DMSO as new, counterintuitive excipient for freeze-drying human keratinocytes

Abstract

DMSO is widely used as powerful cryoprotectant for the storage and transport of frozen cells. Beyond this established application of DMSO, we could now show that it has also promising lyoprotectant effects in the field of lyophilisation of therapeutic cells. Freeze-drying of HaCaT keratinocytes in 10% HES, 5% HE and in presence of DMSO led to an increase in cell membrane integrity from 25.3 ± 2.7 % without DMSO to 41.4 ± 4.3 % with 2% DMSO, as determined by trypan blue exclusion. Interruption of the lyophilisation cycle at different sampling points showed a rapid decrease of cell membrane integrity below a critical residual moisture content. DMSO was able to stabilise cell membranes below this moisture level up to a final residual moisture content of less than 1%. Furthermore, DMSO increased the total protein content of cells after freeze-drying and subsequent SDS PAGE analysis indicated that certain abundant proteins were better preserved with the use of DMSO. Owed to its low vapour pressure, a significant part of DMSO is not removed during freeze-drying and remains as plasticiser in the lyophilised cake. However, a T_g above 60°C for 2% DMSO indicates that samples can still be stored at temperatures of 2-8°C. Also, no macroscopic or microscopic collapse can be observed by SEM or BET measurements and DMSO addition leads even to more elegant cakes with reduced cake cracking. With a better preservation of cell membranes and cellular structures, DMSO can contribute to the still unsolved problem of freeze-drying cells of higher complexity.

Keywords: Cell membrane integrity; DMSO; Freeze-drying; Keratinocytes; Lyophilisation; Organic solvent; Protein stabilisation; Residual moisture content.