Most methods to culture cells in three dimensions depend on a cell-seedable biomaterial to define the global structure of the culture and the microenvironment of the cells. Efforts to tailor these scaffolds have focused on the chemical and mechanical properties of the biomaterial itself. Here, we present a strategy to control the distributions of soluble chemicals within the scaffold with convective mass transfer via microfluidic networks embedded directly within the cell-seeded biomaterial. Our presentation of this strategy includes: a lithographic technique to build functional microfluidic structures within a calcium alginate hydrogel seeded with cells; characterization of this process with respect to microstructural fidelity and cell viability; characterization of convective and diffusive mass transfer of small and large solutes within this microfluidic scaffold; and demonstration of temporal and spatial control of the distribution of non-reactive solutes and reactive solutes (that is, metabolites) within the bulk of the scaffold. This approach to control the chemical environment on a micrometre scale within a macroscopic scaffold could aid in engineering complex tissues.

Geometrically-enhanced differential immunocapture (GEDI) and an antibody for prostate-specific membrane antigen (PSMA) are used for high-efficiency and high-purity capture of prostate circulating tumor cells from peripheral whole blood samples of castrate-resistant prostate cancer patients.Prostate circulating tumor cells (PCTCs) are often found in the blood of patients suffering from metastatic prostate cancer 1, 2. While these PCTCs are rare, as few as one cell per 10 9 hematologic cells in blood 3,4 , they are theorized to contribute to metastatic progression 3,5 . Currently, the enumeration of PCTCs is used clinically as a prognostic indicator of patient survival 2,6,7 . Capture of peripheral blood PCTCs may enable early clinical assessment of metastatic process and chemotherapeutic response, as well as genetic and pharmacological evaluation of cancer cells.Current approaches to isolate circulating tumor cells are complex and produce low yields and purity 5 . Existing commercial and research devices for the immunocapture of rare cancer cells use EpCAM antibodies 2,8,9 , which capture many circulating endothelial cells and large numbers of leukocytes. As a result, purity of captured cells is widely variable and often below 50%. In addition, while previous devices use 3D antibody-coated surfaces for immunocapture 8,9 , these devices are not designed to induce a size-dependent collision frequency. Devices focused on size-dependent particle transport are typically focused on sorting 10 , separation 11,12 , or filtration 13 .In this communication, we demonstrate high-efficient and high-purity capture of PCTCs from peripheral blood samples of castrate-resistant prostate cancer patients using an antibody for prostate-specific membrane antigen (PSMA), a highly prostate-specific cellsurface antigen 14 . In addition, we describe a theoretical framework for the use of staggered § To whom correspondence should be addressed: The GEDI µdevice geometry was designed to maximize streamline distortion and thus bring desired cells in contact with the immunocoated obstacle walls for capture. Blood is a dense heterogeneous cell suspension consisting of cells of various sizes ranging from approximately 4 to 18 µm in size16. PCTCs, in contrast, are larger and range from 15 to 25 µm in diameter 16 . Relative obstacle alignment was chosen so that the displacement caused by cell impact with obstacles (which ranges from zero to one cell radius) increases the likelihood of future cell impacts for large cells more than for small cells. Thus when cellobstacle impact does not lead to capture, larger cells are displaced onto streamlines that impinge onto the next obstacle, while smaller cells are displaced onto streamlines that do not impinge ( Figure 1A). Cell advection was modeled in silico (computational details in supplementary information) to determine obstacle array geometries that optimize PCTCwall interactions and minimize wall shear forces to maximize PCTC capture. For a given obstacle geometry, the frequency of cell-wall...

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents.

Objective. Lubricin, also referred to as superficial zone protein and PRG4, is a synovial glycoprotein that supplies a friction-resistant, antiadhesive coating to the surfaces of articular cartilage, thereby protecting against arthritis-associated tissue wear and degradation. This study was undertaken to generate and characterize a novel recombinant lubricin protein construct, LUB:1, and to evaluate its therapeutic efficacy following intraarticular delivery in a rat model of osteoarthritis (OA).Methods. Binding and localization of LUB:1 to cartilage surfaces was assessed by immunohistochemistry. The cartilage-lubricating properties of LUB:1 were determined using a custom friction testing apparatus. A cell-binding assay was performed to quantify the ability of LUB:1 to prevent cell adhesion. Efficacy studies were conducted in a rat meniscal tear model of OA. One week after the surgical induction of OA, LUB:1 or phosphate buffered saline vehicle was administered by intraarticular injection for 4 weeks, with dosing intervals of either once per week or 3 times per week. OA pathology scores were determined by histologic analysis.Results. LUB:1 was shown to bind effectively to cartilage surfaces, and facilitated both cartilage boundary lubrication and inhibition of synovial cell adhesion. Treatment of rat knee joints with LUB:1 resulted in significant disease-modifying, chondroprotective effects during the progression of OA, by markedly reducing cartilage degeneration and structural damage.Conclusion. Our findings demonstrate the potential use of recombinant lubricin molecules in novel biotherapeutic approaches to the treatment of OA and associated cartilage abnormalities.Osteoarthritis (OA) severely restricts the daily activities, mobility, and overall quality of life of millions of patients worldwide, imposing a high societal burden that reflects the current lack of effective medical therapies. OA is characterized by escalated degeneration and loss of articular cartilage, the specialized connective tissue covering the ends of interfacing bones within joints. To help withstand formidable biomechanical forces and loads, articular cartilage surfaces possess an inherently low coefficient of friction, which is facilitated in part by localization of the boundary lubricant lubricin (1). Lubricin was originally identified as a lubricating glycoprotein present in synovial fluid (2), and it is now recognized to have a major protective role in preventing cartilage wear and synovial cell adhesion and proliferation (3). Lubricin is encoded by the PRG4 gene, and PRG4-nullifying mutations can cause OA-like symptoms in mice and humans (3,4). Lubricin synthesis/localization (and therefore function) is also down-regulated in sheep (5), guinea pig (6), and rat (7)

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This article reviews existing methods for the isolation, fractionation, or capture of rare cells in microfluidic devices. Rare cell capture devices face the challenge of maintaining the efficiency standard of traditional bulk separation methods such as flow cytometers and immunomagnetic separators while requiring very high purity of the target cell population, which is typically already at very low starting concentrations. Two major classifications of rare cell capture approaches are covered: (1) non-electrokinetic methods (e.g., immobilization via antibody or aptamer chemistry, size-based sorting, and sheath flow and streamline sorting) are discussed for applications using blood cells, cancer cells, and other mammalian cells, and (2) electrokinetic (primarily dielectrophoretic) methods using both electrode-based and insulative geometries are presented with a view towards pathogen detection, blood fractionation, and cancer cell isolation. The included methods were evaluated based on performance criteria including cell type modeled and used, number of steps/stages, cell viability, and enrichment, efficiency, and/or purity. Major areas for improvement are increasing viability and capture efficiency/purity of directly processed biological samples, as a majority of current studies only process spiked cell lines or pre-diluted/lysed samples. Despite these current challenges, multiple advances have been made in the development of devices for rare cell capture and the subsequent elucidation of new biological phenomena; this article serves to highlight this progress as well as the electrokinetic and non-electrokinetic methods that can potentially be combined to improve performance in future studies.

We report on the incorporation of microfluidic structure within a high-water-content hydrogel [4% (w/v) calcium alginate]. We used the microfluidic network to control the chemical environment within the hydrogel and demonstrated higher rates of delivery and extraction of solutes than was achievable by diffusion alone.

Lubricin is a secreted, cytoprotective glycoprotein that contributes to the essential boundary lubrication mechanisms necessary for maintaining low friction levels at articular cartilage surfaces. Diminishment of lubricin function is thereby implicated as an adverse contributing factor in degenerative joint diseases such as osteoarthritis. Lubricin occurs as a soluble component of synovial fluid, and is synthesized and localized in the superficial layer of articular cartilage (and thus has also been described as ''superficial zone protein'', or SZP); however, defined interactions responsible for lubricin retention at this site are not well characterized. In the current studies, we identified molecular determinants that enable lubricin to effectively bind to articular cartilage surfaces. Efficient and specific binding to the superficial zone was observed for synovial lubricin, as well as for recombinant full-length lubricin and a protein construct comprising the lubricin Cterminal (hemopexin-like) domain (LUB-C, encoded by exons 7-12). A construct representing the Nterminal region of lubricin (LUB-N, encoded by exons 2-5) exhibited no appreciable cartilagebinding ability, but displayed the capacity to dimerize, and thus potentially influence lubricin aggregation. Disulfide bond disruption significantly attenuated recombinant lubricin and LUB-C binding to cartilage surfaces, demonstrating a requirement for protein secondary structure in facilitating the appropriate localization of lubricin at relevant tissue interfaces. These findings help identify additional key attributes contributing to lubricin functionality, which would be expected to be instrumental in maintaining joint homeostasis. ß