Lab-on-a-chip platforms for understanding neuronal cellular interactions

Neurons are highly polarized cells that connect and communicate with each other via electrical impulses and chemical synapses, usually between dendrites and axons. The brain alone consists of approximately 100 billion neurons that are present at birth and do not divide, and thus cannot be replaced. Damage or loss of neurons has been linked to many diseases of the central nervous system (CNS) including Alzheimer's Disease (AD), Parkinson Disease (PD), Amyotrophic Lateral Sclerosis (ALS) and Traumatic Brain Injury (TBI). In 2005, the World Health Organization (WHO) reported that neurological disorders constitute 6.3% of the global burden of disease and 12% of total global death, making neurological disorders one of the most significant causes of mortality. Studies show that axonal pathology and degeneration can cause significant functional impairment and can precede, and sometimes cause, neuronal death in several neurological disorders, creating a compelling need to understand the mechanisms of axon degeneration. Microfluidic devices that allow manipulation of fluids in channels with typical dimensions of tens to hundreds of micrometers have therefore emerged as a powerful platform for such studies due to their ability to isolate and direct the growth of axons. Chapter 1 offers an introductory overview of the nervous system and also briefly discusses the limitations of traditional primary cell culture methods and the compartmentalized campenot chamber. This is followed by a comprehensive overview on the development of microfluidic devices for neuroscience application. In Chapter 2, a new microfluidic platform to apply very mild (0.5%) and mild (5%) stretch injury to individual cortical axons was developed, allowing characterisation of the neuronal response to axonal stretch injury. This was realised by covering a pneumatic channel (90 ˜í¬¿m wide, 17 ˜í¬¿m high) with a thin, flexible poly (dimethylsiloxane) (PDMS) membrane so that pressurising the pneumatic channel allowed a controlled deflection of the membrane. A compartmentalized microfluidic culturing chamber was positioned to guide axons across the pneumatic channel, allowing the axons across the pneumatic channel to be discretely stretched by pressurising the pneumatic channel. Results show that stretch injury in the range of 0.5% to 5% to a short (90 ˜í¬¿m long) localized region of a cortical axon was able to trigger a degenerative response in both axons and soma. In Chapter 3, the neuronal response to axonal stretch injury was further investigated with a particular focus on repetitive injury. Closer investigations revealed growth cone collapse and significant abnormal cytoskeletal rearrangements in a time frame characteristic for TBI. Interestingly, a second very mild stretch within 24 h significantly exacerbated this response. One of the limitations of the devices described above was the use of a two-step photolithography process to fabricate the compartmentalized microfluidic culturing device for isolation of axons from somas. Therefore, in Chapter 4, a novel, low cost method to fabricate small microchannnels (in the range of 25-300 ˜í¬¿m) on rigid poly(methylmethacylate) (PMMA) microchips by using a low cost direct CO2 laser micromachining system ($2500) was developed to simplify the two-step photolithographic fabrication process of the culturing chambers. A concept from near field scanning optical microscopy (NSOM) was applied to narrow the size of the laser beam using a stainless steel disk containing a small aperture ‚Äö- a pinhole. The pinhole significantly reduced the diameter of the laser spot, decreasing the ablated feature width from around 300 ˜í¬¿m to around 60 ˜í¬¿m when using the 50 ˜í¬¿m diameter pinhole. With a 35 ˜í¬¿m pinhole, the width could be reduced to around 25 ˜í¬¿m. The separation efficiency for the electrophoretic separation of fluorescent dyes more than doubled when using the narrower channels made using the 50 ˜í¬¿m pinhole, probably due to more efficient heat dissipation. The laser engraver system was then used to pattern PDMS microfluidic device for culturing neurons. However, results demonstrated that primary neurons were unhealthy when cultured in laser engraved PDMS culturing chambers. Therefore, further modifications during the laser engraving process are required before these devices can be practically applied for studies using neural cells. Finally in Chapter 5, to further enhance the understanding of the complex signaling cascades that occur in vivo signalling, a novel microfluidic compartmentalized coculture platform was developed using a one step photolithography process. This multichamber device introduces a maze-like structure to restrict axonal outgrowth whilst facilitating the growth of glia, hence physically isolating the neuronal populations. The maze structure consists of multiple offset walls (50 ˜í¬¿m wide, 22 ˜í¬¿m high, 250 ˜í¬¿m long) spaced at regular intervals (50 ˜í¬¿m) with 150 ˜í¬¿m gaps. Initial investigations using this device demonstrate its effectiveness in blocking axonal growth while being permissive to glial growth in a fluidically isolated microenvironment, confirming its potential for the investigation of the role of glial signaling in neuronal communication, for example the investigation of cellular interactions underlying the brains response to trauma and other neurological disorders. In summary, two new culture platforms were developed to advance the understanding of neuronal degeneration and neuron-glia signalling in TBI and other neurological disorders. A fast, inexpensive direct machining method for fabrication of microfluidic devices was also developed. However, further modifications are required before practical implementation for the fabrication of PDMS devices for application in neuroscience research can occur.