity within 5 min. To demonstrate utility, we carried out glucose detection in our device. With 5 μL of whole blood as an input, our microfluidic device enabled a time-to-answer of 10 min with a limit of detection of 0.21 ± 0.04 mM for glucose. This device has immediate applications for rapid and sensitive monitoring of hypoglycemia at the point of care (POC). Furthermore, our automated microfluidic device represents a platform technology that may be used to detect other biomarkers in whole blood.

There is increasing interest in utilizing in vitro cultures as patient avatars to develop personalized treatment for cancer. Typical cultures utilize Matrigel-coated plates and media to promote the proliferation of cancer cells as spheroids or tumor explants. However, standard culture conditions operate in large volumes and require a high concentration of cancer cells to initiate this process. Other limitations include variability in the ability to successfully establish a stable line and inconsistency in the dimensions of these microcancers for in vivo drug response measurements. This paper explored the utility of microfluidics in the cultivation of cancer cell spheroids. Six patient-derived xenograft (PDX) tumors of high-grade serous ovarian cancer were used as the source material to demonstrate that viability and epithelial marker expression in the microfluidic cultures was superior to that of Matrigel or large volume 3D cultures. To further demonstrate the potential for miniaturization and multiplexing, we fabricated multichamber microfluidic devices with integrated microvalves to enable serial seeding of several chambers followed by parallel testing of several drug concentrations. These valve-enabled microfluidic devices permitted the formation of spheroids and testing of seven drug concentrations with as few as 100,000 cancer cells per device. Overall, we demonstrate the feasibility of maintaining difficul-to-culture primary cancer cells and testing drugs in a microfluidic device. This microfluidic platform may be ideal for drug testing and personalized therapy when tumor material is limited, such as following the acquisition of biopsy specimens obtained by fine-needle aspiration. Researchers in the United States have developed a microfluidic system for culturing ovarian cancer organoids. The use of microfluidics makes it possible to create cultures starting from a smaller number of cells than existing approaches. The team compared the new approach to standard techniques and found that the resulting cultures were superior in terms of viability, proliferation, and phenotype expression. Building on this framework, the researchers developed an 8-chamber microfluidic device which could be seeded serially and then used for parallel testing of different drug concentrations. The device required with fewer than 100,000 cells, which is insufficient to test multiple conditions using existing approaches. The ability to work reliably with small cells numbers makes this microfluidic platform well-suited for personalized drug testing when the sample material is limited.

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.

Human pluripotent stem cells (hPSCs) may be differentiated into any adult cell type and therefore hold incredible promise for cell therapeutics and disease modeling. There is increasing interest in three-dimensional (3D) hPSC culture because of improved differentiation outcomes and potential for scale up. Our team has recently described bioactive heparin (Hep)-containing core-shell microcapsules that promote rapid aggregation of stem cells into spheroids and may also be loaded with growth factors for the local and sustained delivery to the encapsulated cells. In this study, we explored the possibility of further modulating bioactivity of microcapsules through the use of an ultrathin coating composed of tannic acid (TA). Deposition of the TA film onto model substrates functionalized with Hep and poly(ethylene glycol) was characterized by ellipsometry and atomic force microscopy. Furthermore, the presence of the TA coating was observed to increase the amount of basic fibroblast growth factor (bFGF) incorporation by up to twofold and to extend its release from 5 to 7 days. Most significantly, TA-microcapsules loaded with bFGF induced higher levels of pluripotency expression compared to uncoated microcapsules containing bFGF. Engineered microcapsules described here represent a new stem cell culture approach that enables 3D cultivation and relies on local delivery of inductive cues., This study was supported in part by the grants from the Mayo Clinic Center for Regenerative Medicine, J.W. Kieckhefer Foundation, Al Nahyan Foundation, Regenerative Medicine Minnesota (RMM 101617 TR 004), and NIH (DK107255). Additional support was provided by an NIH Grant EB021911 to H.B. Additional funding was provided by the Mayo Clinic Center for Cell Signaling in Gastroenterology (P30DK084567).

Background: To evaluate the diagnostic performance of ultrasound risk-stratification systems for the discrimination of benign and malignant thyroid nodules and to determine the optimal cutoff values of individual risk-stratification systems. Methods: PubMed, Embase, SCOPUS, Web of Science, and Cochrane library databases were searched up to August 2022. Sensitivity and specificity data were collected along with the characteristics of each study related to ultrasound risk stratification systems. Results: Sixty-seven studies involving 76,512 thyroid nodules were included in this research. The sensitivity, specificity, diagnostic odds ratios, and area under the curves by K-TIRADS (4), ACR-TIRADS (TR5), ATA (high suspicion), EU-TIRADS (5), and Kwak-TIRADS (4b) for malignancy risk stratification of thyroid nodules were 92.5%, 63.5%, 69.8%, 70.6%, and 95.8%, respectively; 62.8%, 89.6%, 87.2%, 83.9%, and 63.8%, respectively; 20.7111, 16.8442, 15.7398, 12.2986, and 38.0578, respectively; and 0.792, 0.882, 0.859, 0.843, and 0.929, respectively. Conclusion: All ultrasound-based risk-stratification systems had good diagnostic performance. Although this study determined the best cutoff values in individual risk-stratification systems based on statistical assessment, clinicians could adjust or alter cutoff values based on the clinical purpose of the ultrasound and the reciprocal changes in sensitivity and specificity.

Extracellular vesicles (EVs) are lipid bilayer particles secreted from various cells. EVs carry molecular information of parent cells and hold considerable promise for early disease diagnostics. This paper describes a general strategy for multiplexed immunosensing of EV surface proteins, focusing on surface markers CD63, CD81, nephrin, and podocin to prove the concept. This sensing strategy entailed functionalizing gold nanoparticles (AuNPs) with two types of antibodies and then tagging with metal ions, either Pb2+ or Cu2+. The metal ions served as redox reporters, generating unique redox peaks at -0.23 and 0.28 V (vs Ag/AgCl) during electrochemical oxidation of Pb2+ and Cu2+, respectively. Capture of EVs on the working electrode, followed by labeling with immunoprobes and square wave voltammetry, produced redox currents proportional to concentrations of EVs and levels of expression of EV surface markers. Importantly, metal-ion tagging of immunoprobes enabled detection of two EV surface markers simultaneously from the same electrode. We demonstrated dual detection of either CD63/CD81 or podocin/nephrin surface markers from urinary EVs. The NP-enabled immunoassay had a sensitivity of 2.46 × 105 particles/mL (or 40.3 pg/mL) for CD63- and 5.80 × 105 particles/mL (or 47.7 pg/mL) for CD81-expressing EVs and a linear range of four orders of magnitude. The limit of detection for podocin and nephrin was 3.1 and 3.8 pg/mL, respectively. In the future, the capacity for multiplexing may be increased by extending the repertoire of metal ions used for redox tagging of AuNPs.

Three-dimensional (3D) or spheroid cultures of human pluripotent stem cells (hPSCs) offer the benefits of improved differentiation outcomes and scalability. In this paper, we describe a strategy for the robust and reproducible formation of hPSC spheroids where a co-axial flow focusing device is utilized to entrap hPSCs inside core-shell microcapsules. The core solution contained single cell suspension of hPSCs and was made viscous by the incorporation of high molecular weight poly(ethylene glycol) (PEG) and density gradient media. The shell stream comprised of PEG-4 arm-maleimide or PEG-4-Mal and flowed alongside the core stream toward two consecutive oil junctions. Droplet formation occurred at the first oil junction with shell solution wrapping itself around the core. Chemical crosslinking of the shell occurred at the second oil junction by introducing a di-thiol crosslinker (1,4-dithiothreitol or DTT) to these droplets. The crosslinker reacts with maleimide functional groups via click chemistry, resulting in the formation of a hydrogel shell around the microcapsules. Our encapsulation technology produced 400 µm diameter capsules at a rate of 10 capsules per second. The resultant capsules had a hydrogel shell and an aqueous core that allowed single cells to rapidly assemble into aggregates and form spheroids. The process of encapsulation did not adversely affect the viability of hPSCs, with >95% viability observed 3 days post-encapsulation. For comparison, hPSCs encapsulated in solid gel microparticles (without an aqueous core) did not form spheroids and had

Human pluripotent stem cells (hPSC) hold considerable promise as a source of adult cells for treatment of diseases ranging from diabetes to liver failure. Some of the challenges that limit the clinical/translational impact of hPSCs are high cost and difficulty in scaling-up of existing differentiation protocols. In this paper, we sought to address these challenges through the development of bioactive microcapsules. A co-axial flow focusing microfluidic device was used to encapsulate hPSCs in microcapsules comprised of an aqueous core and a hydrogel shell. Importantly, the shell contained heparin moieties for growth factor (GF) binding and release. The aqueous core enabled rapid aggregation of hPSCs into 3D spheroids while the bioactive hydrogel shell was used to load inductive cues driving pluripotency maintenance and endodermal differentiation. Specifically, we demonstrated that one-time 1h long loading of pluripotency signals, fibroblast growth factor (FGF)-2 and transforming growth factor (TGF)-β1, into bioactive microcapsules was sufficient to induce and maintain pluripotency of hPSCs over the course of 5 days at levels similar to or better than a standard protocol with soluble GFs. Furthermore, stem cell-carrying microcapsules that previously contained pluripotency signals could be reloaded with an endodermal cue, Nodal, resulting in higher levels of endodermal markers compared to stem cells differentiated in a standard protocol. Overall, bioactive heparin-containing core-shell microcapsules decreased GF usage five-fold while improving stem cell phenotype and are well suited for 3D cultivation of hPSCs.

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.

Background: To assess the safety and effectiveness of transoral robotic thyroidectomy (TORT) in thyroid tumor. Methods: PubMed, Embase, Web of Science, SCOPUS, Cochrane database, and Google Scholar up to June 2022. Studies comparing outcomes and complications between TORT and control groups (robotic bilateral axillo-breast, trans-axillary, postauricular approach, conventional open thyroidectomy (OT), and transoral endoscopic approach) were analyzed. Results: Ten studies of 1420 individuals. The operative time (SMD 1.15, 95%CI [0.48; 1.89]) was significantly longer and the number of retrieved lymph nodes (LNs) (SMD −0.27, 95%CI [−0.39; −0.16]) was fewer in TORT than in the control group. The postoperative cosmetic satisfaction score (SMD 0.60, 95%CI [0.28; 0.92]) was statistically higher in TORT than in the control group. In subgroup analysis, there was no significant difference between robotic surgeries. However, TORT had significantly longer operative times (SMD 2.08, 95%CI [0.95; 3.20]) and fewer retrieved LNs (SMD −0.32, 95%CI [−0.46; −0.17]) than OT. TORT satisfied significantly more patients in cosmetic view. However, it increased hospitalization days and postoperative pain on the operation day and first day compared to OT. Conclusions: TORT is not inferior to other robotic-assisted approaches. Its operation time and hospitalization days are longer and postoperative pain is greater than OT, although its cosmetic satisfaction is high.

Hepatocytes are parenchymal cells of the liver responsible for drug detoxification, urea and bile production, serum protein synthesis, and glucose homeostasis. Hepatocytes are widely used for drug toxicity studies in bioartificial liver devices and for cell-based liver therapies. Because hepatocytes are highly differentiated cells residing in a complex microenvironment in vivo, they tend to lose hepatic phenotype and function in vitro. This paper first reviews traditional culture approaches used to rescue hepatic function in vitro and then discusses the benefits of emerging microfluidic-based culture approaches. We conclude by reviewing integration of hepatocyte cultures with bioanalytical or sensing approaches.

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 aptamer-based electrochemical biosensor for rapid and sensitive detection of IFN-γ. In this study, we explored the use of silicon nanowires (Si 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 3x faster and were 2x 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 co-infected with HIV/TB.

Bovine tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) in which it is hard to identify the pathological symptoms. Release of bovine interferon gamma (BoIFN-γ) by T-cells provides an important diagnostic marker of MTB infections. In this work, we developed for the first time an electrochemical aptasensor for sensitive and specific determination of BoIFN-γ. A thiolated IFN-γ-binding aptamer was conjugated with methylene blue (MB) and immobilized on a gold electrode by self-assembly. Binding of IFN-γ to the electrode surface caused a conformational change in the aptamer, decreasing electron transfer efficiency. The redox current was quantified using square wave voltammetry (SWV) and was found to be specific for bovine IFN-γ with detection limit of 0.1 nM in pristine buffer and 0.9 nM in blood. The biosensor described here may, in the future, be used for on-site testing of bovine blood to help better identify and contain outbreaks of bovine TB.

We describe an automated microsystem integrating hepatocyte spheroid culture and droplet-based bioanalysis modules. This device was shown to be suitable for long-term maintenance of primary hepatocytes. Importantly, this device was outfitted with microvalves to modulate composition of the input media with high temporal control. This ability was used to stimulate hepatocytes with waives of hormones, insulin and glucagon, so as to mimic physiological composition of hormones in the hepatic portal circulation. Using on-chip bioanalysis module we demonstrate that hepatocytes behave in physiological manner, consuming glucose upon stimulation with insulin and releasing glucose upon exposure to glucagon.

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.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.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.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.

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.

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.

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.

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.

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.

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.

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)-γ 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-γ and anti-CD4 antibodies (Abs). To define sites for single cell attachment, PEG hydrogel microwells (20 μm 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 stre...

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.

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).

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.

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.