*Purpose: Develop organogenesis through ectopic hepatocyte transplantation (HT) into the upper abdominal lymph nodes (LN).

*Methods: Three groups of Mongrel dogs weighting between 25-35 kg (control (C) n=3, autologous (AU) n=6; allogeneic (AL) n=21) underwent heterotopic HT into their LNs through either direct or endoscopic ultrasound (EUS) guided endoluminal injections. The hepatocytes were isolated from the liver left lateral segment of a donor group of animals (n=10) using a modified two-step collagenase perfusion method and transplanted using 3 different doses (low=75M, medium=150M and high=750M) through 1 ml infusions into the LNs. All the recipients underwent an end-to-side portacaval shunt (PCS) as a way to induce sub-acute liver failure and upregulation of hepatotropic factors. These recipients received immune suppressive (IS) therapy (Tacrolimus (Tac) and Prednisone) post-operatively for the duration of the experiments (30-180 days follow up).

*Results: The median cold ischemia time for HT was 25.1 hours, ranging from 5.5 to 54.8 hours. The median cell viability was 81%. The C group had a 66% mortality. The AU and AL groups had survival rates of 75% and 78%, respectively. Early deaths were attributed to acute hepatic failure and ischemic bowel. Both primary end points for safety and efficacy were successfully achieved. The AU group had 100% engraftment and the AL 50%. The effect sizes for transplantation of hepatocytes were large for both the AU group (d=1.12, n=6, with one early death due to infection) and the AL group (d=1.01, n=21). A strong association between effect size and dose was also noted, with the highest dose (750M) having the largest effect size (d=2.65, n=3). Histopathological analysis showed the formation of normal ectopic hepatic tissue within the transplanted LNs, including the presence of sinusoids, portal triads and bile ducts in both AU and AL groups. Most animals were required to be maintained on sub-therapeutic Tac levels due to significant adverse events (neurotoxicity 30%, infections 18% and diabetes 12.5%.

*Conclusions: Hepatocyte transplantation can be performed safely and effectively using this new minimally invasive EUS method under standard IS therapy in a fully mismatched allogeneic combination. Suboptimal IS yielded decreased engraftment in the AL group. The ectopic liver tissue had normal hepatic cytoarchitecture and no signs of acute cellular rejection up to 180 days follow up.

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