RESUMEN
The choroid plexus (CP) is a transplantable cell source secreting tropic and trophic factors for the treatment of brain and peripheral trauma characterized by cellular loss or dysfunction. Here we characterize the expression and secretion of vascular endothelial growth factor (VEGF) from neonatal porcine CP. Light and electron microscopy revealed that enzymatic digestion of the CP produced a preparation consisting primarily of epithelial cells without notable contaminating cells. Microarray analysis, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay were used to quantify the nuclear, cytoplasmic, and secretory compartmentalization of VEGF. In vitro, the kinetics of VEGF release were orderly, with stepwise increases in secretion over time. The secretory profile of VEGF from CP grown in configurations ranging from a simple monolayer to free-floating 3-dimensional clusters to clusters encapsulated within alginate-polyornithine microcapsules was similar. VEGF output was not affected notably when the cells were maintained in 90% stress medium or in other maintenance media devoid of serum proteins. Secreted VEGF was bioactive, as confirmed by demonstrating its continued ability to proliferate co-cultured human umbilical vascular endothelial cells. The robust ability of these cells to continue to secrete VEGF (and presumably other bioactive proteins) across a variety of dimensional configurations and medium types has implications for their use in clinical indications requiring novel and imaginative use of engineered ectopic transplant sites.
Asunto(s)
Plexo Coroideo/citología , Plexo Coroideo/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Péptidos/química , Ingeniería de Tejidos/métodos , Factor A de Crecimiento Endotelial Vascular/metabolismo , Alginatos/química , Animales , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Materiales Biocompatibles Revestidos/química , Ácido Glucurónico/química , Ácidos Hexurónicos/química , Ensayo de Materiales , PorcinosRESUMEN
Alginate-polycation microcapsule systems have been used over decades as delivery vehicles for cell and protein therapy. These systems have been unpredictable across a range of indications with questions resulting around the inherent stability of the alginate polysaccharide and failure mode of the delivery system. The current study focuses on such a system using 5 different alginates, 2 of which are commercially purified, which are crosslinked by polyornithine. Capsules formed by frequency-generated droplet formation were studied in the peritoneal cavity of Long-Evans rats over the course of 3 months by morphometry, Fourier-transform infrared spectroscopy (FTIR), and scanning electron microscopy of the surface. Individual capsule components were also investigated on FTIR and a relative stability index was generated by titration for comparison to explanted samples over time. Using these techniques, a distinct degradation pattern was noted and is compared between the 5 alginate sources.