We have established a protocol for the isolation, maintenance, and differentiation of human stria vascularis from human post-mortem tissues obtained from the Pathology Institute in Basel, Switzerland (Ethical permit: EKNZ 2020-01379). Autopsy-derived post-mortem human temporal bones were used as a tissue source due to the paucity of surgical procedures in which healthy stria vascularis tissue could be obtained. Donors were from both genders and ranged in age from 50 to 75 years, five male and one female. Exclusion criteria were prior hearing disorders or loss (with the exception of age-related hearing loss), recent chemotherapy, and any diseases that could affect hearing. The samples were collected an average 11 h post-mortem, but up to 18 h post-mortem. During autopsy, the skulls were opened as usual by removing the skull cap. After removal of the brain, the dura mater at the base of the skull was pulled off. The petrous part of the temporal bone was removed from the base of the skull en bloc using an oscillating saw, so that the middle and inner ear were not opened during removal. The excised bone fragment was immersed in 70% (v/v) ethanol for 30 s and subsequently placed in sterile PBS containing penicillin/streptomycin (100 U/mL) and stored at 4°C until further processing. The temporal bone specimens were stabilized in a temporal bone holder (Storz & Co., Tuttlingen, Germany). An open tympanoplasty approach was performed. After a subtotal mastoidectomy, the superior and posterior canal wall, tympanic membrane, malleus, and incus were removed. The protympanon was enlarged to the petrous segment of the internal carotid artery. Parts of the tympanic segment of the facial nerve and tendon of the m. tensor tympani were dissected to access the full circumference of the cochlea. The promontory bone was blue-lined with a diamond drill until the cochlear turns were visible. The lateral bone of the turns was completely drilled away and the membranous labyrinth of the cochlea carefully extracted.

Tissue was trypsinized with Trypsin–EDTA 0.25% (Sigma cat#T4049) for 5 min, followed by blocking with 10% human serum solution (Sigma cat#H3667) and centrifugation at 1500 rpm for 5 min. Tissue pieces were distributed in wells coated with 0.03 mg/mL fibronectin in DPBS (ScienCell cat#8248) or 0.01 mg/mL poly-L-lysine in sterile water (ScienCell cat#0403) to seed ECs and PCs, respectively, and then rinsed with sterile water and DPBS.

The cells were seeded and incubated at 37°C in 5% CO₂. The growth medium for ECs consisted of 500 mL EC basal medium, 25 mL human serum (Sigma cat#H3667), 5 mL EC growth supplement (ScienCell cat#1052), and 5 mL penicillin/streptomycin solution (ScienCell cat# 0503). The PC growth medium consisted of 500 mL PC basal medium, 10 mL human serum (Sigma cat#H3667), 5 mL PC growth supplement (ScienCell cat#1252), and 5 mL penicillin/streptomycin solution (ScienCell cat#0503). Before the use in experiments cells are cultured and expanded in T25 or T75 flasks coated with appropriate attachment factor. The cells used for the experiments are at the passage 2 or 3, and after expansion in the flask for the seeding on the transwell they are trypsinized, and in the end re-suspended in the appropriate cell medium.

The medium was changed every 2 days. Before placing the cells on the transwell, the cell type was validated at the gene and protein level using immunostaining and gene expression of marker proteins as described below. Treatments were performed with 50 ng/mL TNF⍺, 20 ng/mL IL-6, and 100 ng/mL LPS for 72 h, and controls were treated with vehicle only.

RNA was isolated from collected cells and extracted using the Direct-Zol RNA MiniPrep kit (Zymo Research, Germany, cat#R2050) according to the manufacturer’s instructions. Total RNA (1,000 ng) was reverse-transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, United States). We analyzed triplicate samples by quantitative PCR on an ABI Prism 7900HT Sequence Detection System (Applied Biosystems, United States) using the Power Sybr Green Master Mix (Applied Biosystems, United States). Primers targeting GAPDH were synthesized by Microsynth (St. Gallen, Switzerland) and added at a final concentration of 250 nM per reaction. The full primer sequences used in this study were (5`-3`): TJP1 (ZO1): Fw – CAA CAT ACA GTG ACG CTT CAC A, Rev. – CAC TAT TGA CGT TTC CCC ACT C; F11R (JAM1): Fw – ATG GGG ACA AAG GCG CAA G, Rev. – CAA TGC CAG GGA GCA CAA CA; OCLN (OCL): Fw – ACA AGC GGT TTT ATC CAG AGT C, Rev. – GTC ATC CAC AGG CGA AGT TAA T; CDH5 (CADH5): Fw – TTG GAA CCA GAT GCA CAT TGA T, Rev. – TCT TGC GAC TCA CGC TTG AC; CLDN5 : Fw – CTC TGC TGG TTC GCC AAC AT, Rev. – CAG CTC GTA CTT CTG CGA CA; GAPDH : Fw – GGA GCG AGA TCC CTC CAA AAT, Rev. – GGC TGT TGT CAT ACT TCT CAT GG. The relative quantities of specifically amplified cDNAs were calculated by the comparative threshold cycle method (2–∆∆Ct), and GAPDH expression was used as the endogenous reference.

Transepithelial electrical resistance (TEER) was measured with a Voltohmmeter EVOM3. After counting the cells using Trypan Blue in the automated cell counter (Bio-Rad, TC20) the cell suspension is placed on each side of the coated transwell membrane in two steps. First, the inserts are flipped upside down and 150 μL pericyte cell suspension is placed on the abluminal side. They are then placed back in the incubator for 3 h to allow cells to settle and attach. After 3 h the inserts are flipped back in their original position and placed in the PC medium containing 24 well slot, then EC cell suspension is added in the luminal side of the insert and cells are allowed 3 h to attach in the small volume of 150 μL and then the rest of the EC medium is added.

For TEER measurements, ECs were seeded at a density of 2 × 10⁵/cm² and grown on membrane inserts (Corning, cat#3470) coated with fibronectin. The PCs were combined with ECs (harvested after 2 and 3 passages and seeded at a density of 1 × 10⁵/cm²) and grown on the poly-l-lysin-coated inserts. The untreated Transwells containing vehicle were used as a control and prepared the same way as the ones treated with TNF-α, IL-6, and LPS. Measurements were obtained following the manufacturer’s protocol. Briefly, electrodes were maintained by soaking the tips once a week in a 1% Tergazyme solution for 15 min and rinsing with sterile water, and the same was done just before disinfection and before beginning an experiment. The STX4 electrode were disinfected in 70% ethanol for no more than 5 min, followed by rinsing with medium or PBS. This was followed by measuring the resistance in treated samples and controls. Cells were allowed to attach and rest for 24 h before the first measurement was done and then measurements were performed once per day and three measurements per well. First, electrodes were placed in the culture medium for a few minutes. To measure the blank resistance, the electrode was placed in a transwell insert without cells filled with cell medium. Further the measurements in experimental wells with cells were performed.

After finishing the measurement, electrodes were disinfected with ethanol, rinsed with sterile water, and allowed to air dry. To calculate TEER, the surface area of the Transwell (in cm²) was multiplied by the net resistance (the resistance of a blank Transwell covered by cell culture media subtracted from the measured resistance).

To test the permeability, 2 μg daptomycin (Sigma, cat#SBR00014) was added to the EC medium for the top chamber, whereas the basolateral bottom chamber contained only PC medium. The untreated Transwells containing vehicle were used as a control and prepared the same way as the TNF-α and LPS-treated Transwells. A 20 μL aliquot was taken from the apical and basolateral chamber reservoirs at defined time points for both the BLB Transwell with cells and control sample: 0, 30, 60, 90, 180, and 440 min. The samples were then evaluated by HPLC to determine daptomycin concentrations at those time points.

For syndecan1 staining endothelial cells were grown on 4-well fibronectin coated glass-bottom dishes (Ibidi, Germany, cat#80426). For EC and PC marker staining cells were co-cultured on each side of the Transwell insert (Corning, cat#CLS3470), EC on the luminal fibronectin coated side and PC on the abluminal poly-l-lysin-coated side. Cells were fixed in 4% paraformaldehyde (Sigma, United States, cat#158127) in PBS (Sigma, United States, cat#P4417), permeabilized with 0.1% Triton X-100 (Sigma, United States, cat#X100) in PBS, and incubated for 1 h at room temperature with anti-von Willebrand factor (vWf, Sigma, United States, cat#F3520), anti-PDGFR-β (Santa Cruz Biotechnology, United States, cat#sc374573), anti-CD138-syndecan1 (Invitrogen, cat#MA53-2600) and ZO-1 (Invitrogen #33-9,100) primary antibodies. Next, samples were washed and incubated with appropriate secondary antibodies (Thermo Fisher, United States, cat#A2124, cat#A-11001, cat#A-11008) in PBS-T for 1 h at room temperature. Samples were washed with PBS and incubated with DAPI for 5 min. The cells were washed with PBS and mounted on microscope slides using Dako Fluorescent Mounting Medium (Dako, Denmark, cat#S3023). Images were captured by a Nikon Eclipse Ti2 inverted widefield microscope. Images were processed and analyzed using Fiji-win 32 software.

ECs were homogenized in cell lysis buffer (Sigma-Aldrich, St. Louis, MO, United States, cat#C3228) with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, United States, cat#C3228, cat#P8340) for 1 min on ice. Protein concentrations were determined by the Bradford method using Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Switzerland, cat#5000006). Protein concentrations were confirmed using a NanoDrop (Thermo Fisher Scientific, Waltham, MA, United States). Samples were mixed with Laemmli sample buffer (Sigma-Aldrich, St. Louis, MO, United States, cat#S3401), heated at 95°C for 5 min, and then resolved on 4–20% Mini-PROTEAN^® TGX^™ precast protein gels (Bio-Rad, Switzerland, cat#4561096). After electrophoresis, separated proteins were transferred onto polyvinylidene fluoride membranes. Membranes were incubated with 5% non-fat dry milk dissolved in PBS-T for 1 h at room temperature to block non-specific protein binding site. Next, the membranes were washed with PBS-T (3 × 10 min) before being incubated overnight at 4°C in 5% non-fat dry milk in PBS with the primary antibody anti-CD138 (syndecan1) (Invitrogen, cat#MA53-2600). The membranes were then washed with PBS-T (3 × 10 min) and incubated for 1 h at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody. After washing, immunoreactive protein bands were visualized using Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Waltham, MA, United States, cat#34076). An anti-B-ACTIN antibody was used as a control to demonstrate equivalent protein loading. The intensity of the immuno-positive bands was determined using Fiji-win 32 software, capturing the identical regions on each blot and deducting the background signal. The intensity of all proteins of interest was normalized to the intensity of B-ACTIN in the same sample.

Statistical analyses were performed in GraphPad Prism software (San Diego, CA, United States). Multiple groups were compared by two-way analysis of variance, and two groups were compared by an unpaired Student’s t-test. Data were confirmed to be normally distributed by a Shapiro–Wilk test. p-values <0.05 were considered significant.

While culturing the ECs and PCs on each side of the Transwell membrane (Figures 1B,C), daily TEER measurements were performed until the values reached a stable threshold. Upon reaching stable values, TNF-α, IL-6, and LPS were added. TNF-α had the greatest impact on barrier integrity, which was reflected in a significant drop in TEER values in treated Transwells from the first 12 h after the treatment begin, which consistently continued throughout the treatment period (Figure 2A). IL-6 treatment had similar trend as TNF-α, but with less pronounced, though still significant, differences between the treatment and control (Figure 2B). LPS treatment had a modest impact on barrier integrity, showing significant differences between the control and treatment only on the last treatment day (Figure 2C).

As junctional proteins are one of the most important factors in maintaining endothelial blood vessel barrier integrity, following our initial integrity measurements, we exposed the endothelial layer to 50 ng/mL TNF⍺ and 100 ng/mL LPS for 72 h and measured the gene expression levels of some of the major junctional genes. The results showed a significant decrease in ZO1, OCL, and CLDN expression levels in samples exposed to TNF-α (Figure 3A). In LPS-treated samples, a slight decrease in JAM1 and slight increase in CLDN expression was observed (Figure 3B). Upon ZO-1 immunostaining, EC cells exposed to TNF⍺ revealed a lower signal compared to the control (Figure 3C).

With previous results indicating barrier changes after exposure to TNF-α and, to a slight extent, LPS, we performed permeability assays using daptomycin (molecular weight 1.6 kDa), which normally does not pass the healthy blood vessel barrier. We added 2000 ng of daptomycin into the top chamber with ECs pre-treated with TNF-α or LPS and measured the concentrations of daptomycin in both chambers at 0, 30, 60, 90, 180, and 440 min and compared them to the control samples. At the initial time points, there was no difference between the control and TNF-α-treated Transwells, and the first significant difference was with daptomycin passing from the top to the bottom chamber at 440 min (Figure 4A). In the LPS pre-treated samples, there was no difference in daptomycin permeability over the measured time (Figure 4B).

Considering previous results and a major impact of TNF-α on endothelial integrity, we explored its effect on syndecan1 expression in our culture conditions. Syndecan1 is expressed in ECs and is one of the main markers of endothelial glycocalyx degradation. After exposure to TNF-α, immunostaining revealed a significant decrease in syndecan1 compared with control samples (Figures 5A,B). The immunostaining results were confirmed by decreased syndecan1 protein levels in TNF-α-treated samples.