Seok Chung

Capillary morphogenesis is a complex cellular process that occurs in response to external stimuli. A number of assays have been used to study critical regulators of the process, but those assays are typically limited by the inability to control biochemical gradients and to obtain images on the single cell level. We have recently developed a new microfluidic platform that has the capability to control the biochemical and biomechanical forces within a three dimensional scaffold coupled with accessible image acquisition. Here, the developed platform is used to evaluate and quantify capillary growth and endothelial cell migration from an intact cell monolayer. We also evaluate the endothelial cell response when placed in co-culture with physiologically relevant cell types, including cancer cells and smooth muscle cells. This resulted in the following observations: cancer cells can either attract (MTLn3 cancer cell line) endothelial cells and induce capillary formation or have minimal effect (U87MG cancer cell line) while smooth muscle cells (10T 1/2) suppress endothelial activity. Results presented demonstrate the capabilities of this platform to study cellular morphogenesis both qualitatively and quantitatively while having the advantage of enhanced imaging and internal biological controls. Finally, the platform has numerous applications in the study of angiogenesis, or migration of other cell types including tumor cells, into a three-dimensional scaffold or across an endothelial layer under precisely controlled conditions of mechanical, biochemical and co-culture environments.

New and more biologically relevant in vitro models are needed for use in drug development, regenerative medicine, and fundamental scientific investigation. While the importance of the extracellular microenvironment is clear, the ability to investigate the effects of physiologically relevant biophysical and biochemical factors is restricted in traditional cell culture platforms. Moreover, the versatility for multi-parameter manipulation, on a single platform, with the optical resolution to monitor the dynamics of individual cells or small population is lacking. Here we introduce a microfluidic platform for 3D cell culture in biologically derived or synthetic hydrogels with the capability to monitor cellular dynamics in response to changes in their microenvironment. Direct scaffold microinjection, was employed to incorporate 3D matrices into microfluidic devices. Our system geometry permits a unique window for studying directional migration, e.g. sprouting angiogenesis, since sprouts grow predominantly in the microscopic viewing plane. In this study, we demonstrate the ability to generate gradients (non-reactive solute), surface shear, interstitial flow, and image cells in situ. Three different capillary morphogenesis assays are demonstrated. Human adult dermal microvascular endothelial cells (HMVEC-ad) were maintained in culture for up to 7 days during which they formed open lumen-like structures which was confirmed with confocal microscopy and by perfusion with fluorescent microspheres. In the sprouting assay, time-lapse movies revealed cellular mechanisms and dynamics (filopodial projection/retraction, directional migration, cell division and lumen formation) during tip-cell invasion of underlying 3D matrix and subsequent lumen formation.

We have developed a microchip for polymerase chain reaction (PCR) with polydimethylsiloxane (PDMS). PDMS has good characteristics: it is cheap, transparent, easy to fabricate and biocompatible. But in micro PCR, the porosity of PDMS causes several critical problems such as bubble formation, sample evaporation and protein adsorption. To solve those problems, we coated the micro PCR chips with Parylene film, which has low permeability to moisture and long-term stability. We investigated the influence of low thermal conductivity of PDMS and Parylene on the thermal characteristics of the PCR chips with numerical analysis. The thermal responses of micro PCR chips were compared for three materials: silicon, glass and PDMS. From the results, we identified appropriate thermal responses of the PDMS-based micro PCR chips by heating both the top and bottom sides. We could successfully amplify the angiotensin converting enzyme gene with as small a volume as 2 μl on the PDMS-based micro PCR chips without any additives.

Harmful materials in the blood are prevented from entering the healthy brain by a highly selective blood-brain barrier (BBB), and impairment of barrier function has been associated with a variety of neurological diseases. In Alzheimer's disease (AD), BBB breakdown has been shown to occur even before cognitive decline and brain pathology. To investigate the role of the cerebral vasculature in AD, a physiologically relevant 3D human neural cell culture microfluidic model is developed having a brain endothelial cell monolayer with a BBB-like phenotype. This model is shown to recapitulate several key aspects of BBB dysfunction observed in AD patients: increased BBB permeability, decreased expression of claudin-1, claudin-5, and VE-cadherin, increased expression of matrix-metalloproteinase-2 and reactive oxygen species, and deposition of β-amyloid (Aβ) peptides at the vascular endothelium. Thus, it provides a well-controlled platform for investigating BBB function as well as for screening of new drugs that need to pass the BBB to gain access to neural tissues.