Lenny Nelson

An improved ex vivo method of primary porcine hepatocyte isolation for use in bioartificial liver systems

Introduction Primary porcine hepatocytes are commonly used in bioartificial liver devices and for in vitro studies of hepatocyte function. Although in vivo isolation of porcine hepatocytes can give high yield and viability, such methods are time‐consuming and expensive, requiring specialist surgical facilities. Aim To develop a simple, low‐cost, high viability, high yield, reproducible ex vivo method for obtaining functional porcine hepatocytes for use in bioartificial liver systems. Methods Weanling piglets (12 kg) were killed with pentobarbitone sodium, the infra‐hepatic inferior vena cava was clamped and the supra‐hepatic inferior vena cava cannulated. The whole liver was retrogradely perfused in situ with cold saline and excised, followed by an ex vivo open‐loop and re‐circulating perfusion method (at 37°C) in five steps. The liver was disrupted, sequentially filtered in washing buffer, purified by centrifugation and resuspended in Williams E medium. Viability and cell number were assessed using trypan blue exclusion. The cells were subsequently cultured in serum‐free chemically‐defined medium and function was assessed. Results The time interval from when the animals were killed to the final cell wash was 105 ± 5 min (n = 20). Cell viability was 85 ± 6% with a yield of (2.4 ± 0.5) × 1010 from 12 ± 1 kg piglets using 0.03% (w/v) collagenase (n = 20). Hepatocytes from all isolations were successfully plated and grown in monolayer culture. In freshly isolated hepatocytes (day 0) total protein content (TP) was 1.2 ± 0.1 mg/106 cells (n = 5) and 1.2 ± 0.3 mg/106 cells (n = 5) for day 2 monolayer cultures, corresponding to approximately 9 × 106 hepatocytes per dish. The percentage of total LDH released into the medium was 13 ± 4% for day 0 and 8 ± 4% at day 2; conversely, intracellular LDH activities were 87 ± 4% and 92 ± 4% of the total, respectively. The urea synthesis rate was 196 ± 36 nmol/h/mg total protein at day 0 (n = 5) and 292 ± 62 nmol/h/mg protein (n = 9) at day 2. The total P450 content was 99 ± 11 pmol/mg total protein for fresh cells (n = 5) and maintained at 89 ± 35 pmol/mg total protein in day 2 cultures. Conclusions This ex vivo method provides a high viability, high yield, cost‐effective and rapid technique for isolating functional porcine hepatocytes with high plating efficiency, which compares favourably with results obtained using complex in vivo techniques. Eur J Gastroenterol Hepatol 12:923‐930

PTU-044 Hepatic/endothelial cell co-culture; establishing optimal conditions for liver tissue engineering

Introduction Development of 3D hepatic organoids utilising human cell derivatives for in vitro drug testing and bioartificial liver support systems is challenging. Tissue engineering thick, complex structures such as human liver organoids (micro-tissue; >500 mm) will require vascularisation of 3D cultures—coupled with biomatrix support scaffolds—to both maintain integrity and promote formation of endothelial channels (sinusoid-like structures) to facilitate oxygen and nutrient transfer to hepatocytes in 3D culture. Key challenges involve directing nascent microvessels into an appropriate environment defined prominently by heterotypic cell–cell contacts and meeting high metabolic demands. In this preliminary study, we aimed to optimise heterotypic co-culture of either Human Umbilical Vein Endothelial Cells (HUVECs) or Endothelial Outgrowth Cells (EOCs) with hepatic C3A cells, using appropriate biomatrix scaffolds. Methods Different ratios of HUVECs: C3A or EOCs:C3A to form co-cultures were studied using contrast/confocal microscopy and flow cytometry following appropriate immunostaining. Flow cytometry was used to study integrin expression (Cd49a, Cd49b, Cd49f, Cd49e), endothelial markers (CD31) and EpCam, as an hepatic marker. Fingerprint unbiased metabolomics analysis was used to assess function of co-cultured cells. Matrigel and MaxGel (Sigma) were tested as candidate bioscaffolds and were compared with standard 2D cultures on plastic and collagen for each cell line as controls. Results A ratio 3:1 (HUVECs:C3A) was optimal for growth using endothelial culture medium (Lonza EGM-2 medium, UK). Cell phenotype was maintained for 7 days in co-culture with strong integrin (Cd49a, Cd49b, Cd49f, Cd49e), and CD31 expression on HUVECs and EpCam on C3A cells, while a reduction of HUVEC cell number was noted with a parallel increase of C3A cells by day 7—to form more sheet-like structures in co-culture. Metabolomics analysis of culture media showed enhanced urea cycle, lipid synthesis and amino acid utilisation of C3A cells in co-culture. Matrigel promoted formation of microvessel structures, with interconnected channels, in both EOCs and HUVECs; and was superior to MaxGel. MaxGel in 3D sandwich culture promoted differentiated (cuboidal) morphology of C3As, but not EoCs. Conclusion Optimisation of both cell ratios and cell numbers, as well as selection of appropriate culture media are critical factors in developing a successful hepatic co-culture system with the ability to form sinusoid-like/microvessel structures. This study represents an early step towards understanding the requirements of vascularised liver tissue for future clinical/pharmaceutical applications. Competing interests None declared.