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Stem cells have attracted increasing research interest in the field of regenerative medicine due to their unique abilities to differentiate into multiple cell lineages. Label-free, real-time, and long-term monitoring for stem cell differentiation is requisite in studying directional differentiation and development mechanisms for tissue engineering applications, but a great challenge because of the rigorous demands for sensitivity, stability and biocompatibility of devices. In this article, a label-free and real-time monitoring approach using a zinc oxide (ZnO) nanorod field effect transistor (FET) is proposed to detect cell traction forces (CTFs) exerted by cells on underlying substrates. The ZnO nanorod FET with the approach of difference-frequency lock-in detection achieves high sensitivity, good stability, and excellent biocompatibility, by which real-time and long-term (over 20 days) monitoring of cellular mechanical changes in osteogenic differentiation of mesenchymal stem cells (MSCs) is successfully achieved. We also employ electrical impedance monitoring using microelectrode array chips and microscopic observation to investigate cell migration and nodular aggregation behaviors of MSCs in osteogenic differentiation. Various biochemical assays including alkaline phosphatase (ALP), osteopontin expression and alizarin red staining are utilized to verify osteogenic differentiation of MSCs. We propose a combination of cell traction force measurement, impedance measurement and microscopic observation to provide multimodal profiling of cell morphology, and cellular biomechanical and electrophysiological phenotypes, which can track cellular dynamics in stem cell development and help to deeply understand the mechanism of osteogenic differentiation.

Electrical stimulation (ES) as an easy and effective inducing method has been widely used in induction differentiation of stem cells, e.g. osteogenic differentiation of mesenchymal stem cells (MSCs) for bone healing and bone tissue therapies. However, the micro-effect of an inhomogeneous electric field has rarely been investigated for ES in induction differentiation, and conventionally used ex situ assays may preclude accurate assessment due to variation from cell inoculation and treatments. Here, a novel electrical stimulation method with a microelectrode array chip is proposed for osteogenic differentiation of MSCs. The electric field applied onto the MSCs by the microelectrode array is designed similarly with a natural aggregation distribution of differentiated MSCs. The proposed ES method accelerates osteoblast proliferation and differentiation in the electrode array region and generates a larger amount of mineralized deposits, which are assayed via in situ alizarin red staining and morphology observation as well as immunocytochemistry. In addition, this method allows a direct in situ assessment to compare the osteogenic differentiation of MSCs with and without ES on a single chip to avoid culture environment difference. The method provides a fundamental platform for investigating induced differentiation of stem cells and allows integration with multifunctional cell assays to achieve in situ tracking for the differentiation process of stem cells.