Abstract
Consistent cell preparation is a fundamental preliminary step for understanding complex cellular mechanisms in various cell-based research fields, including basic cell biology, cancer research, and tissue engineering. However, certain elusive factors, such as cellular de-differentiation and contamination with mycoplasma or other types of cells, have compromised the reproducibility and reliability of cell-based approaches. Here, we propose an epi relief-contrast cellular monitoring system (eRC-CMS) that allows images of cells in a typical culture plate to be acquired, stored, and analysed for daily cell quality control. Due to its full flatbed nature and automated system, cells placed at any location on the stage can be analysed without special attention. Using this system, changes in the size, circularity, and proliferation of endothelial cells in subculture were recorded. Analyses of images of ~9,930,000 individual cells revealed that the growth activity and cell circularity in subcultures were closely correlated with their angiogenic activity in a subsequent hydrogel assay, demonstrating that eRC-CMS is useful for assessing cell quality in advance. We further demonstrated that eRC-CMS was feasible for the imaging of neurite elongation and spheroid formation. This system may provide a robust and versatile approach for daily cell preparation to facilitate reliable and reproducible cell-based studies.
Highlights
There is increasing concern regarding scientific research results that cannot be reproduced, in the fields of basic and preclinical biological research[1]
An ideal system for quality control may be one in which (i) cells can be continuously monitored under stable culture conditions in a CO2 incubator without any handling of the culture plates and disturbance of the culture; (ii) no special skill or additional labour is required, with multiple culture dishes and plates placed in a CO2 incubator being automatically recognized, digitally labelled, and analysed in terms of cell morphology, proliferation, and other parameters; (iii) recorded image data, analysed outcomes, and alarms for cell quality are accessible online; and (iv) the system can be inserted into a typical CO2 incubator and can be produced inexpensively
We proposed an epi relief-contrast cellular monitoring system for the quality control of cells grown in culture
Summary
There is increasing concern regarding scientific research results that cannot be reproduced, in the fields of basic and preclinical biological research[1]. Continuous monitoring of cell morphology and proliferation can be performed using commercially available systems (e.g., IncuCyte, Essen BioScience, USA; BioStation, Nikon, Japan) that include an incubator box mounted on a stage of a standard inverse microscope or a standard incubator with a built-in microscope[7, 8] Both systems are designed for focusing on cellular events rather than for cell quality control and are unfit for the simultaneous monitoring of cells in multiple culture plates. We examined whether eRC-CMS could be used to monitor the behaviour of human umbilical vein endothelial cells (HUVECs) with different passage numbers and to quantify changes in morphology, size, circularity, and proliferation These cell characteristics were linked with endothelial angiogenic activity in a subsequent hydrogel culture that is important for in vitro angiogenic models for the development of anti-angiogenic cancer drugs and for engineering vascularized tissues. We further examined whether eRC-CMS was capable of sufficient resolution to acquire time-lapse images of neurite elongation and three-dimensional spheroid formation
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