Yandong Gao

Co-Culture of Neurons and Glia in a Novel Microfluidic Platform

In this study, we developed a microfluidic cell co-culture platform that permits individual manipulation of the microenvironment of different cell types. Separation of the cell culture chambers is controlled by changing the position of a microfabricated valve, which serves as a barrier between the chambers. This unique feature of our platform allowed us to maintain healthy co-cultures of hippocampal neurons and glia for several weeks under optimal conditions. Controlled fluidic exchange between the cell culture chambers provided neurons with a continuous supply of in situ conditioned glia media that was critical for their survival. Using the barrier valve, we transfected neurons in the adjacent chambers with green fluorescent protein (GFP) and mCherry cDNA, respectively, with a transfection efficiency of approximately 40%. Co-culture with glia further enhanced the transfection efficiency of neurons to almost 60%. Thus the microfluidic devices offer a novel platform for the long-term culture, transfection, and individual treatment of central nervous system cells.

A versatile valve-enabled microfluidic cell co-culture platform and demonstration of its applications to neurobiology and cancer biology

A versatile microfluidic platform allowing co-culture of multiple cell populations in close proximity with separate control of their microenvironments would be extremely valuable for many biological applications. Here, we report a simple and compact microfluidic platform that has these desirable features and allows for real-time, live-cell imaging of cell-cell interactions. Using a pneumatically/hydraulically controlled poly(dimethylsiloxane) (PDMS) valve barrier, distinct cell types can be cultured in side-by-side microfluidic chambers with their optimum culture media and treated separately without affecting the other cell population. The platform is capable of both two-dimensional and three-dimensional cell co-culture and through variations of the valve barrier design, the platform allows for cell-cell interactions through either direct cell contact or soluble factors alone. The platform has been used to perform dynamic imaging of synapse formation in hippocampal neurons by separate transfection of two groups of neurons with fluorescent pre- and post-synaptic protein markers. In addition, cross-migration of 4T1 tumor cells and endothelial cells has been studied under normoxic and hypoxic conditions, which revealed different migration patterns, suggesting the importance of the microenvironments in cell-cell interactions and biological activities.

Simultaneous On-Chip DC Dielectrophoretic Cell Separation and Quantitative Separation Performance Characterization

Through integration of a MOSFET-based microfluidic Coulter counter with a dc-dielectrophoretic cell sorter, we demonstrate simultaneous on-chip cell separation and sizing with three different samples including 1) binary mixtures of polystyrene beads, 2) yeast cells of continuous size distribution, and 3) mixtures of 4T1 tumor cells and murine bone marrow cells. For cells with continuous size distribution, it is found that the receiver operator characteristic analysis is an ideal method to characterize the separation performance. The characterization results indicate that dc-DEP separation performance degrades as the sorting throughput (cell sorting rate) increases, which provides insights into the design and operation of size-based microfluidic cell separation.

Biomolecule kinetics measurements in flow cell integrated porous silicon waveguides

A grating-coupled porous silicon (PSi) waveguide with an integrated polydimethylsiloxane (PDMS) flow cell is demonstrated as a platform for near real-time detection of chemical and biological molecules. This sensor platform not only allows for quantification of molecular binding events, but also provides a means to improve understanding of diffusion and binding mechanisms in constricted nanoscale geometries. Molecular binding events in the waveguide are monitored by angle-resolved reflectance measurements. Diffusion coefficients and adsorption and desorption rate constants of different sized chemical linkers and nucleic acid molecules are determined based on the rate of change of the measured resonance angle. Experimental results show that the diffusion coefficient in PSi is smaller than that in free solutions, and the PSi morphology slows the molecular adsorption rate constant by a factor of 102–104 compared to that of flat surface interactions. Calculations based on simplified mass balance equations and COMSOL simulations give good agreement with experimental data.

The Rho family GEF Asef2 regulates cell migration in three dimensional (3D) collagen matrices through myosin II

Cell migration is fundamental to a variety of physiological processes, including tissue development, homeostasis, and regeneration. Migration has been extensively studied with cells on 2-dimensional (2D) substrates, but much less is known about cell migration in 3D environments. Tissues and organs are 3D, which is the native environment of cells in vivo, pointing to a need to understand migration and the mechanisms that regulate it in 3D environments. To investigate cell migration in 3D environments, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that the Rho family guanine nucleotide exchange factor (GEF) Asef2 inhibits cell migration in 3D type I collagen (collagen I) matrices. Treatment of cells with the myosin II (MyoII) inhibitor blebbistatin abolished the decrease in migration by Asef2. Moreover, Asef2 enhanced MyoII activity as shown by increased phosphorylation of serine 19 (S19). Furthermore, Asef2 increased activation of Rac, which is a Rho family small GTPase, in 3D collagen I matrices. Inhibition of Rac activity by treatment with the Rac-specific inhibitor NSC23766 abrogated the Asef2-promoted increase in S19 MyoII phosphorylation. Thus, our results indicate that Asef2 regulates cell migration in 3D collagen I matrices through a Rac-MyoII-dependent mechanism.

A Study of Small Molecule Absorption in Polydimethylsiloxane

While polydimethylsiloxane (PDMS) has proven to be a popular material in the construction of microfluidic devices, the polymer network structure of PDMS makes it permeable to some molecules. This feature can be problematic for applications where thin PDMS walls are used to separate two different molecular streams or cell populations. Therefore, a better understanding of factors affecting small molecule absorption in PDMS is important for better design and operation of microfluidic devices.We report on studies of various factors in the absorption of fluorescent dyes in PDMS. Results show significant effects of thermal aging of PDMS on the absorption of the hydrophobic molecule Nile Red. In addition, the short-term hydrophobic recovery of the PDMS devices after being plasma bonded to a glass slide was investigated. Furthermore, we have tested the effects of aspiration and flushing, which show that the wetting property (hydrophilic or hydrophobic) of the flushing solvents can significantly affect the absorption of the Nile Red dye. Finally, parallel tests of PDMS absorption of fluorescein isothiocyanate (FITC) were conducted with no absorption observed.Copyright

A Simple Approach to Probe the Extracellular Signaling Pathways Using Ligand Traps

Cells communicate with one another through a huge variety of extracellular soluble signaling molecules. A common method in biology to investigate the signaling pathways is to inactivate the gene coding the interested ligand or receptor from cells using modern DNA technology, known as gene knockout. Even though very effective, however, gene knockout is a time-consuming and cost-prohibitive process and requires huge amount of efforts to conduct. Here we present a simple method to probe the extracellular signaling pathways through engineering a semi-permeable barrier between two cell populations. In this approach, ligand traps, receptor-coated nano/micro-particles, are embedded inside the nanoporous barrier. Because the receptors have the ability to selectively bind to certain ligand(s) with high affinity, the associated ligands can be ‘trapped’ inside the barrier when they try to perfuse from one cell population to the other. As a result, the targeted soluble ligands can be effectively blocked from the molecular exchange between the two cell populations. We have demonstrated the feasibility of this novel approach using fluorescent proteins. An analytical model has also been developed to guide the design of the ligand-trap-embedded barrier.Copyright

Molecular Dynamics Simulation of Homogeneous and Heterogeneous Thermal Bubble Nucleation

Thermal bubble nucleation was studied using molecular dynamics for both homogeneous and heterogeneous systems using isothermal-isobaric (NPT) and isothermal-isostress (NPzz T) ensembles. Simulation results indicate that homogeneous thermal bubble nucleation is induced from cavities occurring spontaneously in the liquid when the temperature exceeds the superheat limit. In contrast to published results using NVE and NVT ensembles, no stable nanoscale bubble exists in NPT ensembles, but instead, the whole system changes into vapor phase. For a heterogeneous system composed of a nanochannel with an initial distance of 3.49 nm between the two solid plates, it is found that if the liquid-solid interaction is equal to or stronger than that between liquid argon atoms, the bubble nucleation temperature of the confined liquid argon can be higher than the corresponding homogeneous nucleation temperature, because of the more ordered arrangement of atoms within two solid walls nanometers apart. This observation is in contradiction to the common understanding that homogeneous bubble nucleation temperature sets an upper limit for thermal phase change under a given pressure. Compared to the system where the liquid-solid interaction is the same as that between liquid argon atoms, the system with reduced liquid-solid interaction possesses a significantly reduced bubble nucleation temperature, while the system with enhanced liquid-solid interaction only has a marginally increased bubble nucleation temperature.Copyright