Comparison of methods for isolating primary hepatocytes from mini pigs

Abstract

Successful porcine hepatocyte isolation is crucial for the development of bioartificial liver devices and hepatocyte transplantation. Serva collagenase NB grades are formulated collagenases that are suitable for various tissue isolation applications. N-acetylcysteine (NAC) can improve the viability of human hepatocytes. The aim of this study was to compare the effectiveness of two collagenases and effect of NAC on hepatocyte isolation from porcine liver tissue. Porcine hepatocytes were isolated using the perfusion method from Bama mini pigs assigned to the Serva NB 4 group (n=6), the Serva NB 8 group (n=6), or the NB 8+NAC group (n=6). Viability and yield were defined as fresh hepatocytes and their spheroids formation after 24-hour rocker culture in serum-free medium. Metabolic function was assessed by gene expression, albumin, and urea synthesis. All procedures resulted in successful hepatocyte isolation. Cells from the NB 8+NAC group had (97.8±1.9)% viability, which was higher than the NB 8 group with (94.4±2.4)% and the NB 4 group with (94.5±3.2)% (P<.001). The final cell yield reached (11.8±1.0)×10(9) cells in the NB 8+NAC group, compared to (9.5±2.1)×10(9) cells in the NB 8 group (P<.01) and (9.1±1.1) ×10(9) cells in the NB 4 group (P<.001). The secretion of albumin was superior in the NB 8+NAC group at a concentration of (425.8±35.3) ng/mL compared to the NB 8 group (339.1±32.6) ng/mL (P <.001) and NB 4 group (293.6±43.3) ng/mL (P <.01). The injury of hepatocytes also decreased in the NB 8+NAC group (P<.01). The data are presented as means ± SD. Formulated collagenase Serva NB 8 and NAC could improve the porcine hepatocyte isolation, resulting in higher yields of viable cells.

Isolations and Cultures of Primary Hepatocytes

“…With upgrade of commercial collagenase products such as Roche Liberase research grades and Serva collagenase NB grades, the isolations are more specific to the species, while the purity, viability, and yield of isolated primary hepatocytes are highly improved [4,18]. Except for the primary component, highly purified collagenaseand Ⅱmixture, we found that the amount of neutral protease thermolysin also determines the viability and cell yield, and especially the further success of spheroid formation.…”

“…It also has direct antioxidant properties and appears to have hepatoprotective effects against liver ischemia/reperfusion injury to improve the viability [9]. It has been proved to improve the viability and conserves the metabolic function of hepatocytes [4,18].…”

“…Mechanical dissociation and filtering is necessary to remove connective tissue, and subsequent centrifugation is required to separate hepatocytes from both dead hepatocytes and nonparenchymal cells [4]. Subsequent centrifugation can enrich hepatocytes, and remove part of contamination of other cell types, but when the yield is low, or when selective removal of all nonparenchymal cells, purification is required.…”

“…Freshly isolated primary hepatocytes retain their liver-specific functions [4]; therefore, they continue to be a valuable cell resource to develop alternative treatments such as hepatocyte transplantation and bioartificial liver support systems for the treatment of acute liver failure, chronic liver diseases and several inherited metabolic disorders of the liver. In addition, primary hepatocytes have been used for many aspects of biochemical and pharmaceutical research to understand the biological processes occurring in the liver.…”

“…They were later isolated from many other species, including mice [6], bovines [7], pigs [4,8] and human [9]. Large-scale of hepatocytes without the loss of essential specific differentiated functions are required to advance above-mentioned tissue engineering, gene therapy and extracorporeal liver support systems [10,11].…”