1,721,040 research outputs found
A picoliter array in a microfluidic device
No full textWe present a simple microfluidic device that uses an array of well-defined chambers to immobilize thousands of femtoliter-to picoliter-scale aqueous drops suspended in inert carrier oil. This device enables timelapse studies of large numbers of individual drops, while simultaneously enabling subsequent drop recovery
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Rapid Biophysical Characterization of Cancer Cells by Quantitative Deformability Cytometry
Full text linkCells are complex, viscoelastic materials that undergo changes in their mechanical phenotype, or ‘mechanotype’, during diverse physiological and disease processes, such as malignant transformation. As cancer cells exhibit altered cell mechanical properties compared to their benign counterparts, cell mechanotype is an emerging hallmark of cancer and demonstrates potential to enhance cancer detection and classification. However, widespread adoption of cell mechanotype as a clinical biomarker for cancer requires standardized metrics for high throughput mechanotyping measurements.This dissertation presents a microfluidic platform, quantitative deformability cytometry (q-DC), for rapid, calibrated measurements of single-cell mechanical properties. Cells are driven to deform through micron- scale constrictions at 100 cells/second, while changes in cell strain are tracked by a high-speed camera. The applied mechanical stresses induced by the driving pressure are determined using gel calibration particles, which enables calibrated measurements of elastic modulus and fluidity from the single-cell stress-strain relationships. Additional physical properties, such as cell size, strain, transit time, and creep time, are also measured for individual cells by q-DC. This dissertation highlights a comprehensive methodology for designing, analyzing, and reducing variability in q-DC measurements; the calibration method for measuring the applied stress in the microfluidic channels; and the influence of stress and strain in q-DC mechanotyping.This dissertation also demonstrates how multiple physical phenotypes from q-DC can be used to distinguish human cancer cell lines and predict the ability of cells to invade through a matrix. A physical phenotyping model of invasion is trained and validated using breast and ovarian human cancer cell lines with both genetic and pharmacologic perturbations, which correctly predicts the invasion of five cancer cell samples; whereas one context is identified where the model does not accurately predict invasion. Taken together, this work lays the groundwork for calibration in high throughput mechanotyping methods, demonstrates the predictive power of multiple physical phenotypes for cell invasion, and incites deeper investigation into additional predictive markers for cancer cell invasion
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Dissecting the role of cell physical properties in the invasion of pancreatic ductal adenocarcinoma
Full text linkMetastasis is a physical process in which cells are required to deform through narrow gaps and generate forces as they invade surrounding tissues. Understanding the relationship between invasion and cell physical properties, such as deformability and contractility, can impart knowledge that guides the development of new therapeutics, yet the physical properties of pancreatic cancer cells are still poorly understood. In many cancers, more invasive cells are more deformable than less invasive cells. However, using atomic force microscopy, I discovered that more invasive pancreatic ductal adenocarcinoma (PDAC) cells tend to have a higher Young’s modulus, indicating that they are stiffer. This finding challenges the oversimplified notion that decreased cell stiffness is a hallmark of cancer invasion. To gain a more complete understanding of why stiffer PDAC cells are more invasive, I examine the ability of PDAC cells to actively generate forces through actomyosin contractility and actin polymerization, as both processes have been associated with cancer cell invasion and stiffness. Using pharmacologic inhibitors, I determined that actomyosin contractility (inhibited with blebbistatin), as well as the polymerization of actin by Arp2/3 (inhibited with CK-666) and formin (inhibited with SMIFH2), contribute to the stiffness of PDAC cells. Interestingly, I observe that these inhibitors are cell line specific, indicating that different PDAC cell lines may rely on different modes of motility to invade the extracellular matrix. In addition, using measurements of cell physical phenotypes obtained by microfluidic quantitative deformability cytometry, my collaborators and I define a minimal set of physical phenotypes that can predict PDAC cell invasion. Taken together, my dissertation work provides valuable insight into the physical mechanisms of cancer cell invasion, and establishes a physical model that can predict cell invasion based on single-cell physical phenotypes including cell stiffness. My results provide the foundation for future studies into the relationship between cell physical properties and the tumor microenvironment, which undergoes substantial physical changes throughout PDAC progression
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From cell to table: Engineering multicomponent tissue by spontaneous adhesion of myogenic and adipogenic microtissues cultured with customized scaffolds for cultured meat applications
No full textThere is increasing consumer demand for alternative animal protein products that are delicious and sustainably produced to address concerns about the impacts of mass-produced meat on human and planetary health. Cultured meat has the potential to provide a source of nutritious dietary protein that is both palatable and has reduced environmental impact. The process of culturing meat involves growing animal cells derived from tissues of interest in vitro in a bioreactor. To further support and guide the growth of cells into tissue, animal cells are typically grown on a matrix, or scaffold, which provides a substrate for attachment and can contribute to developing structure in cultured cuts of meat. However, technical challenges currently limit the scalability of cultured meat including the ability to engineer structured cuts of meat—such as a steak—at a scale compatible with food production. The adoption of cultured meat may also be challenged by negative public perceptions. One approach is to make delicious cultured meat that consumers will crave. For example, the integration of intramuscular fat—or marbling—into cultured meat will be critical for meat texture, mouthfeel, flavor, and thus consumer appeal. In this dissertation, I highlight potential advantages of using edible scaffolds to advance cultured meat production by providing structure to build multicomponent tissues. I demonstrate that edible microcarriers can support the expansion and differentiation of both myogenic and adipogenic cells in a single bioreactor system. My findings also show the potential of tuning physical properties of edible microcarriers to increase the efficiency of adipose tissue generation. Finally, I describe an approach to engineer marbled cultured meat. The key innovation in my approach is the engineering of myogenic and adipogenic microtissues, which I use as building blocks that spontaneously adhere to produce marbled cultured meat. Taken together, these findings provide valuable insights which can be translated for the upscaled production of delicious cultured meat products
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The effect of beta-adrenergic signaling on the mechanics of the breast tumor microenvironment mediated by cancer-associated fibroblasts
No full textActivation of β-adrenergic receptors (βARs)—for example by endogenous stress hormones—is associated with breast cancer progression, but complete mechanistic knowledge that would support the development of more effective treatment strategies is lacking. While biochemical pathways in cancer progression are well characterized, altered tumor mechanics also contribute to cancer progression. We previously discovered that βAR activation alters the mechanical behaviors of breast tumor cells, but if we could understand how βAR signaling regulates tumor mechanics, this would inform opportunities for more effective therapies. To dissect how β-blockade impacts tumor mechanics, we analyzed gene expression profiles of tumor samples from breast cancer patients in a Phase II window-of-opportunity randomized control trial (Hiller et al., 2020). Bulk RNA-sequencing analysis showed that the βAR antagonist, propranolol, downregulates expression of extracellular matrix (ECM) components. Since fibroblasts are major contributors to the tumor microenvironment (TME), we next determined how βAR activation impacts cancer-associated fibroblasts (CAFs) derived from human breast tumors (Tchou et al., 2012). CAFs are primary modulators of ECM composition, and the contractile forces they exert on the surrounding matrix impact tumor stiffness. Bulk RNA-sequencing analysis of CAFs treated with the βAR agonist isoproterenol shows an upregulation of actin-mediated contractility and growth factor genes. To measure how βAR activation affects CAF contractility, we embed cells in 3D collagen hydrogels, treat with isoproterenol, and quantify the hydrogel size after 24 hours. We additionally assess cytoskeletal structure, including dually phosphorylated myosin light chain 2 (ppMLC2) and paxillin. Findings from our ongoing studies should provide further insight into how β-adrenergic signaling impacts tumor mechanics, which could guide optimization of current chemotherapy treatments
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Identification of Small Molecules as Novel Adipogenic Enhancers for Edible Adipose Tissues
No full textWith the global population projected to reach 9.8 billion by 2050, there is a critical need for sustainable and innovative food sources. Cultured meat, an innovative and sustainable approach to food production that allows the production of meat by cultivating animal cells in vitro, exhibits the potential to circumvent the challenges associated with traditional animal agriculture. A key to the success of cultured meat is developing adipose tissue, crucial for achieving the desired taste, texture, and nutritional quality. Accelerating adipogenesis has the potential to improve production efficiency, making large-scale cultured meat manufacturing more viable and cost-effective. This study aims to identify small molecules that accelerate adipogenesis for edible fat tissue production using a high-throughput screen. Our findings identified magnolol and dicoumarol as effective natural compounds that promote lipid accumulation in differentiating mouse, rabbit, and porcine preadipocytes. These compounds have also demonstrated a synergetic effect in accelerating adipogenesis, which highlights the potential of novel treatments in effectively promoting adipocyte differentiation across various species
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Unveiling Novel Regulators of Mechanical Phenotype in Breast and Ovarian Cancer Cells by High-throughput Deformability Screening
No full textDuring cancer metastases, tumor cells migrate through the extracellular matrix (ECM) of connective tissues and traverse even smaller, sub-micron pores to spread to distant sites. The biophysical properties of cells, such as cellular deformability, are implicated in metastasis. In addition, tumor cells with mesenchymal phenotype tend to be more deformable than those with epithelial phenotype. Our current investigation of cellular mechanical phenotype or mechanotype builds on previously reported differences in acquired cisplatin-resistant ovarian adenocarcinoma cells. Our team screened the library of 1280 pharmacologically active compounds (LOPAC) to identify drugs that could reverse the inherent deformability of cisplatin-resistant cancer cells. We identified top hits that significantly decrease cell deformability of OVCAR5-cisplatin resistant (cisR) cells and analyze novel regulators of cellular deformability using high-throughput parallel microfiltration (PMF). We used Mergeomics, a bioinformatics approach to conduct meta-analysis across the molecular targets of the hits, their associated signaling pathways, and subnetwork neighbors, to predict regulators of cellular deformability, and validated these results experimentally. To assess cell deformability we used cellular filtration, where the percent retention represents the fraction of suspended cells retained over our micro-pore membrane filter following PMF. We confirmed the effects of key drivers in regulating cell deformability using pharmacologic and genetic manipulations. We also observed shared molecular mediators of cellular mechanotype, deformability, and invasion in breast and ovarian cancer cell lines, following treatment with another pharmacologically active agent, 4′-hydroxyacetophenone (4-HAP). 4-HAP is known to inhibit non-muscle myosin II (NM2), increasing cell stiffness and cortical tension. We discussed the functional interpretation of cellular deformability and overall cell mechanotype to obtain a mechanistic understanding of the mechanome and shared regulation of metastatic behaviors in breast and ovarian cancer cell lines. Taken together, our data revealed potential targets to modulate cellular mechanotype and highlights cell mechanical behaviors as a target for anti-cancer therapeutics
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Engineering Adipose Tissue: From Microcarriers to Small Molecule Enhancers of Adipogenesis
No full textCultured meat, or the practice of growing animal-based meat ex vivo, has potential to address consumer concerns and complement existing methods of animal protein production to improve resiliency of our food systems. Research and development to date in the cultured meat sector has focused heavily on advancing methods to culture muscle tissue; but fat plays a key role in improving meat palatability, and thus will be integral for consumer adoption of cultured meat products. This dissertation reports efforts towards engineering fat tissue by using mechanobiology and chemical biology approaches to enhance the accumulation of lipids, which are the primary providers of taste and aroma in fat.Chapter 1 details current challenges and opportunities of fabricating edible, bioreactor-compatible scaffolds for scaling up the production of cultured meat. We highlight potential advantages of using edible scaffolds to advance cultured meat production by accelerating cell growth and differentiation, providing structure to build complex 3D tissues, and enhancing the nutritional and sensory properties of cultured meat.
In Chapter 2, we describe the synthesis of polyacrylamide microcarriers, using inverse emulsification, that are customizable in size (20–250 �m) and stiffness, spanning a physiologically relevant range between approximately 0.5 to 75 kPa. We show that the microcarriers support the culture of adherent cell types. We also demonstrate the utility of these microcarriers, using adipocytes, as a tool for imaging-based cellular assays, which make them compatible with high-throughput methods for identifying mediators of cell behaviors.
With the goal of discovering food-safe media additives that increase lipid accumulation in cultured fat, Chapter 3 describes the identification of small molecules that promote differentiation using a high-throughput adipogenic assay. After screening libraries of natural products, metabolites, and FDA-approved small molecules, we validate three novel candidates that exhibit low toxicity to cells and the potential to enhance lipid accumulation in adipocytes
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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