PMID- 22722061 OWN - NLM STAT- MEDLINE DCOM- 20130211 LR - 20131121 IS - 1090-2392 (Electronic) IS - 0011-2240 (Linking) VI - 65 IP - 3 DP - 2012 Dec TI - Effects of cryoprotectant addition and washout methods on the viability of precision-cut liver slices. PG - 179-87 LID - S0011-2240(12)00115-0 [pii] LID - 10.1016/j.cryobiol.2012.05.011 [doi] AB - 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 degrees 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. CI - Copyright (c) 2012 Elsevier Inc. All rights reserved. FAU - Guan, Na AU - Guan N AD - Division of Pharmacokinetics, Toxicology and Targeting, Department of Pharmacy, University of Groningen, The Netherlands. FAU - Blomsma, Sylvia A AU - Blomsma SA FAU - van Midwoud, Paul M AU - van Midwoud PM FAU - Fahy, Gregory M AU - Fahy GM FAU - Groothuis, Geny M M AU - Groothuis GM FAU - de Graaf, Inge A M AU - de Graaf IA LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20120618 PL - Netherlands TA - Cryobiology JT - Cryobiology JID - 0006252 RN - 0 (Cryoprotective Agents) RN - 0 (Organ Preservation Solutions) RN - 8L70Q75FXE (Adenosine Triphosphate) SB - IM MH - Adenosine Triphosphate/metabolism MH - Animals MH - Cryoprotective Agents/*metabolism MH - Equipment Design MH - Liver/*metabolism/ultrastructure MH - Male MH - Organ Preservation/*instrumentation/methods MH - Organ Preservation Solutions/*metabolism MH - Osmosis MH - Osmotic Pressure MH - Permeability MH - Rats MH - Rats, Wistar MH - Vitrification EDAT- 2012/06/23 06:00 MHDA- 2013/02/12 06:00 CRDT- 2012/06/23 06:00 PHST- 2012/02/13 00:00 [received] PHST- 2012/05/11 00:00 [revised] PHST- 2012/05/14 00:00 [accepted] PHST- 2012/06/23 06:00 [entrez] PHST- 2012/06/23 06:00 [pubmed] PHST- 2013/02/12 06:00 [medline] AID - S0011-2240(12)00115-0 [pii] AID - 10.1016/j.cryobiol.2012.05.011 [doi] PST - ppublish SO - Cryobiology. 2012 Dec;65(3):179-87. doi: 10.1016/j.cryobiol.2012.05.011. Epub 2012 Jun 18.