Jennifer T. Blundo

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.

Poly (vinyl alcohol) (PVA) is a versatile polymer that when modified with functional groups can be polymerized to produce hydrogels with a range of mechanical properties. In this study, PVA was modified with pendent acrylamide groups and crosslinked via photopolymerisation. The swelling behavior and tensile properties of the resulting hydrogels were studied as a function of percent macromer at the time of polymerization, functional group density, backbone molecular weight, and percent hydrolysis of the PVA. Percent macromer had the strongest influence, with tensile modulus increasing in direct proportion to increasing percent macromer. Changing the functional group density of the macromers as well as changing the molecular weight of the PVA backbone significantly impacted the swelling and mechanical behavior. Although percent hydrolysis of the PVA backbone resulted only in slight variations in the network, it did prove to be a significant variable. However, it was also found that the tensile modulus was directly related to the amount of polymer in the hydrogel. Rheological studies demonstrated that by increasing the number of chain interactions in solution (i.e., increasing the percent macromer, etc.) the resulting network produced was more interconnected and thus stronger. Overall, it was found that hydrogels produced from PVA macromers that had larger molecular weights and more functional groups per PVA chain and were less hydrophilic and formulated into higher percent macromer solutions were stronger, stiffer materials.

A healthy skeleton relies on bone's ability to respond to external mechanical forces. The molecular mechanisms by which bone cells sense and convert mechanical stimuli into biochemical signals, a process known as mechanotransduction, are unclear. Focal adhesions play a critical role in cell survival, migration and sensing physical force. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that controls focal adhesion dynamics and can mediate reparative bone formation in vivo and osteoblast mechanotransduction in vitro. Based on these data, we hypothesized that FAK plays a role in load-induced bone formation. To test this hypothesis, we performed in vitro fluid flow experiments and in vivo bone loading studies in FAK-/- clonal lines and conditional FAK knockout mice, respectively. FAK-/- osteoblasts showed an ablated prostaglandin E(2) (PGE(2)) response to fluid flow shear. This effect was reversed with the re-expression of wild-type FAK. Re-expression of FAK containing site-specific mutations at Tyr-397 and Tyr-925 phosphorylation sites did not rescue the phenotype, suggesting that these sites are important in osteoblast mechanotransduction. Interestingly, mice in which FAK was conditionally deleted in osteoblasts and osteocytes did not exhibit altered load-induced periosteal bone formation. Together these data suggest that although FAK is important in mechanically-induced signaling in osteoblasts in vitro, it is not required for an adaptive response in vivo, possibly due to a compensatory mechanism that does not exist in the cell culture system.

Therapeutic effects from injection of stem cells are often hampered by acute donor cell death as well as migration away from damaged areas. This is likely due to the fact that injected cells do not have the physical and biochemical cues for ordered engrafment. Here we evaluate 3 common biomatrices (Matrigel, Collagen I, Purmatrix) that has the potential of providing suitable scaffolds needed to enhance stem cell survival. The longitudinal fate of transplanted stem cells was monitored by reporter imaging techniques.

A BioMEMS platform was designed and fabricated to enable electromechanical stimulation of cell cultures. The device consists of a stretchable microelectrode array embedded in a biocompatible elastomeric polymer, which is deformed by vacuum over a glass baseplate. The entire device is optically transparent and ∼1mm thick. Four cell culture wells apply electrical and/or mechanical stimulation in the range of 1–10 V/cm and 0–10% equibiaxial strain. Studies of electromechanical stimulation of cardiac myocyte precursors on this platform are proposed to elucidate mechanisms of differentiation.