This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli can be applied to multiple cell types interacting over distances of <1 mm, thereby replicating many aspects of the in vivo microenvironment. Capabilities exist for time-dependent manipulation of flow and concentration gradients as well as high-resolution real-time imaging for observing spatial-temporal single-cell behavior, cell-cell communication, cell-matrix interactions and cell population dynamics. These heterotypic cell type assays can be used to study cell survival, proliferation, migration, morphogenesis and differentiation under controlled conditions. Applications include the study of previously unexplored cellular interactions, and they have already provided new insights into how biochemical and biophysical factors regulate interactions between populations of different cell types. It takes 3 d to fabricate the system and experiments can run for up to several weeks.

Pancreatic stellate cells (PSCs), a major component of the tumor microenvironment in pancreatic cancer, play roles in cancer progression as well as drug resistance. Culturing various cells in microfluidic (microchannel) devices has proven to be a useful in studying cellular interactions and drug sensitivity. Here we present a microchannel plate-based co-culture model that integrates tumor spheroids with PSCs in a three-dimensional (3D) collagen matrix to mimic the tumor microenvironment in vivo by recapitulating epithelial-mesenchymal transition and chemoresistance. A 7-channel microchannel plate was prepared using poly-dimethylsiloxane (PDMS) via soft lithography. PANC-1, a human pancreatic cancer cell line, and PSCs, each within a designated channel of the microchannel plate, were cultured embedded in type I collagen. Expression of EMT-related markers and factors was analyzed using immunofluorescent staining or Proteome analysis. Changes in viability following exposure to gemcitabine and paclitaxel were measured using Live/Dead assay. PANC-1 cells formed 3D tumor spheroids within 5 days and the number of spheroids increased when co-cultured with PSCs. Culture conditions were optimized for PANC-1 cells and PSCs, and their appropriate interaction was confirmed by reciprocal activation shown as increased cell motility. PSCs under co-culture showed an increased expression of α-SMA. Expression of EMT-related markers, such as vimentin and TGF-β, was higher in co-cultured PANC-1 spheroids compared to that in mono-cultured spheroids; as was the expression of many other EMT-related factors including TIMP1 and IL-8. Following gemcitabine exposure, no significant changes in survival were observed. When paclitaxel was combined with gemcitabine, a growth inhibitory advantage was prominent in tumor spheroids, which was accompanied by significant cytotoxicity in PSCs. We demonstrated that cancer cells grown as tumor spheroids in a 3D collagen matrix and PSCs co-cultured in sub-millimeter proximity participate in mutual interactions that induce EMT and drug resistance in a microchannel plate. Microfluidic co-culture of pancreatic tumor spheroids with PSCs may serve as a useful model for studying EMT and drug resistance in a clinically relevant manner.

Bacterial biofilms—aggregations of bacterial cells and extracellular polymeric substrates (EPS)—are an important subject of research in the fields of biology and medical science. Under aquatic conditions, bacterial cells form biofilms as a mechanism for improving survival and dispersion. In this review, we discuss bacterial biofilm development as a structurally and dynamically complex biological system and propose microfluidic approaches for the study of bacterial biofilms. Biofilms develop through a series of steps as bacteria interact with their environment. Gene expression and environmental conditions, including surface properties, hydrodynamic conditions, quorum sensing signals, and the characteristics of the medium, can have positive or negative influences on bacterial biofilm formation. The influences of each factor and the combined effects of multiple factors may be addressed using microfluidic approaches, which provide a promising means for controlling the hydrodynamic conditions, establishing stable chemical gradients, performing measurement in a high-throughput manner, providing real-time monitoring, and providing in vivo-like in vitro culture devices. An increased understanding of biofilms derived from microfluidic approaches may be relevant to improving our understanding of the contributions of determinants to bacterial biofilm development.

Microenvironmental control of interstitial flow around pancreatic islets effectively maintains islet function in vitro.

Sprouting angiogenesis requires a coordinated guidance from a variety of angiogenic factors. Here, we have developed a unique hydrogel incorporating microfluidic platform which mimics the physiological microenvironment in 3D under a precisely orchestrated gradient of soluble angiogenic factors, VEGF and ANG-1. The system enables the quantified investigation in chemotactic response of endothelial cells during the collective angiogenic sprouting process. While the presence of a VEGF gradient alone was sufficient in inducing a greater number of tip cells, addition of ANG-1 to the VEGF gradient enhanced the number of tip cells that are attached to collectively migrated stalk cells. The chemotactic response of tip cells attracted by the VEGF gradient and the stabilizing role of ANG-1 were morphologically investigated, elucidating the 3D co-operative migration of tip and stalk cells as well as their structures. We found that ANG-1 enhanced the connection of the stalk cells with the tip cells, and then the direct connection regulated the morphogenesis and/or life cycle of stalk cells.

Prominent techniques such as real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and rapid kits are currently being explored to both enhance sensitivity and reduce assay time for diagnostic tests. Existing commercial molecular methods typically take several hours, while immunoassays can range from several hours to tens of minutes. Rapid diagnostics are crucial in Point-of-Care Testing (POCT). We propose an approach that integrates a time-series deep learning architecture and AI-based verification, for the enhanced result analysis of lateral flow assays. This approach is applicable to both infectious diseases and non-infectious biomarkers. In blind tests using clinical samples, our method achieved diagnostic times as short as 2 minutes, exceeding the accuracy of human analysis at 15 minutes. Furthermore, our technique significantly reduces assay time to just 1-2 minutes in the POCT setting. This advancement has the potential to greatly enhance POCT diagnostics, enabling both healthcare professionals and non-experts to make rapid, accurate decisions.