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Generation of encapsulated cells expressing GDNF

To generate cell lines secreting GDNF suitable for encapsulation, stable clones were established from ARPE-19 cells (immortalized retinal pigment epithelial cells) by transfection with two different GDNF expression constructs. More than 200 clones were screened and among those, six clones secreting 90-133 ngGDNF/105 cells/24h in serum supplemented growth medium were chosen for further evaluation. When culturing the clones in a serum-free medium optimized for epithelial cells, GDNF secretion was increased approximately 2 folds for most clones. For all clones, stable GDNF secretion appeared to be maintained over 4 months in culture (and more than 20 passages) under proliferative conditions. Furthermore, GDNF secretion was stable at least 6 weeks after reaching confluency and GDNF bioactivity of conditioned media from the clones has been verified using a cell-based assay. After encapsulation of the clones in a standard device for experimental implantation into rat brains, GDNF release levels between 1-15 ng/device/24h could be achieved. Such devices could be used to obtain mechanistic insights into the neuroprotective role in appropriate animal models.

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