Current Approaches of Preservation of Cells During (freeze-) Drying.

The widespread application of therapeutic cells requires a successful stabilization of cells for the duration of transport and storage. Cryopreservation is currently considered the gold standard for the storage of active cells; however, (freeze-) drying cells could enable higher shelf life stability at ambient temperatures and facilitate easier transport and storage. During (freeze-) drying, freezing, (primary and secondary) drying and also the reconstitution step pose the risk of potential cell damage. To prevent these damaging processes, a wide range of protecting excipients has emerged, which can be classified, according to their chemical affiliation, into sugars, macromolecules, polyols, antioxidants and chelating agents. As many excipients cannot easily permeate the cell membrane, researchers have established various techniques to introduce especially trehalose intracellularly, prior to drying. This review aims to summarize the main damaging mechanisms during (freeze-) drying and to introduce the most common excipients with further details on their stabilizing properties and process approaches for the intracellular loading of excipients. Additionally, we would like to briefly explain recently discovered advantages of drying microorganisms, sperm, platelets, red blood cells, and eukaryotic cells, paying particular attention to the drying technique and residual moisture content.

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

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 Tg 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.