Structurally patterned pyrolysed three‐dimensional carbon scaffolds (p3D‐carbon) are fabricated and applied for differentiation of human neural stem cells (hNSCs) developed for cell replacement therapy and sensing of released dopamine. In the absence of differentiation factors (DF) the pyrolysed carbon material induces spontaneous hNSC differentiation into mature dopamine‐producing neurons and the 3D‐topography promotes neurite elongation. In the presence and absence of DF, ≈73–82% of the hNSCs obtain dopaminergic properties on pyrolysed carbon, a to‐date unseen efficiency in both two‐dimensional (2D) and 3D environment. Due to conductive properties and 3D environment, the p3D‐carbon serves as a neurotransmitter trap, enabling electrochemical detection of a significantly larger dopamine fraction released by the hNSC derived neurons than on conventional 2D electrodes. This is the first study of its kind, presenting new conductive 3D scaffolds that provide highly efficient hNSC differentiation to dopaminergic phenotype combined with real‐time in situ confirmation of the fate of the hNSC‐derived neurons.

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In this work, we present the investigation of the pyrolysis parameters at high temperature (1100ºC) for the fabrication of two-dimensional pyrolytic carbon electrodes. The electrodes were fabricated by pyrolysis of lithographically patterned negative epoxy based photoresist SU-8. A central composite experimental design was used to identify the influence of dwell time at the highest pyrolysis temperature and heating rate on electrical, electrochemical and structural properties of the pyrolytic carbon: Van der Pauw sheet resistance measurements, cyclic voltammetry, electrochemical impedance spectroscopy and Raman spectroscopy were used to characterize the pyrolytic carbon. The results show that the temperature increase from 900ºC to 1100ºC improves the electrical and electrochemical properties. At 1100ºC, longer dwell time leads to lower resistivity, while the variation of the pyrolysis parameters has small influence on electrochemical performance.

An electrochemical detection system specifically designed for multi-parameter real-time monitoring of stem cell culturing/differentiation in a microfluidic system is presented. It is composed of a very compact 24-channel electronic board, compatible with arrays of microelectrodes and coupled to a microfluidic cell culture system. A versatile data acquisition software enables performing amperometry, cyclic voltammetry and impedance spectroscopy in each of the 12 independent chambers over a 100 kHz bandwidth with current resolution down to 5 pA for 100 ms measuring time. The design of the platform, its realization and experimental characterization are reported, with emphasis on the analysis of impact of input capacitance (i.e., microelectrode size) and microfluidic pump operation on current noise. Programmable sequences of successive injections of analytes (ferricyanide and dopamine) and rinsing buffer solution as well as the impedimetric continuous tracking for seven days of the proliferation of a colony of PC12 cells are successfully demonstrated.

In this work, we have developed a microfluidic cytotoxicity assay for a cell culture and detection platform, which enables both fluid handling and electrochemical/optical detection. The cytotoxic effect of anticancer drugs doxorubicin (DOX), oxaliplatin (OX) as well as OX-loaded liposomes, developed for targeted drug delivery, was evaluated using real-time impedance monitoring. The time-dependent effect of DOX on HeLa cells was monitored and found to have a delayed onset of cytotoxicity in microfluidics compared with static culture conditions based on data obtained in our previous study. The result of a fluorescent microscopic annexin V/propidium iodide assay, performed in microfluidics, confirmed the outcome of the real-time impedance assay. In addition, the response of HeLa cells to OX-induced cytotoxicity proved to be slower than toxicity induced by DOX. A difference in the time-dependent cytotoxic response of fibrosarcoma cells (HT1080) to free OX and OX-loaded liposomes was observed and attributed to incomplete degradation of the liposomes, which results in lower drug availability. The matrix metalloproteinase (MMP)-dependent release of OX from OX-loaded liposomes was also confirmed using laryngopharynx carcinoma cells (FaDu). The comparison and the observed differences between the cytotoxic effects under microfluidic and static conditions highlight the importance of comparative studies as basis for implementation of microfluidic cytotoxic assays.