The influence of polyvinylpyrrolidone (PVP) on the toxicity of cryoprotective agents in different liver cell types and on vitrification outcome in precision-cut liver and kidney slices

Cryopreservation of precision-cut tissue slices would strongly improve their application possibilities in ADME-tox studies. Low toxic mixtures of cryoprotectant agents (CPAs) are needed for cryopreservation of cells, and tissues by vitrification. Many of these mixtures contain polyvinylpyrrolidone (PVP), a non-permeable macromolecule, but the effect of this agent on the viability of the tissue is unknown. We investigated the effect of adding or replacing PVP on the viability of precision cut liver and kidney slices before and after vitrification with special emphasis on the susceptibility of the different non-parenchymal cells (NPC) in the liver to PVP toxicity. Methods: PVP in the commercially available CPA M22 was replaced by a polymer that is one of the components of the PVP-free CPA Sol Z; the resulting solution was called CPR, and the polymer in Sol Z was replaced by PVP (called Sol ZP). The viability of PCLS was studied after loading the slices with one of the four solutions and after vitrification. Differential scanning calorimetry showed that adding or omitting PVP did not change the ability to prevent ice-crystal formation during vitrification of the CPAs. Results: ATP levels of PCLS treated with the PVP free Sol CPR were significantly higher than that of PCLS treated with M22 ((98% versus 78% compared to untreated controls), whereas ATP levels of PCLS treated with Sol ZP (85%) were comparable to that of PCLS treated with Sol Z (89%). HE staining of PCLS treated with the CPAs indicated that the viability of hepatocytes after treatment was comparable to that of control slices for all four solutions. However, non-parenchymal cells (NPC) in the sinusoidal space of PCLS showed consistent changes in the morphology. Immunohistological staining for different NPC (stellate, Kupffer and endothelial cells) suggested that the viability of NPC cells was not improved by the new CPAs. After vitrification, the ATP level of PCLS treated with Sol CPR was 61 % and with M22 was 43% compared to slices exposed to the CPAs but not vitrified. This suggests that the removal of PVP from M22 has some protective effect on PCLS at both CPA loading/unloading and vitrification. Moreover, kidney slices treated with Sol CPR also showed higher viability after vitrification than slices that were treated with M22. Conclusion: PVP has no protective effect on the viability of liver and kidney slices after vitrification although it has been widely used for this purpose. We showed for the first time that damage due to CPA toxicity does not only affect the hepatocytes but also the NPC. Sol CPR is a promising CPA for the vitrification-based cryopreservation of PCLS, but further optimization of the vitrification technique is required to improve the viability of the slices after vitrification and re-warming.