Impedance Spectroscopic Characterisation of Porosity in 3D Cell Culture Scaffolds with Different Channel Networks

Over the last two decades, three-dimensional (3D) cell culture systems have gained increasing interest in different fields spanning from biomedical sciences to pharmaceutical research [1]. The 3D format is believed to enable the formation and maintenance of a more in vivo like cellular environment able to supply cells with oxygen, nutrients, and soluble factor gradients (e.g. growth factors and hormones) leading to a more biologically relevant behaviour of cell functionality [2]. Careful considerations need to be taken in the design of 3D scaffolds for supporting cell organisation, proliferation and differentiation [3]. Based on the specific application, they can be fabricated from a variety of biocompatible materials (e.g. biopolymers, synthetic polymers, ceramic, metals). Among these, polydimethylsiloxane (PDMS) is a cheap transparent material with low autofluorescence and can be molded with a resolution down to a few micrometers [4]. It has already been demonstrated to be a suitable cell culture scaffold [5] after chemical and/or physical functionalisation [6] forming a hydrophilic surface, which can be further coated with specific extracellular matrix proteins, such as collagen and laminin. Different scaffold parameters, such as pore size, geometry, orientation and interconnectivity, as well as channel branching can be tuned in order to influence the diffusion of nutrients and cell organisation within the 3D environment [7]. This can be achieved with traditional fabrication technologies (e.g. solvent-casting/particle leaching, gas foaming, fibre bonding, phase separation, and emulsion freeze drying [8]). Recently, 3D printing has also emerged as a promising technique to precisely control the scaffold architecture [9]. A wide range of methods are currently available for characterising scaffold porosity, spanning from scanning electron microscopy (SEM) to gas adsorption and mercury intrusion porosimetry (MIP). All of them strictly depend on the sample exposure to vacuum conditions [10]. SEM allows characterisation of both the degree of scaffold porosity and pore size, although it requires sectioning and gold sputtering of the sample in order to provide relevant information. Furthermore, sputter coating has the potential disadvantage of causing heat damage to the sample. Gas adsorption may be applied to measure the scaffold surface area and probe its entire surface including irregularities and pore interiors. Prior to the measurements, the sample is pretreated at an elevated temperature in vacuum to remove any contaminants, which increases the risk of its degradation. MIP is a widely used approach to measure pore distribution and connectivity, tortuosity and compressibility in biomaterials. It allows the detection of pore size ranging from 2 up to more than 50 nm in diameter by monitoring the proAbstract : We present the application of electrochemical impedance spectroscopy (EIS) as a method for discriminating between different polydimethylsiloxane (PDMS) scaffolds for three-dimensional (3D) cell cultures. The validity of EIS characterisation for scaffolds having different degree of porosity (networks of structured or random channels) is discussed in relation to Archie s law. Guidelines for EIS analysis are presented and demonstrated to provide porosity information in physiological buffer that agrees well with a more conventional weight-based approach. We also propose frequency ranges that may serve as means of single-frequency measurements for fast scaffold characterization combined with in vitro monitoring of 3D cell cultures.