Automated analysis and survival selection of anchorage‐dependent cells under normal growth conditions

Abstract

An instrument is described that can automatically analyze and select for a subpopulation of anchorage‐dependent cells in tissue culture. Cells that label with fluorescently tagged antibodies or demonstrate structural variations are saved from exposure to a destructive high‐intensity argon laser beam. The surviving population may then be cloned. The cell selection may occur in a tissue culture plate or in a microflow incubator which is designed to maintain a constant flow of media at 37°C across cells growing on a glass coverslip. This incubator sits on an inverted microscope which focuses the laser beam to a diameter as small as 1 μm. A high‐speed computer‐controlled two‐dimensional stage moves the cells past the beam for analysis, the results of which determine the fate of each cell: whether it is to be destroyed by radiant energy or selected for survival and subsequent proliferation. Another selection strategy performed by the instrument involves growing the cells on a thin, blackened polyester film which can be cut by the argon laser beam. Cells selected for cloning are then circumscribed. The heat of cutting welds the circumscribed film to a plastic coverslip surface or tissue culture chamber bottom. Nonselected cells may be removed by pulling the unattached polyester sheet from the attachment surface. The selected cells remain on polyester film disks welded to the plastic. Selections may be done automatically under computer control or manually by operator direction of stage movements. This instrument extends the art of automated cell selection and analysis to normal cell lines that must maintain cell‐substratum contact (anchorage dependence) for differentiated cell function, e.g., neurons, fibroblasts, or kidney cells.

High‐throughput laser‐mediated in situ cell purification with high purity and yield

“…Previous efforts toward in situ cell purification have been limited to adherent cell types (12), presumably due to unacceptable cell motion induced by the obligatory frequent and rapid stage movements to bring each cell to the fixed laser position with this approach (unpublished observation). Due to the large field of view and galvanometric scanning optics on LEAP, the infrequently needed stage movements could be implemented at a modest acceleration rate that did not disturb unattached nonadherent cells.…”

“…Flow cytometry works extremely well for most nonadherent cells and many other robust cell types. However, for certain fragile and/or adherent cells (e.g., neuronal, dendritic, and epithelial cells), the process of detachment, sorting, and reattachment can result in low yield (12,13). Further, such cell manipulations may significantly alter cell physiology because it is well known that gene expression is altered upon detachment or attachment of adherent cells (12).…”

“…However, for certain fragile and/or adherent cells (e.g., neuronal, dendritic, and epithelial cells), the process of detachment, sorting, and reattachment can result in low yield (12,13). Further, such cell manipulations may significantly alter cell physiology because it is well known that gene expression is altered upon detachment or attachment of adherent cells (12). Additional challenges of live cell sorting include biohazard risks in the fields of infectious disease and bioterrorismrelated research (14) and maintaining the sterility of sorted cells.…”

“…Building on the knowledge from medical laser uses and robust high-throughput semiconductor manufacturing tools, a novel laser-mediated cell manipulation technology has been developed. The resulting laser-enabled analysis and processing (LEAP) technology can implement cell imaging and multiple forms of laser-mediated cell manipulations including cell purification, optoinjection (i.e., laser-mediated cell transfection) (18,19), and chromophore-assisted laser-inactivation (e.g., direct knockout of protein function) (20) in an automated and highthroughput manner, thereby overcoming certain limitations of previous approaches to these methods (12,(21)(22)(23).…”

Toward complete laser ablation of melanoma contaminant cells in a co‐culture outgrowth model via image cytometry

“…For sorting, Schindler et al described a laser-based instrument with adjustable power to automatically analyze and select for a subpopulation of anchorage-dependent cells in tissue culture (6). More recently, a pulse laser was commercially implemented for removing tumor cells from a cell population (4,7).…”