Electrophysiology of in vitro neuronal and skeletal myotube culture models using microelectrode arrays

Technological advances have transformed motorised prosthetics, enhancing their functionality using myoelectric techniques. Nonetheless, advanced myoelectric prosthetic technology still encounters limitations of signal resolution due to loss of signal discrimination through layers of subcutaneous fat and skin. Various tissue-electrode interfacing prosthetic systems have been investigated to decrease signal loss. Brain-interface prosthetics reduce the effects of limited signal resolution, however, they are disadvantaged through the requirement of invasive surgeries and the foreign body response, limiting their potential as new-age prosthetic systems. Significant research is still required to support advancements in technology that allow for direct signal processing and translation of neural input from motor neurons to implanted microelectrode arrays while also preventing premature device failure and inhibiting the foreign body response. Investigations of targeted re-innervation of truncated motor neurons connected to a layer of skeletal muscle on microelectrode arrays (MEAs) had combined multidisciplinary technology to support early stage methods towards the development of new-age neural interfacing prosthetic devices. Additionally, another early target for the development of implantable MEAs for prosthetic interfaces is the production of translational 2D in vitro cell culture models that mimic the same process of targeted re-innervation of neurons to muscle cells on MEAs. The milestone of this project in support of development of implantable MEAs for prosthetic interfaces was through development of complex in vitro functional neuron-skeletal muscle cultures that exhibited multi-innervated neuronal control of myotubes. As such, in order to achieve the milestone, cell culture studies were performed on each cell type to acquire baseline electrophysiology measurements prior to the integration of the cells into complex multiculture systems.MEA recordings of the electrophysiological activity of primary skeletal myotubes of C57BL/6 mice were recorded until delamination occurred. The myotubes exhibited regular individual spikes of extracellular activity during spontaneous periods of activity and periods of electrical stimulation. A selection of neural cells were assessed for motor neuron-like phenotypes and capability to exhibit spontaneous electrophysiological activity on MEAs. Immortalised neural cell lines provided a cheap and easy to use range of cells as the motor neuron-like model for the multiinnervated cultures. However, the lack of spontaneous extracellular activity on MEAs and nonresponsive characteristics to electrical stimulation resulted in the cells being an inadequate neuronal model. As an alternative, a new neuronal cell type was investigated. Human induced pluripotent stem cells into NGN2 induced neuron-astrocyte cocultures exhibited bursts of spontaneous extracellular activity on MEAs after at least four weeks of differentiation. Additionally, the cultures were very responsive to electrical stimulation, indicating that they could be a good candidate for use in multi-innervated culture studies. To support the development of multi-innervated culture studies, custom designed multicompartment devices were produced using 3D printing of biocompatible materials that were attached to MEAs. The limiting factor that prevented long-term culture studies using 3D printed multicompartment devices was the degradation of the biocompatible adhesive between the 3D printed material and MEA substrates. To work around the adhesion issue, alternative production techniques were required that produced custom designed multicompartment structures and custom designed MEAs as a single device.This thesis provided the foundational steps towards development of implantable MEAs for prosthetic interfaces, however, the milestone of development of complex 2D in vitro multiinnervated cultures was not achieved. The output required to achieve the milestone was more significant than initially expected in a doctoral thesis. Nonetheless, a considerable degree of multidisciplinary research was conducted throughout the thesis to support the progression of prosthesis technology. To reach the target milestone, further optimisation of the multicompartment device on MEAs or an alternate production technique was required to be used to allow long-term electrophysiology studies of multi-innervated cultures to be performed.