Coolsafe 110

Three million cells were washed with PBS and resuspended in 0.5 ml of lyophilization solution which was 0.1 M D-(+)-Trehalose dihydrate (Sigma-Aldrich, cat. T9531) in PBS. Cell suspensions were placed in safe-lock polypropylene microcentrifuge tubes and were snap-frozen by immersing sideways in liquid nitrogen. Immediately before loading into the freeze dryer, the tubes were opened, and a film in which 7 holes were pierced (1 mm diameter each) was placed on top of the tube’s opening. The samples were loaded into a CoolSafe 110 freeze dryer (ScanVac, LaboGene, Denmark) belonging to the Proteomics Core Facility at the University of Debrecen, Hungary, which has a condenser temperature as low as –110° C and a vacuum pump capable of reaching an absolute pressure of 0.004 mBar. Samples were lyophilized for 6 hours (with the environmental temperature conditioned to 22° C) and were either processed immediately or stored for 2 weeks or 2 months at room temperature (23–25° C) in the presence of CaCl₂ dihydrate (desiccant) in a non-transparent, tightly sealed box to prevent moisture absorption and light exposure.

Bare AcDX and PLGA microspheres were prepared by a flow-focusing microfluidic device. AcDX (10 mg mL⁻¹) and PLGA (20 mg mL⁻¹; 5002A, PURAC, Netherlands) were dissolved in a mixture of ethyl acetate-dimethyl sulfoxide and served as the inner fluid. Poloxamer 407 (P-407) (BASF, Germany) solution (10 mg mL⁻¹, pH 7.4) was used as the outer fluid. In the flow–focusing geometry^{56 ,57 (link)}, the inner fluid is flowed inside the outer capillary and the outer fluid is flowed between the inner and outer capillary in the opposite direction. The inner fluid (0.8 mL h⁻¹) was focused by the outer fluid (10 mL h⁻¹) through the narrow orifice of the tapered inner capillary. The outer fluid exerts pressure and shear stress that force the inner fluid into a narrow thread, and generates droplets inside the inner capillary. The formed oil-in-water emulsion droplets were solidified via the diffusion of solvents from droplets to the surrounding outer fluid. At last, the collected microspheres were washed triple thoroughly with water and then freeze dried (CoolSafe 110, LaboGene APS, Denmark).