Fibronectin coated

Prx1CreER-GFP and Prx1CreER-GFP;Ift88^fl/fl juveniles were sacrificed between 3 and 4 weeks of age and their fore- and hindlimbs were dissected. The skin, fascia, connective tissue, and majority of the muscle surrounding the periosteum was removed using a scalpel and the specimens were placed in sterile phosphate-buffered saline (PBS; Life Technologies, Carlsbad, CA) on ice. The epiphyses and remaining muscle surrounding the periosteum were removed, and the specimens were transferred to fresh cold PBS. In a sterile culture hood, the periosteum was scored with a scalpel, peeled off the bone, cut into 1mm² sections, and placed into fibronectin-coated (Sigma-Aldrich) tissue culture dishes containing minimum essential medium (MEM)α (Life Technologies) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S; Life Technologies). Tissue sections were incubated at 37 °C for 7–10 days and the resulting primary periosteal cells were passaged onto fresh fibronectin-coated tissue culture dishes. Cells were then passaged or sorted upon reaching 80% confluence.

This study was approved by the Institutional Review Board of the Seoul National University Hospital (IRB Number). Human peripheral blood samples (10 cc) were obtained from donors after informed consent. PBMCs were isolated from the blood samples using Ficoll-Paque PLUS (GE Healthcare, NJ, USA) according to the manufacturer's recommendations. Freshly isolated PBMCs were suspended in EGM-2MV BulletKit™ (Lonza, Basel, Switzerland) and seeded on the fibronectin-coated (Sigma-Aldrich, MO, USA) six-well plate at 6 × 10⁶ cells per well. The media were changed every single day for up to 2 weeks after plating. Adherent CiMS cells were observed from as early as five days after the start of culture and gradually formed colonies. CiMS were passaged using 0.05% Trypsin-EDTA solution.

Murine HL-1 cardiomyocytes (HL-1), immortalized using the simian virus SV40 T-antigen under the control of an atrial natriuretic factor (ANF) promotor [46 (link)], were generously provided by William C. Claycomb (Louisiana State University, New Orleans, LA, USA) and used at passages 18–44. Cells were cultured in Claycomb medium with 10% HL-1 Cell Screened FBS (Merck), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM L-Glutamine (Gibco, Waltham, MA, USA) and 100 µM norepinephrine, and cultured on 0.02% gelatin/5 µg/mL fibronectin-coated (Sigma-Aldrich) cell culture flasks at 37 °C and 5% CO₂.

Six NPC-derived cell lines (HONE-1 [28 (link)], SUNE-1 [29 (link)], HK1 [30 (link)], TW01 [31 (link)], TW04 [31 (link)], and C666-1 [32 (link)]) and an immortalized nonmalignant nasopharyngeal epithelial cell line (NP69 [33 (link)]) were used in this study. Many of these were originally sourced from the University of Hong Kong (laboratory of Professor George S. W. Tsao) with permission for use granted by the provider. The NPC cell lines were cultured in RPMI-1640 (Gibco, Life Technologies, USA) containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine, and 100 U/mL penicillin-streptomycin (Gibco, USA). The EBV-positive cell line, C666-1, was cultured on fibronectin-coated (Sigma, USA) cell culture flask containing prewarmed (37°C) RPMI-1640 medium. The NP69 cells were cultured in defined keratinocyte-serum-free medium (Invitrogen, USA) supplemented with 0.2 ng/mL growth factors, 5% heat-inactivated FBS, and 100 U/mL penicillin-streptomycin. All cells were maintained at 37°C in a humidified environment containing 5% CO₂. Cells were harvested at a growth confluency of 70–80%.

For the coculture of LECs with melanoma cells, cells were seeded on fibronectin-coated (Sigma-Aldrich) cell culture dishes in a 1:2 or 3:4 melanoma cell/LEC ratio in EGM-2 media. In the first experiments, LEC and melanoma cells were separated using magnetic nanoparticles. For this separation process, melanoma cells were first loaded with dextran-coated nanoparticles at 1 mg/mL (fluid-MAG-DX, Chemicell) for 24 hours. Dextran-loaded melanoma cells were cultured with LECs for 48 hours, after which the cells were sorted using a MidiMacs separator and LS columns (Miltenyi Biotec). Due to the cessation of the production of these magnetic nanoparticles, we had to change the separation to be carried out by FACS (Sony, SH800Z). The sorted cells were used for the subsequent functional or molecular assays.

Human ECFCs were cultured using a protocol approved by the First Affiliated Hospital of Nanjing Medical University. Human peripheral blood‐derived ECFCs were isolated and cultured as described previously 18, 19. Briefly, human peripheral blood (20 ml) obtained from healthy adult donors with informed consent was fractionated via centrifugation in a Ficoll‐Paque (Sigma‐Aldrich) gradient. Isolated mononuclear cells were re‐suspended in endothelial basal medium‐2 (EBM‐2) (Lonza, Walkersville, MD, USA), consisting of 10% foetal bovine serum (FBS), human epidermal growth factor (hEGF), human VEGF1, human fibroblast growth factor (hFGF), insulin‐like growth factor 1 (IGF‐1), ascorbic acid and heparin. Cells were diluted to a final concentration of 7.5 × 10⁵ cells per ml and seeded into 2% fibronectin‐coated (Sigma‐Aldrich) six‐well plates with approximately 2 × 10⁶ cells per well. Medium was replaced after the first 24 hrs. Under daily observation, colonies of ECFCs were obtained after 14–28 days on average. Cells were used in their fourth to sixth passages.
Human pulmonary artery endothelial cells (HPAECs) were purchased from Lonza and cultured in EBM‐2 (Lonza) supplemented with 5% FBS, hEGF, human VEGF, hFGF, IGF‐1, ascorbic acid and heparin. Both ECFCs and HPAECs were cultured in an incubator at 37°C in 5% CO₂ saturated with H₂O.