Human Hepatocyte Isolation: Does Portal Vein Embolization Affect the Outcome?

Abstract

Primary human hepatocytes are widely used for basic research, pharmaceutical testing, and therapeutic concepts in regenerative medicine. Human hepatocytes can be isolated from resected liver tissue. Preoperative portal vein embolization (PVE) is increasingly used to decrease the risk of delayed postoperative liver regeneration by induction of selective hypertrophy of the future remnant liver tissue. The aim of this study was to investigate the effect of PVE on the outcome of hepatocyte isolation. Primary human hepatocytes were isolated from liver tissue obtained from partial hepatectomies (n = 190) using the two-step collagenase perfusion technique followed by Percoll purification. Of these hepatectomies, 27 isolations (14.2%) were performed using liver tissue obtained from patients undergoing PVE before surgery. All isolations were characterized using parameters that had been described in the literature as relevant for the outcome of hepatocyte isolation. The isolation outcomes of the PVE and the non-PVE groups were then compared before and after Percoll purification. Metabolic parameters (transaminases, urea, albumin, and vascular endothelial growth factor secretion) were measured in the supernatant of cultured hepatocytes for more than 6 days (PVE: n = 4 and non-PVE: n = 3). The PVE and non-PVE groups were similar in regard to donor parameters (sex, age, and indication for surgery), isolation parameters (liver weight and cold ischemia time), and the quality of the liver tissue. The mean initial viable cell yield did not differ between the PVE and non-PVE groups (10.16 ± 2.03 × 10(6) cells/g vs. 9.70 ± 0.73 × 10(6) cells/g, p = 0.499). The initial viability was slightly better in the PVE group (77.8% ± 2.03% vs. 74.4% ± 1.06%). The mean viable cell yield (p = 0.819) and the mean viability (p = 0.141) after Percoll purification did not differ between the groups. PVE had no effect on enzyme leakage and metabolic activity of cultured hepatocytes. Although PVE leads to drastic metabolic alterations and changes in hepatic blood flow, embolized liver tissue is a suitable source for the isolation of primary human hepatocytes and is equivalent to untreated liver tissue in regard to cell yield and viability.

The isolation of primary hepatocytes from human tissue: optimising the use of small non-encapsulated liver resection surplus

“…Using our methodology we found an average total yield of viable hepatocytes to be 28,000,000 cells, which is 80 times higher than that reported by Bhogal et al ( 2011 ). In contrast, others have reported viable yields between 2.6 × 10 6 and 18.7 × 10 6 /g tissue (Alexandre et al 2002 ; Alexandrova et al 2005 ; Vondran et al 2008 ; Kluge et al 2016 ) using perfusion whilst we report here an average viable cell yield of 0.64 ± 0.19 × 10 6 /g tissue. The discrepancy in reported yields between studies using perfusion and our methodology may be in part explained by the efficiency of perfusion, collagenase used, and the quality of liver tissue.…”

“…The first reported isolation of human primary hepatocytes was published just over two decades ago (Guguen-Guillouzo et al 1982 ). In recent years, there have been an increasing number of reports of human hepatocyte isolation using the two-step perfusion method and these studies have predominantly used fragments of resected liver tissue (usually removed due to primary or secondary tumours) (Bhogal et al 2011 ; Lee et al 2013 , 2014 ; Pfeiffer et al 2015 ; Kegel et al 2016 ; Kluge et al 2016 ).…”

“…Many factors may influence the viability and yield of hepatocytes from human liver tissue including donor age and gender, presence of liver steatosis or fibrosis, and donor serum enzymes (Bhogal et al 2011 ; Lee et al 2014 ). However, Kluge et al ( 2016 ) reported the perfusion method gave high yields of hepatocytes (9.56 × 10 6 /g tissue) with high viability (74%) in liver tissue from individuals with or without portal vein embolization. In contrast, Bhogal et al ( 2011 ) suggested that median viability of hepatocytes isolated from healthy and disease tissue was 40%, with a total hepatocyte yield of 350,000 cells.…”

Cell sources forin vitrohuman liver cell culture models

“…due to clamping during surgery, can lead to an impaired yield and viability of PHH. 8 In contrast, portal vein embolization showed no negative influence on isolation outcome, 10 and the yield of PHH was even increased with warm ischemia times ex vivo , and when the patients received chemotherapeutic treatment. 9 Furthermore, the type of disease was identified to have a significant impact upon the cell yield.…”

Impact of Percoll purification on isolation of primary human hepatocytes

“…An average of 56.4% of the cells could be recovered from Percoll-purification, whereas yield was significantly diminished from 25.90 (±9.72) × 10 6 to 15.3 (±8.66) × 10 6 cells per gram of liver tissue (Table 2). When analyzing the features of this subgroup, the three cases differed from the overall group in a few known factors 24,31–33 that are known to affect the isolation outcome, including preoperative levels of ALT, AST, AP, GGT and in the operating technique (Table 3).…”