Gastric submucosa as the safer and repeatable site for hepatocyte transplantation

To establish a safe repeatable method for hepatocyte transplantation avoiding serious complications, such as portal thrombosis in the case of the intraportal route of transplantation, we attempted liver cell transplantation into the submucosal layer of the stomach wall. Hepatocytes were isolated from the Lewis rats by a two-step collagenase perfusion method. The final hepatocyte suspension containing 2 x 10(7) viable hepatocytes in 1 mL of 0.2% collagen gel solution. Recipient rats underwent 20% partial hepatectomy and the hepatocyte suspension (2 x 10(7) cells) was injected into the submucosal layer of the anterior wall of the stomach. Rats were humanely killed and histologically examined at days 1, 3, 7, 30, or 180. Most transplanted hepatocytes remained in the submucosal layer until day 7. The surviving hepatocytes were arranged in clusters in the submucosa on day 30; 5-bromo-2'-deoxy-uridine (BrdU)-positive cells were observed. Also, the function of glycogen storage was detected by Periodic acid-Schiff (PAS) reactions on days 7, 30, and 180. The transplanted hepatocytes proliferated, reconstructing liver tissue-like structures in the gastric submucosa on day 180. The gastric submucosa is easily, repeatedly accessible by the gastro-endoscope. Thus, these results suggest that the gastric submucosa is a possible site for safe repetitions hepatocyte transplantation using endoscopic injection.