Automated cell isolation from photodegradable hydrogel based on fluorescence image analysis.

We report an automated cell-isolation system based on fluorescence image analysis of cell aggregates cultured in a photodegradable hydrogel. The system incorporates cell culture in a humidified atmosphere with controlled CO2 concentration and temperature, image acquisition and analysis, micropatterned light exposure, and cell collection by pipetting. Cell aggregates were cultured on hydrogels, and target cells were selected by phase contrast and fluorescence image analysis. After degradation of the hydrogel by exposure to micropatterned UV light, cell aggregates were transferred to a collection vessel by robotic pipetting. We assessed the system for hydrogel degradation, recovery of target cells, and contamination by off-target cells. We demonstrated two practical applications of our method: (i) in cell aggregates from MCF-7-RFP strains in which 18.8% of cells produced red fluorescent protein (RFP), we successfully obtained 14 proliferative fluorescence-positive cell aggregates from 31-wells, and all of the isolated strains produced a higher proportion of RFP production than the original populations; (ii) after fluorescent immunostaining of human epidermal growth factor receptor 2 (HER2) in cancer cells, we successfully isolated HER2-positive cells from a mixed population of HER2-positive and -negative cells, and gene sequence analysis confirmed that the isolated cells mainly contained the target cells.

Perfusion culture of endothelial cells under shear stress on microporous membrane in a pressure-driven microphysiological system.

This paper reports perfusion culture of human umbilical vein endothelial cells (HUVECs) on a microporous membrane in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. We designed fluidic culture unit with microporous membrane to culture HUVECs under fluidic shear stress and constructed a perfusion culture model in the PD-MPS platform. Four fluidic culture units were arranged in the microplate-sized device, which enables four-throughput assay for characterization of HUVECs under flow. Medium flow was generated above and below the membrane by sequential pneumatic pressure to apply physiological shear stress to HUVECs. HUVECs exhibited aligned morphology to the direction of the flow with shear stress of 11.5-17.7 dyn/cm2 under the flow condition, while they randomly aligned under static culture condition in a 6 well plate. We also observed 3.3- and 5.0-fold increase in the expression levels of the thrombomodulin and endothelial nitric oxide synthase mRNAs, respectively, under the flow condition in the PD-MPS compared to the static culture in 6 well plate. We also observed actin filament aligned to the direction of flow in HUVECs cultured under the flow condition.

Perfusion culture of multi-layered HepG2 hepatocellular carcinoma cells in a pressure-driven microphysiological system.

Here we report the perfusion culture of a multi-layered tissue composed of HepG2 cells (a human hepatoma line) in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. The perfusion culture of multi-layered tissue model was constructed by inserting a modified commercially available permeable membrane insert into the PD-MPS. HepG2 cells were layered on the membrane, and culture medium was perfused both through and below the membrane. The seeded density (number of cells/cm2) of the culture model is 70 times that of static culture in a conventional 35-mm culture dish. Pressure-driven circulation of the medium in our compact device (8.6 × 7.0 × 4.5 cm3), which comprised two perfusion-culture modules and a pneumatic connection port, enabled perfusion culture of two multi-layered tissues (initially 1 × 105 cells). To obtain insight into the basic functionality of the multi-layered tissues as hepatocytes, we compared albumin production and urea synthesis between perfusion cultures and static cultures. The HepG2 cells grew and secreted increasing amounts of albumin throughout 20 days of perfusion culture, whereas albumin secretion did not increase under static culture conditions. In addition, on day 20, the amount of albumin secreted by the HepG2 cells in the microfluidic device was 68% of that in the conventional culture dish, which was seeded with the same number of cells but had a 70 times larger culture area. These features of high-density culture of functioning cells in a compact device support the application of PD-MPS in single- and multi-organ MPS.

Kinetic analysis of sequential metabolism of triazolam and its extrapolation to humans using an entero-hepatic two-organ microphysiological system.

The microphysiological system (MPS) is a promising tool for predicting drug disposition in humans, although limited information is available on the quantitative assessment of sequential drug metabolism in MPS and its extrapolation to humans. In the present study, we first constructed a mechanism-based pharmacokinetic model for triazolam (TRZ) and its metabolites in the entero-hepatic two-organ MPS, composed of intestinal Caco-2 and hepatic HepaRG cells, and attempted to extrapolate the kinetic information obtained with the MPS to the plasma concentration profiles in humans. In the two-organ MPS and HepaRG single culture systems, TRZ was found to be metabolized into α- and 4-hydroxytriazolam and their respective glucuronides. All these metabolites were almost completely reduced in the presence of a CYP3A inhibitor, itraconazole, confirming sequential phase I and II metabolism. Both pharmacokinetic model-dependent and -independent analyses were performed, providing consistent results regarding the metabolic activity of TRZ: clearance of glucuronidation metabolites in the two-organ MPS was higher than that in the single culture system. The plasma concentration profile of TRZ and its two hydroxy metabolites in humans was quantitatively simulated based on the pharmacokinetic model, by incorporating several scaling factors representing quantitative gaps between the MPS and humans. Thus, the present study provided the first quantitative extrapolation of sequential drug metabolism in humans by combining MPS and pharmacokinetic modeling.

Glass-based organ-on-a-chip device for restricting small molecular absorption.

The use of organ-on-a-chip (OOC) devices is a promising alternative to existing cell-based assays and animal testing in drug discovery. A rapid prototyping method with polydimethylsiloxane (PDMS) is widely used for developing OOC devices. However, because PDMS tends to absorb small hydrophobic molecules, the loss of test compounds in cell-based assays and increases in background fluorescence during observation often lead to biased results in cell-based assays. To address this issue, we have fabricated a glass-based OOC device and characterized the medium flow and molecular absorption properties in comparison with PDMS-based devices. Consequently, we revealed that the glass device generated a stable medium flow, restricted the absorption of small hydrophobic molecules, and showed enhanced cell adhesiveness. This glass device is expected to be applicable to precise cell-based assays to evaluate small hydrophobic molecules, for which PDMS devices cannot be applied because of their absorption of small hydrophobic molecules.