Hanks balanced salt solution (hbss)

Primary DFSCs were derived from donors. The dental follicle cell was isolated and enzymatically dissociated in Hank's balanced saline solution buffer (HBSS) (WISENT, Canada) containing 1 mg/ml trypsin (WISENT) at 37°C for 10 min followed by 5 min of centrifugation at 350g upon trypsin inhibition (Invitrogen). The isolated cells were washed with HBSS and resuspended in DMEM/F12 medium (WISENT). DFSC formed over 7 days of incubation with 5% CO2 at 37°C. Subculturing was done every 3-4 days. Characterisation of DFSCs was performed using Human MSC Analysis Kit (BD) by flow cytometry (Unpublished data were provided by H.W.). Experiments were performed with cultured cells between passages 2 and 12 except those otherwise indicated.

Spleens were excised, collected in Hank’s balanced salt solution without calcium and magnesium (HBSS) (Wisent, St. Bruno, QC, Canada), and processed individually. Homogenous cell suspensions were prepared by passing organs through a 70 μm cell strainer (BD Biosciences, Mississauga, ON, Canada). Cells were treated with ACK buffer (0.15M NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA; pH 7.2), and then washed with HBSS. Splenocytes were resuspended in RPMI supplemented with 10% fetal bovine serum (FBS), 1 mM penicillin/streptomycin (all from Wisent), and 0.5 mM β-mercaptoethanol (Sigma) (complete RPMI, cRPMI).

To evaluate whether CAR-T/T cells had an effect on the permeability of the iBEC monolayer, NaFl permeability (Pe) (luminal to abluminal) was performed after 24 h. Briefly, the iBEC transwell inserts were washed with 1 ml 1 × Hank’s buffered saline solution (HBSS) (Wisent) to remove residual T cells and medium. The inserts were then placed into plates with 1 ml of transport buffer (5 mM MgCl₂ and 10 mM HEPES in HBSS, pH 7.4) and incubated at 37 °C for 10 min and then 250 µl of the transport buffer was removed from the luminal chamber of each insert and replaced with 250 µl of NaFl (50 µg/ml) in transport buffer. The plates were then incubated at 37 °C with gentle rotation (20 rpm/min) and 100 µl of transport buffer was collected from the bottom of the wells at 15, 30, 45 and 60 min intervals for permeability analysis; 100 µl transport buffer were added back to the wells and the plates were returned to the incubator. Inserts without iBEC were used for the background controls. The quantitation of NaFl was performed using a fluorescent plate reader (ex., 485 nm and em., 530 nm) and plotted against a standard curve (0–50 ng NaFl solution in transport buffer), as previous described [35 ].