Response of Domestic Cat Primary Fibroblasts to Trehalose-Loading Methods,Air-Drying,and Storage Conditions at Non-freezing Temperatures

Abstract

Introduction:

Long-term preservation of biological material for biomedical or animal conservation purposes currently relies on ultracold temperatures. However, almost complete dehydration of cells in the presence of trehalose could allow cost-effective and flexible storage at ambient temperatures.

Objective:

The study aimed to characterize optimal trehalose-loading methods before passive air-drying and explore different storage options using domestic cat primary fibroblasts as a model.

Methods:

In Experiment 1, cells were loaded with trehalose using optimized electroporation (EP), thermal shock, or cold-responsive nanoparticles (CRNPs) containing trehalose. An evaluation of the cell viability after freezing and thawing was first conducted to compare trehalose protections conveyed by the different loading methods. In Experiment 2a, cells loaded with trehalose were passively air-dried for 2 days (once dry state was confirmed) and then assessed for viability, DNA integrity, or proliferative ability. In Experiment 2b, dried cells were stored for up to 4 weeks either at ambient temperatures, ambient temperatures under vacuum, 4°C, or −20°C while being assessed for viability every 24 hours for the first 5 days of storage and at the 2- or 4-week timepoint.

Results:

Loading cells with trehalose using EP and thermal shock methods achieved cryoprotective levels, but CRNPs did not enable them to reach that. After reaching the dry state, cells from all trehalose-loading methods and untreated controls had viable cells. Additionally, trehalose-loaded cells from all methods had no increase in DNA damage after drying when compared with fresh controls. Following each method, a small number of cells retained proliferative ability. Storage at 4°C helped prolong viability through the initial 5 days compared with other storage conditions; however, no cells were viable after 2 weeks.

Conclusion:

Cells maintain viability, DNA integrity, and proliferative ability after reaching the dry state; however, storage conditions beyond 5 days must be improved.