RNAi screening reveals a large signaling network controlling the Golgi apparatus in human cells.

The Golgi apparatus has many important physiological functions, including sorting of secretory cargo and biosynthesis of complex glycans. These functions depend on the intricate and compartmentalized organization of the Golgi apparatus. To investigate the mechanisms that regulate Golgi architecture, we developed a quantitative morphological assay using three different Golgi compartment markers and quantitative image analysis, and performed a kinome- and phosphatome-wide RNAi screen in HeLa cells. Depletion of 159 signaling genes, nearly 20% of genes assayed, induced strong and varied perturbations in Golgi morphology. Using bioinformatics data, a large regulatory network could be constructed. Specific subnetworks are involved in phosphoinositides regulation, acto-myosin dynamics and mitogen activated protein kinase signaling. Most gene depletion also affected Golgi functions, in particular glycan biosynthesis, suggesting that signaling cascades can control glycosylation directly at the Golgi level. Our results provide a genetic overview of the signaling pathways that control the Golgi apparatus in human cells.

Laminar-flow immediate-overlay hepatocyte sandwich perfusion system for drug hepatotoxicity testing.

Drug hepatotoxicity testing requires in vitro hepatocyte culture to maintain the long-term and stable liver specific functions. We developed a drug testing platform based on laminar-flow immediate-overlay hepatocyte sandwich perfusion culture. The immediate-overlay sandwich (collagen-coated porous polymeric membrane as top overlay) protects the cells and integrity of the top collagen matrix from the impact of flow. A bioreactor was designed that allowed proper control of shear stress and mass transfer. The culture parameters such as the optimal perfusion initiation time and flow rate were systematically and mechanistically determined. The optimized system could re-establish hepatocyte polarity to support biliary excretion and to maintain other liver specific functions, such as the biotransformation enzyme activities, for two weeks that extended the usable in vitro hepatocyte-based drug testing window. When the perfusion cultured hepatocytes from days 7 or 14 were used for drug testing, the APAP-induced hepatotoxicity measurements were more sensitive and consistent over time than the static culture control, enabling further exploitations in large-scale drug testing applications.

Identification and characterization of a novel prespheroid 3-dimensional hepatocyte monolayer on galactosylated substratum.

Three-dimensional (3D) hepatocyte spheroids mimicking the structural and functional characteristics of hepatocytes in vivo were self-assembled onto a galactosylated polyethylene terephthalate (PET) substratum, and the dynamic process of spheroid formation was investigated using time-lapse confocal microscopy. Hepatocytes cultured on this galactosylated substratum formed small cell-aggregates within 12 h, which gradually merged into "island-like" clusters at approximately 1 day and spread to form prespheroid monolayer within 2 days; the prespheroid monolayer was stretched to fold into compact and larger 3D spheroids after 3 days. We compared the expressions of F-actin (cytoskeleton), phosphorylated focal adhesion kinase (p-FAK, cell-substratum interactions) and E-cadherin (cell-cell interactions) during the dynamic process of 3D hepatocyte spheroid formation with the dynamic process of 2D hepatocyte monolayer formation on collagen substratum. Hepatocytes in the prespheroid monolayer stage exhibited the strongest cell-substratum interactions of all 4 stages during spheroid formation with cell-cell interactions and F-actin distribution comparable with those of the 3D hepatocyte spheroids. The prespheroid monolayer also exhibited better hepatocyte polarity (multidrug resistance protein 2) and tight junction (zonula occludens-1) formation, more-differentiated hepatocyte functions (albumin production and cytochrome P450 1 A activity), and higher sensitivity to hepatotoxicity than the conventional 2D hepatocyte monolayer. The transient prespheroid 3D monolayer could be stabilized on a hybrid glycine-arginine-glycine-aspartic acid-serine (GRGDS)/galactose-PET substratum for up to 1 week and destabilized to form 3D spheroids in excess soluble GRGDS peptide.

Improved hepatocyte excretory function by immediate presentation of polarity cues.

Liver tissue constructs with excretory function are crucial to developing realistic hepatocyte models for engineering effective bioartificial liver-assisted devices and for modeling the in vivo tissue. Current hepatocyte in vitro models suffer from limited or inefficient hepatocyte repolarization, which results in poor removal of xenobiotics and other waste products from the cells. We hypothesized that the temporal and spatial presentation of the cell matrix and cell-cell contacts as polarity cues would be important to define the axis of polarization to improve the excretory function of hepatocytes. The spatial presentation of polarity cues can be best achieved with sandwich configuration. We improve the temporal presentation of polarity cues by introducing the collagen overlay immediately in synchrony with cell-cell contacts instead of after 24 h in conventional sandwich culture. We demonstrate that the immediate presentation of the collagen matrix overlay enhances the formation of apicobasolateral domains, tight junctions, and the recovery of the functional activity of 2 canalicular transporters, the multidrug resistance-associated protein (Mrp2) and P-glycoprotein (P-gp) at 48 h of culture, and enhances the albumin secretion, urea production, and 7-ethoxyresorufin-O-deethylation cytochrome P450 activities of hepatocytes over 14 days of culture as compared to the 24-h overlay controls. The improvement in the excretory function of hepatocytes for the removal of waste products deleterious to cells may improve the functional maintenance and the in vivo fidelity of tissue-engineered liver constructs.

Encapsulating live cells with water-soluble chitosan in physiological conditions.

A new class of microcapsules was prepared under physiological conditions by polyelectrolyte complexation between two oppositely-charged, water-soluble polymers. The microcapsules consisted of an inner core of half N-acetylated chitosan and an outer shell of methacrylic acid (MAA) (20.4%)-hydroxyethyl methacrylate (HEMA) (27.4%)-methyl methacrylate (MMA) (52.2%) (MAA-HEMA-MMA) terpolymer. Both 400 and 150 kDa half N-acetylated chitosans maintained good water solubility and supplied enough protonated amino groups to coacervate with terpolymer at pH 7.0-7.4, in contrast to other chitosan-based microcapsules which must be prepared at pH <6.5. The viscosity of half N-acetylated chitosan solutions between 80 and 3000 cPas allowed the formation of microcapsules with spherical shape. Molar mass, pH and concentration of half N-acetylated chitosan, and reaction time, influenced the morphology, thickness and porosity of the microcapsules. Microcapsules formed with high concentration of half N-acetylated chitosan exhibited improved mechanical stability, whereas microcapsules formed with low concentration of half N-acetylated chitosan exhibited good permeability. This 3D microenvironment has been configured to cultivate sensitive anchorage-dependent cells such as hepatocytes to maintain high level of functions.

A configurable three-dimensional microenvironment in a microfluidic channel for primary hepatocyte culture.

We have developed a technique for the in situ three-dimensional (3D) immobilization of primary rat hepatocytes within a localized matrix in a microfluidic channel that provides a 3D microenvironment incorporating both a configurable 3D matrix and fluid perfusion. This is based on the laminar flow complex coacervation of a pair of oppositely charged polyelectrolytes, i.e., methylated collagen and a terpolymer of HEMA-MMA-MAA. 3D collagen matrices were formed with minimal gelation times (<8 min), were able to entrap cells under aqueous noncytotoxic conditions, and permitted culture media to be perfused in the microchannel by virtue of the spatial confinement of the 3D matrix on one side of the channel. The architecture and stability of the collagen matrix could be configured by the use of different material combinations and changes in the polyelectrolyte flow rates and retention time. Primary rat hepatocytes cultured for 24 h in the 3D matrix within the microchannel showed comparable or enhanced cytochrome P450 7-ethoxyresorufin-O-deethylation activity with static controls. The configurable 3D microenvironment in the microfluidic channel may be a potential 3D culture model of primary hepatocytes for drug testing applications.

Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices.

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate-methyl methacrylate-methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.