Table 6.

Functional validation of 3D in vitro neural systems and associated challenges. Amy Hopkins, Elise DeSimone, Karolina Chwalek and David Kaplan, Progress in Neurobiology.

Physiological Process	Analysis Method	Challenges	3D in vitro Approaches
BBB integrity	Compound permeability	• Robust perfusion cultures • Reproducible measurements	• Perfusion bioreactor consisting of bundles of hollow channels, (Cecchelli et al., 2007; Cucullo et al., 2002, 2013)
	TEER	• Placement of electrodes within vessels • Reproducible electrode placement & readings	• Custom-built chambers, electrodes, and power/recording systems, (Cucullo et al., 2002, 2013; McAllister et al., 2001; Santaguida et al., 2006)
Neural electrical activity	Patch-clamping	• Penetration of glass micropipette through scaffold • Visualization through scaffold	• Increase strength of small electrodes (Tien et al., 2013) • Optically transparent biomaterials (Balint et al., 2014) • Slice preparations (Hanson et al., 2010; Kapfhammer, 2010; Stavridis et al., 2005)
	Extracellular recordings	• Culturing high neural cell densities • Construction of polarized neural populations	• Optimization of 3D in vitro culture systems in regards to cell density and 3D architecture (Cullen et al., 2006; East et al., 2010; Miller, 2014)
Neuronal and glial signaling	Calcium imaging, VSDs	• Visualization through 3D biomaterials • Identification of different cell types • Diffusion of dyes through 3D tissues • Signal-to-noise ratios	• Optically transparent biomaterials (Dana et al., 2014) • Ratiometric dyes (Homma et al., 2009) • Slice preparations (Dana et al., 2014)

BBB = blood-brain barrier; TEER = transendothelial electrical resistance; VSD = voltage-sensitive dye