Egm 2 kit

Human RPTEC (Renal Primary Tubule Epithelial Cell) (LONZA, CC-2553) was cultured through an REGM BulletKit (LONZA, SWISS, Basal) and used within passages 5–7. HUVEC (Human Umbilical Vein Endothelial Cell) (LONZA, C2519A) were cultured through EGM-2 KIT (LONZA, SWISS, Basal) and used within passage numbers 4–7. All cells were cultured from a 75-Tflask to approximately 90% of the total area, which was reproduced at a concentration of 3 × 10⁶ cells/mL, and seeded into membranes. The badges were replaced once every 1–2 days.

Human embryonic kidney (HEK) 293 T cells (ThermoFisher Scientific, Rockford, IL) were cultured in Dulbecco's Modified Eagle's Medium (Gibco; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS). Primary human retinal microvascular endothelial cells (hREC) were purchased from Cell Systems (Kirkland, WA) and cultured in an endothelial growth medium (EGM)-2 kit (Lonza, Walkersvile, MD) and 2% FBS (Atlanta Biologicals, Flowery Branch, GA). All cells were maintained at 37 °C in a humidified incubator with 5% CO₂.

Primary human umbilical vein endothelial cells (HUVEC) (Lonza, cod. C2517A) were cultured in EBM-2 (Lonza) medium supplemented with EGM-2 kit (Lonza). Colon cancer HCT-116 (ATCC, cod. CCL-247) cells were cultured in McCoy 5A medium (Sigma-Aldrich) supplemented with 10% (v/v) fetal bovine serum (FBS) (Lonza), 2-mM L-glutamine (Sigma-Aldrich), 1% (v/v) penicillin/streptomycin solution (Sigma-Aldrich). Cell cultures were maintained in an incubator at 37 °C and in humidified atmosphere with 5% CO₂.

Conditionally immortalized murine LECs (imLECs) [27 (link)], which express a heat-labile version of the large T antigen, were cultured at 33 °C in medium supplemented with 40% DMEM (low glucose), 40% F12-Ham, 20% FBS (all from Gibco, Waltham, MA, USA), 56 μg/mL heparin (Sigma-Aldrich), 10 μg/mL endothelial cell growth supplement (Sigma-Aldrich), 1% antibiotic antimycotic solution (Fluka, Buchs, Switzerland) and 2 nM L-glutamine (Fluka), as previously described [27 (link),29 (link)]. Notably, cell culture dishes precoated with 10 μg/mL collagen (Purecol, Advanced Biomatrix) and 10 μg/mL fibronectin (Millipore) were used. Murine IFNγ (1 U/mL, Peprotech, London, UK) was added to the cultured cells to induce large T-antigen expression [37 (link)]. After reaching confluency, the medium was exchanged without adding IFNγ, and the cell culture dish was transferred to 37 °C. Forty-eight hours later, imLECs were washed with PBS and detached with Accutase (Sigma-Aldrich) for flow cytometry analysis.
Human primary dermal LECs (PromoCell-Lot. 439Z007.2, p4-p6) were cultured on collagen-coated cell culture dishes (10µg/mL, Purecol, Advanced BioMatrix) in EBM medium (Lonza, Basel, Switzerland) containing supplements and growth factors provided in the EGM-2 kit (Cat#: CC-4176, Lonza). The cells were incubated at 37 °C until reaching the confluency.

Human umbilical vein endothelial cells (HUVECs, Lonza, C2519A) were cultured in EGM-2 medium. Cells were maintained at 37 ℃ with 5% CO₂. During OCT4-30Kc19 and BMP4 growth factor treatment, cells were maintained in EBM-2 with 1% FBS without growth factor supplements from EGM-2 kit (Lonza). Osteogenic differentiation was carried out in StemPro™ Osteogenesis medium (Thermo Fisher) and the medium was changed every two to three days.