Endothelial cell growth medium 2

Primary jawbone-derived human osteoblasts (JHOBs) were isolated following slightly modified protocols^{36 (link),55 (link)}. Briefly, small bone pieces (approximately 8 mm³) of human mandibular jawbone were obtained from healthy donors during surgery (as approved by Charité’s Ethics Committee, EA1/038/19; informed consent of all participating subjects was obtained and all methods were performed in accordance with the relevant guidelines and regulations) and stored in growth medium [high-glucose DMEM supplemented with 10% fetal calf serum, 100 IE mL⁻¹ penicillin, 100 µg mL⁻¹ streptomycin and 2 mM GlutaMAX (Gibco, Thermo Fisher Scientific, Waltham, USA)] at 4 °C until isolation. Jawbone pieces were rinsed thoroughly with PBS and soft tissue was removed using a scalpel. To reduce the germ load, samples were incubated in Betaisodona iodide solution (Mundipharma, Frankfurt am Main, Germany) for 60 s followed by repeated rinsing with PBS. Bone pieces were put in a 60 mm tissue culture-treated petri dish covered with growth medium. Cells were expanded upon confluency around the bone pieces and used at passage 5. Human umbilical vein endothelial cells (HUVECs) were cultured in Endothelial Cell Growth Medium 2 (PromoCell, Heidelberg, Germany) and used at passage 3. All consumables were obtained from Corning Inc. (Corning, USA) unless stated otherwise.

HUVECs were purchased from Lonza (from pooled donors) and cultured in Endothelial Cell Growth Medium 2 (EGM2, PromoCell). Cells were used between P3 and P6 for experiments. Human pericytes (from placenta) were purchased from PromoCell and cultured in Pericytes Growth Medium 2 (PGM2, PromoCell). Cells were used between P3 and P6 for experiments. Human cardiac endothelial cells and fibroblasts were purchased from PromoCell and cultured in Endothelial Cell Growth Medium MV 2 and Fibroblast Growth Medium 3 respectively (PromoCell). Endothelial cells were cultured on 0.1% gelatin treated T75 flasks. We note that although placental pericytes and HUVECs are not normally found in the cardiac microvasculature, they constitute a reliable source of relevant cells allowing the reproducible assembly of complex in vitro models.

iSLK BAC16 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/high glucose (Welgene, Gyeongsan, South Korea) with 10% fetal bovine serum (FBS; GenDEPOT, Katy, TX, United States) and 1% antibiotic-antimycotic (Invitrogen, Waltham, MA, United States). Hygromycin B (1.2 mg/mL; Invitrogen), geneticin (250 μg/mL; Invitrogen), and puromycin (1 μg/mL; Invitrogen) were added and cultured to maintain the latent infection of iSLK BAC16. Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany) and cultured in endothelial cell growth medium 2 (PromoCell) containing cell growth supplements. BCBL-1 cells were cultured in RPMI 1640 (Biowest, Riverside, MO, United States) supplemented with 10% FBS. All cells were cultured at 37°C in a humidified atmosphere containing 95% air and 5% CO₂. SP600125 was purchased from Calbiochem (San Diego, CA, United States). DMSO was purchased from LPS Solution (Daejeon, South Korea).

Two pools of HUVEC (from three donors, Promocell) and HSaVEC from two single donors, Promocell), between passage three and eight, were cultured in Endothelial Cell Growth Medium 2 (Promocell), supplemented with 100 μg/ml streptomycin and 100 U/ml penicillin in a humidified atmosphere with 5 % CO₂ at 37 °C. The clinical isolate RVi/Wuerzburg.DEU/47.11 (Wb-12, genotype 2B) was provided by Dr. Benedikt Weissbrich at the University of Wuerzburg, propagated on Vero76 cells (ATCC) and titered by the immunocolorimetric plaque assay [39 (link)].
For growth curve analysis, cells were seeded in 48-well culture plates at a density of 5°10⁴ cells per well and infected with Wb-12 at an MOI of 5 the following day. After adsorption for 2 h at 35 °C, the viral inoculum was removed, the cells were washed three times with 1x PBS and overlaid with 500 μl fresh medium. Supernatants and cells were collected at the indicated time points. The amount of intracellular virus was determined by dissolving the cell pellet in 500 μl fresh medium followed by three subsequent freeze-thaw cycles and centrifugation for 5 min at 400∙g to remove debris. Virus titer of the supernatant was determined by immunocolorimetric plaque assay on Vero76 cells in duplicate.

Cells were grown on 1% gelatin‐coated tissue culture (TC)‐treated dishes (BD Falcon) at 37°C and 5% CO₂ in Endothelial Cell Growth Medium 2 (PromoCell) (for HUVEC and HBMEC) or Endothelial Cell Growth Medium MV2 (PromoCell) (for MS1 and HCMEC) supplemented with the respective SupplementMix (containing growth factors and 2 or 5% FBS, respectively) and 10 Units/ml penicillin/streptomycin.