True monolayer cell culture in a confined 3D microenvironment enables lineage informatics

Background: There is a need for methods to (1) track cells continuously to generate lineage trees; (2) culture cells in in vivo‐like microenvironments; and (3) measure many biological parameters simultaneously and noninvasively. Herein, we present a novel imaging culture chamber that facilitates “lineage informatics,” a lineage‐centric approach to cytomics. Methods: We cultured cells in a confined monolayer using a novel “gap chamber” that produces images with confocal‐like qualities using standard DIC microscopy. Lineage and other cytometric data were semiautomatically extracted from image sets of neural stem and progenitor cells and analyzed using lineage informatics. Results: Cells imaged in the chamber every 3 min could be tracked for at least 6 generations allowing for the construction of extensive lineage trees with multiparameter data sets at hundreds of time points for each cell. The lineage informatics approach reveals relationships between lineage, phenotype, and microenvironment. Mass transfer characteristics and 3D geometry make the chamber more in vivo‐like than traditional culture systems. Conclusions: The gap chamber allows cells to be cultured, imaged, and tracked in true monolayers permitting detailed informatics analysis of cell lineage, phenotype, and fate determinants. The chamber is biomimetic and straightforward to build and use, and should find many applications in long‐term cell imaging. © 2006 International Society for Analytical Cytology