پرفیوژن و جداسازی هپاتوسیت از کبد رات

Background and Purpose: Perfused rat liver and isolated hepatocytes are useful to study specific metabolism of this organ. Traditionally, mechanical, chemical and enzymatic methods have been used to isolate hepatocytes, and it appears that a mixed method is the preferred choice. Materials and Methods: The rat liver perfused in situe in three subsequent steps, 10 minutes with Krebs-Ringer solution containing ethylene diamine tetra-acetic acid (EDTA, 2mM) without calcium and magnesium, and 1 minute with the same solution, however, without EDTA in open non-recirculatory pathway. This followed by 10 minutes with Krebs-Ringer solution, containing collagenase (200 IU/mL) in closed recirculatory system. The hepatocytes were isolated, collected and incubated (6-8 × 106 cells/mL) for 3-hours in Krebs-Ringer solution, under the atmosphere of O2:CO2 (95:5). The cell viability was assessed with tripan blue exclusion and lactate dehydrogenase (LDH) leakage. Results: Assay of LDH activity, the marker of cytosolic enzyme, show that more than 92% and 88% of the enzyme had been retained in the cells, with less than 8% and 12% leakage from the damaged cells in the beginning and end of incubation, respectively. Microscopic evaluation showed that the majority of the cells had intact plasma membrane without vacuoles. When the cells were stained with dye, less than 5% of vital dye included these cells. Conclusion: The results showed that perfusion of the rat liver with the solution containing calcium chealator and collagenase can yield about 10 mL of hepatocytes with a viability of more than 90%.