Mouse Fetal Liver Cells in Artificial Capillary Beds in Three-Dimensional Four-Compartment Bioreactors

Abstract

Bioreactors containing porcine or adult human hepatocytes have been used to sustain acute liver failure patients until liver transplantation. However, prolonged function of adult hepatocytes has not been achieved due to compromised proliferation and viability of adult cells in vitro. We investigated the use of fetal hepatocytes as an alternative cell source in bioreactors. Mouse fetal liver cells from gestational day 17 possessed intermediate differentiation and function based on their molecular profile. When cultured in a three-dimensional four-compartment hollow fiber-based bioreactor for 3 to 5 weeks these cells formed neo-tissues that were characterized comprehensively. Albumin liberation, testosterone metabolism, and P450 induction were demonstrated. Histology showed predominant ribbon-like three-dimensional structures composed of hepatocytes between hollow fibers. High positivity for proliferating cell nuclear antigen and Ki-67 and low positivity for terminal dUTP nick-end labeling indicated robust cell proliferation and survival. Most cells within these ribbon arrangements were albumin-positive. In addition, cells in peripheral zones were simultaneously positive for ␣-fetoprotein, cytokeratin-19, and c-kit, indicating their progenitor phenotype. Mesenchymal components including endothelial, stellate, and smooth muscle cells were also observed. Thus, fetal liver cells can survive, proliferate, differentiate, and function in a three-dimensional perfusion culture system while maintaining a progenitor pool, reflecting an important advance in hepatic tissue engineering.

Identification of Oxysterol 7α-Hydroxylase (Cyp7b1) as a Novel Retinoid-Related Orphan Receptor α (RORα) (NR1F1) Target Gene and a Functional Cross-Talk between RORα and Liver X Receptor (NR1H3)

“…Human livers were obtained through the Liver Tissue Procurement and Distribution System (Pittsburgh, PA), and hepatocytes were isolated by three-step collagenase perfusion (Strom et al, 1999). Mouse primary hepatocytes and stellate cells were isolated from 8-week-old female C57BL/6J mice by collagenase perfusion and differential centrifugation (Monga et al, 2005;Mu et al, 2005). Cells were plated on six-well plates and maintained in hepatocyte maintenance medium from Lonza Walkerville, Inc. (Walkersville, MD) supplemented with dexamethasone (10 Ϫ7 M), insulin (10 Ϫ7 M), and gentamicin (50 g/ml).…”

Adipogenesis of Human Adipose-Derived Stem Cells Within Three-Dimensional Hollow Fiber-Based Bioreactors

“…Moreover, the use of dynamic 3D perfusion bioreactors to expand and differentiate cells is currently being studied by several groups. [14][15][16] Bioreactor designs based on interwoven sets of semi-permeable hollow fibers in a multicompartment configuration compared with direct perfusion and rotating wall vessel suspension designs have the advantage of providing uniform nutrient and gas exchange with more physiological gradients and integral oxygenation, and-via the bicarbonate buffer system and CO 2 gas exchange-pH regulation to the entire cell compartment volume with negligible shear stresses. Such bioreactors offer a better environment for sustained long-term cultures of cell types not requiring mechanical stimuli.…”

“…14 Various types of bioreactors can be utilized, such as the hollow fiber-based bioreactors, [15][16] continuous stirred tank bioreactors, 22 rotating wall bioreactors, 23 Couette-Taylor bioreactors, 24 and shear flow perfusion bioreactors. 25 There is a need for improved 3D systems to be used as high-throughput tools for drug discovery.…”

β-Catenin deletion in hepatoblasts disrupts hepatic morphogenesis and survival during mouse development

“…We examined immunolocalization of ␤-catenin, hepatocyte nuclear factor-4␣, cytokeratin-19 and platelet endothelial cell adhesion molecule (PECAM; Santa Cruz Biotechnology). 21 For negative control, we incubated the sections with secondary antibodies (Chemicon) only.…”