Human neuroblastoma (SH-SY5Y) cell culture and differentiation in 3-D collagen hydrogels for cell-based biosensing.
Cell-based three-dimensional systems are desirable in the field of high throughput screening assays due to their potential similarity to in vivo environment. We have used SH-SY5Y human neuroblastoma cells cultured in 3-D collagen hydrogel, confocal microscopy and immunofluorescence staining, to assess the merit of the system as a functional, cell-based biosensor. Our results show differences between 2-D and 3-D resting membrane potential development profile upon differentiation. There was no statistically significant difference in SH-SY5Y proliferation rate between 2-D monolayer and 3-D collagen culture formats. A large percentage of cells (2-D, 91.30% and 3-D, 84.93%) did not develop resting membrane potential value equal to or lower than -40 mV; instead cells exhibited a heterogeneous resting membrane potential distribution. In response to high K(+) (50 mM) depolarization, 3-D cells were less responsive in terms of increase in intracellular Ca(2+), in comparison to 2-D cells, supporting the hypothesis that 2-D cell calcium dynamics may be exaggerated. L-Type Ca(2+) expression levels based on staining results was inconsistent with Bay K 8644 channel activation results, strongly suggesting that either the majority of the channels were non-functional or could not be activated by Bay K 8644. In general, the results in this study confirm the depolarization-induced differences in intracellular calcium release when cultured using a 2-D versus a 3-D matrix.
Desai
Anu
A
Cellular Bioengineering Laboratory, Faculty of Engineering, University of Georgia, Athens, 30602, USA.
Kisaalita
William S
WS
Keith
Charles
C
Wu
Z-Z
ZZ
D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D024042:Collagen Type I; D006801:Humans; D020100:Hydrogels; D008564:Membrane Potentials; D009447:Neuroblastoma / Q000473:pathology* / Q000503:physiopathology*
D015220:Calcium Channels; D024042:Collagen Type I; D020100:Hydrogels; D002118:Calcium