Dynamic seeding and in vitro culture of hepatocytes in a flow perfusion system.

Our laboratory has investigated hepatocyte transplantation using biodegradable polymer matrices as an alternative treatment to end-stage liver disease. One of the major limitations has been the insufficient survival of an adequate mass of transplanted cells. This study investigates a novel method of dynamic seeding and culture of hepatocytes in a flow perfusion system. In experiment I, hepatocytes were flow-seeded onto PGA scaffolds and cultured in a flow perfusion system for 24 h. Overall metabolic activity and distribution of cells were assessed by their ability to reduce MTT. DNA quantification was used to determine the number of cells attached. Culture medium was analyzed for albumin content. In Experiment II, hepatocyte/polymer constructs were cultured in a perfusion system for 2 and 7 days. The constructs were examined by SEM and histology. Culture medium was analyzed for albumin. In experiment I, an average of 4.4 X 10(6) cells attached to the scaffolds by DNA quantification. Cells maintained a high metabolic activity and secreted albumin at a rate of 13 pg/cell/day. In experiment II, SEM demonstrated successful attachment of hepatocytes on the scaffolds after 2 and 7 days. Cells appeared healthy on histology and maintained a high rate of albumin secretion through day 7. Hepatocytes can be dynamically seeded onto biodegradable polymers and survive with a high rate of albumin synthesis in the flow perfusion culture system.

Effects of anastomosis of tissue-engineered neointestine to native small bowel.

BACKGROUND: Our laboratory is investigating the tissue engineering of small intestine using intestinal epithelial organoid units seeded onto highly porous biodegradable polymer matrices. This study investigated the effects of anastomosis of tissue-engineered intestine to native small bowel alone or combined with small bowel resection on neointestinal regeneration. METHODS: Intestinal epithelial organoid units harvested from neonatal Lewis rats were seeded onto biodegradable polymer tubes and implanted into the omentum of adult Lewis rats as follows: (1) implantation alone (n = 9); (2) implantation followed by anastomosis to native small bowel at 3 weeks (n = 11); and (3) implantation after small bowel resection and anastomosis to native small bowel at 3 weeks (n = 8). All constructs were harvested at 10 weeks and examined by histology. Morphometric analysis of the neomucosa was obtained using a computer image analysis program. RESULTS: Cyst development was noted in all animals. All anastomoses were patent at 10 weeks. Histology revealed the development of a vascularized tissue with a neomucosa lining the lumen of the cyst with invaginations resembling crypt-villus structures. Morphometric analysis demonstrated significantly greater villus number, villus height, crypt number, crypt area, and mucosal surface length in groups 2 and 3 compared with group 1, and significantly greater villus number, villus height, crypt area, and mucosal surface length in group 3 compared with group 2 (P < 0.05, ANOVA, Tukey test). CONCLUSION: Intestinal epithelial organoid units transplanted on biodegradable polymer tubes can regenerate into complex tissue resembling small intestine. Anastomosis to native small bowel combined with small bowel resection and anastomosis alone contribute significant regenerative stimuli for the morphogenesis and differentiation of tissue-engineered neointestine.

Small intestinal submucosa as a small-caliber venous graft: a novel model for hepatocyte transplantation on synthetic biodegradable polymer scaffolds with direct access to the portal venous system.

BACKGROUND/PURPOSE: Hepatotrophic factors in the portal blood are critically important for the survival of heterotopically transplanted hepatocytes. Currently, no model exists for the implantation of hepatocytes on biodegradable polymer scaffolds with direct access to the portal blood. This study investigates the use of small intestinal submucosa (SIS) as a small-caliber venous conduit that may be used for the implantation of tissue-engineered liver. METHODS: SIS was prepared from segments of rat jejunum and implanted as a venous conduit between the portal vein and inferior vena cava in 26 heparinized Lewis rats. Venograms were performed periodically, and the grafts were harvested at various time-points and examined by scanning electron microscopy (SEM) and histology. Von Willebrand Factor (vWF) staining was performed to assess endothelialization. RESULTS: Five rats died of technical complications. Seventeen of 21 rats (81%) maintained patent grafts at the time of death up to 8 weeks. Venograms demonstrated patent grafts at 3 and 8 weeks. SEM results showed a smooth luminal surface with endothelial-like cells by 3 weeks. Histology demonstrated a confluent luminal endothelial monolayer, absence of thrombus, and neovascularization in the SIS graft. VWF staining results were positive, confirming the growth of endothelial cells on the luminal surface. In preliminary studies, implantation of hepatocytes seeded on biodegradable polymer tubes into the SIS graft demonstrated clusters of viable cells after 2 days. CONCLUSIONS: Rat SIS can be prepared readily, maintains high patency as a small-caliber venous graft, and may be a useful model for the transplantation of tissue-engineered liver with access to the portal circulation.

Regenerative signals for intestinal epithelial organoid units transplanted on biodegradable polymer scaffolds for tissue engineering of small intestine.

BACKGROUND: Our laboratory is investigating the tissue engineering of small intestine using intestinal epithelial organoid units seeded onto highly porous biodegradable polymer tubes. This study investigated methods of stimulation for optimizing neointestinal regeneration. METHODS: Intestinal epithelial organoid units harvested from neonatal Lewis rats were seeded onto porous biodegradable polymer tubes and implanted into the omentum of adult Lewis rats in the following groups: (1) the control group (group C), implantation alone (n=9); (2) the small bowel resection (SBr) group, after 75% SBr (n=9); (3) the portacaval shunt (PCS) group, after PCS (n=8); and (4) the partial hepatectomy (PH) group, after 75% PH (n=8). Neointestinal cyst size was recorded using ultrasonography. Constructs were harvested at 10 weeks and were examined using histology. Morphometric analysis of the neomucosa was obtained using a computer image analysis program (NIH Image, version 1.59). RESULTS: Cyst development was noted in all animals. Cyst lengths and diameters were significantly larger in the SBr group at 7 and 10 weeks compared with the other three groups (P<0.05; analysis of variance [ANOVA], Fisher's protected least significant difference). Histology revealed a well-vascularized tissue with a neomucosa lining the lumen with invaginations resembling crypt-villus structures. Morphometric analysis demonstrated a significantly greater villus number, height, area, and mucosal surface in the SBr group compared with the other three groups and a significantly greater crypt number and area in the PCS group compared with group C (P<0.05; ANOVA, Fisher's protected least significant difference). CONCLUSIONS: Intestinal epithelial organoid units transplanted on porous biodegradable polymer tubes can successfully vascularize, survive, and regenerate into complex tissue resembling small intestine. SBr and, to a lesser extent, PCS provide significant regenerative stimuli for the morphogenesis and differentiation of tissue-engineered small intestine.