Cryoprotectant cocktail solution preparation and stepwise cryoprotectant loading protocol

Multicryoprotectant cocktail solutions were made from three cryoprotectants: EG (Fisher), DMSO (Fisher), and PG (Fisher). Fresh cryoprotectant cocktail solutions were prepared in 50 mL final volumes with Dulbecco’s Modified Eagle Medium F12 (DMEM) (Gibco) for porcine cartilage or with X-VIVO 10 SFM for human cartilage on the same day of the experiment, using the following concentrations of cryoprotectants (M = molar): Protocol E–D–P: solution one [3 M EG + 3 M DMSO] and solution two [3 M EG + 3 M DMSO + 2 M PG] and Protocol E–D: solution one [3 M EG] and solution two [4 M EG + 4 M DMSO]. After weighing, cartilage cubes were transferred into the prepared 50 mL cryoprotectant cocktail solutions for cryoprotectant permeation at specific temperatures and times: Protocol E–D–P: solution one at 0 °C for 10 min, followed by solution two at −10 °C for 20 min and Protocol E–D: solution one at 0 °C for 20 min, followed by solution two at −5 °C for 15 min. After the cryoprotectant loading into the cartilage, the particulated cartilage cubes were quickly removed from the Falcon tubes with a mesh strainer and transferred into a sterile 1.8 mL Cryovial tube using a chemical spoon. After closing the vial lid, the Cryovial tube was placed onto a Cryovial cane and quickly plunged into liquid nitrogen for vitrification.