Comparison of isolated hepatocytes and tissue slices for study of liver hypothermic preservation/reperfusion injury.
Simple models are needed that effectively test the variables that may be important in liver preservation. Two such models are isolated hepatocytes and tissue slices. In this study the effects of hypothermic preservation on the viability of hepatocytes (HC) and tissue slices (TS) from rat livers were measured by LDH leakage after cold storage and rewarming. We compared how glycine, calcium, and fasting, shown previously to affect preservation injury in hepatocytes, affected both HC and TS viability. Hepatocytes were cold-stored in University of Wisconsin organ preservation solution for up to 48 h and rewarmed in Krebs-Henseleit Bicarbonate (KHB) for 120 min. Tissue slices were studied in two ways. Either livers were cold-stored intact and then tissue slices (TS-A) prepared and rewarmed in KHB, or tissue slices were prepared from the fresh liver, cold-stored, and then rewarmed (TS-B). The latter method may be similar to cold storage of HC. Freshly prepared samples (HC, TS-A, or TS-B) showed < 15% LDH leakage during the rewarming phase. Cold storage for 24 h resulted in < 30% LDH leakage in all preparations. After 48 h cold storage there was a significant increase in LDH leakage (HC, 65.1 +/- 5.1%; TS-A, 52.9 +/- 0.8%, TS-B, 53.6 +/- 2.6%). Glycine (3 mM) or calcium (1.5 mM) included in the KHB significantly reduced LDH leakage from 48 h cold-stored HC to 20.7 +/- 1.8 and 26.3 +/- 2.4%, respectively. These agents caused a smaller decrease in LDH release from tissue slices (around 40%). Hepatocytes appear more susceptible to preservation/reperfusion damage than the more structurally intact tissue slices as suggested by the greater release of LDH. Another difference was that the agents which improved preservation quality of HC were not as effective in TS. Hepatocytes may be more vulnerable to preservation/reperfusion damage because of the harsh methods used in their preparation. The damage induced during preparation appears amenable to suppression by glycine or calcium. Tissue slices, which are intact pieces of liver tissue, may be more suitable for studies related to development of better methods for liver preservation. The intact cells in TS have not been exposed to harsh conditions and maintain a more natural cell-cell relationship.
Vreugdenhil
P K
PK
Department of Surgery, University of Wisconsin, Madison, USA.
Marsh
D C
DC
Southard
J H
JH
D000241:Adenosine; D000493:Allopurinol; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002469:Cell Separation; D003080:Cold Temperature; D005215:Fasting; D005978:Glutathione; D005998:Glycine / Q000494:pharmacology; D066298:In Vitro Techniques; D007328:Insulin; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000098:blood supply / Q000166:cytology / Q000293:injuries*; D019188:Organ Preservation Solutions; D011887:Raffinose; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000209:etiology* / Q000517:prevention & control; D014021:Tissue Preservation / Q000379:methods*
D007328:Insulin; D019188:Organ Preservation Solutions; C055206:University of Wisconsin-lactobionate solution; D000493:Allopurinol; D007770:L-Lactate Dehydrogenase; D005978:Glutathione; D000241:Adenosine; D011887:Raffinose; D002118:Calcium; D005998:Glycine