Fig 4. Titan cells generation in vitro is impacted by various environmental conditions.

(A) The sequence of media used at steps 2 and 3 of the protocol was crucial for titan cells generation: yeasts cultured in Yeast Peptone Dextrose (YPD) and transferred to minimal medium (MM) produced significantly more titan cells (cells >10μm, dotted grey line) than yeasts cultured in MM or YPD and transferred in MM or YPD, respectively; (B) Exposure to d light at step 3 had a positive impact on titan cells generation compared to incubation in the dark; (C) Raising the incubation temperature to 37°C at step 3 decreased titan cells formation compared to 30°C; (D) Modification of the initial pH of the MM used at step 3 impacted titan cells formation with pH 5.5 being optimal while a more acidic (pH = 4), a neutral (pH = 7) or an alkaline (pH = 8.5) pH inhibited titan cells formation; (E) The impact of hypoxia was tested by physical (closed cap) and by chemical (COCl₂ in MM at 1 nM) method and compared to normoxia (21% oxygen). Physically- and chemically-induced hypoxia enhances the production of titan cells compared to normoxia with a higher proportion of titan cells in chemically- compared to physically-induced hypoxia. All experiments were performed in triplicate and pooled (mean cell counted ± SD = 2305±1438). Median and IQR are shown in black for each condition (* p<0.0001 vs reference condition). The percentages above each condition represents the % of titan cells observed.