Development of a miniaturized 96-Transwell air–liquid interface human small airway epithelial model

In order to overcome the challenges associated with a limited number of airway epithelial cells that can be obtained from clinical sampling and their restrained capacity to divide ex vivo, miniaturization of respiratory drug discovery assays is of pivotal importance. Thus, a 96-well microplate system was developed where primary human small airway epithelial (hSAE) cells were cultured at an air–liquid interface (ALI). After four weeks of ALI culture, a pseudostratified epithelium containing basal, club, goblet and ciliated cells was produced. The 96-well ALI cultures displayed a cellular composition, ciliary beating frequency, and intercellular tight junctions similar to 24-well conditions. A novel custom-made device for 96-parallelized transepithelial electric resistance (TEER) measurements, together with dextran permeability measurements, confirmed that the 96-well culture developed a tight barrier function during ALI differentiation. 96-well hSAE cultures were responsive to transforming growth factor β1 (TGF-β1) and tumor necrosis factor α (TNF-α) in a concentration dependent manner. Thus, the miniaturized cellular model system enables the recapitulation of a physiologically responsive, differentiated small airway epithelium, and a robotic integration provides a medium throughput approach towards pharmaceutical drug discovery, for instance, in respect of fibrotic distal airway/lung diseases.


Details of the customized constructed transepithelial electrical resistance (TEER)
device TEER values have proven to be simple and rapid indicators of cellular barrier integrity and can be collected in real time without cell damage 1 . Therefore, this non-invasive technique has been miniaturized to suit well for the continuous monitoring of the barrier function of hSAECs during their various stages of growth and differentiation in 96 Transwell plates.
In general, TEER is the measurement of electrical resistance across any cellular layer and is a very sensitive and reliable method. The electrode components in the here implemented novel TEER device are constructed based on literature 1 and in following the structure of the classical STX2 "chopstick" electrodes.
Primarily, the collection of TEER values works based on the Ohm's Law Method. For electrical measurements, two individual electrode pairs are used, with 2 electrodes place in the upper compartment and the other two in the lower compartment, separated by the cellular layer. One pair is made out of silver for applying the alternating current voltage throughout the cell layer. An alternating current square wave at a frequency of 12.5 Hz is applied. An additional silver electrode pair for the measurement of the resulting potential differences is placed into the individual two compartments respectively. Subsequently the ohmic resistance is calculated based on Ohm's law as the ration of the voltage and current. The novel TEER device has a measurement range of 1-10000 Ω with a resolution of 1 Ω.
The calculation of the actual TEER values include measuring the blank resistance of the semipermeable membrane only (without cells), just medium/ buffer (was determined at the setup of the device). Determination of the resistance across the cell layer in 96 Transwell plates is performed simultaneously and three times in a row. The cell-specific resistance is subsequently calculated as the actual tissue resistance subtracted by the blank resistance. The average out of the three independent measurements per Transwell are displayed in the implemented data acquisition tool.
Typically TEER values are reported in units of Ω x cm 2 to incorporate the growth area.
In general, TEER readings are highly dependent on the positioning of the electrodes, temperature and the current density generated by the electrodes across the cell layer.
The here introduced self-constructed TEER device circumvents these difficulties due to the speed of each measurement and the reproducible placement of the electrodes within the Transwells. Thus, this novel technique offers the possibility to enable TEER measurements on an increased throughput.

Details of the cell isolation and characterization process of primary human small airway epithelial cells
Human small airway epithelial cells were isolated through an enzymatic digestion protocol from normal cadaveric lung tissue samples located at the lower end of either left or right lung (in the 1mm bronchiole area). Isolated cells were expanded in standard submerged culture, passaged once and subsequently cryopreserved at 500,000 cells per vial. All lots are tested for cytokeratin 19, desmoplactin and occludin in early and later stages of the differentiation process. The within this studies used donor (CC-2547S, Batch No: 0000501937) was also further characterized by mucin expression starting at day 20. TEER was detectable by day 7-10 and beta-Tubulin (Cilia) was determined by day 20. Whereas ZO-1 staining was already measurable on day 9.