Gene Activation of the Apoptotic Caspase Cascade Following Cryogenic Storage

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Abstract
The improvement in preservation is now critical for support of the newly emerging fields of tissue engineering and regenerative medicine. The expanding number of complex biologics banked for therapeutic applications necessitates the development of new preservation technologies. Recent studies in biologic preservation have implicated apoptosis as a contributing factor to cell death. These studies have described the role and extent of apoptotic cell death following cryopreservation but have not addressed investigation into the signaling pathways responsible for program execution. Accordingly, we investigated the role of apoptotic gene activation of caspase3 at the genetic and protein levels following cryopreservation (culture media + dimethyl sulfoxide) in a human fibroblast cell model. Additionally, investigation into the extent of caspase-3 involvement in cell death following preservation in a newly developed cryopreservation medium was performed. In this study we report on the upregulation of transcriptional and proteolytic activity of caspase-3 following "classic" cryopreservation and a reduction in caspase-3 up-regulation through the utilization of an intracellular-like cryopreservation medium (HypoThermosol®). We further report on the transcriptional up-regulation of caspase-8 and-9 following thawing leading to caspase-3 activation and cellular demise. This report represents the first investigation into the identification of the mechanistic pathways of apoptotic induction following cryopreservation. These findings provide insight into the basis of activation and progression of apoptosis following preservation and identify further approaches to enhance cryopreservation efficacy. Identification of these processes as well as their control has led to the development of new biopreservation technologies that may provide a foundation for the preservation of more complex biologics.