Cold preservation of isolated hepatocytes in UW solution: experimental studies on the respiratory activity at 0 degrees C.
To date, little attention has been paid to the role of the gas milieu in preservation solutions and its effect on cell viability. Dissolved O2 in the preservation media may be an important parameter to consider. In this study we polarographically measured the O2 concentration in air-equilibrated UW solution at 0 degrees C, as well as the respiratory activity of isolated hepatocytes cold-preserved in this solution up to 72 hours. To perform measurements at 0 degrees C, it was first necessary to characterize the sensor behavior at low temperatures. We verified that the sensor response is still linear at this temperature but the rate of response is significantly slower. The O2 solubility in UW-air solution at 0 degrees C was determined using a modified physical method and it was 410 microM O2, which, as expected, is lower than the solubility in water at the same temperature (453 microM O2). Isolated hepatocytes cold-stored in UW-air solution retained a measurable respiratory activity during a period of 72 hours. The O2 consumption rate was 0.48 +/- 0.13 nmol/O2/min/10(6) cells, which represents 1% of the control value at 36 degrees C (61.46 +/- 14.61 nmol/O2/min/10(6) cells). The respiratory activity and cell viability were well maintained during the preservation period. At present, preservation conditions need to be improved for cells to remain functionally active. Dissolved O2 may be required for energy re-synthesis but it also leads to an increment in reactive oxygen species. The O2 concentration in the preservation solution should be carefully controlled, reaching a compromise between cell requirement and toxicity.
Llarrull
Maria Soledad
MS
Farmacologia, departamento de Ciencias Fisiologicas; Facultad de ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Argentina.
Pizarro
Maria Dolores
MD
Scandizzi
Angel L
AL
Bottai
Hebe
H
Guibert
Edgardo E
EE
Rodriguez
Joaquin V
JV
D000241:Adenosine; D000493:Allopurinol; D000818:Animals; D019069:Cell Respiration / Q000502:physiology*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents; D005978:Glutathione; D022781:Hepatocytes / Q000378:metabolism*; D007328:Insulin; D008297:Male; D019188:Organ Preservation Solutions; D010100:Oxygen / Q000032:analysis; D010101:Oxygen Consumption; D011887:Raffinose; D051381:Rats; D017208:Rats, Wistar; D013696:Temperature
D003451:Cryoprotective Agents; D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose; D010100:Oxygen