John Dayron Rivera, Jonathan Soucy, Volha Malechka, Emil Kriukov, Petr Y Baranov | 2023
Abstract
Purpose : Optimization of retinal ganglion cell (RGC) survival and maturation is essential to improve the efficacy of RGC replacement therapy for patients with optic neuropathies. Laminin, a protein in the CNS and retinal extracellular matrix (ECM), can bind and activate the β1 Integrin receptor on RGCs and is required to maintain RGC culture in 2D in vitro. It is necessary for cell adhesion and survival to promote the extension of neurites, synapse formation, and intercellular signaling. In this study, we explored minor laminin-derived protein (iMatrix 511) and other ECM ligands as a substrate and supplement for 2D RGC culture.
Methods : Human RGCs for the studies were differentiated and purified from brn3b-tdTomato-hESC using a 2D RGC differentiation protocol. To study RGC-substrate interaction, we seeded cells (13.9k/well) into substrate-coated wells of 96 well plates containing: iMatrix511, matrigel, laminin, poly-d-lysine (PDL)+laminin, PDL+BB(borate buffer), PDL alone, geltrex, cultrex, collagen 1, fibronectin, and synthemax. The attachment of the RGCs was measured after 30 minutes of seeding. Cell viability was assessed with Calcein blue at 1 hr post-plating and neurite outgrowth at three days. For data analysis, ANOVA was used to measure significance.
Results : There was no attachment difference between different substrates besides PDL+BB showing a 22% RGC decrease compared to the control (p = 0.008). We observe a 20% survival increase for iMatrix 511 compared to laminin at a coat concentration of 4 ug/cm2. Besides PDL and PDL+BB, all cell culture substrates had a 40% increase in RGC neurite growth compared to the negative control. Our studies show a 48% population increase of RGC with three neurite processes for the cell culture substrate, iMatrix 511, compared to laminin (p = 0.04). Finally, the only cell culture substrates resulting in four or more primary neurites were iMatrix 511, laminin, and matrigel. Nidogen, collagen, and heparan sulfate proteoglycans—all ingredients found in matrigel, geltrex, and cultrex — did not outcompete the survival and neurite outgrowth benefits of laminin.
Conclusions : We show that iMatrix 511 is sufficient and superior to laminin as an RGC substrate in its ability to support cell survival and neurite outgrowth. Adding glycoproteins (fibronectin and synthemax), collagen, and unfixed ingredients in Matrigel, geltrex, and cultrex may induce neurite outgrowth.
Methods : Human RGCs for the studies were differentiated and purified from brn3b-tdTomato-hESC using a 2D RGC differentiation protocol. To study RGC-substrate interaction, we seeded cells (13.9k/well) into substrate-coated wells of 96 well plates containing: iMatrix511, matrigel, laminin, poly-d-lysine (PDL)+laminin, PDL+BB(borate buffer), PDL alone, geltrex, cultrex, collagen 1, fibronectin, and synthemax. The attachment of the RGCs was measured after 30 minutes of seeding. Cell viability was assessed with Calcein blue at 1 hr post-plating and neurite outgrowth at three days. For data analysis, ANOVA was used to measure significance.
Results : There was no attachment difference between different substrates besides PDL+BB showing a 22% RGC decrease compared to the control (p = 0.008). We observe a 20% survival increase for iMatrix 511 compared to laminin at a coat concentration of 4 ug/cm2. Besides PDL and PDL+BB, all cell culture substrates had a 40% increase in RGC neurite growth compared to the negative control. Our studies show a 48% population increase of RGC with three neurite processes for the cell culture substrate, iMatrix 511, compared to laminin (p = 0.04). Finally, the only cell culture substrates resulting in four or more primary neurites were iMatrix 511, laminin, and matrigel. Nidogen, collagen, and heparan sulfate proteoglycans—all ingredients found in matrigel, geltrex, and cultrex — did not outcompete the survival and neurite outgrowth benefits of laminin.
Conclusions : We show that iMatrix 511 is sufficient and superior to laminin as an RGC substrate in its ability to support cell survival and neurite outgrowth. Adding glycoproteins (fibronectin and synthemax), collagen, and unfixed ingredients in Matrigel, geltrex, and cultrex may induce neurite outgrowth.