Research in cell biology greatly relies on cell-based in vitro assays and models that facilitate the investigation and understanding of specific biological events and processes under different conditions. The quality of such experimental models and particularly the level at which they represent cell behavior in the native tissue, is of critical importance for our understanding of cell interactions within tissues and organs. Conventionally, in vitro models are based on experimental manipulation of mammalian cells, grown as monolayers on flat, two-dimensional (2D) substrates. Despite the amazing progress and discoveries achieved with flat biology models, our ability to translate biological insights has been limited, since the 2D environment does not reflect the physiological behavior of cells in real tissues. Advances in 3D cell biology and engineering have led to the development of a new generation of cell culture formats that can better recapitulate the in vivo microenvironment, allowing us to examine cells and their interactions in a more biomimetic context. Modern biomedical research has at its disposal novel technological approaches that promote development of more sophisticated and robust tissue engineering in vitro models, including scaffold- or hydrogel-based formats, organotypic cultures, and organs-on-chips. Even though such systems are necessarily simplified to capture a particular range of physiology, their ability to model specific processes of human biology is greatly valued for their potential to close the gap between conventional animal studies and human (patho-) physiology. Here, we review recent advances in 3D biomimetic cultures, focusing on the technological bricks available to develop more physiologically relevant in vitro models of human tissues. By highlighting applications and examples of several physiological and disease models, we identify the limitations and challenges which the field needs to address in order to more effectively incorporate synthetic biomimetic culture platforms into biomedical research.

The blood-brain barrier (BBB) restricts paracellular and transcellular diffusion of compounds and is part of a dynamic multicellular structure known as the “neurovascular unit” (NVU), which strictly regulates the brain homeostasis and microenvironment. Several neuropathological conditions (e.g., Parkinson’s disease and Alzheimer’s disease), are associated with BBB impairment yet the exact underlying pathophysiological mechanisms remain unclear. In total, 90% of drugs that pass animal testing fail human clinical trials, in part due to inter-species discrepancies. Thus, in vitro human-based models of the NVU are essential to better understand BBB mechanisms; connecting its dysfunction to neuropathological conditions for more effective and improved therapeutic treatments. Herein, we developed a biomimetic tri-culture NVU in vitro model consisting of 3 human-derived cell lines: human cerebral micro-vascular endothelial cells (hCMEC/D3), human 1321N1 (astrocyte) cells, and human SH-SY5Y neuroblastoma cells. The cells were grown in Transwell hanging inserts in a variety of configurations and the optimal setup was found to be the comprehensive tri-culture model, where endothelial cells express typical markers of the BBB and contribute to enhancing neural cell viability and neurite outgrowth. The tri-culture configuration was found to exhibit the highest transendothelial electrical resistance (TEER), suggesting that the cross-talk between astrocytes and neurons provides an important contribution to barrier integrity. Lastly, the model was validated upon exposure to several soluble factors [e.g., Lipopolysaccharides (LPS), sodium butyrate (NaB), and retinoic acid (RA)] known to affect BBB permeability and integrity. This in vitro biological model can be considered as a highly biomimetic recapitulation of the human NVU aiming to unravel brain pathophysiology mechanisms as well as improve testing and delivery of therapeutics.

3D cell culture formats more closely resemble tissue architecture complexity than 2D systems, which are lacking most of the cell-cell and cell-microenvironment interactions of the in vivo milieu. Scaffold-based systems integrating natural biomaterials are extensively employed in tissue engineering to improve cell survival and outgrowth, by providing the chemical and physical cues of the natural extracellular matrix (ECM). Using the freeze-drying technique, porous 3D composite scaffolds consisting of poly(3,4-ethylene-dioxythiophene) doped with polystyrene sulfonate (PEDOT:PSS), containing ECM components (i.e., collagen, hyaluronic acid, and laminin) are engineered for hosting neuronal cells. The resulting scaffolds exhibit a highly porous microstructure and good conductivity, determined by scanning electron microscopy and electrochemical impedance spectroscopy, respectively. These supports boast excellent mechanical stability and water uptake capacity, making them ideal candidates for cell infiltration. SH-SY5Y human neuroblastoma cells show enhanced cell survival and proliferation in the presence of ECM compared to PEDOT:PSS alone. Whole-cell patch-clamp recordings acquired from differentiated SHSY5Y cells in the scaffolds demonstrate that ECM constituents promote neuronal differentiation in situ. These findings reinforce the usability of 3D conducting supports as engineered highly biomimetic and functional in vitro tissue-like platforms for drug or disease modeling.

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Three-dimensional in vitro stem cell models has enabled a fundamental understanding of cues that direct stem cell fate and be used to develop novel stem cell treatments. While sophisticated 3D tissues can be generated, technology that can accurately monitor these complex models in a high-throughput and non-invasive manner is not well adapted. Here we show the development of 3D bioelectronic devices based on the electroactive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) - PEDOT:PSS and their use for non-invasive, electrical monitoring of stem cell growth. We show that the electrical, mechanical and wetting properties as well as the pore size/architecture of 3D PEDOT:PSS scaffolds can be fine-tuned simply by changing the processing crosslinker additive. We present a comprehensive characterization of both 2D PEDOT:PSS thin films of controlled thicknesses, and 3D porous PEDOT:PSS structures made by the freeze-drying technique. By slicing the bulky scaffolds we show that homogeneous, porous 250 um thick PEDOT:PSS slices are produced, generating biocompatible 3D constructs able to support stem cell cultures. These multifunctional membranes are attached on Indium-Tin oxide substrates (ITO) with the help of an adhesion layer that is used to minimize the interface charge resistance. The optimum electrical contact result in 3D devices with a characteristic and reproducible, frequency dependent impedance response. This response changes drastically when human adipose derived stem cells grow within the porous PEDOT:PSS network as revealed by fluorescence microscopy. The increase of these stem cell population within the PEDOT:PSS porous network impedes the charge flow at the interface between PEDOT:PSS and ITO, enabling the interface resistance to be extracted by equivalent circuit modelling, used here as a figure of merit to monitor the proliferation of stem cells. The strategy of controlling important properties of 3D PEDOT:PSS structures simply by altering processing parameters can be applied for development of a number of stem cell in vitro models. We believe the results presented here will advance 3D bioelectronic technology for both fundamental understanding of in vitro stem cell cultures as well as the development of personalized therapies.