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Prolonged hypothermic cardiac storage with University of Wisconsin solution. An assessment with human cell cultures
S E Fremes1, R K Li, R D Weisel
1Division of Cardiovascular Surgery and Clinical Biochemistry, University of Toronto, Ontario, Canada.
Insights
University of Wisconsin solution and 0°C storage best preserve cardiac cells during hypothermic preservation, extending viability beyond 4 hours. This research offers a model for improving organ transplant storage solutions and temperatures.
Area of Science:
- Cardiology
- Cell Biology
- Transplantation Science
Background:
- Hypothermic storage of cardiac allografts is standard for transplantation.
- Current methods limit ischemic times to 4 hours, increasing mortality.
- Optimizing preservation solutions and temperatures can extend cold ischemia limits.
Purpose of the Study:
- To screen various preservation solutions and temperatures for cardiac allograft organ preservation.
- To evaluate the efficacy of different storage conditions on human endothelial cells and ventricular myocytes.
Main Methods:
- Four solutions (modified Euro-Collins, PBS, Stanford cardioplegia, UW) and temperatures (0°C, 4°C, 8°C) were tested.
- Cell viability assessed via morphology, trypan blue exclusion, cell adhesion, and ATP analysis.
- Endothelial cells stored for 36 hours; myocytes for 24 hours.
Main Results:
- University of Wisconsin (UW) solution best preserved endothelial cell morphology and viability at 0°C.
- UW solution also best preserved myocyte morphology and ATP levels at 0°C.
- Myocytes were more sensitive to prolonged storage than endothelial cells.
Conclusions:
- UW solution is the most effective tested for cardiac cell preservation.
- Colder temperatures (0°C-4°C) offer better protection than 8°C.
- The study provides a model for assessing interventions in prolonged hypothermic storage.
Abstract:
Hypothermic storage of cardiac allografts is routinely used for transplantation but is associated with an increased mortality when ischemic times are greater than 4 hours. The ideal storage conditions (solution and temperature) could extend the current limits of cold ischemia. Human endothelial cells and ventricular myocytes were studied to screen various solutions and temperatures for organ preservation. Four solutions (modified Euro-Collins, phosphate-buffered saline, Stanford cardioplegia, and University of Wisconsin) were evaluated. Endothelial cells were evaluated after prolonged hypothermic storage consisting of 0 degree, 4 degrees, and 8 degrees C for 36 hours, and ventricular myocytes were stored at 0 degree and 8 degrees C for 24 hours. Cell viability was determined by morphology (10 dishes per group), and trypan blue exclusion (5 dishes per group) in addition to a cell adhesion assay (endothelial cells 5 dishes per group) and adenine nucleotide analysis with high-performance liquid chromatography techniques (ventricular myocytes 5 dishes per group). Endothelial cell morphology was best preserved by University of Wisconsin solution (p less than 0.001, chi 2) and at 0 degree C (p less than 0.01, chi 2). Endothelial cells stored with University of Wisconsin solution excluded trypan blue better (1.0% +/- 0.5% cells stained, p less than 0.001. Analysis of variance [ANOVA]). Cell adhesion was poorly protected with Stanford cardioplegia (p less than 0.001, ANOVA). Myocyte morphology was preserved best with University of Wisconsin solution at 0 degree C (p less than 0.001, chi 2). According to trypan blue staining, Euro-Collins and University of Wisconsin solutions were superior to Stanford cardioplegia or phosphate-buffered solutions (p less than 0.001, ANOVA). Temperature did not influence the trypan blue results. Adenosine triphosphate was maintained best with University of Wisconsin solution at 0 degree C (p less than 0.01, ANOVA). Myocytes were more sensitive to the effects of prolonged storage compared with endothelial cells by morphologic criteria and trypan blue staining characteristics, irrespective of the shorter preservation times. University of Wisconsin solution was the most effective solution tested. Colder temperatures (0 degree to 4 degrees C) provided better protection than 8 degrees C. Myocytes were more sensitive to prolonged preservation than endothelial cells. Furthermore, the technique used appears helpful as a model of prolonged hypothermic storage and could be expanded to assess other interventions.