Coolsafe 4 15l freeze dryers

AHCFs (5 × 10⁵ cells/dish) were cultured in 100 mm-diameter Petri dishes with FGM-3 medium for up to 21 days. The decellularization process was performed following a previously described protocol [21 (link)]. Briefly, cells were removed by incubation with a solution of 0.25% Triton X- 100 and 10 mM NH₄OH in phosphate buffered saline (PBS, Gibco, Waltham, MA, USA) prewarmed to 37 °C. Petri dishes were washed twice with PBS and then frozen for 24 h and lyophilized using CoolSafe 4-15L freeze-dryers (Labogene, Scandinavia) for 24 h. Afterwards, lyophilized BM was collected from wells in the form of powder and stored at −80 °C.

Three-dimensional hydrogel and 3D BM hydrogel internal sections were examined by SEM. Hydrogel samples were formed as described above and fixed with 4% paraformaldehyde at room temperature, then frozen for 24 h and lastly lyophilized using CoolSafe 4-15L freeze-dryers, Labogene (Scandinavia) for 24 h. The samples were fractured with the aid of a bistoury in liquid nitrogen, and then sputter coated with gold. SEM images of the sections were acquired at different magnifications.