Beneficial Effects of Human Mesenchymal Stromal Cells on Porcine Hepatocyte Viability and Albumin Secretion

Porcine hepatocytes transplanted during acute liver failure might support metabolic functions until the diseased liver recovers its function. Here, we isolated high numbers of viable pig hepatocytes and evaluated hepatocyte functionality after encapsulation. We further investigated whether coculture and coencapsulation of hepatocytes with human multipotent mesenchymal stromal cells (MSC) are beneficial on hepatocyte function. Livers from 10 kg pigs (n=9) were harvested, and hepatocytes were isolated from liver suspensions for microencapsulation using alginate and poly(ethylene-glycol)- (PEG-) grafted alginate hydrogels, either alone or in combination with MSC. Viability, albumin secretion, and diazepam catabolism of hepatocytes were measured for one week. 9.2 ± 3.6 × 109hepatocytes with 95.2 ± 3.1% viability were obtained after isolation. At day 3, free hepatocytes displayed 99% viability, whereas microencapsulation in alginate and PEG-grafted alginate decreased viability to 62% and 48%, respectively. Albumin secretion and diazepam catabolism occurred in free and microencapsulated hepatocytes. Coencapsulation of hepatocytes with MSC significantly improved viability and albumin secretion at days 4 and 8 (p<0.05). Coculture with MSC significantly increased and prolonged albumin secretion. In conclusion, we established a protocol for isolation and microencapsulation of high numbers of viable pig hepatocytes and demonstrated that the presence of MSC is beneficial for the viability and function of porcine hepatocytes.

Contribution of bone marrow mesenchymal stem cells to porcine hepatocyte culture in vitro

One of the greatest challenges in the attempt to create functional bioartificial liver designs is the maintenance of porcine hepatocyte differentiated functions in vitro. Co-cultivation of hepatocytes with nonparenchymal cells may be beneficial for optimizing cell functions via mimicry of physiological microenvironment. However, the underlying mechanisms remain to be elucidated. An equal number of freshly isolated porcine hepatocytes and purified bone marrow mesenchymal stem cells (MSCS) was randomly co-cultured and the morphological and functional changes of heterotypic interactions were characterized. Furthermore, contributions of soluble factors involved in the separated co-culture system were evaluated. The purity of the third-passage MSCS and primary hepatocytes was more than 90% and 99%, respectively. Hepatocyte viability was greater than 95%. A rapid attachment and self-organization of three-dimensional hepatocyte spheroids were encouraged, which was due to the supporting MSCS of high motility. The elevated induction of both albumin production and urea synthesis was achieved in co-culture (P < 0.05). Data from semipermeable membrane cultures suggested that interleukin-6 is one of the key stimulators in hepatic functional enhancement. These results demonstrate for the first time that soluble factors have beneficial effects on the preservation of hepatic morphology and functionality in the co-culture of hepatocytes with MSCS in vitro, which could represent a promising tool for tissue engineering, cell biology, and bioartificial liver devices.