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Olfactory ensheathing cells (OECs) promote axonal regeneration and improve locomotor function when transplanted into the injured spinal cord. A recent clinical trial demonstrated improved motor function in domestic dogs with spinal injury following autologous OEC transplantation. Their utility in canines offers promise for human translation, as dogs are comparable to humans in terms of clinical management and genetic/environmental variation. Moreover, the autologous, minimally invasive derivation of OECs makes them viable for human spinal injury investigation. Genetic engineering of transplant populations may augment their therapeutic potential, but relies heavily on viral methods which have several drawbacks for clinical translation. We present here the first proof that magnetic particles deployed with applied magnetic fields and advanced DNA minicircle vectors can safely bioengineer OECs to secrete a key neurotrophic factor, with an efficiency approaching that of viral vectors. We suggest that our alternative approach offers high translational potential for the delivery of augmented clinical cell therapies.

Abstract:The development of safe technologies to genetically modify neurons is of high interest in regenerative neurology, for both translational and basic science applications. Such approaches have conventionally been heavily reliant on viral transduction methods which have safety and production limitations. Magnetofection (magnet-assisted gene transfer using iron oxide nanoparticles as vectors) has emerged as a highly promising non-viral alternative for safe and reproducible genetic modification of neurons. Despite the high promise of this technology, there is an important gap in our knowledge of the safety of this approach, namely, whether it alters neuronal function in adverse ways such as by altering neuronal excitability and signalling. We have investigated the effects of magnetofection in primary cortical neurons by examining neuronal excitability using the whole cell patch clamp technique. We found no evidence that magnetofection alters the voltage-dependent sodium and potassium ionic currents that underpin excitability. Our study provides important new data supporting the concept that magnetofection is a safe technology for bioengineering of neuronal cell populations.Response to Reviewers: The reviewer comments have been addressed in the previous revision submitted. An unmarked version of the manuscript, version V3 is included here. Received: day month year / Revised: day month year / Accepted: day month year (automatically inserted by the publisher) © Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2011 Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation ABSTRACTThe development of safe technologies to genetically modify neurons is of high interest in regenerative neurology, for both translational and basic science applications. Such approaches have conventionally been heavily reliant on viral transduction methods which have safety and production limitations. Magnetofection (magnet-assisted gene transfer using iron oxide nanoparticles as vectors) has emerged as a highly promising non-viral alternative for safe and reproducible genetic modification of neurons. Despite the high promise of this technology, there is an important gap in our knowledge of the safety of this approach, namely, whether it alters neuronal function in adverse ways such as by altering neuronal excitability and signalling. We have investigated the effects of magnetofection in primary cortical neurons by examining neuronal excitability using the whole cell patch clamp technique. We found no evidence that magnetofection alters the voltage-dependent sodium and potassium ionic currents that underpin excitability. Our study provides important new data supporting the concept that magnetofection is a safe technology for bioengineering of neuronal cell populations.

Tissue engineering methodologies for various physiological systems are seeing a significant trend towards 3D cell culture in or on ‘soft’ polymeric hydrogel materials, widely considered to provide a more biomimetic environment for cell growth versus ‘hard’ materials such as glass or plastic. Progress has been slower with 3D neural cell culture with current studies overwhelmingly reliant on hard substrates. Accordingly, our knowledge of the alterations in electrochemical properties of neurons propagated in soft materials is relatively limited. In this study, primary cortical neurons and glial cells were seeded onto the surface of collagen hydrogels and grown in vitro for 7–8 days. At this time, neurons had formed a complex neurite web interspersed with astrocytes. Neuronal patch clamp recordings revealed voltage-gated Na+ and K+ currents in voltage clamp and action potentials in current clamp. When measured at voltages close to maximum activation, both currents were >1 nA in mean amplitude. When compared to primary cortical neurons cultured on glass coverslips, but otherwise under similar conditions (Evans et al., 2017), the Na+ current from hydrogel neurons was found to be significantly larger although there were no differences in the K+ current amplitude, membrane potential, input resistance or cell capacitance. We speculate that the larger size of the neuronal voltage-dependent Na+ current in the hydrogels is related to the better biomimetic properties of the soft material, being close to values reported for neurons recorded in brain slices. The results highlight the potential benefits offered by neuronal culture on soft and biomimetic polymeric materials for neural tissue engineering studies.