We performed cell-based drug combination screening using an integrated droplet-based microfluidic system based on the sequential operation droplet array (SODA) technique. In the system, a tapered capillary connected with a syringe pump was used for multistep droplet manipulations. An oil-covered two-dimensional droplet array chip fixed in an x-y-z translation stage was used as the platform for cell culture and analysis. Complex multistep operations for drug combination screening involving long-term cell culture, medium changing, schedule-dependent drug dosage and stimulation, and cell viability testing were achieved in parallel in the semiopen droplet array, using multiple droplet manipulations including liquid metering, aspirating, depositing, mixing, and transferring. Long-term cell culture as long as 11 days was performed in oil-covered 500 nL droplets by changing the culture medium in each droplet every 24 h. The present system was applied in parallel schedule-dependent drug combination screening for A549 nonsmall lung cancer cells with the cell cycle-dependent drug flavopiridol and two anticancer drugs of paclitaxel and 5-fluorouracil. The highest inhibition efficiency was obtained with a schedule combination of 200 nM flavopiridol followed by 100 μM 5-fluorouracil. The drug consumption for each screening test was substantially decreased to 5 ng-5 μg, corresponding to 10-1000-fold reductions compared with traditional drug screening systems with 96-well or 384-well plates. The present work provides a novel and flexible droplet-based microfluidic approach for performing cell-based screening with complex and multistep operation procedures.

Three-dimensional (3D) cell culture provides an effective way over conventional two-dimensional (2D) monolayer culture to more closely imitate the complex cellular organization, heterogeneity, and interactions as well as tissue microenvironments in vivo. Here we present a novel droplet-based 3D cell culture method by using droplet array attached on the sidewall of a PDMS piece. Such an arrangement not only avoids cells from adhering on the chip surface for achieving 3D cell culture in nanoliter-scale droplets, but also facilitates performing multiple operations to cells in droplets, including cell suspension droplet generation, drug treatment, and cell staining with a capillary-based liquid handling system, as well as in situ observation and direct scanning with a confocal laser scanning microscope. We optimized the system by studying the effects of various conditions to cell culture including droplet volume, cell density and fabrication methods of the PDMS pieces. We have applied this system in the 3D culture of HepG2 cells and the stimulation testing of an anticancer drug, doxorubicin, to 3D cell spheroids.

Abstract. Hepatocellular carcinoma (Hcc) is one of the most common types of cancer worldwide. The initial hepatocellular alterations that precede the appearence of Hcc include chronic viral hepatitis/cirrhosis, foci of phenotypically altered hepatocytes and, subsequently, dysplastic hepatocytes that form foci and nodules. These changes cause a discrepancy in the microenvironment of liver cells, which may result in changes in the protein expression profile of the cells. The aim of the present study was to investigate differences between the protein expression profiles at various stages of liver disease in order to better understand the mechanisms of Hcc and to identify potential biomarkers for its early diagnosis. The proteins of specific cells were obtained from Hcc tissue sections and pre-cancerous lesions using a manual microdissection technique, and were investigated by a two dimensional gel electrophoresis (2-de) Maldi-ToF MS proteomics approach. Select identified proteins were reconfirmed by immunohistochemistry. A total of 95 differentially expressed proteins, with an over 2-fold disparity in expression levels between cells of varying morphology during the stages of hepatocarcinogenesis, were detected by 2-de. among these 95 proteins, 80 were determined to be involved in numerous cell functions, including cell growth and proliferation, protein synthesis and metabolism, apoptosis and signal transduction. These identified proteins, which include stratifin (14-3-3), transgelin 2, heat-shock protein (HSP)70, HSP27, manganese superoxide dismutase, prohibitin, dJ1, α-enolase, peroxiredoxin 6, aldo-keto reductase family member B10, phosphoglycerate kinase 1, α-1-antitrypsin and nm23-H1, may play a role in the development of Hcc. Protein expression profiles differed markedly between the HCC tissue samples and pre-cancerous lesions, suggesting that alterations in protein expression occurred frequently during the process of hepatocarcinogenesis. analysis of the differential expression of proteins related to the development of Hcc may help elucidate the molecular mechanisms of the disease. These proteins may also serve as candidate biomarkers for early Hcc diagnosis. IntroductionHepatocellular carcinoma (Hcc) is one of the world's most common malignancies. The majority of Hcc cases are associated with chronic hepatitis or cirrhosis. Pathological changes in Hcc are involved in liver cell degeneration and necrosis, inflammatory cell infiltration, hepatocyte regeneration and fibrous tissue proliferation. During the development of Hcc, the liver undergoes a series of morphological changes, including chronic viral hepatitis/cirrhosis, foci of phenotypically altered hepatocytes and atypical hyperplasia (1-4). These changes result in an altered microenvironment, in which the intracellular protein expression pattern is changed as well.The proteomics approach allows for the analysis of changes in the protein expression profile of cells during disease progression. By comprehensively analyzing changes in protein expression patt...

Establishing cell migration assays in multiple different microenvironments is important in the study of tissue repair and regeneration, cancer progression, atherosclerosis, and arthritis. In this work, we developed a miniaturized and massive parallel microfluidic platform for multiple cell migration assays combining the traditional membrane-based cell migration technique and the droplet-based microfluidic technique. Nanoliter-scale droplets are flexibly assembled as building blocks based on a porous membrane to form microdroplet chains with diverse configurations for different assay modes. Multiple operations including in-droplet 2D/3D cell culture, cell co-culture and cell migration induced by a chemoattractant concentration gradient in droplet chains could be flexibly performed with reagent consumption in the nanoliter range for each assay and an assay scale-up to 81 assays in parallel in one microchip. We have applied the present platform to multiple modes of cell migration assays including the accurate cell migration assay, competitive cell migration assay, biomimetic chemotaxis assay, and multifactor cell migration assay based on the organ-on-a-chip concept, for demonstrating its versatility, applicability, and potential in cell migration-related research.

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