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Charlotte Fischer, Florian Rehfeldt, Mai-Linh Kortleben, Mohammed Khattab, Christian van Oterendorp; Influence of substrate elasticity on primary retinal ganglion cell culture. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2040.
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© ARVO (1962-2015); The Authors (2016-present)
Cell culture substrate stiffness has been increasingly recognised as an influencing factor on cell growth, morphology and survival. We performed retinal ganglion cell (RGC)- culture on polyacrylamide (PA) hydrogel substrates and analysed cell morphology comparing to conventional cell culture on glass cover slips.
Primary cultures of retinae of young (day 7-13) and old (6 month) Wistar-rats were set up using a Papain Dissociation Kit (Worthington Biochem, USA) and B27-supplemented neurobasal-A medium. Cells were seeded on coated (poly-l-lysine and laminin) PA gels with a Young’s elastic modulus of 0.75kPa (equivalent to neural tissue, ‘soft’) and 10kPa (equivalent to muscle tissue, ‘intermediate stiffness') and on coated glass coverslips. RGC number and morphology (total length of neurite tree and neurite number) was analysed by immunohistochemical stainig for bIII-tubulin and image analysis with ImageJ / Simple neurite tracer plugin. Activation of pro-survival signalling pathways (phospho-AKT, phospho-CREB, phospho- ERK 1/2) was assessed by western blots.
The median total neurite length from young rats (n=12 cells per dish; n=6 dishes) was highest on 10kPa gels (1855 ± 272 (0.75kPa) vs. 3628 ± 436 (10kPa); vs 2476 ± 242 (glass), p=0.01 for 10kPa vs. 0.75kPa and vs. glass) In cultures from old rats total neurite length was higher on 10kPa than 0.75kPa, but not different to glass (740 ± 42 (0.75kPa) vs. 995 ± 61 (10kPa), p=0.016). The RGC number showed a trend towards higher numbers in the 10kPa group, but the difference to 0.75kPa and glass was not statistically significant. Higher donor age was significantly connected with smaller RGC number and neurite length on all substrates (p=0.02 to p<0.0001 in ANOVA). No significant difference was observed for the neurite number in either age group (p>0.05). Activation of pro-survival signalling pathways was significantly stronger in young compared to old animals, but not significantly different between 10kPa and glass for all three pathways (ANOVA with Holm-Sidak post-hoc test).
RGCs grown on intermediate stiff gels showed significantly longer neurites than on soft gels or glass. However, the age of the donor animal had a stronger influence on neurite length and pro-survival pathways than the substrate elasticity. The enhancement of neurite outgrowth on substrates stiffer than neural tissue was surprising and requires further investigation.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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