Imaging of 3D neuronal cell cultures in hydrogels under iSPIM with an electrically tunable lens
Three-dimensional in vitro neuronal networks could give better insights to the functioning of the brain, mimicking extracellular conditions more accurately than twodimensional ones. Moreover, the development of genetically encoded calcium indicators together with fluorescence microscopy has facilitated the recording of neuronal activity as sharp calcium changes upon neuronal firing. Nonetheless, imaging such fast events, in threedimensional cultures and at high resolution, requires instrumentation capable of acquiring wide fields of view along large depths and high frame rates.
In our study we image 3D neurons from rat primary cell cultures placed in hydrogels.
We use an inverted SPIM configuration, allowing for the imaging of samples on standard petri dishes. In our iSPIM, the sample remains static while we scan the light sheet along it with a galvanometric mirror. By including an electrically tunable lens in the detection path of the microscope, we rapidly refocus on the illuminated section of the sample, avoiding the need to displace the detection objective and reducing vibrations on the sample. We present our observations of these three-dimensional cultures under our optical setup.
Building a 3-Dimensional human iPSC-derived cortical neuronal network: You need a scaffold to build a brain
The cerebral neocortex is the most newly-evolved region of the brain. In humans it has undergone dramatic progenitor expansion, subtype specification, and network formation.
Human induced pluripotent stem cell (iPSC)-derived neural progenitors have shown an in vivo-like ability to self-organise & promote cerebral cortical lineage determination under minimal growth factor conditions (Shi et al., 2012a; Renner et al., 2017). However, although the cortex’s laminated organisation represents a great functional significance in vivo, neocortical networks are rarely modelled in vitro using 3D cell culture, or omit key neural subtypes (Kirwan et al 2015).
Despite recent developments in tissue modelling, e.g. organoids (Kelava & Lancaster, 2016), 3D models exhibit high variability – impeding exploration of network topography.
Alongside advancements in stem cell biology, progress in nanoscale printing, volumetric imaging & network analysis presents a unique opportunity to combine these fields in search of a better model. Building on this, the MESO-BRAIN project aims to develop a 3D cortical model using expertise from each field (Figure 1) – here we present initial findings.
Our first aim was to determine a suitable material for use in scaffold fabrication – this included investigating potential toxicity, optimal coating reagents, compatibility with optical imaging & two-photon polymerisation processing.