The influence of polyacrylamide gel substrate elasticity on primary cultures of rat retinal ganglion cells

• Published in: Experimental eye research 2024-12, Vol.249, p.110116, Article 110116
• Main Authors: Strake, Matthias, Fischer-Wedi, Charlotte V., Khattab, Mohammed H., Lauermann, Peer, Wollnik, Carina, Stanischa, Christina, Hoerauf, Hans, Rehfeldt, Florian, van Oterendorp, Christian
• Format: Article
• Language: English
• Subjects: Acrylic Resins; Animals; Animals, Newborn; Blotting, Western; Cell culture substrate; Cell Survival - physiology; Cells, Cultured; Cyclic AMP Response Element-Binding Protein - metabolism; Elastic Modulus - physiology; Elasticity - physiology; Mitochondria; Mitochondria - metabolism; Neurite outgrowth; Neurites - physiology; Polyacrylamide gel; Polylysine - pharmacology; Proto-Oncogene Proteins c-akt - metabolism; Rats; Rats, Wistar; Retinal ganglion cell; Retinal Ganglion Cells - cytology; Retinal Ganglion Cells - metabolism; Retinal Ganglion Cells - physiology; Tubulin - metabolism
• ISSN: 0014-4835; 1096-0007; 1096-0007
• DOI: 10.1016/j.exer.2024.110116

In vitro primary cell culture models of retinal ganglion cells (RGC) are widely used to study pathomechanisms of diseases such as glaucoma. The biomechanic interaction with the culture substrate is known to influence core cellular functions. RGC cultures, however, are usually grown on rigid plastic or glass substrates. We hypothesized that soft polyacrylamide gel substrates may alter survival and neurite outgrowth of primary cultured RGC. Primary retinal cultures from postnatal (day 1–6) Wistar rats were grown on glass coverslips or polyacrylamide (PA) gel substrate with different Young's elastic moduli (0.75, 10 or 30 kPa). Substrates were coated with Poly-l-lysine and/or laminin. RGC were immunostained with anti-beta-III-tubulin. Total neurite length, growth cone morphology, RGC density, mitochondrial morphology and transport as well as pro-survival pathways (Erk1/2, Akt, CREB) were assessed. PA gel substrates of E = 10 kPa significantly increased the total neurite length by factor 1.5 compared to glass (p = 0.02). The growth cone area was significantly larger by factor 5.3 on 30 kPa gels (p = 0.01). The presence of a substrate coating was more important for neurite outgrowth and RGC survival on PA gels (poly-l-lysine > laminin) than on glass. Neither mitochondrial morphology and motility nor the activation of pro-survival pathways significantly differed between the four substrates. PA gel substrates significantly enhanced RGC neurite outgrowth. The signaling cascades mediating this effect remain to be determined. •Polyacrylamide gels were used as substrate for primary retinal ganglion cell culture.•Gels of 10 kPa significantly enhanced neurite outgrowth compared to glass.•Higher gel stiffness positively correlated with the neurite growth cone size.•Poly-l-lysine coating was an important mediator of this effect.