Identification of kinases and regulatory proteins required for cell migration using a transfected cell-microarray system.

BACKGROUND: Cell migration plays a major role in a variety of normal biological processes, and a detailed understanding of the associated mechanisms should lead to advances in the medical sciences in areas such as cancer therapy. Previously, we developed a simple chip, based on transfected-cell microarray (TCM) technology, for the identification of genes related to cell migration. In the present study, we used the TCM chip for high-throughput screening (HTS) of a kinome siRNA library to identify genes involved in the motility of highly invasive NBT-L2b cells. RESULTS: We performed HTS using TCM coupled with a programmed image tracer to capture time-lapse fluorescence images of siRNA-transfected cells and calculated speeds of cell movement. This first screening allowed us to identify 52 genes. After quantitative PCR (qPCR) and a second screening by a conventional transfection method, we confirmed that 32 of these genes were associated with the migration of NBT-L2b cells. We investigated the subcellular localization of proteins and levels of expression of these 32 genes, and then we used our results and databases of protein-protein interactions (PPIs) to construct a hypothetic but comprehensive signal network for cell migration. CONCLUSIONS: The genes that we identified belonged to several functional categories, and our pathway analysis suggested that some of the encoded proteins functioned as the hubs of networks required for cell migration. Our signal pathways suggest that epidermal growth factor receptor (EGFR) is an upstream regulator in the network, while Src and GRB2 seem to represent nodes for control of respective the downstream proteins that are required to coordinate the many cellular events that are involved in migration. Our microarray appears to be a useful tool for the analysis of protein networks and signal pathways related to cancer metastasis.

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.

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.

On-chip screening method for cell migration genes based on a transfection microarray.

Cell migration plays a major role in a variety of biological processes and a detailed understanding of associated mechanisms should lead to advances in the medical sciences, for example, in drug discovery for cancer therapy. However, the traditional methods used for analysis of cell migration cannot easily be scaled up for high-throughput screening. In this study, we have attempted to develop a novel simple method for high-throughput phenotypic screening for the identification of genes that are required for cell migration. As the appropriate cell line for the method, we found NBT-L2b cells that would be suitable for screening of migration-related genes in our method without influence by other cellular processes. Moreover, the idea for printing both the labeled fibronectin, for identification of the starting region of a cell, and the green fluorescent protein (GFP) expression vector, for identification of cells that had been transfected with siRNA and of the end point of migration, brings a rapid and efficient high-throughput screening procedure. Our new method will lead to an enhanced understanding of cell migration.

A simple method for producing multiple copies of controlled release small molecule microarrays for cell-based screening.

Traditional drug discovery involves the screening of lead compounds from a chemical library by using cell-based high throughput screening (HTS) procedures. This has created a demand for the development of cell-based microarray chips for HTS of compounds. Although several cell-based microarray devices and procedures for screening of chemical libraries have been reported, each has limitations in terms of simplicity, speed, and cost. Here, we sought to make a simple method for producing multiple copies of microarray chips for the controlled release of small molecules during cell-based screening. Arrays of polytetrafluoroethylene microchannels were set in poly(dimethylsiloxane) and were formed in a metal mold. Subsequently, a biodegradable polymer, PLGAs, with chemical compounds was injected into each channel, and the array was sliced perpendicular to the channels to create multiple copies of the microarray chip. After seeding the cells on the microarray chip, we were able to successfully control the diffusion of small molecules and locally introduce the compounds into cells. The described method enables the production of multiple copies of the chip by using an easy, rapid, and inexpensive array fabrication without any specialized devices. Moreover, screening using the microarray chip minimizes the consumption of cells and chemicals. Both the biodegradable material and compound injected into each channel can be individually tuned for optimized performance. Therefore, we expect that this method will be useful for developing cell-based HTS assays for small chemical compounds to find drug candidates.

Development of super-dense transfected cell microarrays generated by piezoelectric inkjet printing.

Super-dense transfected cell microarrays (TCMs) were created by a piezoelectric inkjet printer on a glass substrate that had been grafted with poly(ethylene glycol) (PEG). The micro-spots that contained plasmid and extra-cellular matrix (ECM) protein were separated from one another by a hydrophilic barrier generated by PEG. We successfully constructed the densest TCMs with spots of 50 µm in diameter and 150 µm in pitch.

Transfection microarrays for high-throughput phenotypic screening of genes involved in cell migration.

Cell migration is important in several biological phenomena, such as cancer metastasis. Therefore, the identification of genes involved in cell migration might facilitate the discovery of antimetastatic drugs. However, screening of genes by the current methods can be complicated by factors related to cell stimulation, for example, abolition of contact inhibition and the release inflammatory cytokines from wounded cells during examinations of wound healing in vitro. To overcome these problems and identify genes involved in cell migration, in this chapter we describe the use of transfection microarrays for high-throughput phenotypic screening.