RAPID DRUG DELIVERY TO IN VITRO NEURONAL CULTURES N. Herzog, A. Johnstone, S. Idinyang, T. C. Bellamy and N. A. Russell University of Nottingham, UK ABSTRACT Cellular signalling in vivo often involves agonist concentrations changing on a time scale that is difficult to achieve using open-bath preparations in vitro. We have therefore developed a microfluidic system to deliver agonists to cells on biologically relevant timescales. Switching is achieved by transiently shifting the interface between two parallel laminar flows in a microfluidic chamber containing cultured cells. Cholinergic stimulation was delivered to cultured neuronal networks and the response observed using a calcium-sensitive fluorescent dye. This LabVIEW controlled system can be easily integrated with other equipment, including imaging systems or electrode arrays, to allow cell-triggered drug delivery experiments to be conducted. KEYWORDS: Flow switching, Neuronal culture, Pressure-driven perfusion INTRODUCTION Signalling between cells in vivo typically involves rapidly changing concentrations of agonist molecules [1]. To study these agonist effects in vitro involves washing drugs on and off a cell culture at a rate not feasible with open-bath preparations. Consequently we have developed a microfluidics-based system to enable transient drug exposure on biologically-relevant timescales. By rapidly switching the relative flow rates of two parallel streams entering a channel, one containing culture media and the other media plus agonist, the cells grown in the channel are transiently exposed to the agonist as the interface shifts. This method has the potential for high response rate and precision [2]. Pressure driven pumps were used with fluorescein in water to characterise the system. Cholinergic stimulation was then delivered to cultured neuronal networks under constant flow, and the cell response was observed using a calcium-sensitive fluorescent dye. EXPERIMENTAL The drug delivery system (figure 1) consisted of either a commercial (OBI, Elveflow), or custommade multichannel pressure-driven pump connected to a custom microfluidic culture chamber. The chamber had two inlets and one outlet, and was produced with polydimethylsiloxane (PDMS) bonded to glass using standard soft-lithography methods. The custom-made pump consisted of a manifold with several integrated pressure-controlled fluid reservoirs under the control of proportional-integralderivative (PID) algorithm implemented in LabVIEW. This program allows the option of real-time pump command to be sent from other equipment. The system was first characterised by delivering water and fluorescein in water into the chamber inlets, and shifting the position of the interface between the laminar flows. The response of a cultured neuronal network to a brief pulse of agonist was then tested. Primary rat hippocampal cells were prepared and grown in the microfluidic culture chamber. After 15 days in vitro in media (Neurobasal, GIBCO), the chamber was flushed with a fluorescent calcium-sensitive dye (OGB-I, Invitrogen) and incubated. Under constant media flow, brief pulses of Carbachol (20µM) were applied and the response observed.
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18th International Conference on Miniaturized Systems for Chemistry and Life Sciences October 26-30, 2014, San Antonio, Texas, USA
RESULTS AND DISCUSSION A schematic of the custom pump and culture chamber is shown in figure 1, together with the travelling pulse of fluorescein as it passes a fixed point in the chamber. At a linear velocity of 1 mm/s the pulse lasted 0.58 ± 0.05 seconds (mean ± standard deviation, n=10).
Figure 1: a) Custom Perfusion pump); b) Microfluidic culture chamber c) travelling pulse achieved with the system
Figure 2: Cultured cells in chamber (left); Relative cell calcium levels over time, after Carbachol pulse (right)
In figure 2 the time evolution of Calcium activity in cultured neuronal cells is shown after exposure to a brief Carbachol pulse. Each image is an average of 5 trials. Calcium levels clearly rise in response to the Carbachol and persist after it is removed.
CONCLUSION We have demonstrated controlled, repeatable drug delivery to neural cultures on a biologically relevant timescale. The perfusion system under LabVIEW control allows for a range of control algorithms (including PID, adaptive or fuzzy controllers) to improve the response time. It also enables the system to be integrated with other equipment such as Multi-Electrode Arrays (MEAs) or imaging systems, and can therefore be used to conduct cell-triggered drug delivery experiments. ACKNOWLEDGEMENTS This work is funded by the EPSRC (EP/H022112/1) and Marie-Curie ITN (NETT). Thanks to Sorka Abanu for his contribution to the initial perfusion system development. REFERENCES [1] Arbuthnott and Wickens. "Space, time and dopamine." Trends in neurosciences 30.2 (2007): 62-69. [2] Bae et al, “Rapid switching of chemical signals in microfluidic devices” Lab Chip, 2009, 9, 3059-65 CONTACT: Nitzan Herzog, (+44) 115 84 68848,
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