The Path from Nasal Tissue to Nasal Mucosa on Chip: Part 2-Advanced Microfluidic Nasal In Vitro Model for Drug Absorption Testing

Abstract

The nasal mucosa, being accessible and highly vascularized, opens up new opportunities for the systemic administration of drugs. However, there are several protective functions like the mucociliary clearance, a physiological barrier which represents is a difficult obstacle for drug candidates to overcome. For this reason, effective testing procedures are required in the preclinical phase of pharmaceutical development. Based on a recently reported immortalized porcine nasal epithelial cell line, we developed a test platform based on a tissue-compatible microfluidic chip. In this study, a biomimetic glass chip, which was equipped with a controlled bidirectional airflow to induce a physiologically relevant wall shear stress on the epithelial cell layer, was microfabricated. By developing a membrane transfer technique, the epithelial cell layer could be pre-cultivated in a static holder prior to cultivation in a microfluidic environment. The dynamic cultivation within the chip showed a homogenous distribution of the mucus film on top of the cell layer and a significant increase in cilia formation compared to the static cultivation condition. In addition, the recording of the ciliary transport mechanism by microparticle image velocimetry was successful. Using FITC-dextran 4000 as an example, it was shown that this nasal mucosa on a chip is suitable for permeation studies. The obtained permeation coefficient was in the range of values determined by means of other established in vitro and in vivo models. This novel nasal mucosa on chip could, in future, be automated and used as a substitute for animal testing.

Keywords: microfluidics; mucociliary clearance; nasal drug administration; nasal mucosa; organ on chip; permeation test.

Figure 1

Fluidic design of the NM-chip. (a) Exploded view showing a basal compartment for continous cell culture medium supply, a porous PET membrane interlayer and an apical compartment to guide the periodic airflow. (b) Top view with geometrical dimensions of the cell cultivation chamber in the center together with inlets and outlets. (c) Side view of the chip design with a detail view of the transition with a step h between membrane surface and apical inlet channel roof. (d) Cross-section of the cell cultivation chamber with upside-down hanging P1 cell layer (g indicates the direction of gravity).

Figure 2

The microfabricated NM-chip. (a) (I) Exploded view illustrating the sandwich construction with eight layers. The basal compartment consists of (from top to bottom): a glass slide with electrodes made of ITO; a contoured layer of DSA; and a glass slide layer with microchannel, through-holes and the cell cultivation chamber in the center. The apical compartment consists of (from bottom to top): a glass slide with ITO counter electrodes, vias and through-holes; a contoured DSA layer; and a glass slide layer with microchannel, through-holes and the cell cultivation chamber in the center. The basal and apical system halves are connected by the contoured silicone layer with through-holes and the contoured PET nanoporous membrane placed in the center. (II) Sketch of the assembled chip. (b) Photograph of the NM-chip system halves with the contoured silicone layer with PET membrane.

Figure 3

Technical drawings of the devices for membrane transfer and chip mounting. (a) Exploded view of the transfer holder, showing the upper and lower part of the transfer tooling with the membrane in between. (b) Chip in transfer holder ready for cell seeding using a pipette. (c) Assembled transfer holder with cell culture media wetting in the apical compartment. (d) Membrane aligned on the apical chip compartment and ready for transfer using the ejection tool. (e) Closed NM-chip within the chip mount ready for dynamic cultivation and permeation experiments.

Figure 4

Schematic view of the experimental setup. The mounting bracket holding the NM-chip was equipped with inlets I1 and I2, outlets O1 and O2, side ports S1 and S2 and contact holes C1-4 to the ITO-microelectrodes. The cell culture medium was pumped by syringe pump SP1 through the chip and collected in the waste reservoir W1 as indicated by the red arrows. The periodic bidirectional airflow indicated by the blue arrows was generated by the opposing syringes in syringe pump SP2, which was microprocessor-controlled. The chip mounting bracket was positioned on top of an inverted fluorescence microscope with a camera. The setup was placed in a self-built incubator (red frame) in which the temperature was stabilized by a panel heater that was controlled by a thermo-controller coupled to a Pt-100 temperature sensor. The TEER value could be read out using EVOM^2® electronics connected to the ports C1-4. The ports S1 and S2 were only opened while drawing fluid samples (yellow arrow) and replenishing media (green arrow) for permeation analysis.

Figure 5

Simulation of the airflow inside the NM-chip at 110 mL/min. (a) Velocity profile at medium channel height with the airflow direction from left to right at a slightly tilted view. (b) Airflow velocity cross-sectional profiles at the inflow (A-1), in the center (A-2) and at the outflow of the chamber (A-3). (c) Airflow distribution of resulting WSS (top view) at the bottom of the cell cultivation chamber. (d) Histogram showing the distribution of the WSS (and indication of underlying Gaussian distributions) in the cell cultivation chamber during inhalation.

Figure 6

Confocal microscopy bright field images of alcian blue staining of mucus, performed at time stages 7 (a,b), 10 (c,d) and 14 (e,f) days of static cultivation at ALI in the transfer holder with membrane, and for each case after subsequent dynamic cultivation with periodic airflow for 3 h in the NM-chip. (g) The area fraction covered with mucus was obtained by image analysis for the same cultivation conditions (n = 10). The center lines in the box plots represent the median, the outer box edges show the 25% and 75% percentiles, and the whiskers show the outliers. The larger variations after 14 days result from small uncovered islands (* indicates p < 0.05). (h) NM-chip apical compartment (after membrane removal) with mucus stained from cell culture after 7 days ALI with NM-chip cultivation.

Figure 7

Immunohistochemical fluorescence staining of P1 cells by antibody staining (red) for γ-tubulin as evidence for ciliated- cells combined with cell nuclei staining (blue) with Hoechst 33342. Fluorescence images of P1 cells after (a) 7, (c) 10 and (e) 14 days in static ALI cultivation in the transfer holder and (b,d,f) with additional short dynamic cultivation of 3 h in the NM-chip in each case. (g) Area fractions with cilia (red) and cell nuclei (blue), which were determined via image processing for all cases considered (n = 1).

Figure 8

Vector fields (arrows indicate direction and magnitude of local velocity) together with color-coded velocity magnitude obtained by PIV analysis from the time lapse recordings of an ANaMuc model cell layer within the NM-chip at (a) 7 days, (b) 10 days and (c) 14 days of static cultivation, followed by 3 h of dynamic cultivation on-chip in each case.