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

Abstract

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.

Preservation of Embryonic Stem Cells

“…Consequently, the biostability of the freeze-dried biologicals could be significantly compromised (Hancock et al 1995;He et al 2008a;He et al 2006b). More importantly, a recent study reported that the collapse temperature of cell culture medium-based trehalose solutions important for freeze-drying mammalian cells can be much lower than that of a simple binary trehalose-water solution (T C = ~ -30 o C) and trehalose solutions used for freeze-drying pharmaceuticals and prokaryotes (Yang et al 2010a), as shown in Fig. 7E.…”

A freeze-drying microscopy study of the kinetics of sublimation in a model lactose system

“…Pharmaceutical applications include the freeze-drying of living cells, vaccines, enzymes, and biological media. In the food industry, freeze-drying is applied to the manufacture of products such as coffee, milk powder and infant formula (Chow et al, 2008;Fonseca et al, 2004;Nasirpour et al, 2007;Pardo et al, 2002;Thomas et al, 2004;Yang et al, 2010). The process involves freezing the desired product solution, followed by sublimation and secondary drying (Oetjen, 1999).…”

“…There are many reports in the literature regarding the use of FDM to determine the T c of various products, for which it is now a well-established technique (Adams and Ramsay, 1996;Meister and Gieseler, 2009;Meister et al, 2009;Yang et al, 2010;Zhai et al, 2003).…”

Thermostability of Biological Systems: Fundamentals, Challenges, and Quantification

“…Otherwise, the sample may collapse (can’t maintain the morphology of the frozen sample) resulting in incomplete drying and heterogeneity in the freeze-dried product, which could significantly decrease the biostability of the freeze-dried biologicals [107-109]. More importantly, a recent study reported that the collapse temperature of cell culture medium-based trehalose solutions important for freeze-drying mammalian cells can be much lower than that of a simple binary trehalose-water solution and trehalose solutions used for freeze-drying pharmaceuticals and prokaryotes [110]. …”