Tools for the Advancement of Cell-based Screening for Neurological Disorders

The pharmaceutical industry has changed in multiple ways. Major consolidation has continued with increasing mergers and acquisitions. Concurrently, there has been a decreasing trend in the number of new drugs being commercialized. Of this reduced output, new drug discovery has increasingly focused on treatment of neurological disorders, and R&D outsourcing has increased in the form of partnerships with academic drug discovery centers. As public institutions, it is critical for these partnerships to have low cost solutions for their drug discovery needs. In this spirit new focus has been directed at developing technology to improve drug screening for neurological diseases. This new technology includes microfluidic devices for increasing throughput as well as the use of human induced pluripotent stem cell (iPSC) derived neurons which present an advantage over animals for modeling human diseases. Although they show great promise, human iPSC-derived neurons are still hindered by many challenges, including long differentiation times and low yields of homogenous neuronal subtypes. These challenges along with the post-mitotic nature of other mature primary neurons limit the pool of available cells for screening. Historically this has been addressed by using immortalized non-neuronal cell lines in neurological screening; however screening on neurons represents the possibility of better outcomes due to their phenotypic and morphological accuracy. Thus there is an increased demand for technology to expand neuron throughput for screening. This work explores the use of microraft arrays to increase throughput for neuron-based neurological disorder drug screening. Microraft arrays are culture devices consisting of an array of 1,600 releasable, paramagnetic, polystyrene microrafts (500 µm x 500 µm x 100 µm) each serving as an individual culture surface. The device is used to culture both primary rat neurons as well as human neurons derived from embryonic stem cells, and new tools are created to support this device for screening applications. Individual microraft cultures were maintained in multi-well plates and tools were developed to isolate and transport of individual microrafts to facilitate screening studies. Centering and quantification of these microrafts was achieved and together these results show a strong potential for the use of this device in neurological screening. Finally, in order to demonstrate the high-throughput potential of this technology, scalable assays including a bead-based ELISA and an immunofluorescence assay are devised to detect fragile X mental retardation protein which is reduced in patients with fragile X syndrome – a well-known neurodevelopmental disorder.Doctor of Philosoph