Multiphase matrix of silica, culture medium and air for 3D mammalian cell culture.

The craving for multiphase materials with adjustable properties for mammalian cell encapsulation persists despite intensive research on 3D cell culture and tissue engineering. This interest is incited by the complex interaction between cells and different materials, various manufacturing methods, cell chip applications, and the aspiration to abolish animal experiments. This study aims to show the feasibility of preparing a stable multiphase material for prolonged mammalian cell embedment and 3D cell culture. The material comprises silica as the solid phase, cell culture medium with serum as the main liquid phase and air as the gas phase. The silica sol-cell culture medium-serum mixture was foamed, and it turned into a stable foamed hydrogel. The stability, flow properties and foaming parameters were studied by rheological and surface tension measurements. The viability of embedded cells was studied by measuring the metabolic activity at different time points. Their sensitivity to the surrounding conditions was compared to cells grown in monolayers by exposing them to a toxic compound. A stable foamed hydrogel with cell culture medium as the main liquid phase was prepared. Based on oscillatory measurements, the foamed hydrogel stays stable for at least 6-7 weeks and the embedded mammalian cells remain viable for the same time period. Appropriate surface tension and viscosity were crucial for an at least twofold volume increase by foaming, which is necessary for the mammalian cells to survive and proliferate. A test with a toxic compound reveals a difference in the sensitivity of cells in monolayer cultures versus embedded cells.

Arrayed adenoviral expression libraries for functional screening.

With the publication of the sequence of the human genome, we are challenged to identify the functions of an estimated 70,000 human genes and the much larger number of proteins encoded by these genes. Of particular interest is the identification of gene products that play a role in human disease pathways, as these proteins include potential new targets that may lead to improved therapeutic strategies. This requires the direct measurement of gene function on a genomic scale in cell-based, functional assays. We have constructed and validated an individually arrayed, replication-defective adenoviral library harboring human cDNAs, termed PhenoSelect library. The adenoviral vector guarantees efficient transduction of diverse cell types, including primary cells. The arrayed format allows screening of this library in a variety of cellular assays in search for gene(s) that, by overexpression, induce a particular disease-related phenotype. The great majority of phenotypic assays, including morphological assays, can be screened with arrayed libraries. In contrast, pooled-library approaches often rely on phenotype-based isolation or selection of single cells by employing a flow cytometer or screening for cell survival. An arrayed placental PhenoSelect library was screened in cellular assays aimed at identifying regulators of osteogenesis, metastasis, and angiogenesis. This resulted in the identification of known regulators, as well as novel sequences that encode proteins hitherto not known to play a role in these pathways. These results establish the value of the PhenoSelect platform, in combination with cellular screens, for gene function discovery.