There is a high demand for in vitro models of the central nervous system (CNS) to study neurological disorders, injuries, toxicity, and drug efficacy. Three-dimensional (3D) in vitro models can bridge the gap between traditional two-dimensional culture and animal models because they present an in vivo-like microenvironment in a tailorable experimental platform. Within the expanding variety of sophisticated 3D cultures, scaffold-free, self-assembled spheroid culture avoids the introduction of foreign materials and preserves the native cell populations and extracellular matrix types. In this study, we generated 3D spheroids with primary postnatal rat cortical cells using an accessible, size-controlled, reproducible, and cost-effective method. Neurons and glia formed laminin-containing 3D networks within the spheroids. The neurons were electrically active and formed circuitry through both excitatory and inhibitory synapses. The mechanical properties of the spheroids were in the range of brain tissue. These in vivo-like features of 3D cortical spheroids provide the potential for relevant and translatable investigations of the CNS in vitro.

Inverse emulsification was used to fabricate polyacrylamide (PAAm) microbeads with size and elastic properties similar to typical, mammalian cells. These biomimicking microbeads could be fluorescently stained and functionalized with a collagen type-I coating, post-polymerization, for tracking bead locations and promoting cell recognition/binding, respectively. By occupying a previously unfilled range of sizes and mechanical properties, these microbeads may find unique use in both biomedical and materials applications.

Adipose-derived stem/stromal cells (ASCs) respond heterogeneously when exposed to lineage-specific induction medium. Variable responses at the single-cell level can be observed in the production of lineage-specific metabolites, expression of mRNA transcripts, and adoption of mechanical phenotypes. Understanding the relationship between the biological and mechanical characteristics for individual ASCs is crucial for interpreting how cellular heterogeneity affects the differentiation process. The goal of the current study was to monitor the gene expression of peroxisome proliferator receptor gamma (PPARG) in adipogenically differentiating ASC populations over two weeks, while also characterizing the expression-associated mechanical properties of individual cells using atomic force microscopy (AFM). Results showed that ASC mechanical properties did not change significantly over time in either adipogenic or control medium; however, cells expressing PPARG exhibited significantly greater compliance and fluidity compared to those lacking expression in both adipogenic and control media environments. The percent of PPARG+ cells in adipogenic samples increased over time but stayed relatively constant in controls. Previous reports of a slow, gradual change in cellular mechanical properties are explained by the increase in the number of positively differentiating cells in a sample rather than being reflective of actual, single-cell mechanical property changes. Cytoskeletal remodeling was more prevalent in adipogenic samples than controls, likely driving the adoption of a more compliant mechanical phenotype and upregulation of PPARG. The combined results reinforce the importance of understanding single-cell characteristics, in the context of heterogeneity, to provide more accurate interpretations of biological phenomena such as stem cell differentiation.