Lab on a Chip最新文献

Pancreatic ductal adenocarcinoma (PDAC) is the most common and lethal form of pancreatic cancer. One major cause for a fast disease progression is the presence of a highly fibrotic tumor microenvironment (TME) mainly composed of cancer-associated fibroblasts (CAF), and various immune cells, especially tumor-associated macrophages (TAM). To conclusively evaluate drug efficacy, it is crucial to develop in vitro models that can recapitulate the cross talk between tumor cells and the surrounding stroma. Here, we constructed a fit-for-purpose biochip platform which allows the integration of PDAC spheroids (composed of PANC-1 cells and pancreatic stellate cells (PSC)). Additionally, the chip design enables dynamic administration of drugs or immune cells via a layer of human umbilical vein endothelial cells (HUVEC). As a proof-of-concept for drug administration, vorinostat, an FDA-approved histone deacetylase inhibitor for cutaneous T cell lymphoma (CTCL), subjected via continuous flow for 72 h, resulted in a significantly reduced viability of PDAC spheroids without affecting vascular integrity. Furthermore, dynamic perfusion with peripheral mononuclear blood cells (PBMC)-derived monocytes resulted in an immune cell migration through the endothelium into the spheroids. After 72 h of infiltration, monocytes differentiated into macrophages which polarized into the M2 phenotype. The polarization into M2 macrophages persisted for at least 168 h, verified by expression of the M2 marker CD163 which increased from 72 h to 168 h, while the M1 markers CD86 and HLA-DR were significantly downregulated. Overall, the described spheroid-on-chip model allows the evaluation of novel therapeutic strategies by mimicking and targeting the complex TME of PDAC.

Polymer-based microwell platforms have garnered much interest due to their usefulness in culturing and analyzing small quantities of biological cells and spheroids. Existing methods for fabricating polymer microwell arrays involve complex fabrication processes and/or are limited in their ability to create dense arrays of very small (<50 μm in diameter) microwells. Here, we present a simple and rapid technique for fabricating high-density arrays of microwells ranging from 20 to 160 μm in diameter on a variety of polymer substrates. In this approach, a polymer surface is ablated using a CO₂ laser that is rastered over a stainless steel mesh, which serves as a shadow mask. A theoretical laser–polymer interaction model was developed for predicting the microwell volume based on the substrate properties and laser settings. Microwell volumes predicted by the model were within 5.4% of fabricated microwell volumes determined experimentally. Cellulose acetate microwell arrays fabricated using this technique were used to culture Lewis lung carcinoma cells expressing ovalbumin (LLC-OVA), which were maintained for up to 72 h with a negligible (<5%) loss in viability. As a second proof of principle demonstration, LLC-OVA cells grown in microwell arrays were co-cultured with OT-I T cells and measurements of interferon gamma (IFN-γ), a marker for T cell activation, were performed which revealed a positive correlation between LLC-OVA cell-T cell interaction time and T cell activation. These two in vitro demonstrations showcase the capability of this technique in generating polymer microwell arrays for high-throughput cellular studies, including cell growth dynamics studies and cell interaction studies. Furthermore, we envision that these platforms can be used with different cell types and for other biological applications, such as spheroid formation and single cell analysis, further expanding the utility of this technique.

Despite contributing to cancer progression, extracellular vesicles (EVs) could serve as potential drug delivery systems in cancer treatment, having the ability to dissolve water-insoluble drugs and facilitate targeted delivery. However, the clinical translation of EVs is still in its infancy. While traditional methods for EV modifications will remain relevant, microfluidic approaches are expected to replace benchtop methods. Taking advantage of lab-on-chip devices, passive cargo loading through microfluidic mixing and incubation may be an important strategy to produce functional engineered EVs. This study focuses on developing a microfluidic device to generate EVs loaded with verteporfin (VP), a hydrophobic porphyrin with potential applications in neuroblastoma (NB) therapy, aiming to enhance its therapeutic effectiveness. The platform ensures perfect mixing and tunable incubation time for mesenchymal stem cell-derived EVs and VP, demonstrating a significantly higher loading efficiency than traditional methods, while operating under gentle conditions that preserve EV integrity and functionality, unlike other microfluidic techniques that involve harsh mechanical or chemical treatments. The VP-loaded EVs (VP-EVs) can then be easily recovered, making them available for subsequent analysis and use. MTT assay confirmed that VP-EVs are more efficient than free VP in reducing the viability of a NB cell line. Finally, immunofluorescence assay and western blot demonstrated a greater reduction in YAP expression after treatment with VP-EVs in an NB cell line when compared to free VP. Being both non-destructive and straightforward, this microfluidic loading technique facilitates its adaptability to a wide spectrum of therapeutic compounds. As a versatile tool, microfluidic technology will help to fully unlock the potential of EVs for speeding up precision medicine and disease treatment.

We experimentally investigate droplet pattern formation in coaxial microchannels using ternary mixtures of two immiscible fluids and a miscible solvent. The influence of solvent concentration is examined through periodic pattern analysis of droplet flow and functional relationships are developed to determine the initial interfacial tension of dispersions made of aqueous mixtures of solvent and oil at short timescales, i.e., when solvent diffusion into the continuous phase has a negligible effect on flow morphologies. We examine a wide range of flow rates and delineate vast flow maps of droplet regimes, including dripping and jetting flows, to clarify the hydrodynamic behavior of conjugate fluid mixtures in square microcapillaries. A method based on analysis of droplet size and spacing is implemented to predict the role of the miscible fluid additive concentration in microfluidic multiphase flows of water–isopropanol and ethanol–isopropanol blends in viscous silicone oil. This approach enables measurement of extremely small values of interfacial tension at large solvent concentrations. This work shows a technique for exploring and characterizing numerous ternary flow systems of interest with a variety of organic solvents and oils.

Molecular networks of organelle membranes are involved in many cell processes. However, the nature of plasma membrane as a barrier to various analytical tools, including antibodies, makes it challenging to examine intact organelle membranes without affecting their structure and functions via membrane permeabilization. Therefore, in this study, we aimed to develop a microfluidic method to unroof cells and observe the intrinsic membrane molecules in organelles. In our method, single cells were precisely arrayed on the bottom surface of microchannels in a light-guided manner using a photoactivatable cell-anchoring material. At sufficiently short cell intervals, horizontal stresses generated by the laminar flow instantly fractured the upper cell membranes, without significantly affecting some organelles inside the fractured cells. Subsequently, nucleus and other organelles in unroofed cells were observed via confocal fluorescence and scanning electron microscopy. Furthermore, distribution of the mitochondrial membrane protein, translocase of outer mitochondrial membrane 20, on the mitochondrial membrane was successfully observed via immunostaining without permeabilization. Overall, the established cell unroofing method shows great potential to examine the localization, functions, and affinities of proteins on intact organelle membranes.

Conductive hydrogels based on liquid metal microdroplets are widely used as wearable electronic devices. Droplet uniformity affects sensor sensitivity for weak signals, such as heart rate and pulse rate. Surface acoustic waves at micrometer wavelengths allow precise control of a single droplet, and have the potential to make uniformly discrete liquid metal droplets and distribute them in hydrogels. But the control law of liquid metal droplet size and its spatial configuration by acoustic surface waves is not clear. The aim of this paper is to present an analysis of the acoustic regulation mechanism in the interfacial evolution of fluids with high interfacial tension coefficients, and to investigate the influence of microdroplet generation characteristics (size and spacing) on the conductive and mechanical properties of conductive hydrogels. The results showed that the combined action of acoustic radiation force, shear force and pressure difference force helped to overcome interfacial tension and speed up the interfacial necking process during the filling and squeezing stages. The use of acoustic surface waves serves to diminish the influence of droplet size on the two-phase flow rate. This provides an effective approach for achieving decoupled control of microdroplet size and spacing, alongside the formation of a homogenous array of liquid metal droplets. The acoustic surface wave effect makes the liquid metal microdroplets more uniform in size and spacing. As the liquid metal content relative to the hydrogel substrate solution increases, the liquid metal size decreases. The hydrogel's initial conductivity and conductivity after self-healing increase by 10% and 25%, respectively, which can realize the effective monitoring of ECG and EMG signals. This study helps to reveal the evolution mechanism of liquid-metal interfaces induced by acoustic surface waves, elucidate the effects of microdroplet size and spacing on the conductive and mechanical properties of hydrogels, and provide theoretical guidance for the high-precision preparation of wearable electronic devices.

Correction for ‘In vitro vascularized liver tumor model based on a microfluidic inverse opal scaffold for immune cell recruitment investigation’ by Pingwei Xu et al., Lab Chip, 2024, 24, 3470–3479, https://doi.org/10.1039/D4LC00341A

On-chip infrared spectroscopic gas sensing using a hollow-core anti-resonant reflecting optical waveguide (ARROW) with a large external confinement factor (ECF) was rarely reported due to the complex fabrication process and polarization dependence. Alternatively, we proposed ARROW gas sensors using chalcogenide (ChG) anti-resonant layers which require thermal evaporation and epoxy resin bonding for fabrication instead of the complicated wafer bonding process. Polarization characteristics and ethylene (C₂H₂) sensing performance at 1.532 μm were measured for two ARROW sensors with four-side (WG_A) and three-side (WG_B) anti-resonant layers around the hollow-core. Due to a symmetric structure, the 1 cm-long WG_A sensor exhibits polarization-insensitive characteristics, which does not require an additional polarization controller for integrated on-chip sensors and enhances the stability and reliability of the sensor under fluctuating polarization states. A high ECF of 71% and a 1σ limit of detection (LoD) of ∼23 parts-per-million (ppm) for WG_A were achieved at an averaging time of 39.2 s. The broadband multi-gas detection capability of WG_A was verified through C₂H₂ detection at 1.532 μm and CH₄ at 1.654 μm, highlighting the potential of ARROWs for on-chip multi-gas sensing.

Bacterial cells organize their genomes into a compact hierarchical structure called the nucleoid. Studying the nucleoid in cells faces challenges because of the cellular complexity while in vitro assays have difficulty in handling the fragile megabase-scale DNA biopolymers that make up bacterial genomes. Here, we introduce a method that overcomes these limitations as we develop and use a microfluidic device for the sequential extraction, purification, and analysis of bacterial nucleoids in individual microchambers. Our approach avoids any transfer or pipetting of the fragile megabase-size genomes and thereby prevents their fragmentation. We show how the microfluidic system can be used to extract and analyze single chromosomes from B. subtilis cells. Upon on-chip lysis, the bacterial genome expands in size and DNA-binding proteins are flushed away. Subsequently, exogeneous proteins can be added to the trapped DNA via diffusion. We envision that integrated microfluidic platforms will become an essential tool for the bottom-up assembly of complex biomolecular systems such as artificial chromosomes.

A subset of coronavirus disease 2019 (COVID-19) patients develops severe symptoms, characterized by acute lung injury, endothelial dysfunction and microthrombosis. Viral infection and immune cell activation contribute to this phenotype. It is known that systemic inflammation, evidenced by circulating inflammatory factors in patient plasma, is also likely to be involved in the pathophysiology of severe COVID-19. Here, we evaluate whether systemic inflammatory factors can induce endothelial dysfunction and subsequent thromboinflammation. We use a microfluidic Vessel-on-Chip model lined by human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs), stimulate it with plasma from hospitalized COVID-19 patients and perfuse it with human whole blood. COVID-19 plasma exhibited elevated levels of inflammatory cytokines compared to plasma from healthy controls. Incubation of hiPSC-ECs with COVID-19 plasma showed an activated endothelial phenotype, characterized by upregulation of inflammatory markers and transcriptomic patterns of host defense against viral infection. Treatment with COVID-19 plasma induced increased platelet aggregation in the Vessel-on-Chip, which was associated partially with formation of neutrophil extracellular traps (NETosis). Our study demonstrates that factors in the plasma play a causative role in thromboinflammation in the context of COVID-19. The presented Vessel-on-Chip can enable future studies on diagnosis, prevention and treatment of severe COVID-19.