Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices

• Published in: Cryobiology Vol. 65; no. 3; pp. 179 - 187
• Main Authors: Guan, Na, Blomsma, Sylvia A., van Midwoud, Paul M., Fahy, Gregory M., Groothuis, Geny M.M., de Graaf, Inge A.M.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier Inc 01.12.2012; Elsevier
• Subjects: Adenosine Triphosphate - metabolism; Animals; Biological and medical sciences; Chemical toxicity; Cryopreservation; Cryoprotectant toxicity; Cryoprotectants; Cryoprotective Agents - metabolism; Digestive system; Equipment Design; Gradient-wise cryoprotectant loading; Ice-free cryopreservation; Investigative techniques, diagnostic techniques (general aspects); Liver - metabolism; Liver - ultrastructure; Male; Medical sciences; Organ Preservation - instrumentation; Organ Preservation - methods; Organ Preservation Solutions - metabolism; Osmosis; Osmotic Pressure; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques; Permeability; Rats; Rats, Wistar; Step-wise cryoprotectant loading; Tissue banking; Vitrification; UW; Step-wise cryoprotectant loading; Vitrification; Cryoprotectants; Tissue banking; Osmosis; Cryoprotectant toxicity; WME; CPA; Chemical toxicity; Cryopreservation; Ice-free cryopreservation; Me2SO; PCLS; Gradient-wise cryoprotectant loading; Digestive system; Toxicity; Liver; Biology; Histological section; Cryobiology; Cryosection; Cryoprotective agent; Technique; Osmotic pressure; Biomedical engineering
• ISSN: 0011-2240; 1090-2392
• DOI: 10.1016/j.cryobiol.2012.05.011

Successful vitrification of organ slices is hampered by both osmotic stress and chemical toxicity of cryoprotective agents (CPAs). In the present study, we focused on the effect of osmotic stress on the viability of precision-cut liver slices (PCLS) by comparing different CPA solutions and different methods of loading and unloading the slices with the CPAs. For this purpose, we developed a gradient method to load and unload CPAs with the intention of minimizing sudden changes in osmolarity and thereby avoiding osmotic stress in the slices in comparison with the commonly used step-wise loading/unloading approach. With this gradient method, the CPA solution was introduced at a constant rate into a specially designed mixing chamber containing the slices. We showed that immediate mixing of the infused CPA and the chamber constituents occurred, which enabled us to control the CPA concentration to which PCLS were exposed as a function of time. With this method, CPA concentration versus time profiles were varied using various commercially available CPA mixtures [VMP, VM3, M22, and modified M22 (mM22)]. The viability of PCLS was determined after CPA loading and unloading and subsequent incubation during 3h at 37°C. Despite the reduction of osmotic stress, the viability of slices did not improve with gradual loading and unloading and remained considerably lower than that of untreated slices. The toxicity of the three CPA solutions did not correlate with either their potential osmotic effects or their total concentrations, and did not change strongly with exposure time in 100% CPA. The most likely explanation for these observations is that PCLS are not very sensitive to osmotic changes of the magnitude imposed in our study, and chemical toxicity of the CPA solutions is the main barrier to be overcome. The chemical toxicity of the CPAs used in this study probably originates from a source other than the total concentration of the solutions. The presented gradient method using the specially designed chamber is more time and cost effective than the step-wise method and can be universally applied to efficiently evaluate different CPA solutions.