Staining and Analysis of hiPSC-Derived Cortical Neurons Cultured in Hydrogels

Abstract

Cell culturing is a common and reproducible way to study cell maturation and behavior in simple, manmade environments. Traditionally, the culturing has been done by plating cells on a planar surface. However, this does not allow the cells to assemble the way they would in vivo. Different types of three-dimensional (3D) platforms have been developed to create biologically more relevant growth environments for the cells. These environments can be created using extracellular matrix (ECM) -resembling structures such as hydrogels as scaffolds for the cells. Such scaffolds are also utilized in neuroscience. This allows modeling and studying neuronal growth patterns and networking in a way that would not be possible with conventional platforms. In this work, different hydrogels were used as scaffolds for human induced pluripotent stem cell (iPSC) -derived cortical neurons. The two aims of the work were to optimize immunocytochemical (ICC) staining of the cells and to quantitatively analyze neuronal network formation in 3D. The former involved comparing staining protocols with different fixing-, antibody incubation- and antibody washing times. The latter involved surveying and comparing software for neuronal reconstruction and analysis in 3D. In addition, the hydrogels were compared in terms of their stability and ability to support neuronal growth. The most important outcome from the staining protocol optimization was improvement of sample cleanness. Background artifacts could efficiently be removed from the samples by prolonging the antibody washing time. Prolongation of fixing and antibody incubation times did not provide improvements. Although multiple available neuronal reconstruction tools were found, only few of them were suitable for analyzing neuronal populations in 3D. Two software: Imaris and Avizo, were tested in practice. From these two options, Imaris was chosen for quantitative analysis of neuronal network formation in different hydrogels. As a result, differences in neuronal growth in the hydrogels could be shown with numerical data. The best performing gel in terms of both gel stability and neuronal growth was Collagen. In addition, good results were obtained with Hyaluronan - polyvinyl alcohol - collagen (HPC) gel after changing the well plate format.