BACKGROUND: In vivo cell therapies can potentially be used to generate new tissue in cases of tissue/organ loss due to trauma, or disease. In studies of in vivo liver tissue engineering our aim is to increase implanted cell survival, which is very low using conventional single cell implantations. In this study we evaluated the formation of multi-cellular liver progenitor cell (LPC) spheroids in vitro, and, subsequently implanted the spheroids in a vascularised murine tissue engineering chamber. Spheroid LPC survival and differentiation was compared with LPC dissociated single cell implantations of the same cell number. METHODS & RESULTS: In vitro: 20,000 LPCs in methylcellulose media uniformly produced one spheroid/well. Over 10 days, day 2 spheroids had the greatest proliferation (Ki67 immunolabelling) and the least apoptosis (caspase 3 immunolabelling), and were considered optimal for implantation. Day 2 spheroids contained 22,500 cells. In vivo: Bilateral silicon chambers were established on SCID mouse epigastric pedicles. Chambers were filled with Matrigel containing 200,000 dissociated single cells on one side and 9 spheroids in the opposite chamber. Fourteen and 45 day chambers were evaluated by immunohistochemistry/morphometry to determine LPC survival/differentiation. At 45 days significantly increased numbers of panCK+ve LPCs (p<0.006, 16 fold increase) and CK18+ve hepatocyte-like cells (p<0.019, 6 fold increase) were found in spheroid chambers compared to control (dissociated single cell) chambers. CK18+ve cells formed hepatic acini and cords. Ongoing in vitro studies have isolated liver sinusoidal endothelial cells (LSECs) which have been used to generate spheroids with and without LPCs. In vivo implantations indicate that liver specific vasculature (LYVE-1 +ve/CD31-ve) can also be grown in the chambers. CONCLUSION: Spheroid LPC delivery into a vascularised tissue engineering chamber significantly increases cell survival/differentiation, compared to dissociated cells. By increasing spheroid numbers, this spheroid/chamber technology offers promise in generating new liver tissue for treating liver failure.