Vitrification of ECV304 cell suspensions using solutions containing propane-1,2-diol and trehalose

In this paper, we report on the suitability of solutions containing propane-1,2-diol (propylene glycol, PD), sugars, and salts for the vitrification of the human cell line, ECV304. Cooling (at 10 degrees C/min) and rewarming (at 80 degrees C/min) were at rates that are practicable for the tissues to be studied later. Under these conditions, 45% PD in phosphate-buffered saline (PBS) sometimes froze during cooling and always devitrified during rewarming but both events were avoided if the PBS salts were replaced by an osmotically equivalent concentration of sucrose or trehalose. The effect of such solutions on cells was evaluated using a cell culture assay in which the number of cells recovered after 3 days of culture was divided by the number cells plated, giving a cell multiplication factor or CMF. In the absence of PD the cells tolerated a low-salt concentration in solutions that were made isotonic with sugars, but they recovered poorly when 45% PD was also present. Trehalose gave significantly better recovery than sucrose. When 39% PD and 15% trehalose were included in a low-salt vehicle solution (LSV) that contained approximately 5% of the total salt concentration of PBS (this solution was designated LSV/39/15), the cells exhibited approximately 40% of untreated control CMF following exposure for 9min. LSV/39/15 vitrifies with a glass transition temperature of -102 degrees C, does not devitrify when warmed at 80 degrees C/min, and has suitable dielectric properties for uniform and rapid dielectric heating. An improved method for adding and removing LSV/39/15 gave a CMF of approximately 55% of untreated controls. Using this method, 1.0ml suspensions of ECV304 cells was cooled to, and stored briefly at, -120 degrees C and then rewarmed by immersion in a 37 degrees C water bath ( approximately 75 degrees C/min). The CMF of the cooled samples was similar to that of the exposure-only controls, approximately 50% of the untreated control CMF in both cases.