Quantification of cell proliferation and monitoring its kinetics are essential in fields of research such as developmental biology, oncology, etc. Although several proliferation assays exist, monitoring cell proliferation kinetics remains challenging. We present a novel cell proliferation assay based on real-time monitoring of cell culture inside a standard incubator using a lensfree video-microscope, combined with automated detection of single cell divisions over a population of several thousand cells. Since the method is based on direct visualization of dividing cells, it is label-free, continuous, and not sample destructive. Kinetics of cell proliferation can be monitored from a few hours to several days. We compare our method to a standard assay, the EdU proliferation assay, and as proof of principle, we demonstrate concentration-dependent and time-dependent effect of actinomycin D—a cell proliferation inhibitor.
Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree video microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.
Owing to its simplicity, lensless imaging system is adept at continuous monitoring of adherent cells inside the incubator.
The setup consists of a CMOS sensor with pixel pitch of 2.2 μm and field of view of 24 mm2, LED with a dominating
wavelength of 525 nm, along with a pinhole of 150 μm as the source of illumination. The in-line hologram obtained
from cells depends on the degree of cell-substrate adhesion. Drastic difference is observed between the holographic
patterns of floating and adherent cells. In addition, the well-established fact of reduction of cell-substrate contact during
cell division is observed with our system based on corresponding spontaneous transition in the holographic pattern. Here,
we demonstrate that by recognizing this specific holographic pattern, number of cells undergoing mitosis in a cell culture
with a population of approximately 5000 cells, can be estimated in real-time. The method is assessed on comparison with
Edu-based proliferation assay. The approach is straightforward and it eliminates the use of markers to estimate the
proliferation rate of a given cell culture. Unlike most proliferation assays, the cells are not harvested enabling continuous
monitoring of cell culture.