Results and Discussion
To test the overall effects of cryopreservation media and post-thaw culture configuration, LNCaP samples were cryopreserved in traditional preservation solutions or CryoStor CS5 and plated on either the CellBIND Surface or a TCT surface.
Analysis of cell survival immediately post-thaw revealed no significant difference in cell viability regardless of the preservation media (Table). This observation was expected and consistent with previous reports given that the timing of assessment does not account for CIDOCD. As observed in numerous other cell systems, such as hepatocytes, it is only after 24 hours culture post-thaw that a true level of survival is reached (Figure 1). When samples were allowed to recover in culture for 24 hours and then assessed for survival, significant differences were seen in the effectiveness of both the preservation media and the culture conditions (Figure 2).
Overall, the combination of CryoStor CS5 and the CellBIND Surface resulted in a significant increase in the survival and attachment of LNCaP cells over traditional solutions (media/DMSO or Serum/DMSO) and TCT surface. This observation was made in both the T-25 flask (Figure 2a) and 96-well plate (Figure 2b) culture configurations. The CellBIND Surface alone had a positive impact on the recovery of LNCaP cells (increase of 28% over TCT using standard freeze medium).
Further, the use of CryoStor in place of media and DMSO significantly increased, by 45%, the recovery of LnCAP cells on the TCT surface (p<0.001). The use of serum/DMSO as the cryopreservation media had a slight positive effect on overall survival but remained significantly below that of CryoStor in all conditions.
On average, the combined CryoStor/ CellBIND surface approach yielded a 58% increase in cell survival over traditional freeze media and standard tissue culture treated surfaces (p=0.0013). Importantly, when cultures were visualized by light microscopy, the CryoStor CS5/CellBIND surface approach showed an increase in attached cell number and improved cell morphology relative to traditional media/DMSO and tissue culture ware approaches (Figure 3). Analysis of cell membrane integrity using the fluorescent stain Calcein-AM corroborated the light microcopy data showing that the surviving cells were more numerous and retained a high degree of membrane integrity (Figure 4).
Through this study we tested the hypothesis that both the freezing media formulation and post-thaw culture surface might significantly impact cryopreservation outcome. It was further determined that by combining the use of novel cryopreservation media and cell culture surfaces, one can positively impact the overall level of cell survival and attachment.
Specifically, the CryoStor/CellBIND Surface approach resulted in a significant increase in LNCaP cell viability following cryopreservation and provided for the retention of a more native cellular attachment morphology. This is important for numerous reasons including the fact that these two factors have been correlated directly with overall functionality and usability of cryopreserved cellular systems.
The CryoStor/CellBIND Surface combination approach now offers researchers a leg up in this area by reducing much of the time and effort presently required thereby increasing productivity and cost-effectiveness of in vitro screening processes.