During development of the nervous system the response of growing axons to their environment is critical to the formation of the complex wiring pattern between neurons. Growth and guidance factors combined with extracellular matrices influence the speed and direction of axonal growth. Although much progress has been made in identifying the factors that influence axonal growth, as well as how axons respond to these factors individually, much less is known about how axons behave in response to the combined effects of multiple factors. To fully understand how axonal growth is regulated ideally this should be studied in vivo. The ability to express fluorescent protein markers in neurons has made such an approach possible. However, it is still limited by a variety of factors. Imaging individual live axons deep in brain tissue is still restricted to layers less than 600 microns. Phototoxicity limits the time lengths of such observations. More importantly, the environment is difficult to characterize or control.
As a complementary approach, we are developing in vitro environments that potentially mimic some of the complexity found in vivo, in particular the development of the anterior visual pathway. In this system, the axon trajectories are simple, multiple relevant guidance molecules have been identified already (many tested with explants in vitro), and a common cause of blindness (Optic Nerve Hypoplasia) is associated with defects in this process. Additionally, the patterns of guidance molecules found on the flat anatomy of the retina is ideally suited to mimicking by micropatterning and microfluidics techniques. This mimicry is accomplished by combining microfluidics patterning of diffusible gradients and substrate patterning of axon pathfinding cues, including axon guidance factors and extracellular matrix molecules. As a source of highly homogeneous cell populations, we use isolated mouse retinal ganglion cells (RGCs), a cell type that responds to Netrin-1 gradients. For experiments designed to maximize the integrity of the cells (isolation procedures are damaging to cells), we will use retinal explants and we microfluidically isolate the axons from their somas. RGCs (or their axons) are exposed to various soluble factors that have previously been shown to affect their axon growth in vivo. Combinatorial microfluidic mixers allow us to test the effects of multiple factors on the direction and speed of axonal growth of RGCs. These experiments allow us to quantitatively examine the basic principles that govern axon pathfinding in the development of the anterior visual pathway and help to better understand the basis of developmental defects in axon growth that alter the organization and function of the nervous system.
Bhattacharjee, N., Li, N., Keenan, T.M., and Folch, A. “A Neuron-Benign Microfluidic Gradient Generator for Studying the Growth of Mammalian Neurons towards Axon Guidance Factors”, Integrative Biology 2, 669 (2010).
Keenan, T.M. and Folch, A. “Biomolecular gradients in cell culture systems”, Lab Chip 8, 34 (2008).
Keenan, T.M., Hsu, C.-H., and Folch, A. “Microfluidic “Jets” for Generating Steady-State Gradients of Soluble Molecules on Open Surfaces”, Appl. Phys. Lett. 89: 114103 (2006).
Keenan, T.M., Hooker, A., Spilker, M. E., Boggy, G. J., Li, N., Vicini, P., and Folch, A. “Automated Identification of Axonal Growth Cones in Time-Lapse Image Sequences”, J. Neurosci. Methods 151: 232 (2006).
Li, N. and Folch, A. “Integration of topographical and biochemical cues by axons during growth on microfabricated 3-D substrates”, Experimental Cell Research 311: 307 (2005).
Despite decades of intensive research and clinical trials, glioblastoma multiforme (GBM) remains uniformly lethal within 12-16 months of diagnosis. This grim reality reflects the marginal effectiveness of current treatments that are applied generically to GBM patients without consideration of the enormous impact of individual variations in tumor phenotypes. The most commonly proposed screening methods for GBM utilize isolated tumor cell cultures or patient-derived xenografts (PDXs). However, cultured tumor cells do not retain phenotypic heterogeneity or interactions with the tumor microenvironment, both critical factors in treatment response and resistance. In contrast, while PDX models retain these features, they are cumbersome and expensive. Critically, variable and lengthy lead times for growth make it impractical for PDX testing to inform on a timescale rapid enough to guide decisions for patient-specific therapy.
A relatively unexplored alternative system for patient-specific drug sensitivity profiling, patient-derived organotypic slice cultures (PDSCs), addresses many of these challenges. Compared to cell cultures, PDSCs retain both tumor cell heterogeneity and tumor microenvironment. In contrast to PDX, PDSCs can be tested in a clinically relevant time frame. Our preliminary studies demonstrate that PDSCs are readily established from human GBMs and conserve proliferation and morphologic integrity for up to 21 days. Unlike cell cultures, PDSCs lack a standardized high-throughput drug delivery system. To address this need, we have developed a microfluidic device that permits regioselective drug delivery with spatiotemporal control in a slice culture derived from human glioma stem cell xenografts. Our prototype platform is based on a 96 well plate interface and applies drug stripes to slices and allows sequential drug tests in orthogonal locations, which enables serial or even combinatorial drug delivery. Finally, administration of markers for cell death and for viability yields a simple and quantitative imaging readout that permits real-time automated analysis. Therefore, we hypothesize that microfluidic drug delivery applied to PDSC can provide a robust paradigm to profile patient-specific drug responses. The innovative combination of microfluidic technology and PDSCs for patient-specific drug screening has the potential to create a revolutionary tool to guide patient-specific therapies. Ultimately, by validation in actual patient samples, our microfluidic slice-based assay should not only complement genomic approaches that identify the best therapies for individual patients, but also serve as a novel platform for screening and development of new therapeutic agents.
Tim C. Chang, Andrei M. Mikheev, Wilson Huynh, Raymond J. Monnat, Jr., Robert C. Rostomily, and Albert Folch, "Parallel Microfluidic Chemosensitivity Testing on Individual Slice Cultures", Lab on a Chip, accepted for publication (2014).
Tim C. Chang, Weiliang Tang, William Jen Hoe Koh, Alexander J. E. Rettie, Mary J. Emond, Raymond J. Monnat, Jr., and Albert Folch, "Microwell Arrays Reveal Cellular Heterogeneity During the Clonal Expansion of Transformed Human Cells", Technology, accepted for publication (2014).
Sidorova, J.M. Li, N., Schwartz, D.C., Folch, A., and Monnat Jr., R.J. “Microfluidic-assisted analysis of replicating DNA molecules”, Nature Protocols 4: 849 (2009).
Sidorova, J.M., Li, N., Folch, A., and Monnat Jr., R.J. “The RecQ helicase WRN is required for normal replication fork progression after DNA damage or replication fork arrest”, Cell Cycle 7, 796 (2008).