Growth within small diameter micro-columns induced bi-polar morphology in astrocytes. (A) Representative confocal reconstruction of aligned astrocytes seeded at a medium density in 350 μm ID hydrogel micro-columns, stained to denote astrocyte somata/processes (GFAP; green), possible neuronal contamination (β-tubulin-III; red), and a Hoechst nuclear counterstain (blue). Astrocytes grown in hydrogel micro-columns of 350 μm (pictured) and 180 μm (not pictured) ID consistently formed a bi-polar morphology nearly perfectly aligned with the central axis of the micro-column. These bi-polar astrocytes often formed continuous chains (B) as shown in zoomed region of interest. (C, D) In contrast, astrocytes grown in a traditional planar culture environment on collagen-coated polystyrene exhibited an archetypal stellate morphology as shown in (C) phase contrast microscopy and (D) following immunocytochemistry (GFAP, green; Hoechst, blue). Of note, robust GFAP expression also confirmed the astrocytic phenotype of cells cultured in 2-D and the bi-polar aligned cells cultured within 3-D micro-columns as seen in (A, B, D). (E, F) Long, aligned individual processes were observed from the bi-polar astrocytes within the micro-columns as shown in (E) phase and (F) following GFAP immunolabeling. (G) The effect of micro-column diameter on induction of bi-polar morphology was quantified, as shown graphically as the percentage of bi-polar astrocytes for the various micro-column/culture conditions. Micro-columns with an ID of 180 μm (n = 121 astrocytes from N = 6 micro-columns) or 350 μm (n = 134; N = 6) resulted in a significant increase in the presence of bi-polar astrocytes compared to 1 mm ID micro-columns (n = 146; N = 5) or 2-D sister cultures on collagen-coated polystyrene (n = 102; N = 5 cultures) (p < 0.001 each). Error bars represent standard deviation. Scale bars A, C, D: 100 μm; Scale bars B, E, F: 20 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)