Decrease In Total Serum Bilirubin Research Articles

Selective intraportal hepatocyte transplantation in analbuminemic and Gunn rats.

Although significant progress has been achieved in isolated hepatocyte transplantation, the optimal site of cell implantation has not yet been determined. We have developed a novel experimental method of intraportal hepatocyte transplantation that allows easy assessment of the morphology and function of transplanted hepatocytes. Donor hepatocytes were harvested from Sprague-Dawley rats by in situ EDTA/collagenase perfusion. Fifteen recipient Nagase analbuminemic rats (NAR) underwent cannulation of the gastroduodenal vein under ether anesthesia. Either the posterior or anterior liver lobes were selectively infused with cells by occluding the portal venous supply of the nontransplanted liver lobes. Normal donor hepatocytes (2 x 10(7)) suspended in normal saline were infused over 1 min (4 ml). Recipients were treated with cyclosporine for the duration of the experiment. Plasma albumin levels were determined by ELISA, before and at various intervals after transplantation. In NAR rats transplanted with normal hepatocytes, there was a significant (P < 0.003) and sustained (12 weeks) increase in plasma albumin levels. Control NAR rats transplanted with NAR hepatocytes (n = 8) showed no significant changes in plasma albumin levels. Similarly, normal Wistar hepatocytes were infused intraportally into the posterior lobes of Gunn rats (n = 4), which lack the ability to conjugate bilirubin. Pre- and posttransplantation bile was collected following bile duct cannulation. Bile analysis by HPLC, demonstrated a significant (P = 0.04) increase in the level of bilirubin conjugates following transplantation and a corresponding decrease in total serum bilirubin (P = 0.04). Our experimental data demonstrate that direct selective intraportal infusion of hepatocytes is an effective technique of hepatocyte transplantation in the rat.

Delivery of whole liver-equivalent hepatocyte mass using polymer devices and hepatotrophic stimulation.

Using hepatocytes injected into prevascularized polymer sponge devices, we studied hepatocyte survival and function after delivery of a whole liver-equivalent of cells into rats. LEW rats and enzyme-deficient Gunn rats served as recipients, respectively. Totally, 28.5 cm2 (0.5-cm thick) of polyvinyl alcohol sponges were implanted per animal. Hepatotrophic stimulation was induced by portacaval shunt and partial (70% or 30%) hepatectomy. Recipient rats received 5 x 10(8) hepatocytes (equivalent to whole rat liver) which were harvested from LEW and Wistar donors, respectively. After engraftment, histologic examination revealed hepatocyte remodeling in the device with capillaries lining plates of hepatocytes, and also tubular structures resembling early biliary radicles. BrdU staining revealed DNA synthesis in hepatocytes, providing evidence of regeneration within the grafts. Quantification of viable hepatocyte area at various time points was performed using computer-assisted morphometry. We then estimated a range of cell numbers from the quantitated cell area. The number of hepatocytes viable at day 7 was estimated at 27.5-46.0% and 6.6-11.0% in the mesentery and subcutaneous site, respectively. Thus the average number was estimated between 10.8% and 18.0% of initially injected hepatocytes. In the Gunn rat experiment, experimental rats that received normal Wistar hepatocytes showed a significantly greater decrease in total serum bilirubin compared with the concurrent control Gunn rats (P < 0.01). At week 1, serum bilirubin in experimental rats decreased to 74.7% (6.80 +/- 0.46 mg/dl) of pretransplantation level (9.10 +/- 0.47 mg/dl) and this was 71.4% of the control rats' bilirubin level (9.53 +/- 0.37 mg/dl). In conclusion, a hepatocyte mass equivalent to a whole rat liver can be delivered into prevascularized polymer sponge devices. At day 7 between 10.8% and 18.0% of these hepatocytes were estimated to be engrafted and functioning. Further optimization of this technique is necessary before clinical application is considered.

A Morphological and Functional Evaluation of Transplanted Isolated Encapsulated Hepatocytes Following Long-Term Transplantation in Gunn Rats

In this study we investigated the fate of microencapsulated hepatocytes following long-term (6 months) transplantation in Gunn rats. Isolated hepatocytes were microencapsulated with a collagen matrix within an alginate-poly L-lysine composite membrane. Isolated, encapsulated hepatocytes (IEH) or free (unencapsulated) isolated hepatocytes were intraperitoneally transplanted into homozygous Gunn rats that exhibit congenital hyperbilirubinemia. Control Gunn rats received empty microcapsules. Total serum bilirubin was measured at weekly intervals for one month post-IEH transplantation, every two weeks for the next month, and monthly thereafter for up to six months. IEH samples were biopsied from the Gunn rats at monthly intervals and analyzed by light and electron microscopy. A significant (p < 0.01) decrease in total serum bilirubin was observed in IEH transplanted animals during the first month of transplantation. Thereafter, total serum bilirubin levels gradually returned to pre-transplantation levels. A mild, transient decrease in total serum bilirubin was seen in animals transplanted with free (unencapsulated) hepatocytes. No decrease in total serum bilirubin levels was seen in the Gunn rats transplanted with control (empty) microcapsules. Transplanted IEH retained its normal ultrastructure for up to one month and intact microcapsules showed no evidence of hepatocyte rejection, at this time. Degenerative changes observed in the IEH beginning at 2 months post-transplantation, suggests that repeated transplantations may be necessary for long-term effectiveness of IEH therapy.