Sihong Wang

A high-throughput microfluidic real-time gene expression living cell array

The dynamics of gene expression are fundamental to the coordination of cellular responses. Measurement of temporal gene expression patterns is currently limited to destructive low-throughput techniques such as northern blotting, reverse transcription polymerase chain reaction (RT-PCR), and DNA microarrays. We report a scalable experimental platform that combines microfluidic addressability with quantitative live cell imaging of fluorescent protein transcriptional reporters to achieve real-time characterization of gene expression programs in living cells. Integrated microvalve arrays control row-seeding and column-stimulation of 256 nanoliter-scale bioreactors to create a high density matrix of stimulus-response experiments. We demonstrate the approach in the context of hepatic inflammation by acquiring approximately 5000 single-time-point measurements in each automated and unattended experiment. Experiments can be assembled in hours and perform the equivalent of months of conventional experiments. By enabling efficient investigation of dynamic gene expression programs, this technology has the potential to make significant impacts in basic science, drug development, and clinical medicine.

Three-dimensional primary hepatocyte culture in synthetic self-assembling peptide hydrogel.

Drug metabolism studies and liver tissue engineering necessitate stable hepatocyte cultures that express liver functions for a minimum of 4 days to 3 weeks. Current techniques, using different biomaterials and geometries, that maintain hepatocellular function in vitro exhibit a low cell density and functional capacity per unit volume. Herein we investigated a well-defined synthetic peptide that can self-assemble into three-dimensional interweaving nanofiber scaffolds to form a hydrogel, PuraMatrix, as a substrate for hepatocyte culture. Freshly isolated primary rat hepatocytes attached, migrated, and formed spheroids within 3 days after seeding on PuraMatrix. Hepatocytes expressed the apical membrane marker dipeptidyl peptidase IV at cell-cell contacts. Compared to the collagen sandwich, albumin and urea secretion on PuraMatrix were higher for the first week, and cytochrome P450IA1 activity was higher throughout the culture period. Mitochondrial membrane potential 1 day after seeding was higher on PuraMatrix than in the collagen sandwich, suggesting better preservation of the metabolic machinery. PuraMatrix and Matrigel showed similar albumin and urea production. PuraMatrix is an attractive system for generating hepatocyte spheroids that quickly restore liver functions after seeding. This system is also amenable to scale-up, which makes it suitable for in vitro toxicity, hepatocyte transplantation, and bioartificial liver development studies.

Correlation of HSP70 Expression and Cell Viability Following Thermal Stimulation of Bovine Aortic Endothelial Cells

Thermal preconditioning protocols for cardiac cells were identified which produce elevated HSP70 levels while maintaining high cell viability. Bovine aortic endothelial cells were heated with a water bath at temperatures ranging from 44 to 50 degrees C for periods of 1-30 min. Thermal stimulation protocols were determined which induce HSP70 expression levels ranging from 2.3 to 3.6 times the control while maintaining cell viabilities greater than 90%. An Arrhenius injury model fit to the cell damage data yielded values of A = 1.4 X 10(66) s(-1) and Ea = 4.1 X 10(5) J/mol. Knowledge of the injury parameters and HSP70 kinetics will enhance dosimetry guideline development for thermal stimulation of heat shock proteins expression in cardiac tissue.

Three dimensional microfluidic cell arrays for ex vivo drug screening with mimicked vascular flow.

Currently, there are no reliable ex vivo models that predict anticancer drug responses in human tumors accurately. A comprehensive method of mimicking a 3D microenvironment to study effects of anticancer drugs on specific cancer types is essential. Here, we report the development of a three-dimensional microfluidic cell array (3D μFCA), which reconstructs a 3D tumor microenvironment with cancer cells and microvascular endothelial cells. To mimic the in vivo spatial relationship between microvessels and nonendothelial cells embedded in extracellular matrix, three polydimethylsiloxane (PDMS) layers were built into this array. The multilayer property of the device enabled the imitation of the drug delivery in a microtissue array with simulated blood circulation. This 3D μFCA system may provide better predictions of drug responses and identification of a suitable treatment for a specific patient if biopsy samples are used. To the pharmaceutical industry, the scaling-up of our 3D μFCA system may offer a novel high throughput screening tool.

Periodic Heat Shock Accelerated the Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Pellet Culture

Osteoarthritis (OA) is one of diseases that seriously affect elderly peoples quality of life. Human mesenchymal stem cells (hMSCs) offer a potential promise for the joint repair in OA patients. However, chondrogenic differentiation from hMSCs in vitro takes a long time (∼6 weeks) and differentiated cells are still not as functionally mature as primary isolated chondrocytes, though chemical stimulations and mechanical loading have been intensively studied to enhance the hMSC differentiation. On the other hand, thermal stimulations of hMSC chondrogenesis have not been well explored. In this study, the direct effects of mild heat shock (HS) on the differentiation of hMSCs into chondrocytes in 3D pellet culture were investigated. Periodic HS at 41°C for 1 hr significantly increased sulfated glycosaminoglycan in 3D pellet culture at Day 10 of chondrogenesis. Immunohistochemical and Western Blot analyses revealed an increased expression of collagen type II and aggrecan in heat-shocked pellets than non heat-shocked pellets on Day 17 of chondrogenesis. In addition, HS also upregulated the expression of collagen type I and X as well as heat shock protein 70 on Day 17 and 24 of differentiation. These results demonstrate that HS accelerated the chondrogenic differentiation of hMSCs and induced an early maturation of chondrocytes differentiated from hMSCs. The results of this study will guide the design of future protocols using thermal treatments to facilitate cartilage regeneration with human mesenchymal stem cells.

Microfluidic cell chips for high-throughput drug screening

The current state of screening methods for drug discovery is still riddled with several inefficiencies. Although some widely used high-throughput screening platforms may enhance the drug screening process, their cost and oversimplification of cell-drug interactions pose a translational difficulty. Microfluidic cell-chips resolve many issues found in conventional HTS technology, providing benefits such as reduced sample quantity and integration of 3D cell culture physically more representative of the physiological/pathological microenvironment. In this review, we introduce the advantages of microfluidic devices in drug screening, and outline the critical factors which influence device design, highlighting recent innovations and advances in the field including a summary of commercialization efforts on microfluidic cell chips. Future perspectives of microfluidic cell devices are also provided based on considerations of present technological limitations and translational barriers.

Heat Shock Protein 70 Expression Kinetics

HSP70 is well known for its major role in cardiac ischemia protection. The purpose of this study was to determine the HSP70 expression kinetics for new protocol design in cardiac surgery, based on HSP70 protection function in clinical applications. Bovine aortic endothelial cells (BAEC) were used in experiments. Cells were heated at 42°C at different time intervals up to 5 hours and subsequently incubated at 37°C for up to 48 hours. Western blot and quantitative protein analysis were performed to measure HSP70 expression. The expression kinetics is a function of thermal stress time as well as poststress time. At least three stages were identified for the kinetics curve: increasing, maximum plateau and decreasing regions. The peak HSP70 concentration is 10 times the basal level for western blot analysis in BAECs. Two hours incubator heating followed by twelve hours post-heating falls in the plateau region. This research result provides information applicable to evaluation of energy sources and heating methods to induce optimal HSP70 expression in a target tissue.Copyright

Liver Tissue Engineering

The development of liver support systems has been in intensive investigation for over 40 years. The main driving force is the shortage of donor organs for orthotopic liver transplantation. Liver cell transplantation and extracorporeal bioartificial livers (BAL) may bridge patients with end-stage liver diseases to successful orthotopic liver transplantation, support patients with acute liver failure to recover, and provide a curing method to patients with certain liver metabolic diseases. Another frontier of current liver tissue engineering is to construct many functional liver units in vitro for drug toxicity and metabolism screening. Much progress has been made, with several artificial liver dialysis devices on the market, a few BAL systems in clinical trials, and other in vitro micro-liver models in development. On the other hand, many lessons have been learned as well. In this chapter, we will focus on the review of advancement, challenges and the critical issues that have to be solved in the development of BAL systems and hepatic cell transplantation as well as in vitro micro-liver models from a tissue engineering perspective.

A multifunctional microfluidic platform for generation, trapping and release of droplets in a double laminar flow

Droplet microfluidics, involving micrometer-sized emulsion of droplets is a growing subfield of microfluidics which attracts broad interest due to its application on biological assays. Droplet-based systems have been used as microreactors as well as to encapsulate many biological entities for biomedical and biotechnological applications. Here, a novel microfluidic device is presented for the generation, trapping and release of aqueous including hydrogel droplets in a double laminar oil flow. This platform enables the storage and release of picoliter-sized droplets in two different carrier oils by using hydrodynamic forces without the need of electrical forces or optical actuators. Furthermore, this design allows droplets to be selectively and simultaneously exposed to two different conditions and collected on demand. Successful encapsulation of hepatoma H35 cells was performed on-chip. Viability of cell-laden droplets was performed off-chip to assess the potential applications in 3D encapsulation cell culture and drug discovery assays.

Real-time detection of cellular death receptor-4 activation by fluorescence resonance energy transfer†

Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL–DR4 in live cells in real‐time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4‐CFP and DR4‐YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4‐CFP and DR4‐YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegels YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4‐CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4‐CFP/YFP reporter cells exhibited a colocalized expression of DR4‐CFP and DR4‐YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi‐fluorescence microscopy for FRET analysis. The real‐time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time‐lapse fluorescence microscopy. Therefore, DR4‐CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti‐cancer drug screening to identify compounds that are capable of activating TRAIL pathways. Biotechnol. Bioeng. 2013; 110: 1396–1404.