Dynamics of self-propelled particles in vibrated dense granular media.

We study a system consisting of a few self-propelled particles (SPPs) placed among a crowd of densely packed granular particles that are vertically vibrated in a two-dimensional circular confinement. Our experiments reveal two important findings. First, an SPP exhibits a fractal renewal process within the dense granular medium, which induces a superdiffusive behavior whose diffusion exponent increases with its aspect ratio. Second, the SPPs eventually reach the boundary and form a moving cluster, which transitions from the moving state to the static state as the number of SPPs is increased. These results suggest a simple and effective method of modulating the fluidity and directionality of granular systems via controlling the shape and the number of SPPs.

Angiogenesis-on-a-chip coupled with single-cell RNA sequencing reveals spatially differential activations of autophagy along angiogenic sprouts.

Several functions of autophagy associated with proliferation, differentiation, and migration of endothelial cells have been reported. Due to lack of models recapitulating angiogenic sprouting, functional heterogeneity of autophagy in endothelial cells along angiogenic sprouts remains elusive. Here, we apply an angiogenesis-on-a-chip to reconstruct 3D sprouts with clear endpoints. We perform single-cell RNA sequencing of sprouting endothelial cells from our chip to reveal high activation of autophagy in two endothelial cell populations- proliferating endothelial cells in sprout basements and stalk-like endothelial cells near sprout endpoints- and further the reciprocal expression pattern of autophagy-related genes between stalk- and tip-like endothelial cells near sprout endpoints, implying an association of autophagy with tip-stalk cell specification. Our results suggest a model describing spatially differential roles of autophagy: quality control of proliferating endothelial cells in sprout basements for sprout elongation and tip-stalk cell specification near sprout endpoints, which may change strategies for developing autophagy-based anti-angiogenic therapeutics.

Aspiration-mediated hydrogel micropatterning using rail-based open microfluidic devices for high-throughput 3D cell culture.

Microfluidics offers promising methods for aligning cells in physiologically relevant configurations to recapitulate human organ functionality. Specifically, microstructures within microfluidic devices facilitate 3D cell culture by guiding hydrogel precursors containing cells. Conventional approaches utilize capillary forces of hydrogel precursors to guide fluid flow into desired areas of high wettability. These methods, however, require complicated fabrication processes and subtle loading protocols, thus limiting device throughput and experimental yield. Here, we present a swift and robust hydrogel patterning technique for 3D cell culture, where preloaded hydrogel solution in a microfluidic device is aspirated while only leaving a portion of the solution in desired channels. The device is designed such that differing critical capillary pressure conditions are established over the interfaces of the loaded hydrogel solution, which leads to controlled removal of the solution during aspiration. A proposed theoretical model of capillary pressure conditions provides physical insights to inform generalized design rules for device structures. We demonstrate formation of multiple, discontinuous hollow channels with a single aspiration. Then we test vasculogenic capacity of various cell types using a microfluidic device obtained by our technique to illustrate its capabilities as a viable micro-manufacturing scheme for high-throughput cellular co-culture.

Agile reversible shape-morphing of particle rafts.

Materials that transform shapes responding to external stimuli can bring unprecedented innovations to soft matter physics, soft robotics, wearable electronics, and architecture. As most conventional soft actuation technologies induce large deformations only in a preprogrammed manner at designated locations, the material systems capable of agile reversible deformations without prescribed patterns are strongly desired for versatile mechanical morphing systems. Here we report a morphable liquid interface coated with dielectric particles, or a particle raft, which can reversibly change its topography under an external electric field. The rafts change from flat floors to towers within seconds, and the morphed structures are even capable of horizontal translation. Our experiments and theory show that the raft deformation is driven by electrostatic attraction between particles and electrodes, while being modulated by electric discharge. A broad range of materials serving as electrodes, e.g., human fingers and transparent polymers, suggests this system's diverse applications, including the human-machine interface and the three-dimensional physical display.

High-Throughput Microfluidic 3D Cytotoxicity Assay for Cancer Immunotherapy (CACI-IMPACT Platform).

Adoptive cell transfer against solid tumors faces challenges to overcome tumor microenvironment (TME), which plays as a physical barrier and provides immuno-suppressive conditions. Classical cytotoxicity assays are widely used to measure killing ability of the engineered cytotoxic lymphocytes as therapeutics, but the results cannot represent the performance in clinical application due to the absence of the TME. This paper describes a 3D cytotoxicity assay using an injection molded plastic array culture (CACI-IMPACT) device for 3D cytotoxicity assay to assess killing abilities of cytotoxic lymphocytes in 3D microenvironment through a spatiotemporal analysis of the lymphocytes and cancer cells embedded in 3D extra cellular matrix (ECM). Rail-based microfluidic design was integrated within a single 96-well and the wells were rectangularly arrayed in 2 × 6 to enhance the experimental throughput. The rail-based microstructures facilitate hydrogel patterning with simple pipetting so that hydrogel pre-solution aspirated with 10 µl pipette can be patterned in 10 wells within 30 s. To demonstrate 3D cytotoxicity assay, we patterned HeLa cells encapsulated by collagen gel and observed infiltration, migration and cytotoxic activity of NK-92 cells against HeLa cells in the collagen matrix. We found that 3D ECM significantly reduced migration of cytotoxic lymphocytes and access to cancer cells, resulting in lower cytotoxicity compared with 2D assays. In dense ECM, the physical barrier function of the 3D matrix was enhanced, but the cytotoxic lymphocytes effectively killed cancer cells once they contacted with cancer cells. The results implied ECM significantly influences migration and cytotoxicity of cytotoxic lymphocytes. Hence, the CACI-IMPACT platform, enabling high-throughput 3D co-culture of cytotoxic lymphocyte with cancer cells, has the potential to be used for pre-clinical evaluation of cytotoxic lymphocytes engineered for immunotherapy against solid tumors.

Multiple roles of lymphatic vessels in peripheral lymph node development.

The mammalian lymphatic system consists of strategically located lymph nodes (LNs) embedded into a lymphatic vascular network. Mechanisms underlying development of this highly organized system are not fully understood. Using high-resolution imaging, we show that lymphoid tissue inducer (LTi) cells initially transmigrate from veins at LN development sites using gaps in venous mural coverage. This process is independent of lymphatic vasculature, but lymphatic vessels are indispensable for the transport of LTi cells that egress from blood capillaries elsewhere and serve as an essential LN expansion reservoir. At later stages, lymphatic collecting vessels ensure efficient LTi cell transport and formation of the LN capsule and subcapsular sinus. Perinodal lymphatics also promote local interstitial flow, which cooperates with lymphotoxin-ß signaling to amplify stromal CXCL13 production and thereby promote LTi cell retention. Our data unify previous models of LN development by showing that lymphatics intervene at multiple points to assist LN expansion and identify a new role for mechanical forces in LN development.

Microfluidics within a well: an injection-molded plastic array 3D culture platform.

Polydimethylsiloxane (PDMS) has been widely used in fabricating microfluidic devices for prototyping and proof-of-concept experiments. Due to several material limitations, PDMS has not been widely adopted for commercial applications that require large-scale production. This paper describes a novel injection-molded plastic array 3D culture (IMPACT) platform that incorporates a microfluidic design to integrate patterned 3D cell cultures within a single 96-well (diameter = 9 mm) plate. Cell containing gels can be sequentially patterned by capillary-guided flow along the corner and narrow gaps designed within the 96-well form factor. Compared to PDMS-based hydrophobic burst valve designs, this work utilizes hydrophilic liquid guides to obtain rapid and reproducible patterned gels for co-cultures. When a liquid droplet (i.e. cell containing fibrin or collagen gel) is placed on a corner, spontaneous patterning is achieved within 1 second. Optimal dimensionless parameters required for successful capillary loading have been determined. To demonstrate the utility of the platform for 3D co-culture, angiogenesis experiments were performed by patterning HUVEC (human umbilical endothelial cells) and LF (lung fibroblasts) embedded in 3D fibrin gels. The angiogenic sprouts (with open lumen tip cells expressing junctional proteins) are comparable to those observed in PDMS based devices. The IMPACT device has the potential to provide a robust high-throughput experimental platform for vascularized microphysiological systems.

Microstructure guided multi-scale liquid patterning on an open surface.

Liquid patterning is a quintessential aspect in cell-based screening. While there are a variety of methods to handle microliquids utilizing surface treatments, complex microfluidic systems, and automated dispensing, most of the stated methods are both expensive and difficult to implement. Here, we present a fast multi-scale microliquid-patterning method on an open surface using embossed microstructures without surface modification. Arrays of micropillars can trap microliquids when a bulk drop is swept by an elastic sweeper on polystyrene (PS) substrates. The patterning mechanism on a basic form of a 2 × 2 rectangular array of circular pillars is analyzed theoretically and verified with experiments. Nanoliter-to-microliter volumes of liquids are patterned into various shapes by arranging the pillars based on the analysis. Furthermore, an array of geometrically modified pillars can capture approximately 8000 droplets on a large substrate (55 mm × 55 mm) in one step. Given the simplistic method of wipe patterning, the proposed platform can be utilized in both manual benchtop and automated settings. We will provide proof of concept experiments of single colony isolation using nanoliter-scale liquid patterning and of human angiogenic vessel formation using sequential patterning of microliter-scale liquids.

Wet-AMD on a Chip: Modeling Outer Blood-Retinal Barrier In Vitro.

Choroidal neovascularization (CNV) in the retinal pigment epithelium (RPE)-choroid complex constituting outer blood retinal barrier (oBRB) is a critical pathological step in various ophthalmic diseases, which results in blindness, such as wet type age-related macula degeneration. Current in vitro experimental models using petri dishes or transwell are unable to study CNV morphogenesis. Here, a unique organotypic eye-on-a-chip model is described that mimics the RPE-choroid complex in vitro. This model consists of an RPE monolayer and adjacent perfusable blood vessel network, which is supporting barrier function of oBRB. The intact barrier function of the RPE-choroid complex is reconstituted while maintaining important structural features. Further, this model can successfully mimic the pathogenesis of CNV especially in terms of morphogenesis, which is penetrating angiogenic sprouts from pre-existing choroidal vessels that result in breakdown of RPE monolayer. The alleviation of the pathological angiogenesis can be modeled with bevacizumab, a clinical drug for CNV treatment. It is believed that this model can be used to aid in the development of advanced in vitro eye drug evaluation in conjunction with animal models.

A Low Permeability Microfluidic Blood-Brain Barrier Platform with Direct Contact between Perfusable Vascular Network and Astrocytes.

A novel three dimensional blood brain barrier (BBB) platform was developed by independently supplying different types of media to separate cell types within a single device. One channel (vascular channel, VC) is connected to the inner lumen of the vascular network while the other supplies media to the neural cells (neural channel, NC). Compared to co-cultures supplied with only one type of medium (or 1:1 mixture), best barrier properties and viability were obtained with culturing HUVECs with endothelial growth medium (EGM) and neural cells with neurobasal medium supplemented with fetal bovine serum (NBMFBS) independently. The measured vascular network permeability were comparable to reported in vivo values (20 kDa FITC-dextran, 0.45 ± 0.11 × 10-6 cm/s; 70 kDa FITC-dextran, 0.36 ± 0.05 × 10-6 cm/s) and a higher degree of neurovascular interfacing (astrocytic contact with the vascular network, GFAP-CD31 stain overlap) and presence of synapses (stained with synaptophysin). The BBB platform can dependably imitate the perivascular network morphology and synaptic structures characteristic of the NVU. This microfluidic BBB model can find applications in screening pharmaceuticals that target the brain for in neurodegenerative diseases.

Biomimetic Model of Tumor Microenvironment on Microfluidic Platform.

The "Tumor microenvironment" (TME) is a complex, interacting system of the tumor and its surrounding environment. The TME has drawn more attention recently in attempts to overcome current drug resistance and the recurrence of cancer by understanding the cancer and its microenvironment systematically, beyond past reductionist approaches. However, a lack of experimental tools to dissect the intricate interactions has hampered in-depth research into the TME. Here, a biomimetic TME model using a microfluidic platform is presented, which enables the interaction between TME constituents to be studied in a comprehensive manner. Paracrine interactions of cocultured tumor cell lines (SK-OV-3, MKN-74, and SW620) with primary fibroblasts show marked morphological changes in the tumor cells, depending on the type of tumor cells, and, importantly, the composition of the extracellular matrix. Furthermore, this model allows direct observation of angiogenesis induced by the tumor-stroma interaction. Finally, reconstituting simultaneous angiogenesis and lymphangiogenesis induced by the tumor-stromal interaction with TME mimicking extrinsic factors is enabled. It is believed that the in vitro biomimetic model and the experimental concepts described will help to shed light on the complex biology of the TME.