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We report the fabrication of a scaffold (hereafter referred to as AngioChip) that supports the assembly of parenchymal cells on a mechanically tunable matrix surrounding a perfusable, branched, three-dimensional microchannel network coated with endothelial cells. The design of AngioChip decouples the material choices for the engineered vessel network and for cell seeding in the parenchyma, enabling extensive remodelling while maintaining an open-vessel lumen. The incorporation of nanopores and micro-holes in the vessel walls enhances permeability, and permits intercellular crosstalk and extravasation of monocytes and endothelial cells on biomolecular stimulation. We also show that vascularized hepatic tissues and cardiac tissues engineered by using AngioChips process clinically relevant drugs delivered through the vasculature, and that millimetre-thick cardiac tissues can be engineered in a scalable manner. Moreover, we demonstrate that AngioChip cardiac tissues implanted with direct surgical anastomosis to the femoral vessels of rat hindlimbs establish immediate blood perfusion. Biodegradable, perfusable scaffolds are able to generate both in vitro cardiac and hepatic vascularized tissue models and in vivo implants for direct surgical anastomosis.

We introduce Digital microfluidic Isolation of Single Cells for -Omics (DISCO), a platform that allows users to select particular cells of interest from a limited initial sample size and connects single-cell sequencing data to their immunofluorescence-based phenotypes. Specifically, DISCO combines digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to collect the contents of single cells from heterogeneous populations, followed by analysis of single-cell genomes and transcriptomes by next-generation sequencing, and proteomes by nanoflow liquid chromatography and tandem mass spectrometry. The results described herein confirm the utility of DISCO for sequencing at levels that are equivalent to or enhanced relative to the state of the art, capable of identifying features at the level of single nucleotide variations. The unique levels of selectivity, context, and accountability of DISCO suggest potential utility for deep analysis of any rare cell population with contextual dependencies. Multi-Omic approaches are a powerful way for obtaining in-depth understanding of a cell’s state. Here the authors present DISCO, combining digital microfluidics, laser cell lysis, and artificial intelligence-driven image processing to analyze single-cell genomes, transcriptomes and proteomes in a mixed population.

Microrobotics extends the reach of human-controlled machines to submillimeter dimensions. We introduce a microrobot that relies on optoelectronic tweezers (OET) that is straightforward to manufacture, can take nearly any desirable shape or form, and can be programmed to carry out sophisticated, multiaxis operations. One particularly useful program is a serial combination of “load,” “transport,” and “deliver,” which can be applied to manipulate a wide range of micrometer-dimension payloads. Importantly, microrobots programmed in this manner are much gentler on fragile mammalian cells than conventional OET techniques. The microrobotic system described here was demonstrated to be useful for single-cell isolation, clonal expansion, RNA sequencing, manipulation within enclosed systems, controlling cell–cell interactions, and isolating precious microtissues from heterogeneous mixtures. We propose that the optoelectronic microrobotic system, which can be implemented using a microscope and consumer-grade optical projector, will be useful for a wide range of applications in the life sciences and beyond.

There is great interest in the development of micromotors which can convert energy to motion in sub-millimeter dimensions. Micromachines take the micromotor concept a step further, comprising complex systems in which multiple components work in concert to effectively realize complex mechanical tasks. Here we introduce light-driven micromotors and micromachines that rely on optoelectronic tweezers (OET). Using a circular micro-gear as a unit component, we demonstrate a range of new functionalities, including a touchless micro-feed-roller that allows the programming of precise three-dimensional particle trajectories, multi-component micro-gear trains that serve as torque- or velocity-amplifiers, and micro-rack-and-pinion systems that serve as microfluidic valves. These sophisticated systems suggest great potential for complex micromachines in the future, for application in microrobotics, micromanipulation, microfluidics, and beyond. Light-driven micromotors can convert energy to motion in sub-millimeter dimensions. Here, the authors extend this concept and introduce reconfigurable micromachines with multiple components, driven by optoelectronic tweezers, and demonstrate new functionalities.

We report a new technique called Digital microfluidic Immunocytochemistry in Single Cells (DISC). DISC automates protocols for cell culture, stimulation and immunocytochemistry, enabling the interrogation of protein phosphorylation on pulsing with stimulus for as little as 3 s. DISC was used to probe the phosphorylation states of platelet-derived growth factor receptor (PDGFR) and the downstream signalling protein, Akt, to evaluate concentration- and time-dependent effects of stimulation. The high time resolution of the technique allowed for surprising new observations—for example, a 10 s pulse stimulus of a low concentration of PDGF is sufficient to cause >30% of adherent fibroblasts to commit to Akt activation. With the ability to quantitatively probe signalling events with high time resolution at the single-cell level, we propose that DISC may be an important new technique for a wide range of applications, especially for screening signalling responses of a heterogeneous cell population. The ability to measure signalling responses in single cells following short pulses of stimulus would shed insight into temporal thresholds for cell activation. Here the authors introduce a microfluidic platform that allows downstream phosphorylation cascades to be observed following as little as one second of stimulus exposure.

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is deregulated in many human diseases including cancer, diabetes, obesity, and autoimmunity. PI3K consists of a p110 catalytic protein and a p85α regulatory protein, required for the stabilization and localization of p110-PI3K activity. The p110-PI3K enzyme generates the key signaling lipid phosphatidylinositol 3,4,5-trisphosphate, which is dephosphorylated by the PI3-phosphatase PTEN. Here we show another function for the p85α regulatory protein: it binds directly to and enhances PTEN lipid phosphatase activity. We demonstrate that ectopically expressed FLAG-tagged p85 coimmunoprecipitates endogenous PTEN in an epidermal growth factor dependent manner. We also show epidermal growth factor dependent coimmunoprecipitation of endogenous p85 and PTEN proteins in HeLa cells. Thus p85 regulates both p110-PI3K and PTEN-phosphatase enzymes through direct interaction. This finding underscores the need for caution in analyzing PI3K activity because anti-p85 immunoprecipitations may contain both p85:p110-PI3K and p85:PTEN-phosphatase enzymes and thus measure net PI3K activity. We identify the N-terminal SH3-BH region of p85α, absent in the smaller p55α and p50α isoforms, as the region that mediates PTEN binding and regulation. Cellular expression of p85ΔSH3-BH results in substantially increased magnitude and duration of pAkt levels in response to growth factor stimulation. The ability of p85 to bind and directly regulate both p110-PI3K and PTEN-PI3-phosphatase allows us to explain the paradoxical insulin signaling phenotypes observed in mice with reduced PI3K or PTEN proteins. This discovery will impact ongoing studies using therapeutics targeting the PI3K/PTEN/Akt pathway.

Metastasis remains a major challenge in treating breast cancer. Breast tumors metastasize to organ-specific locations such as the brain, lungs, and bone, but why some organs are favored over others remains unclear. Breast tumors also show heterogeneity, plasticity, and distinct microenvironments. This contributes to treatment failure and relapse. The interaction of breast cancer cells with their metastatic microenvironment has led to the concept that primary breast cancer cells act as seeds, whereas the metastatic tissue microenvironment (TME) is the soil. Improving our understanding of this interaction could lead to better treatment strategies for metastatic breast cancer. Targeted treatments for different subtypes of breast cancers have improved overall patient survival, even with metastasis. However, these targeted treatments are based upon the biology of the primary tumor and often these patients’ relapse, after therapy, with metastatic tumors. The advent of immunotherapy allowed the immune system to target metastatic tumors. Unfortunately, immunotherapy has not been as effective in metastatic breast cancer relative to other cancers with metastases, such as melanoma. This review will describe the heterogeneic nature of breast cancer cells and their microenvironments. The distinct properties of metastatic breast cancer cells and their microenvironments that allow interactions, especially in bone and brain metastasis, will also be described. Finally, we will review immunotherapy approaches to treat metastatic breast tumors and discuss future therapeutic approaches to improve treatments for metastatic breast cancer.

A critical aspect of creating vascularized tissues is the remodelling that occurs in vivo , driven in large part by the host response to the tissue construct. Rather than a simple inflammatory response, a beneficial tissue remodelling response results in the formation of vascularised tissue. The characteristics and dynamics of this response are slowly being elucidated, especially as they are modulated by the complex interaction between the biomaterial and cellular components of the tissue constructs and the host. This process has elements that are similar to both wound healing and tumour development, and its features are illustrated by reference to the bottom-up generation of a tissue using modular constructs. These modular constructs consist of mesenchymal stromal cells (MSC) embedded in endothelial cell (EC)-covered collagen gel rods that are a few hundred microns in size. Particular attention is paid to the role of hypoxia and macrophage recruitment, as well as the paracrine effects of the MSC and EC in this host response.

An in vitro tissue construct amenable to perfusion was formed by randomly packing mesenchymal stromal cell (MSC)-embedded, endothelial cell (EC)-coated collagen cylinders (modules) into a microfluidic chamber. The interstices created by the random packing of the submillimeter-sized modules created EC-lined channels. Flow caused a greater than expected amount of contraction and remodeling in the modular constructs. Flow influenced the MSC to develop smooth muscle cell markers (smooth muscle actin-positive, desmin-positive, and von Willebrand factor-negative) and migrate toward the surface of the modules. When modules were coated with EC, the extent of MSC differentiation and migration increased, suggesting that the MSC were becoming smooth muscle cell– or pericyte-like in their location and phenotype. The MSC also proliferated, resulting in a substantial increase in the number of differentiated MSC. These effects were markedly less for static controls not experiencing flow. As the MSC migrated, they created new matrix that included the deposition of proteoglycans. Collectively, these results suggest that MSC-embedded modules may be useful for the formation of functional vasculature in tissue engineered constructs. Moreover, these flow-conditioned tissue engineered constructs may be of interest as three-dimensional cell-laden platforms for drug testing and biological assays.