Tc insert

Undifferentiated human bone-marrow-derived mesenchymal stem cells (hBMSC, ATCC-PCS-500-012) were cultured in mesenchymal stem cell basal medium (BM, ATCC PCS500030) supplemented with 7% FBS, 100 IU/mL penicillin/streptomycin, 2.4 mM, 125 pg/mL FGF-b, and 15 ng/mL IGF-1 (ATCC, Milan, Italy), at a density of 5 × 10³ cells/cm² and incubated for 24 h at 37 °C under 5% CO₂. To induce osteogenic differentiation, hBMSC were cultured in osteogenic medium (OM), (DMEM with 10% FBS, 100 IU/mL penicillin/streptomycin, 2 mM L-glutamine (Corning, Manassas, VA, USA), 10 mM β-glycerophosphate (BGP), 100 nM dexamethasone, 100 μM ascorbic acid 2-phosphate (AA) (Sigma Chemical Co., Milan, Italy), or cultured in BM with the presence of implant plus CGF supplemented with 7% FBS, 100 IU/mL penicillin/streptomycin, 10 mM BGP, and 100 μM AA (Device) for 21 days. The medium was replaced at a rate of 50% every 3 days. In all experiments, the implant coated with CGF was placed into a sterile transwell insert (TC-inserts, Sarstedt, Nümbrecht Germany) with a semipermeable membrane at the bottom (pores of 0.4 μm) and inserted into the 12-well culture plates (an insert in each well).

ALIs were
grown on 24-well Tissue Culture (TC) Inserts (Sarstedt, 83.3932.041)
with a pore size of 0.4 μm and a pore density of 2 × 10⁶ pores/cm² (Figure S1). The TC inserts were precoated with matrix proteins from 804G conditioned
medium, and the apical compartment of each was seeded with 100,000
basal cells in 300 μL of basal cell expansion medium (Table S1). Basal cell medium (800 μL) was
also added to the basolateral compartments of the wells. The cells
were incubated at 37 °C and 5% CO₂ for 24–48
h until a confluent layer had formed. The culture medium was then
removed from the apical and basolateral compartments, and 500 μL
of ALI medium (Table S2) was added to the
basolateral compartment of the wells while the apical compartment
was left empty. This step is referred to as the air-lift. The cells
were incubated for a further 21 days (medium refreshed every 48–72
h) following the air-lift to achieve differentiation into a pseudostratified
epithelium.

hAEC cultures were prepared on Transwell inserts (Corning, Glendale, AZ) as described above. Before the transcytosis assay, the functionality of the tight junctions was tested by measuring the transepithelial electrical resistance (TEER). MDCK II cells were seeded on TC inserts (Sarstedt) with a porous membrane (0.4 μm pore diameter, 2x10⁶ pores/cm², and 12 μm thickness). The inserts were placed in a 24-well plate with medium and incubated at 37°C with 5% CO₂. The functionality of the tight junctions was assessed at increasing days post-seeding with B19V (3x10⁹) at pH 7.4, where no interaction with globoside is possible, and with bacteriophage PP7 (3x10¹⁰), which does not interact with eukaryotic cells. PP7 RNA was quantified by RT-PCR as previously described [14 (link)].
Cells were washed once with PBS and layered with 200 μl of apical buffer consisting of PiBS pH 6.1 (20 mM piperazine-N,N′-bis[2-ethanesulfonic acid], 121 mM NaCl, 2.5 mM KCl) or MEM. The inserts were placed in a 24-well plate with 600 μl MEM. To quantify viruses in the apical and basolateral compartments, 50 μl medium was harvested and used for DNA extraction and quantification by qPCR, as described above. Results were normalized for the differences in volume between apical and basolateral compartments.

All drugs were purchased from Sigma. Pitstop 2 and genistein were dissolved in DMSO, whereas chlorpromazine (CPZ), methyl-β-cyclodextrin (mβCDX), and dynasore were dissolved in water. Briefly, MDCK II cells were seeded in TC inserts (Sarstedt). The cells were pre-treated with the inhibitors at pH 7.4 and incubated at 37°C for 1 h. Next, the cells were incubated with the drugs and B19V (3x10⁹) in PiBS (pH 6.1) at 37°C to allow virus attachment and uptake. After 3h, the cells were washed with PBS (pH 7.4) to remove attached but not internalized viruses. B19V was detected in cells by IF with an antibody against capsids and quantified in the basolateral compartment by qPCR, as described above.