Conclusions
The Puck can be used to measure spatiotemporal protein secretion by polarized cells into apical and basal microniches in response to environmental conditions. Computational model results support the absence of biologically significant shear stress to the cells caused by the device. Translational relevance: The Puck can be used validate the mature phenotypic health of autologous induced pluripotent stem cells (iPSC)-derived RPE cells prior to transplantation.
Methods
We designed and built a novel microfluidic "Puck" assembly using laser-cut pieces from preformed sheets of silicone and commercial off-the-shelf parts. Transwell membranes containing polarized retinal pigment epithelial (RPE) cells were reversibly sealed within the Puck and used to study polarized protein secretion. Protein secretion from the apical and basal surfaces in response to hypoxic conditions was quantified using an immunoassay method. Computational fluid modeling was performed on the chamber design.
Purpose
Cells grown in milliliter volume devices have difficulty measuring low-abundance secreted factors due to low resulting concentrations. Using microfluidic devices increases concentration; however, the constrained geometry makes phenotypic characterization with transepithelial electrical resistance more difficult and less reliable. Our device resolves this problem.
Results
Under hypoxic culture conditions (7% O2), basal vascular endothelial growth factor (VEGF) secretion by polarized RPE cells increased significantly from 1.40 to 1.68 ng/mL over the first 2 hours (P < 0.0013) and remained stably elevated through 4 hours. Conversely, VEGF secretion from the apical side remained constant under the same hypoxic conditions. Conclusions: The Puck can be used to measure spatiotemporal protein secretion by polarized cells into apical and basal microniches in response to environmental conditions. Computational model results support the absence of biologically significant shear stress to the cells caused by the device. Translational relevance: The Puck can be used validate the mature phenotypic health of autologous induced pluripotent stem cells (iPSC)-derived RPE cells prior to transplantation.