The Optimization of Short-Term Hepatocyte Preservation Before Transplantation

Abstract

Background: No optimal methods for short-term hepatocyte preservation have been established. We have recently developed a prominent oxygen-permeable bag (Tohoku Device [TD]) for pancreatic islet culture and transplantation. In this study, we investigated whether TD is also effective for hepatocyte preservation and tried to optimize other conditions.

Methods: Hepatocytes were preserved in the following conditions, and their outcomes were observed. First, the effectiveness of TD was investigated. Second, hepatocyte medium (HM) and organ preservation solutions with or without fetal bovine serum (FBS) were compared. Third, as supplementations, FBS and human serum albumin (HSA) were compared. Fourth, low, room and high temperature were compared. And finally, hepatocytes preserved in various conditions were transplanted into the subrenal capsule space of nonalbumin rats and engrafted areas were assessed.

Results: The survival rate of hepatocytes preserved in TD tended to be higher and their viability and function were maintained significantly greater than those of non-TD group. Irrespective of FBS supplementation, the survival rate of HM group was significantly higher than those of organ preservation solution group while viabilities and plating efficiency were similar among them. Although survival rates of groups without FBS were extremely low, results of HSA supplemented group were not inferior to FBS supplemented group. Hepatocytes preserved at high temperature had the worst results. The engrafted area of TD group tended to be higher than those of other groups.

Conclusions: TD is effective for short-term hepatocyte preservation. HSA is a useful substitute for FBS, and preserving in HM at low temperature is recommended.

FIGURE 1

The effects of the TD and a commonly used plastic tube on the efficiency of short-term hepatocyte preservation. A, The survival rate of isolated hepatocytes after 2-hour preservation in the Non-TD group (black bar: 70.3 ± 14.4%, n = 8), TD-shaking group (gray bar: 77.4 ± 8.6%, n = 8) and the TD-quiet group (dotted bar: 82.3 ± 12.0%, n = 8). B, The viability of the isolated hepatocytes after 2-hour preservation in the Non-TD group (black bar: 80.4 ± 5.0%, n = 8), the TD-shaking group (gray bar: 86.5 ± 4.3%, n = 8) and the TD-quiet group (dotted bar: 88.5 ± 3.7%, n = 8). *P < 0.01, the non-TD group versus the TD-quiet group. **P < 0.05, the non-TD group vs. the TD-shaking group. C, The PE of 18-hour cultured hepatocytes after 2-hour preservation in the non-TD group (black bar: 52.0 ± 8.3%, n = 8), TD-shaking group (gray bar: 61.8 ± 9.0%, n = 8) and the TD-quiet group (dotted bar: 65.0 ± 10.4%, n = 8). **P < 0.05, the Non-TD group vs. the TD-quiet group. D, The DNA quantitation of 5-day cultured hepatocytes after 2-hour preservation in the Non-TD group (black bar: 13.1 ± 3.7 μg, n = 8), the TD-shaking group (gray bar: 11.3 ± 1.2 μg, n = 8) and the TD-quiet group (dotted bar: 11.8 ± 1.5 μg, n = 8).

FIGURE 2

The effects of solutions with/without FBS supplementation on the efficiency of short-term hepatocyte preservation. A, The survival rate of isolated hepatocytes after 4-hour preservation in the HM/FBS(+) group (black bar: 76.5 ± 15.9%, n = 8), the ETK/FBS(+) group (dotted bar: 53.7 ± 18.3%, n = 8), the UW/FBS(+) group (oblique line bar: 33.8 ± 9.8%, n = 8), the HM/FBS(−) group (gray bar: 33.3 ± 19.0%, n = 8), the ETK/FBS(−) group (horizontal line bar: 7.4 ± 2.9%, n = 8) and the UW/FBS(−) group (vertical line bar: 9.4 ± 4.5% n = 8). *P < 0.01, the HM/FBS(+) group vs. the ETK/FBS(+), and the UW/FBS(+) group; the HM/FBS(−) group vs. the ETK/FBS(−) group. **P < 0.05, the ETK/FBS(+) group vs. the UW/FBS(+) group; the HM/FBS(−) group vs. the UW/FBS(−) group. B, The viability of the isolated hepatocytes after 4-hour preservation in the HM/FBS(+) group (black bar: 89.5 ± 2.7%, n = 8), the ETK/FBS(+) group (dotted bar: 88.3 ± 3.0%, n = 8), the UW/FBS(+) group (oblique line bar: 89.1 ± 2.9%, n = 8), the HM/FBS(−) group (gray bar: 71.5 ± 9.6%, n = 8), the ETK/FBS(−) group (horizontal line bar: 68.0 ± 10.2%, n = 8) and the UW/FBS(−) group (vertical line bar: 73.1 ± 10.1%, n = 8). C, The PE of 24-hour cultured hepatocytes after 4-hour preservation in the HM/FBS(+) group (black bar: 65.4 ± 10.4%, n = 8), the ETK/FBS(+) group (dotted bar: 58.9 ± 11.8%, n = 8), UW/FBS(+) group (oblique line bar: 62.3 ± 12.7%, n = 8), the HM/FBS(−) group (gray bar: 52.5 ± 10.2%, n = 8), ETK/FBS(−) group (horizontal line bar: 51.0 ± 6.0%, n = 8) and the UW/FBS(−) group (vertical line bar: 48.7 ± 8.5%, n = 8).

FIGURE 3

The effects of FBS and albumin supplementation on the efficiency of short-term hepatocyte preservation. A, The survival rate of isolated hepatocytes after 4-hour preservation in the HM/FBS(+) group (black bar: 67.9 ± 15.6%, n = 8) and the HM/Alb(+) group (gray bar: 66.8 ± 14.1%. n = 8). B, The viability of isolated hepatocytes after 4-hour preservation in the HM/FBS(+) group (black bar: 89.8 ± 3.2%, n = 8) and the HM/Alb(+) group (gray bar: 86.6 ± 2.7%, n = 8). **P < 0.05, the HM/FBS(+) group vs. the HM/Alb(+) group. C, The PE of 24-hour cultured hepatocytes after 4-hour preservation in the HM/FBS(+) group (black bar: 58.8 ± 6.7%, n = 8) and the HM/Alb(+) group (gray bar: 60.1 ± 10.6%, n = 8). D, In the ammonia removal test, the HM/FBS(+) and HM/Alb(+) groups showed almost the same tendency (1-, 2-, and 3-hour percentages of remaining ammonia concentrations, HM/FBS(+) group: 43.9 ± 7.2, 25.5 ± 6.8 and 16.4 ± 6.2%, respectively; HM/Alb(+) group: 46.2 ± 4.3, 22.6 ± 0.1 and 16.0 ± 4.6%, respectively).

FIGURE 4

The effects of temperature on the efficiency of short-term hepatocyte preservation. A, The survival rate of isolated hepatocytes after 4-hour preservation in the LT group (black bar: 73.9 ± 10.9%, n = 8), the RT group (gray bar: 75.2 ± 11.1%, n = 8) and the HT group (dotted bar: 55.8 ± 12.6%, n = 8). *P < 0.01, the LT group vs. the HT group. **P < 0.05, the RT group vs. the HT group. B, The viability of isolated hepatocytes after 4-hour preservation in the LT group (black bar: 86.1 ± 4.0%, n = 8), the RT group (gray bar: 85.9 ± 3.8%, n = 8) and the HT group (dotted bar: 80.2 ± 5.5%, n = 8). **P < 0.05, the LT group vs. the HT group. C, The PE of 24-hours cultured hepatocytes after 4-hour preservation in the LT group (black bar: 53.8 ± 7.6%, n = 8), the RT group (gray bar: 45.9 ± 12.0%, n = 8) and the HT group (dotted bar: 18.8 ± 12.3%, n = 8). *P < 0.01, the HT group vs. the LT and RT groups. D, The isolated hepatocytes of the 3 groups showed a similar pattern in the ammonia removal test (1-, 2-, and 3-hour percentage of remaining ammonia concentrations, LT group: 31.1 ± 6.4%, 11.2 ± 6.1%, and 8.6 ± 2.2%, respectively; RT group: 25.0 ± 4.0%, 10.9 ± 5.0%, and 8.5 ± 1.6%, respectively; HT group: 23.7 ± 5.3%, 11.3 ± 3.1%, and 8.6 ± 2.2%, respectively).

FIGURE 5

The immunohistochemical analysis of hepatocytes transplanted into the renal subcapsular space. A, Hematoxylin and eosin staining of the hepatocyte grafts transplanted into the renal subcapsular space. B, Albumin staining of the same hepatocyte grafts as (A). The albumin-positive hepatocytes engrafted under the renal subcapsular space are shown with orange arrows. The engrafted area was estimated by calculating the area under the curve of the albumin-positive hepatocytes on 8 pathologic sections per recipient kidney. C, The comparison of engraftment efficiency after short-term hepatocyte preservation under several conditions, including the HM/Tube group (black bar: 2.53 ± 0.87 gigapixels, n = 8), the HM + Alb/Tube group (gray bar: 2.89 ± 0.94 gigapixels, n = 8), the HM + FBS/Tube group (dotted bar: 3.27 ± 1.32 gigapixels, n = 8) and the HM + Alb/TD group (horizontal line bar: 3.06 ± 0.78 gigapixels, n = 8), HM + FBS/TD group (vertical line bar: 3.95 ± 1.13 gigapixels, n = 8).