Figure 1

Schematic diagram of the experimental set-up and temperature profiles for two-step freezing and graded freezing.

(a) HUVECs in suspension were subjected to interrupted cooling in a methanol bath and either directly thawed, or plunged and stored in liquid nitrogen before thawing in a 37 °C water bath. (b) Schematic diagram of two-step freezing which involves: (i) rapid cooling to intermediate sub-zero temperatures (hold temperatures), (ii) induced ice formation (☆) using liquid nitrogen-cooled forceps, (iii) holding for 3 minutes at intermediate sub-zero temperatures (iv) rapid plunge into liquid nitrogen, (v) storage in liquid nitrogen and (vi) rapid thawing. Steps (i), (ii), (iii) and (vi) are performed for direct thaw and all steps are performed for plunge thaw. (c) Representative temperature trace of two-step freezing for –3 °C plunge thaw. (d) Schematic diagram of graded freezing which involves: (i) induced ice formation (☆) using liquid nitrogen-cooled forceps, (ii) holding for 3 minutes at the first experimental temperature, (iii) controlled cooling at 1 °C/min or 0.2 °C/min to intermediate sub-zero temperatures (experimental temperatures), (iv) rapid plunge into liquid nitrogen, (v) storage in liquid nitrogen and (vi) rapid thawing. Steps (i), (ii), (iii) and (vi) are performed for direct thaw and all steps are performed for plunge thaw. (e) Representative temperature trace of graded freezing at 0.2 °C/min for −6 °C plunge thaw.