A high-throughput microfluidic assay to study neurite response to growth factor gradients

Studying neurite guidance by diffusible or substrate bound gradients is challenging with current techniques. In this study, we present the design, fabrication and utility of a microfluidic device to study neurite guidance under chemogradients. Experimental and computational studies demonstrated the establishment of a steep gradient of guidance cue within 30 min and stable for up to 48 h. The gradient was found to be insensitive to external perturbations such as media change and movement of device. The effects of netrin-1 (0.1–10 µg mL−1) and brain pulp (0.1 µL mL−1) were evaluated for their chemoattractive potential on neurite turning, while slit-2 (62.5 or 250 ng mL−1) was studied for its chemorepellant properties. Hippocampal or dorsal root ganglion (DRG) neurons were seeded into a micro-channel and packed onto the surface of a 3D collagen gel. Neurites grew into the matrix in three dimensions, and a gradient of guidance cue was created orthogonal to the direction of neurite growth to impact guidance. The average turning angle of each neurite was measured and averaged across multiple devices cultured under similar conditions to quantify the effect of guidance cue gradient. Significant positive turning towards gradient was measured in the presence of brain pulp and netrin-1 (1 µg mL−1), relative to control cultures which received no external guidance cue (p < 0.001). Netrin-1 released from transfected fibroblasts had the most positive turning effect of all the chemoattractive cues tested (p < 0.001). Slit-2 exhibited strong chemorepellant characteristics on both hippocampal and DRG neurite guidance at 250 ng mL−1 concentration. Slit-2 also showed similar behavior on DRG neuron invasion into 3D collagen gel (p < 0.01 relative to control cultures). Taken together, the results suggest the utility of this microfluidic device to generate stable chemogradients for studying neurobiology, cell migration and proliferation, matrix remodeling and co-cultures with other cell lines, with potential applications in cancer biology, tissue engineering and regenerative medicine.Seoul R&BD Support Center (program PA090930

A Microfluidic Platform to Quantify Spatio-Temporal Diffusion of Chemo-Gradients Within 3D Scaffolds: Applications in Axonal Biology

Axonal outgrowth and guidance play an important role in wiring the developing and regenerating nervous system. The critical role of biomolecular gradients in facilitating this axonal sensitivity and directionality along specific trajectories needs to be elucidated for designing effective therapeutic treatments under injury or disease conditions. However, previous in vitro approaches based on micropipette assay or gel-turning assay proved to be unsuitable or inefficient for precise generation and quantification of diffusive gradients. In this study, we utilized a microfluidic device to generate and quantify physiologically-relevant biomolecular gradients in a simple and reliable manner. Using a combination of computational and experimental techniques, we designed and developed a microfluidic platform to study the synergistic effects of 3D scaffold concentration (0 - 3 mg/mL), molecular weight of the diffusing molecule (1-1000 kDa) as well as its dosage (0.1-10 æM), on gradient generation and steady-state spatio-temporal evolution. The device was fabricated using standard soft-lithography techniques, and has three separate chambers, flanked by two media channels on the sides. The scaffold (gel) of interest was filled in the left and right chambers (L = 3.6 mm, thickness = 150 æm), and the biomolecule of interest was loaded in the middle chamber to facilitate diffusion through the gel on both sides. The channels on both sides act as sink for the diffusing biomolecule, creating a gradient across the 3D gel. Two different types of scaffolding materials were used in these studies -- collagen-1 or matrigel®. The viscosities of these gels at various concentrations were obtained from commercial vendors, and diffusion coefficients of biomolecules within these gels calculated using the Stokes-Einstein equation. Computational simulations were performed using the finite element methods (COMSOL® Multiphysics), to obtain a gradient profile across the chamber in all three dimensions. The numerical gri

Exploiting Click-Chemistry and Microfluidics to Map the Neuronal Itinerary of APP Processing and Amyloid-Beta Generation

Alzheimer’s disease (AD) is a chronic neurodegenerative disease and is the sixth leading cause of death in the United States with approximately 5.5 million Americans diagnosed with it. The neuropathological hallmark includes extracellular senile plaques and intraneuronal neurofibrillary tangles. Recent GWAS studies have identified genes associated with AD, and have revealed several classes of genes implicated in disease pathogenesis. In particular, three general pathways associated with an increased risk of AD included: 1) cholesterol metabolism, innate immune system, and the membrane trafficking. Our lab has focused on intracellular trafficking as it relates to the processing of amyloid precursor protein (APP), the precursor protein for the Aβ peptide—a critical component of the senile plaque. Much is still unknown about the intracellular itinerary of APP, and the cellular location of Aβ production. Here, I describe the use of click-chemistry applied to an APP construct to further study APP processing. In addition, I describe work that I did to develop a microfluidic system to enable visualizing APP processing within the unique cytoarchitecture of primary neurons. Microfluidics helps in isolating the soma and axon to understand APP processing in these specific compartments

A microfluidic culture for two populations of dorsal root ganglia for differential staining

The goal of this study was to design and fabricate a microfluidic system that can be used to visibly distinguish the two populations of dorsal root ganglia (DRGs) by differential staining. Polydimethylsiloxane (PDMS) is the most widely used silicon-based organic polymer, and is particularly known for its wide spread use in microfluidics. Various methods have been employed to pump fluids in these channels for applications ranging from patterning of cells and biomolecules to control of local environment factors such as temperature, which requires external pumping or other applied forces. We demonstrated a pump-free device that exploits the surface energy stored in a liquid droplet to pump liquid in the channels. The fluid was pumped by using two droplets of unequal sizes connected via fluid filled channel. The flow was generated from smaller droplet to larger droplet. This passive pumping technique was used to simultaneously stain the two cultured DRGs in connected channels. The in vitro system can be further exploited to study the guided growth in axons. This study provides a cost effective method to detect the influence of the presence of pioneer neuron on the growth patterns of the new generation of neurons. It eliminates the need of using transgenic cells to study the guided growth in axons, thereby giving some insight for the repair of spinal cord injuries and the understanding of the early growth model

A handcrafted multi-branch nerve scaffold for in vivo peripheral nerve regeneration

Arm and leg peripheral nerve injuries contribute largely to functional disability. These injuries seldom affect a single-line nerve; rather the injury goes beyond and disrupts the nerve tissue across multiple nerve paths. Here we investigate handcrafted PDMS based multi-branch nerve guidance conduit, which addresses this crucial branching from the sciatic nerve to aid with the regeneration to their appropriate distal targets. The design concept of the developed multiple branch nerve scaffolds is not specific for the sciatic nerve but can be modified for general use of all peripheral nerves and additional applications. Microchannels are also fabricated into a device in order to guide axons to reach the distal ends, which in turn allow regenerating nerves to reach the target muscles. Experiments are performed on Lewis rats to demonstrate the effectiveness of this nerve conduit. Successful nerve regeneration was confirmed from histological analysis of the harvested nerves from all four branches

Microenvironments Matter:Advances in Brain-on-Chip

To highlight the particular needs with respect to modeling the unique and complex organization of the human brain structure, we reviewed the state-of-the-art in devising brain models with engineered instructive microenvironments. To acquire a better perspective on the brain’s working mechanisms, we first summarize the importance of regional stiffness gradients in brain tissue, varying per layer and the cellular diversities of the layers. Through this, one can acquire an understanding of the essential parameters in emulating the brain in vitro. In addition to the brain’s organizational architecture, we addressed also how the mechanical properties have an impact on neuronal cell responses. In this respect, advanced in vitro platforms emerged and profoundly changed the methods of brain modeling efforts from the past, mainly focusing on animal or cell line research. The main challenges in imitating features of the brain in a dish are with regard to composition and functionality. In neurobiological research, there are now methods that aim to cope with such challenges by the self-assembly of human-derived pluripotent stem cells (hPSCs), i.e., brainoids. Alternatively, these brainoids can be used stand-alone or in conjunction with Brain-on-Chip (BoC) platform technology, 3D-printed gels, and other types of engineered guidance features. Currently, advanced in vitro methods have made a giant leap forward regarding cost-effectiveness, ease-of-use, and availability. We bring these recent developments together into one review. We believe our conclusions will give a novel perspective towards advancing instructive microenvironments for BoCs and the understanding of the brain’s cellular functions either in modeling healthy or diseased states of the brain.</p

Microfluidic Platforms to Unravel Mysteries of Alzheimer's Disease: How Far Have We Come?

Alzheimer’s disease (AD) is a significant health concern with enormous social and economic impact globally. The gradual deterioration of cognitive functions and irreversible neuronal losses are primary features of the disease. Even after decades of research, most therapeutic options are merely symptomatic, and drugs in clinical practice present numerous side effects. Lack of effective diagnostic techniques prevents the early prognosis of disease, resulting in a gradual deterioration in the quality of life. Furthermore, the mechanism of cognitive impairment and AD pathophysiology is poorly understood. Microfluidics exploits different microscale properties of fluids to mimic environments on microfluidic chip-like devices. These miniature multichambered devices can be used to grow cells and 3D tissues in vitro, analyze cell-to-cell communication, decipher the roles of neural cells such as microglia, and gain insights into AD pathophysiology. This review focuses on the applications and impact of microfluidics on AD research. We discuss the technical challenges and possible solutions provided by this new cutting-edge technique to understand disease-associated pathways and mechanisms