Freezone 2.5 l benchtop freeze dryer

To examine changes in the clay composition over time, 2 g of each sample was diluted with 3 mL of distilled water and homogenised by benchtop vortex (~2000 rpm) for 5 min. The resulting mixture was transferred to a Ziplock bag and stored at −80 °C. To remove water content without losing N and S as volatiles, samples were subsequently dried for 48 h in a FreeZone 2.5 L benchtop freeze dryer (Labconco, Kansas City, MO, USA) at −56 °C and <0.02 mbar. Once dry, samples were crushed into a fine powder and transferred into 1.5 mL vials. The vials were stored in air-locked containers with activated desiccant under vacuum. To measure the change in total carbon, nitrogen, and sulphur, samples were analysed using a Thermo Finnigan EA 1112 Series Flash Elemental Analyser after being weighed in a Sartorius Microbalance SE2 w. Following blank correction, the total elemental content data were normalised to cell density.

The obtained nanosuspension was frozen at −20 °C (overnight), and the samples were freeze-dried for 72 h using a Labconco Freezone 2.5-L benchtop freeze dryer (Kansas City, MI, USA). The amount of moisture in the powder was measured using an OHAUS M45 moisture analyzer (D.F.; Mexico) that uses halogen heating. The analysis of the moisture content of the samples was carried out right after the freeze-drying process.

Gelatine was functionalised as described before with small modifications to the original protocol (Shirahama et al., 2016 (link)). Ten grams medical grade 262 Bloom Type A gelatine (Gelita, Ter Apelkanaal, the Netherlands) was added to 1 L 0.25 M carbonate-bicarbonate (CB, Sigma-Aldrich, Zwijndrecht, the Netherlands) buffer pH 9.0 and allowed to dissolve at 50°C for 60 min. Heating was turned off and then 563 µL methacrylic anhydride (MAA; Sigma-Aldrich, CCID: 12974) was added dropwise in three steps. Ten minutes after each step the pH was measured and corrected using a 3 M NaOH (Sigma-Aldrich, CCID: 14798) solution to maintain pH 9.0. The gelatine-MAA solution was allowed to react overnight at room temperature (RT) while stirring. The next day the GelMA solution was dialysed using dialysis with molecular weight cut-off of 12–14 kDa (Sigma-Aldrich) for up to 5 days against deionised water pH 9.0. After dialysis, GelMA solution was aliquoted in 50 ml tubes, frozen with liquid nitrogen and lyophilised on a Labconco Freezone 2.5 L benchtop freeze-dryer (Labconco, Kansas City, MO, United States). Samples were considered dry when weights of the tubes were stable. Three batches were produced, lyophilised, re-dissolved into one large batch and then lyophilised again to minimise batch-to-batch variation.