Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience

Abstract

Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.

Figure 1. Preparation of the Encapsulated Liver Wedge Prior to Perfusion.

Isolation of human hepatocytes was performed from liver wedges (50–413 g). Liver wedges had one cut face (CF) with the remainder of the hepatic capsule being left intact. After washing the liver through exposed vessels with PBS, 20 G cannulae were sutured into suitable vessels with 3/0 prolene purse string sutures. The remaining vessels were oversewn using continuous 3/0 prolene sutures (arrowed). The liver wedge was then ready to be used for the perfusion isolation protocol.

Figure 2. Morphology of Primary Human Hepatocytes after Isolation in Culture.

(a) Comparison of the morphological appearance of primary human hepatocytes isolated from primary biliary cirrhosis (PBC) attached to collagen-coated plates at (i) 2 hours after isolation (magnification 20×), (ii) 1 day after isolation, (iii) 2 days after isolation, (iv) 3 days after isolation and (v) 7 days after isolation using light microscopy. The representative images show the change in morphology of primary human hepatocytes isolated from PBC livers during 1-week in culture. Immediately after isolation, cells appear ovoid and phase bright. Following 1 day in culture the cells appear binucleate and have formed a confluent monolayer. Primary human hepatocytes maintain this morphology throughout the one week culture period. Similar morphological changes were observed for human hepatocytes isolated from normal, ALD and normal resected liver tissue (data not shown). (b) Representative images of the morphology of human hepatocytes isolated from normal, PBC/PSC, ALD and normal resected liver tissue are shown in culture 3 days after successful cell isolation. Human hepatocytes demonstrated and maintained this morphology for at least one week following successful isolation. (i) Demonstrates the morphology of human hepatocytes isolated from normal liver tissue and macro-steototic liver tissue (magnification 20×). Primary human hepatocytes isolated from normal liver tissue display the typical cubic binucleate cell morphology. In contrast, primary human hepatocytes isolated from overtly macro-steatotic livers demonstrate obvious micro-vesicular steotosis. (ii) Human hepatocytes isolated from end stage biliary cirrhosis (PBC/PSC) again show the typical features of cells in culture with a cubic and binucleate morphology. (iii–iv) Cells isolated from ALD and normal resected liver show similar morphological features to human hepatocytes isolated from normal non-steatotic livers and biliary cirrhosis but also exhibit micro-vesicular steatosis.

Figure 3. Immunostaining of Cytokeratin 18, Cytokeratin 19 and CD326 and Metabolic Activity of Primary Human Hepatocytes Isolated from Normal and Diseased Liver Tissue.

(a) Human hepatocytes isolated from normal, PBC, ALD and normal resected liver tissue show strong staining for the intracellular hepatocyte marker Cytokeratin 18 (CK18). In contrast, isotype matched controls immunoglobulin showed no positivity. Furthermore, isolated human hepatocytes showed no staining with the biliary epithelial cell marker Cytokeratin 19 (CK19) or hepatic progenitor cell marker CD326 (EpCAM). Cytospins of human hepatocytes were made immediately after successful cell isolation, fixed in acetone for 5 min and stored at −20°C until immuno-staining was performed. (b) Following successful isolation, human hepatocytes isolated from normal, PBC/PSC, ALD or normal resected liver tissue were able to synthesise albumin (i) for at least one week demonstrating active metabolic activity in culture. Human hepatocytes isolated from normal, PBC/PSC and normal resected liver tissue show significantly greater albumin synthesis after two days in culture (*p<0.05). Morover, human hepatocytes isolated from ALD livers synthesised significantly less albumin than other types of human hepatocytes (p<0.05). In addition, human hepatocytes isolated from normal resected liver tissue synthesised significantly greater amounts of albumin than normal hepatocytes (p<0.037). (n = 3–4 separate samples). Human hepatocytes isolated from normal, PBC/PSC, ALD and normal resected liver tissue were also able to synthesise urea (ii) for at least one week following successful cell isolation. Human hepatocytes isolated from both normal and diseased liver tissue synthesised significantly lower levels of urea one day after isolation (§p<0.001) compared to the remainder of the culture period, but all human hepatocytes produced significantly more urea on day 2 when compared to other days (*p<0.001). Finally, PBC/PSC and normal resected human hepatocyte synthesis significantly greater amounts of urea when compared to the other human hepatocytes (p<0.05) but there was no significant difference between PBC/PSC and resected human hepatocytes. (n = 3–4 separate samples).