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

Abstract

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 × 10⁹ hepatocytes 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.

Figure 1

Liver perfusion and digestion system. The isolated liver is maintained at 37°C by water circulation (in blue) supplied by a water bath kept at 37°C. Perfusion and digestion media are maintained at 37°C and oxygenated through the oxygen cylinder (in green). Thereafter, infusion of perfusion media followed by digestion media (in red) into the liver is performed via the vena cava. Flow-through of both media (in brown) is recovered and discarded.

Figure 2

Hepatocyte viability and function after freezing and thawing. (a) The upper panel presents the bright-field image of thawed hepatocytes in culture after 3 days, the middle panel presents viable cells (FDA staining), and the lower panel presents nonviable cells (PI staining). (b) Albumin was measured by ELISA in the supernatant of cultured hepatocytes at days 4, 8, and 11. The experiment is performed in duplicates.

Figure 3

Viability of free and microencapsulated hepatocytes. (a) Representative images at day 3 for free, Alg-microencapsulated, and Alg-PEG-SH-microencapsulated hepatocytes. Upper panels present bright-field images, middle panels present viable cells (FDA staining), and lower panels present nonviable cells (PI staining). (b) Quantification of viable cells (FDA) and nonviable cells (PI) at day 3. Values are expressed as % of the total cell area (n = 6). Quantification has been performed using ImageJ.

Figure 4

Albumin secretion from free and microencapsulated hepatocytes. Albumin was measured by ELISA in the supernatant of free (n = 9) (a), alginate-encapsulated (n = 3) (b), and Alg-PEG-SH-encapsulated (n = 3) (c) hepatocytes. White bars represent albumin secretion during 24 h from day 2 to day 8, and grey bars represent the total albumin secreted during 8 days.

Figure 5

Diazepam metabolism in free and alginate-microencapsulated hepatocytes. Diazepam (white bars) was added at days 1, 3, and 7 on free and alginate-encapsulated hepatocytes, and supernatant was retrieved after 6 hours of culture. Diazepam and its metabolites nordiazepam and temazepam were measured by LC-MS/MS in 4 independent experiments. ^∗ p < 0.05, ^∗∗ p < 0.01.

Figure 6

Viability of coencapsulated hepatocytes and MSC. (a) Representative images of Alg-encapsulated hepatocytes alone or with MSC after 4 days of culture. Upper panels present bright-field images, middle panels present viable cells (FDA staining), and lower panels present nonviable cells (PI staining). (b) Quantification at day 3. Values are expressed as % of the total cell area (n = 2). Quantification has been performed using ImageJ.

Figure 7

Albumin secretion of hepatocytes coencapsulated and cocultured with MSC. Albumin secretion was measured by ELISA in the supernatant of cell culture after 24 hours from day 2 to day 8. (a) Alg-microencapsulated hepatocytes alone (white bars) or with MSC (grey bars), measured in 2 independent experiments. (b) Free hepatocytes alone (white bars) or with MSC (grey bars), measured in 5 independent experiments. ^∗ p < 0.05, ^∗∗∗ p < 0.001.

Figure 8

Distribution of porcine hepatocytes and human MSC in coculture. Cells were cultured for 2 days and then serum-starved to avoid nonspecific staining. Immunofluorescence was performed on cells after 3 days of culture. (a) Hepatocytes are stained for porcine albumin (in red). (b) Coculture of hepatocytes and MSC. MSC are stained for human vimentin (in green). (c) Coculture of hepatocytes and MSC in a higher magnification (×40). Images are representative of 2 independent pig hepatocyte isolations.

Figure 9

Profiling of human cytokines using cell culture supernatants of MSC alone and MSC cocultured with hepatocytes. (a) Human cytokines were analyzed using antibody membranes in MSC alone and MSC cocultured with hepatocytes (0.2 × 10⁶ cells/mL/24-well plate) obtained from 2 different donors. (b) Spots were quantified using ImageJ and expressed as mean pixel density. Cytokines secreted from MSC cultured alone (white bars) and cytokines secreted from MSC cocultured with hepatocytes (black bars). ^∗∗ p < 0.01.