Skip to main content
. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Prog Neurobiol. 2014 Nov 22;0:1–25. doi: 10.1016/j.pneurobio.2014.11.003

Table 5.

Major methods used to assess in vitro neurological tissues. Amy Hopkins, Elise DeSimone, Karolina Chwalek and David Kaplan, Progress in Neurobiology.

Technique Description 2D or 3D Functional Assay? Challenges in 3D cultures Reference
Live/Dead fluorescent assays Indicates intracellular esterase activity (live cells) vs. loss of plasma membrane integrity Both No Diffusion through 3D constructs; Optical clarity of biomaterial substrates; Loss of viability (O'Connor et al., 2001)
Immuno-histological / cytological fluorescent assays Phenotypic characterization of cultures by targeting antigens of interest with fluorescent-labeled antibodies Both Indirect; Cells will synthesize certain proteins or sugars if they are serving a particular function Diffusion through 3D constructs; Non-specific fluorescence; Optical clarity of biomaterial substrates; Loss of viability (Krassioukov et al., 2002; Stavridis et al., 2005; Whittemore et al., 1994)
Flow cytometry Phenotypic characterization of cultures by targeting antigens of interest with fluorescent-labeled antibodies; Allows for cell counting, sorting and purification Both Indirect; Cells will synthesize certain proteins or sugars if they are serving a particular function Destruction of 3D architecture; Interference of biomaterials with cell counting / sorting (Emery and Dugas, 2013; Foo, 2013)
Molecular techniques (e.g. qPCR, ELISA, proteomics) Phenotypic or genotypic characterization based on detection of genomic DNA, mRNA, or protein Both Indirect; Cells will synthesize certain proteins or sugars if they are serving a particular function Destruction of 3D architecture; Interference of biomaterials with assay; Low cell density does not yield sufficient titers of protein or genetic material (Maximino et al., 2014; Noda et al., 2014; Wesseling et al., 2013)
Patch-clamping Single cell recordings of membrane potential in real time 2D Yes Micro-electrodes too delicate to penetrate biomaterials; Poor optical clarity of biomaterials hinders identification of single cells during patching; Electrical insulation properties of biomaterials may affect recordings; Lack of knowledge of signal processing of 3D in vitro (Kolaj et al., 2014; Neher and Sakmann, 1992; Ward, 1997)
Fluorescent calcium indicators Fluorescent markers that bind calcium and that bind calcium and allow for measurement of calcium flux indicating neurotransmitter release and other intracellular processes Both Yes Diffusion through 3D constructs; Non-specific fluorescence; Optical clarity of biomaterial substrates; Loss of viability; Highspeed capture camera required in case of action potential propagation (Catterall et al., 2013; Frank, 2014; Kim and Jun, 2013)
Permeability studies Continuous flow through cultures to measure permeability of epithelial/endothelial barrier Both Yes Bioreactor required to ensure equal flow through entire construct; Reproducibility of biomaterial scaffold to eliminate variability among samples (Cucullo et al., 2013; Deli et al., 2005; Sun and Pang, 2008)
TEER Measurement of electrical resistance of epithelial/endothelial barrier Both Yes Placement of electrodes on either side of cell layer requires custom culture chamber design which depends on 3D architecture; Reproducibility of biomaterial scaffold critical to eliminate variability among samples (Cucullo et al., 2013)
Multi-electrode arrays Extracellular recordings; Small (~1 μm) arrays implanted in brain tissue to individually record single neurons present in a particular locale; May be flexible and conform to complex surfaces 3D Yes Significant spike sorting required; Reproducibility difficult; Limited to recordings of groups of single cells = may be missing significance of whole network (Claverol-Tinture et al., 2005; Izhikevich, 2004)
Flexible electrodes Extracellular recordings; Biomaterial reinforcements of thin electrodes for penetration of dense tissue and prevention of glial scar formation 3D Yes Significant spike sorting required; Reproducibility difficult; Must take into account biomaterial degradation effects on signal processing (Aregueta-Robles et al., 2014; Kim et al., 2010)

qPCR = Quantitative polymerase chain reaction; ELISA = enzyme-linked immunosorbent assay; BBB = blood-brain barrier; TEER = trans-endothelial electrical resistance