Microfluidic Array Platform Research Articles

Multiplexed miRNA and Protein Analysis Using Digital Quantitative PCR in Microwell Arrays.

Proteins and microRNAs (miRNAs) act in tandem within biological pathways to regulate cellular functions, and their misregulation has been correlated to numerous diseases. Because of their interconnectedness, both miRNAs and proteins must be evaluated together to obtain accurate insights into the molecular pathways of pathogenesis. However, few analytical techniques can measure both classes of biomolecules in parallel from a single biological sample. Here, microfluidic digital quantitative PCR (dqPCR) was developed to simultaneously quantify miRNA and protein targets in a multiplexed assay using a single detection chemistry. This streamlined analysis was achieved by integrating base-stacking PCR and immuno-PCR in a microfluidic array platform. Analyses of let-7a (miRNA) and IL-6 (protein) were first optimized separately to identify thermocycling and capture conditions amenable to both biomolecules. Singleplex dqPCR studies exhibited the expected digital signals and quantification cycles for both analytes over a range of concentrations. Multiplexed analyses were then conducted to quantify both let-7a and IL-6 with high sensitivity (LODs ∼ 3 fM) over a broad dynamic range (5-5000 fM) using only standard PCR reagents. This multiplexed dqPCR was then translated to the analysis of HEK293 cell lysate, where endogenous let-7a and IL-6 were measured simultaneously without sample purification or pretreatment. Collectively, these studies demonstrate that the integration of BS-PCR and immuno-PCR achieves a sensitive and streamlined approach for multiplexed analyses of miRNAs and proteins, which will enable researchers to gain better insights into disease pathogenesis in future applications.

Integrated Bacterial Identification and Antimicrobial Susceptibility Testing for Polymicrobial Infections Using Digital PCR and Digital High-Resolution Melt in a Microfluidic Array Platform.

In diagnosing bacterial infection, rapid bacterial identification (ID) and antimicrobial susceptibility testing (AST) are critical to clinicians in order to provide an effective treatment in a timely manner. The gold standard, culture-based approach provides both ID and antimicrobial susceptibility but requires several days of turnaround time. Especially in polymicrobial infections, where there are more than one organisms interacting collectively that can complicate the treatment. Here, we demonstrate a rapid bacterial diagnostic approach that is capable of bacterial ID/AST in heterogeneous samples within less than 4 hours by using digital PCR (dPCR) and digital high-resolution melt via microfluidic devices. By utilizing dPCR, we are able to quantify amount of nucleic acid, which correlates to phenotypic responses of {\bf individual pathogens in a mixed sample and also shorten the required time of antibiotic exposure. In addition, we employ a machine learning algorithm to automatically identify bacterial species based on melt profiles of 16S rRNA gene.

Recapitulation of in vivo-like paracrine signals of human mesenchymal stem cells for functional neuronal differentiation of human neural stem cells in a 3D microfluidic system

Paracrine signals produced from stem cells influence tissue regeneration by inducing the differentiation of endogenous stem or progenitor cells. However, many recent studies that have investigated paracrine signaling of stem cells have relied on either two-dimensional transwell systems or conditioned medium culture, neither of which provide optimal culture microenvironments for elucidating the effects of paracrine signals in vivo. In this study, we recapitulated in vivo-like paracrine signaling of human mesenchymal stem cells (hMSCs) to enhance functional neuronal differentiation of human neural stem cells (hNSCs) in three-dimensional (3D) extracellular matrices (ECMs) within a microfluidic array platform. In order to amplify paracrine signaling, hMSCs were genetically engineered using cationic polymer nanoparticles to overexpress glial cell-derived neurotrophic factor (GDNF). hNSCs were cultured in 3D ECM hydrogel used to fill central channels of the microfluidic device, while GDNF-overexpressing hMSCs (GDNF-hMSCs) were cultured in channels located on both sides of the central channel. This setup allowed for mimicking of paracrine signaling between genetically engineered hMSCs and endogenous hNSCs in the brain. Co-culture of hNSCs with GDNF-hMSCs in the 3D microfluidic system yielded reduced glial differentiation of hNSCs while significantly enhancing differentiation into neuronal cells including dopaminergic neurons. Neuronal cells produced from hNSCs differentiating in the presence of GDNF-hMSCs exhibited functional neuron-like electrophysiological features. The enhanced paracrine ability of GDNF-hMSCs was finally confirmed using an animal model of hypoxic-ischemic brain injury. This study demonstrates the presented 3D microfluidic array device can provide an efficient co-culture platform and provide an environment for paracrine signals from transplanted stem cells to control endogenous neuronal behaviors in vivo.

Circulating MicroRNA Is a Biomarker of Biliary Atresia

The lack of reliable noninvasive diagnostic biomarkers of biliary atresia (BA) results in delayed diagnosis and worsened patient outcome. Circulating microRNAs (miRNAs) are a new class of noninvasive biomarkers with encouraging diagnostic utility. We examined the ability of serum miRNAs to distinguish BA from other forms of neonatal hyperbilirubinemia. BA-specific serum miRNAs were identified using a microfluidic array platform and validated in a larger, independent sample set. The miR-200b/429 cluster was significantly increased in the sera of patients with BA relative to infants with non-BA cholestatic disorders. Circulating levels of the miR-200b/429 cluster are elevated in infants with BA and have promising diagnostic clinical performance.

Development of a multi-layer microfluidic array chip to culture and replate uniform-sized embryoid bodies without manual cell retrieval

We have developed a multi-layer, microfluidic array platform containing concave microwells and flat cell culture chambers to culture embryonic stem (ES) cells and regulate uniform-sized embryoid body (EB) formation. The main advantage of this platform was that EBs cultured within the concave microwells of a bottom layer were automatically replated into flat cell culture chambers of a top layer, following inversion of the multi-layer microfluidic array platform. This allowed EB formation and EB replating to be controlled simultaneously inside a single microfluidic device without pipette-based manual cell retrieval, a drawback of previous EB culture methods.

A self-contained microfluidic cell culture system

Conventional in vitro cell culture that utilizes culture dishes or microtiter plates is labor-intensive and time-consuming, and requires technical expertise and specific facilities to handle cell harvesting, media exchange and cell subculturing procedures. A microfluidic array platform with eight microsieves in each cell culture chamber is presented for continuous cell culture. With the help of the microsieves, uniform cell loading and distribution can be obtained. Within the arrays, cells grown to the point of confluency can be trypsinized and recovered from the device. Cells trapped in the microsieves after trypsinization function to seed the chambers for subsequent on-chip culturing, creating a sustainable platform for multiple cycles. The capability of the microfluidic array platform was demonstrated with a BALB/3T3 (murine embryonic fibroblast) cell line. The present microfluidic cell culture platform has potential to develop into a fully automated cell culture system integrated with temperature control, fluidic control, and micropumps, maximizing cell culture health with minimal intervention.

High-density microfluidic arrays for cell cytotoxicity analysis

In this paper, we report on the development of a multilayer elastomeric microfluidic array platform for the high-throughput cell cytotoxicity screening of mammalian cell lines. Microfluidic channels in the platform for cell seeding are orthogonal to channels for toxin exposure, and within each channel intersection is a circular chamber with cell-trapping sieves. Integrated, pneumatically-actuated elastomeric valves within the device isolate the microchannel array within the device into parallel rows and columns for cell seeding and toxin exposure. As a demonstration of the multiplexing capability of the platform, a microfluidic array containing 576 chambers was used to screen three cell types (BALB/3T3, HeLa, and bovine endothelial cells) against a panel of five toxins (digitonin, saponin, CoCl(2), NiCl(2), acrolein). Evaluation of on-chip cell morphology and viability was carried out using fluorescence microscopy, with outcomes comparable to microtiter plate cytotoxicity assays. Using this scalable platform, cell seeding and toxin exposure can be carried out within a single microfluidic device in a multiplexed format, enabling high-density parallel cytotoxicity screening while minimizing reagent consumption.