Assessment of endothelial preservation in human cell cultures.
BACKGROUND
Impairment of microcirculation due to endothelial cell damage must be considered a limiting factor in organ preservation. The present study aims at a quantitative assessment of preservation-induced injury in cultured human endothelial cells.


METHODS
Monolayer cultures of human umbilical vein endothelial cells were exposed to cold (40 degrees C) hypoxic storage in University of Wisconsin solution, histidine-tryptophane-ketoglutarate solution, Euro-Collins solution, and saline solution. Cellular integrity was evaluated by viable cell count, ultrastructural analysis, and prostacyclin release after 24, 48, and 72 hours of storage and subsequent 6 hours of reincubation in culture medium at 37 degrees C. Expression of intercellular adhesion molecule-1 was investigated after 6, 12, and 24 hours of cold preservation and after 6 hours of rewarming.


RESULTS
Cellular viability was best maintained with University of Wisconsin and histidine-tryptophane-ketoglutarate solutions with no significant reduction of cell count up to 72 hours; Euro-Collins solution and saline solution caused a significant decline in cell numbers after 24 hours (p < 0.05). Morphology was best preserved by University of Wisconsin solution. Prostacyclin values were elevated after 24 hours in Euro-Collins solution and saline solution, after 48 hours in histidine-tryptophane-ketoglutarate, Euro-Collins, and saline solutions, and after 72 hours in Euro-Collins solution (p < 0.05, compared with University of Wisconsin solution). ICAM expression was weak after cold storage (24 hours) in University of Wisconsin solution, moderate after incubation in histidine-tryptophane-ketoglutarate and Euro-Collins solutions and intensive after storage in saline solution. In contrast, rewarming caused intensive expression of intercellular adhesion molecule-1 in all experimental groups as compared with controls, which showed baseline expression at any time.


CONCLUSIONS
From our results we conclude that in this model cellular integrity is best protected by University of Wisconsin solution, increased prostacyclin release is consistent with morphologic alterations and intercellular adhesion molecule-1 expression is clearly up-regulated in endothelial cells under reperfusion conditions after cold hypoxic storage.
Eberl
T
T
Department of Transplant Surgery, University Hospital Innsbruck, Austria.
Steinlechner
R
R
Hengster
P
P
Herold
M
M
Schröcksnadel
H
H
Salvenmoser
W
W
Rhomberg
M
M
Gnaiger
E
E
Margreiter
R
R
D000241:Adenosine; D000493:Allopurinol; D002452:Cell Count; D015687:Cell Hypoxia; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation; D003470:Culture Media; D004730:Endothelium, Vascular / Q000166:cytology*; D011464:Epoprostenol / Q000032:analysis; D015870:Gene Expression; D005947:Glucose; D005978:Glutathione; D006801:Humans; D006982:Hypertonic Solutions; D007328:Insulin; D018799:Intercellular Adhesion Molecule-1 / Q000032:analysis / Q000235:genetics; D008353:Mannitol; D008833:Microcirculation; D009926:Organ Preservation; D019188:Organ Preservation Solutions; D010975:Platelet Aggregation Inhibitors / Q000032:analysis; D011189:Potassium Chloride; D011343:Procaine; D011887:Raffinose; D015424:Reperfusion; D017694:Rewarming; D012965:Sodium Chloride; D014021:Tissue Preservation / Q000379:methods; D014471:Umbilical Veins / Q000166:cytology; D015854:Up-Regulation
C039984:Bretschneider cardioplegic solution; D003470:Culture Media; C037096:Euro-Collins' solution; D006982:Hypertonic Solutions; D007328:Insulin; D019188:Organ Preservation Solutions; D010975:Platelet Aggregation Inhibitors; C055206:University of Wisconsin-lactobionate solution; D018799:Intercellular Adhesion Molecule-1; D008353:Mannitol; D012965:Sodium Chloride; D011343:Procaine; D000493:Allopurinol; D011189:Potassium Chloride; D011464:Epoprostenol; D005978:Glutathione; D005947:Glucose; D000241:Adenosine; D011887:Raffinose