Cell shield

According to standard protocols W4 murine embryonic stem cells (mESCs), originally isolated from the 129S6 mouse strain, were grown as described previously [9 (link)]. In brief, cells were cultured in DMEM supplemented with 15% FBS Superior (Biochrom AG, Berlin, Germany), 1% Cell Shield® (Minerva Biolabs GmbH, Berlin, Germany), 100 µM non-essential amino acids, 1000 U/mL leukemia inhibitory factor (Phoenix Europe GmbH, Mannheim, Germany) and 100 µM β-mercaptoethanol (Sigma-Aldrich GmbH, Steinheim, Germany) at 37 °C, 5% CO₂, and 20% O₂. We performed cardiovascular differentiation using cardiogenic differentiation medium, containing IBM (Iscove’s Basal Medium, Biochrom AG, Berlin, Germany) supplemented with 10% FBS Superior, 1% Cell Shield®, 100 µM non-essential amino acids, 450 µm 1-thioglycerol (Sigma-Aldrich GmbH, Steinheim, Germany), and 213 µg/mL ascorbic acid (Sigma-Aldrich GmbH, Steinheim, Germany). Cardiovascular differentiation was initiated by hanging-drop culture for two days at 37 °C, 5% CO₂, and 20% O₂. To start formation of embryoid bodies (EB) 400 cells per drop were plated on the cover of a square petri dish and grown for 2 days. Subsequently, EB grew for four more days in suspension culture and were then harvested for transplantation. These cells will be referred to as cardiac induced cells (CiC) [8 (link)].

We utilized three well-characterized HNSCC cell lines: UDSCC1, UDSCC5, and UDSCC6 for our experiments. Cells were cultured in both 2D and 3D models. For 2D culture, cells were seeded in Gibco DMEM medium (ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Bio&SELL GmbH, Feucht, Germany), 1% cell shield (Minerva Biolabs, Berlin, Germany), and 1% NEAA (100×, ThermoFisher Scientific, Waltham, MA, USA) in regular culture flasks. For 3D culturing, the floating sphere formation approach using Ultra-Low Attachment (ULA) surface plates (Corning, New York, NY, USA) was applied: cells were resuspended in medium containing DMEM/F12 (Sigma-Aldrich, St. Louis, MO, USA) with B27 Supplement (50×, Sigma-Aldrich), supplemented with 0.4% BSA (Sigma Aldrich), 20 ng/mL EGF (Sigma Aldrich), 10 ng/mL bFGF (Thermo Scientific), and 5 µg/mL human Insulin (Roche, Basel, Switzerland) at a density of 20,000 cells per well on 6-well ULA surface plates and incubated for 72 h at 36 °C.