A New Technique for Isolating and Culturing Human Hepatocytes from Whole or Split Livers not Used for Transplantation

Abstract

Large numbers of human hepatocytes were obtained from split and whole livers by using an adaptive form of the collagenase perfusion technique employed in rodent and human biopsies. In order to guarantee a homogenous distribution of the perfusate within the whole specimen, major hepatic veins were cannulated with large bore catheters. This technique allowed for the isolation of human hepatocytes on a large scale (up to 18.5 x 10(9) in one case) from normal and diseased liver specimens. The yield of isolated normal viable hepatocytes is inversely proportional to the donor age. In addition, it was noted that a short time between declared death and organ harvest (cross clamp time) results in higher viability of hepatocytes. In contrast, the time of cold organ preservation did not correlate with the viability or the yield of isolated hepatocytes. We conclude that the technique presented here allows isolation of large numbers of human hepatocytes from specimens unsuitable for transplantation but very valuable for biomedical research.

A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle

“…Hepatocytes were isolated as previously described. 26 Confocal Laser Scanning Microscopy. Huh7 cells (5 ϫ10 4 ) or primary human hepatocytes were grown on cover slides in 24-well plates and incubated with 100 L purified wild-type or TLM nucleocapsids (20 nmol/L) isolated from triple-infected Sf9 cells that were diluted in DMEM.…”

Coexpression of p21WAF1/CIP1 in adenovirus vector transfected human primary hepatocytes prevents apoptosis resulting in improved transgene expression

“…In accordance with institutional guidelines, human hepatocytes were isolated from normal liver wedge resections by collagenase-P (Roche Diagnostics, Grenzau, Germany) digestion as recently described. 44,45 Briefly, after enzymatic digestion hepatocytes were separated from non-parenchymal cells by differential centrifugation at 50 g and then passed over a 30% Percoll (Pharmacia, Freiburg, FRG) gradient at a concentration of 10 6 cells/ml Percoll to obtain a highly purified cell population. Hepatocyte purity and viability assessed by microscopy was greater than 95% and viability consistently exceeded 90% by trypan blue exclusion.…”

“…Hepatocyte purity and viability assessed by microscopy was greater than 95% and viability consistently exceeded 90% by trypan blue exclusion. 45 The primary hepatocytes were cultured in Williams Medium E, supplemented with 10% heatinactivated (90 min at 701C) FBS, 1% penicillin/streptomycin, 15 mM hepes buffer (all Life Technologies, Karlsruhe, FRG), 2.0 mg/ml insulin and 0.35 nM Hydrocortisone (both Sigma, Deisenhofen, FRG) at 371C, 5% CO 2 in a humidified atmosphere.…”

Adenovirus-mediated gene transfer of P16INK4/CDKN2 into bax-negative colon cancer cells induces apoptosis and tumor regression in vivo

“…11,49 In accordance with institutional guidelines, human hepatocytes were isolated from normal liver wedge resections by collagenase -P (Roche Diagnostics, Grenzau, Germany ) digestion as recently described. 50,51 Briefly, after enzymatic digestion hepatocytes were separated from nonparenchymal cells by differential centrifugation at 50Âg and then passed over a 30% Percoll (Pharmacia, Freiburg, Germany ) gradient at a concentration of 10 6 cells / mL Percoll to obtain a highly purified cell population. Hepatocyte purity and viability assessed by microscopy was greater than 95% and viability consistently exceeded 90% by trypan blue exclusion.…”

“…Hepatocyte purity and viability assessed by microscopy was greater than 95% and viability consistently exceeded 90% by trypan blue exclusion. 51 The primary hepatocytes were cultured in Williams Medium E, supplemented with 10% heat -inactivated (90 minutes at 708C ) FBS, 1% penicillin /streptomycin, 15 mM Hepes buffer (all Life Technologies, Karlsruhe, Germany ), 2.0 g /mL insulin, and 0.35 nM hydrocortisone ( both Sigma, Deisenhofen, Germnay ) at 378C, 5% CO 2 in a humidified atmosphere.…”