Introduction: The understanding of the pathomechanisms of age-related macular degeneration (AMD) remains a challenge. Typical features of the disease such as drusen and the role of growth factors have been extensively studied[1]-[3], but it remains unclear what causes drusen formation and a growth factor imbalance. Research is directed towards animal models[4] and more recently towards stem cell based therapies which are still in their infancy[5]. Monolayer culture is a widely used alternative to animal studies, but is limited by the relative lack of sophistication when compared to the human retina, since the pathogenesis of AMD involves complex interplay between the retinal pigment epithelium (RPE), Bruch’s membrane and the choroid. It is becoming apparent that 3D culture systems are better suited for complex disease models to allow the study of cellular crosstalk within the microenvironment with advanced co-culture system[6]. Therefore, this study aimed at developing a novel humanized trilayer culture model by culturing human fetal RPE and primate choroidal cells on electrospun medical-grade polycaprolactone (PCL)-gelatin meshes to study the initial changes in AMD.
Materials and Methods: Electrospun PCL-gelatin meshes were made by a custom solution electrospinning apparatus as described previously[7] (Figure 1). The overall architecture of the scaffolds, including the porosity and the surface area-to-volume ratio, was analyzed using the nano-computed tomography (nano-CT) system using a resolution of 1 µm, a voltage of 45 kVp and a current of 177 mA (n=4). The porous nature of the scaffold was an important consideration in studying the effect of co-culture and the possible crosstalk between the RPEs and the choroidal cells. The trilayer model was prepared by seeding the primate choroid endothelial cell line (RF/6A) on the bottom surface of the PCL-gelatin scaffold coated with human recombinant laminin followed by seeding human RPE cells on the top surface of the scaffold[8] (ratio 1:1; 104 cells/cm2), mimicking the retina in vivo microenvironment.
Results and Discussion: Scanning electron microscopy images confirmed that PCL-gelatin electrospun scaffolds allowed excellent cell attachment and infiltration (Figure 2). Functional and morphological analysis of the human RPE cells showed that they were able to phagocytose vitronectin-coated microbeads and that they formed microvilli (Figure 2B). The quantitative analysis of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), factors critical in pathophysiological condition of AMD, indicated significant increases in production of both VEGF and PEDF in RPE/choroidal co-cultures compared to mono-cultures. Our findings indicate the functionality of the current model and provide a strong argument into the advantages of using a 3D trilayer in vitro culture model to study early changes involving the shift in the PEDF/VEGF equilibrium that occurs in AMD.
Conclusion: In summary, we provide the initial evidence that a three dimensional in vitro model can be fabricated to exhibit functional characteristics typical for human RPE and choroidal cells. We believe that this novel bioengineered 3D construct can provide a platform for further studies in the pathomechanisms of AMD.
This study was funded by National Health and Medical Research Council (NHMRC).
References:
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