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BioMedicine Projects

Axon Guidance On a Chip

 

​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.

References:

​​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).

Cancer

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.

References:

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).​

Microfluidic Synaptogenesis

A major goal in neuroscience is to understand the formation and development of synapses, the tiny membrane specializations that enable nerve cells to communicate with each other. The sequence of molecular signals leading to synapse formation (“synaptogenesis”) is qualitatively well known for the more accessible neuromuscular junction (NMJ). However, very little is known of the quantities (concentration, duration, onset, etc.) of the various neurochemical signals involved in synaptogenesis. Intriguingly, all but one of the axons innervating a given myotube at birth retract after a period of ~1 week as a result of a synaptic competition process that remains, for lack of quantitative methods, poorly understood. Our overall objective is to uncover some of the rules governing the formation and elimination of synapses at the NMJ using a microfluidic cell culture system. Our approach is based on substituting the presynaptic neuron by an artificial microfluidic device that delivers known doses of various synaptogenic neurochemicals to micrometer-scale areas of the membrane of cultured myotubes. We focus on the three key factors – agrin, neuregulin, and the neurotransmitter acetylcholine (ACh) – secreted by the nerve tip during synaptogenesis. We measure muscle cell responses that are specific to ACh receptors (AChRs), such as AChR aggregation/disaggregation, degradation/synthesis, insertion, co-localization with other receptors and cytoskeletal proteins, intracellular signaling pathways, etc.


We have developed a microfluidic mimic of the innervation process that allows for focally stimulating microarrays of single, isolated (“microengineered”) myotubes using laminar flow streams (orthogonal to the myotubes). We have found that a) focal application of agrin entices myotubes to recruit new AChRs to the stimulated area; b) when the microtracks are formed with Matrigel, a basal lamina extract, the microengineered myotubes display AChR clusters of complex, in-vivo-like morphologies even before agrin is applied, similarly to what happens in vivo; and c) when agrin is focally applied to those agrin-predating clusters, AChRs are degraded at reduced rates, suggesting that a putative role for agrin in vivo is to help stabilize AChRs. We seek to continue these investigations by studying the dynamics and spatial patterns of various AChR-specific responses upon (competitive, synergistic, or combinatorial) stimulation with agrin, neuregulin, and ACh.

References:

Tourovskaia, A., Li, N., and Folch, A., “Localized acetylcholine receptor clustering dynamics in response to microfluidic focal stimulation with agrin”, Biophys. J. 95: 3009 (2008).

Tourovskaia, A., Figueroa-Masot, X., and Folch, A. “Long-term Microfluidic Cultures of Myotube Microarrays for High-Throughput Focal Stimulation”, Nature Protocols 1: 1092 (2006).

Kosar, T.F. Tourovskaia, A., Figueroa-Masot, X., Adams, M. and Folch, A. “A Nanofabricated Planar Aperture as a Mimic of the Nerve-Muscle Contact During Synaptogenesis”, Lab Chip 6: 632-638 (2006).

Tourovskaia, A., T.F. Kosar, and Folch, A. “Local Induction of Acetylcholine Receptor Clustering in Myotube Cultures Using Microfluidic Application of Agrin”, Biophys. J. 90: 2192-2198 (2006).

Tourovskaia, A., Figueroa-Masot, X. and Folch, A., “Differentiation-on-a-chip: A Microfluidic Platform for Long-Term Cell Culture Studies”, Lab on a Chip 5:14 (2005).

Micro-bioreactors

Biologists and doctors have attempted to recreate "organ-like" conditions in petri dishes. In these conditions, cells dissociated from the organ are seeded on a homogeneous plastic surface (usually coated with protein) and homogeneously batheed in cell culture medium. These conditions do not reproduce the microscale gradients and substrate heterogeneity present in vivo, which can affect critical cellular functions negatively and irreversibly. They also require considerable human labor, so they produce results at very low throughput. Hence, we use microfluidic technology for mimicking the physiological conditions on a cellular scale and for automating high-throughput cell culture experiments that can provide low-cost alternatives to animal and clinical studies.  

References:

​Ellen Tenstad, Anna Tourovskaia, A. Folch, Ola Myklebost, and Edith Rian, “Extensive adipogenic and osteogenic differentiation of patterned human mesenchymal stem cells in a microfluidic device”, Lab Chip 10: 1401 (2010).

​Keenan, T.M., Frevert, C.W., Wu, A., Wong, V., and Folch, A. “A New Method for Studying Gradient-Induced Neutrophil Desensitization Based on an Open Microfluidic Chamber”, Lab Chip 10: 116 (2010).

​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).

​​​Tourovskaia, A., Li, N., and Folch, A., “Localized acetylcholine receptor clustering dynamics in response to microfluidic focal stimulation with agrin”, Biophys. J. 95: 3009 (2008).

​Chen, H.-H., Shen, H., Heimfeld, S., Tran, K.K., Reems, J., Folch, A., and Gao, D. “A microfluidic study of mouse dendritic cell membrane transport properties of water and cryoprotectants”, Int. J. Heat and Mass Transfer 51: 5687 (2008).

​Chen, H.-H., Purtteman, J.J.P., Heimfeld, S., Folch, A., and Gao, D. “Development of a Microfluidic Device for Determination of Cell Osmotic Behavior and Membrane Transport Properties”, Cryobiol. 55: 200 (2007).

​Tourovskaia, A., Figueroa-Masot, X., and Folch, A. “Long-term Microfluidic Cultures of Myotube Microarrays for High-Throughput Focal Stimulation”, Nature Protocols 1: 1092 (2006).

​Tourovskaia, A., T.F. Kosar, and Folch, A. “Local Induction of Acetylcholine Receptor Clustering in Myotube Cultures Using Microfluidic Application of Agrin”, Biophys. J. 90: 2192-2198 (2006).

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