Freeze-drying of mammalian cells using trehalose: preservation of DNA integrity

Abstract

Abstract The aim of this study was to investigate preservation of biomolecular structures, particularly DNA, in freeze-dried fibroblasts, after loading with trehalose via freezing-induced uptake. Cells were freeze-dried with trehalose alone or in a mixture of albumin and trehalose. Albumin was added to increase the glass transition temperature and storage stability. No viable cells were recovered after freeze-drying and rehydration. FTIR studies showed that membrane phase behavior of freeze-dried cells resembles that of fresh cells. However, one day after rehydration membrane phase separation was observed, irrespective of the presence or absence of trehalose during freeze-drying. Freeze-drying did not affect the overall protein secondary structure. Analysis of DNA damage via single cell gel electrophoresis (‘comet assay’) showed that DNA damage progressively increased with storage duration and temperature. DNA damage was prevented during storage at 4 °C. It is shown that trehalose reduces DNA damage during storage, whereas addition of albumin did not seem to have an additional protective effect on storage stability (i.e. DNA integrity) despite the fact that albumin increased the glass transition temperature. Taken together, DNA in freeze-dried somatic cells can be preserved using trehalose as protectant and storage at or below 4 °C.