Collapse temperature of solutions important for lyopreservation of living cells at ambient temperature

In this study, the collapse temperature was determined using the freeze-drying microscopy (FDM) method for a variety of cell culture medium-based solutions (with 0.05-0.8 M trehalose) that are important for long-term stabilization of living cells in the dry state at ambient temperature (lyopreservation) by freeze-drying. Being consistent with what has been reported in the literature, the collapse temperature of binary water-trehalose solutions was found to be similar to the glass transition temperature (T'(g) approximately -30 degrees C) of the maximally freeze-concentrated trehalose solution (approximately 80 wt% trehalose) during the freezing step of freeze-drying, regardless of the initial concentration of trehalose. However, the effect of the initial trehalose concentration on the collapse temperature of the cell culture medium-based trehalose solutions was identified to be much more significant, particularly when the trehalose concentration is less than 0.2 M (the collapse temperature can be as low as -65 degrees C). We also determined that cell density from 1 to 10 million cells/mL and ice seeding at high subzero temperatures (-4 and -7 degrees C) have negligible impact on the solution collapse temperature. However, ice seeding does significantly affect the ice crystal morphology formed during the freezing step and therefore the drying rate. Finally, bulking agents (mannitol) could significantly affect the collapse temperature only when trehalose concentration is low (<0.2 M). However, improving the collapse temperature by using a high concentration of trehalose might be preferred to the addition of bulking agents in the solutions for freeze-drying of living cells. We further confirmed the applicability of the collapse temperature measured with small-scale (2 microL) samples using the FDM system to freeze-drying of large-scale (1 mL) samples using scanning electron microscopy (SEM) data. Taken together, the results reported in this study should provide useful guidance to the development of optimal freeze-drying protocols for lyopreservation of living cells at ambient temperature for easy maintenance and convenient wide distribution to end users, which is important to the eventual success of modern cell-based medicine.