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"n Normal 0 false false false EN-US X-NONE AR-SA MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Arial; mso-bidi-theme-font:minor-bidi;} Background: Proteolytic enzymes, especially collagenases, are used for digestion of extracellular matrix, cell isolation and primary culture. Because of the problems in purification and low amount of collagenases in bacterial or animal sources, it is important to find new sources of the enzymes. So, in the present study actinidin, a plentiful protein in kiwifruit was purified and a mixture of actinidin and trypsin was applied to isolate rat aortic endothelial cells."n"nMethods: Aortic endothelial cells were isolated using digestion solution containing different concentrations of actinidin (from 2 to 16 mg/ml) and trypsin (0.3, 0.6, 1.2 and 2.4 mg/ml) in different times (from 15 to 90 minute). Isolated cells were cultured in DMEM culture medium. Isolated cells were identified by morphological characteristics and immunocytochemical staining; viability of separated cells was estimated by trypan blue exclusion test."n"nResults: Actinidin in concentration of 10 mg/ml with trypsin in concentration of 1.2 mg/ml for one hour could isolate rat aortic endothelial cells. In this condition the viability of cells was estimated 90%. Morphological and immunocytochemical charac-teristics confirmed the isolated cells as endothelial cells."n"nConclusion: The results showed that the mentioned mixture of actinidin and trypsin has not considerable toxic effects on separated cells and is a novel and suitable option for isolation of rat aortic endothelial cells.

AbstractBackground 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%.Key words: Calcium chelator, Collagenase, Hepatocytes, Perfusion, Rat, LiverJ Mazand Univ Med Sci 2008; 18(64): 71-80 (Persian)

Introduction & Objective: Isolation of cells from different tissues rely on proteolytic enzymes mainly collagenases that selectively digest collagen fibers of extra-cellular matrix. It is important to find new and proper collagenases from plant sources. In the present research actinidin, a cysteine protease abundant in Kiwifruit, was used to isolate and culture of rat hepatocytes. Materials & Methods: Different concentrations of actinidin was used to isolate rat hepatocytes by one or two-step perfusion method. The viability of the separated cells was examined by the trypan blue test. The isolated rat hepatocytes were cultured on collagen coated plates in William´s E medium. The morphology of hepatocytes was examined microscopically after staining with the Papanicolaou method. Results: Actinidin in the concentration of 0.4 mg/ml in two-step perfusion method properly isolated hepatocytes from rat liver. The viability of isolated hepatocytes that successfully cultured in collagen coated plates was 90-95 percent. Conclusion: These results showed that actinidin is a proper protease for isolation of hepatocytes. In addition, purification of this enzyme is simpler than the collagenases.