Abstract
Purpose :
Microfluidic platforms have proven useful for constructing hierarchical in vitro tissue models. However, it is important that cultured cells maintain the same phenotype seen in vivo. Establishing and maintaining a polarized RPE monolayer using current devices is problematic. Knowing that RPE cells grow well and maintain tight junctions on transwell plates, we designed and created an alternative approach, a microfluidic PDMS chamber built to resemble a hockey “puck” that can be screwed together in a multilayer assemblage using phenotypically defined RPE cells on a transwell membrane.
Methods :
Layers for the "puck" elements were cut from pre-formed acrylic and silicone sheets using an infrared laser. A transwell membrane supporting a polarized monolayer of RPE cells was cut to size and compressed between the silicone layers and a glass disk. A microflow pump containing DMEM media and bubble trap was connected, with flow yielding serial ~5.0 ul drop volumes. The device and media reservoir were placed into an EVOS micro-incubator simulating a hypoxic environment and 5.0 ul samples were collected over 4 hours. The membrane was then inverted and thus, the apical and basal compartments were serially analyzed under the same experimental conditions. We employed a bead-based immunofluorescence method, AlphaLISA, to detect and quantitate vascular endothelial growth factor (VEGF) secretion using a plate reader at 615 nm. The signal had a limit of detection of ~1.0 pg/mL.
Results :
Under hypoxic conditions, basal VEGF secretion by polarized RPE cells increased significantly from 1788 pg/ml to 2156 pg/ml at 2-hours (p<0.002) and remained elevated through the 4-hour time point. Conversely, VEGF secretion from the apical side remained constant under the same hypoxic conditions, from 1870 pg/ml to 1936 pg/ml (p<0.05).
Conclusions :
The “puck” is a platform assembly capable of sampling discrete reaction volumes from apical or basal secretions by polarized RPE cells in situ. This platform and method establish a real time biochemical mapping methodology, permitting polarized sampling with high spatiotemporal resolution from small ensembles of phenotypically defined RPE cells reflecting the in situ tissue microniche for experimental manipulation. This was confirmed by the polarized, basal secretion of VEGF under conditions of experimental hypoxia.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.