Conducting Polymer‐ECM Scaffolds for Human Neuronal Cell Differentiation

Abstract

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.

Microelectrode Arrays Measure Blocking of Voltage‐Gated Calcium Ion Channels on Supported Lipid Bilayers Derived from Primary Neurons

“…These results are consistent with the literature and our previous study of SH‐SY5Y differentiation on PEDOT:PSS‐based extracellular matrix (ECM) 3D scaffolds. [ 29 , 34 ] CaV2.2 ion channels, one of the most highly expressed ion channels on SH‐SY5Y, were found to have significant increased expression after the differentiation process as assessed via Western blot (Figure 2f ). Within the differentiated and non‐differentiated SH‐SY5Y lysate, numerous types of proteins were present (See Coomassie blue stain in Figure S2a , Supporting Information).…”

“…The differentiation protocol was performed based on previous studies, [ 34 , 45 , 46 ] with slight modifications: Briefly, 2 × 10 4 cells cm −2 SH‐SY5Y were seeded in each well of 6‐well plate with or without poly‐L‐lysine (Sigma) pre‐coated glass coverslips in 15% FBS culture media. Until 70% confluency (day 0), cells were washed once with DPBS, and cell media were replaced with 3% FBS culture media +10 µ m retinoic acid (RA, Sigma‐Aldrich) and changed every other day (day 2 and day 4).…”

Conducting polymer-based scaffolds for neuronal tissue engineering

“…For example, Barberio et al recently fabricated 3D composite CP-based scaffolds made of PEDOT and polystyrene sulfonate (PSS) with natural ECM proteins such as collagen (COL), hyaluronic acid (HA), and laminin (LAM) for human neuronal cell differentiation. 65 They used a freeze-drying (ice-templating) technique to engineer porous 3D scaffolds with mechanically stable, biomimetic, and biocompatible features. A schematic representation for the preparation of scaffolds with different composition scaffolds and the corresponding SEM images are summarized in Fig.…”

A human-derived neurovascular unit in vitro model to study the effects of cellular cross-talk and soluble factors on barrier integrity

“…Potential troubleshooting could be provided by replacing or combining immortalized cells with primary cell lines or iPSCs, both contributing to higher TEER values closer to the in vivo BBB. Furthermore, culturing the cells in their native 3D architecture to better mimic in vivo tissue physiology facilitates better intercellular signaling networks, cell-to-cell contact, and developmental processes ( Moysidou et al, 2021 ; Barberio et al, 2022 ). 3D models composed of hydrogels or other biomaterials that mimic native biomechanical stiffness are more representative and have better BBB formation.…”