Cryostor cs10 cryopreservation medium

Day 16 EMLOs were dissociated to single cells using a cold activated protease method previously described^{45 (link)} and essential details are provided here. Dissociation solution was comprised of 10 mg/ml psychrophilic Bacillus licheniformis (Creative Enzymes) protease and 125 U/ml DNase (Millipore Sigma) in ice-cold 1× PBS supplemented with 5 mM CaCl₂. In all, ~35 EMLOs were pooled into a 2 ml centrifuge tube and allowed to settle. Culture medium was aspirated and 1 ml ice-cold dissociation solution was added to EMLOs and incubated on ice. Every 30–60 s, EMLOs were triturated with a P-1000 pipette and returned to ice. After 15 min, single-cell suspension was validated by optical inspection. 1 ml of ice-cold 1× PBS with 10% fetal bovine serum (FBS) was added and cells were pelleted by centrifugation at 1200 × g for 5 min. The supernatant was completely aspirated. Cells were resuspended in 1× PBS/10% FBS and counted, centrifuged once more, aspirated completely and resuspended to ~1 × 10⁶ cells/ml in CryoStor CS10 cryopreservation medium (BioLife Solutions) that was filtered through a 40 μm cell strainer and then added to a 1.8 ml Nunc cryo-storage tube. Cells were frozen at −80 °C and shipped overnight on dry ice to University of Buffalo Genomics and Bioinformatics Core.