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D.W. Rickman, M.J. Mahoney, P. Saloupis; Development of a Growth Factor-Enriched Synthetic Microenvironment for Retinal Transplantation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):493.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To assess the utility of a novel cellular "niche" for retinal transplantation in which neural stem cells, synthetic extracellular matrix components and sustained release microspheres are assembled prior to transplantation to form a three-dimensional "neo-tissue." Methods: Neurotrophic factors (NGF or BDNF) were prepared using a double emulsion technique to form biodegradable polymeric (50:50 poly-lactic glycolic acid) microparticles (~1 µm diam). Release rates from microparticles were assessed by SDS PAGE and ELISA; bioactivity by a PC12 cell neurite extension assay. Microparticles were combined with GFP-expressing neural progenitor cells derived from the adult hippocampus (gift from Dr. F. Gage) for 24 hr in rotational culture to form spherical neo-tissues (~100-200 µm diam). For delivery, neo-tissues were seeded on top of postnatal day-0 retinal explants. After 3, 5, 7 or 14 days, cultures were fixed, cryosectioned and visualized by confocal microscopy. Results: Quantitative differences in growth factor release rates were achieved by varying the loading of growth factors. Release was greatest during the first 48 hr; followed by a sustained period of slower release. Release of ng levels (per mg microspheres) of bioactive growth factor persisted for up to 28 days. Single as well as aggregated microparticles were distributed uniformly throughout neo-tissues. A fraction of cells within neo-tissues extended processes. By 3-5 days after seeding, few transplanted cells had engrafted, primarily remaining assembled within the neo-tissue in close proximity to sources of growth factor. After 7 days, some cells had migrated out of the neo-tissues, engrafted into the explant and extended processes; most cells remained within the neo-tissue. By 14 days, all transplanted cells had migrated out of the neo-tissue and into the explanted retina, where they underwent limited proliferation and further differentiation. Some cells displayed long processes. Neo-tissue spherical geometry was no longer apparent. Conclusions: Here we demonstrate a method for transplantation of neural stem cells within a controllable synthetic microenvironment. Dispersed cells from these neo-tissues readily engraft in retinal explant cultures where they undergo differentiation. This method of delivery allows for variation in several parameters including growth factor dose and spatial positioning. Furthermore, multiple agents can be delivered in an effort to more closely replicate the composition of the extracellular microenvironment present during development.
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