Fig 1.
Design and assembly of the microfluidic culture system.
a: Schematic section of a culture well: cells are cultured on a Transwell® inserted in the holder. A V-ring is used to seal the system. b: Schematic of the setup: each well is connected to the syringe pump. A plate is placed under the device and is moved to allow for the time-resolved fraction collection. c: Exploded view of the assembly of a single well. Five layers of dry film photoresist are laminated on top of a support layer. A holder is screwed on top of the support layer and a Transwell® is subsequently inserted together with the V-ring. The microfluidic inlet is screwed in the holder while the outlet needle is glued on the bottom of the support layer.
Fig 2.
Simulation of the microfluidic flow in the chamber.
a: Top view of the results of the simulation of half channel. Stream lines are represented in grey. The colours and the scale bar are relative to the pressure along the channel [Pa]. b: 3D view of the simulated system. Stream lines are represented in grey. A heat map of the values of the x component of the velocity in the widest section of the channel is reported together with the colour scale bar [μm/s]. c: Plot of the gradient of the x component of the velocity along the y-axis versus the distance from the centre of the channel.
Fig 3.
Characterization of microfluidic culture system.
Differentiated PBECs were cultured in the microfluidic culture system and after an equilibration phase of 1h apically exposed to pollen extract. a: The flow rate of pollen-challenged and control wells was determined by measuring the outlet volume per hour for a period of 12h. n = 7 independent experiments with different donors. b: The transepithelial electrical resistance (TER) of differentiated PBECs was measured before and after microfluidic culture conditions without or with pollen challenge. Cells cultured in common culture conditions without flow were used as static controls. TER was normalized to the respective value before the start of the experiment. n = 15 independent experiments using 13 different donors; *: p≤0.05.
Fig 4.
Cell viability and barrier integrity of differentiated PBECs in microfluidic compared to static culture conditions.
After 24h in microfluidic or static culture conditions, actin filaments (yellow) and the tight junction protein occludin (green) were stained by immunofluorescence. Images are representative of 3 independent experiments using 3 different donors.
Fig 5.
Release of IL–8 in microfluidic compared to static culture conditions.
Basolateral release of IL–8 by differentiated PBECs after challenge with pollen was analysed by ELISA. n = 6 independent experiments using 5 different donors. a: Overall amount of IL–8 released by differentiated PBECs in a period of 24h after pollen treatment. b: Comparison of pollen induced IL–8 release in static and microfluidic culture condition. The x-fold change in pollen induced IL–8 release compared to untreated control is shown. Linked data points represent experiments run in static and microfluidic culture conditions in parallel with matching PBEC donor. *: p≤0.05 (Wilcoxon).
Fig 6.
Time-dependent release of IL–8 by differentiated PBECs induced by pollen.
a: Using the microfluidic culture system, basolateral flow was collected for 2h periods over 24h. Release of IL–8 was analysed by ELISA. n = 15 independent experiments using 13 different donors. b: Comparison of pollen induced IL–8 release in static and microfluidic culture conditions in 2h intervals over a 12h period. In static culture conditions, aliquots of basolateral medium were taken at matching time points. Release of IL–8 was analysed by ELISA. Microfluidic culture (MF) n = 15; static n = 5 independent experiments using 13 and 4 different donors respectively.