Development of a Novel Cell Monitoring System Based on Lens-Less Imaging Toward Cultivation of Circulating Tumor Cells

Abstract

Circulating tumor cells (CTCs) are defined as tumor cells circulating in the peripheral blood of patients with metastatic cancer. As CTCs could provide useful information regarding malignancy and metastatic property of primary tumor, as well as its response to anticancer agents, fundamental techniques for recovery and analysis of CTCs have been intensively studied in this decade. Although state-of-the-art microfluidic tools and whole genome amplification techniques are now available to investigate single CTCs, it is still extremely difficult to cultivate CTCs that, for example, enables examination of multiple anticancer agents toward identical CTCs. This could be due to the lack of knowledge of division and proliferation behavior of CTCs. The critical difficulty in investigation of CTC proliferation is that cell division events of CTCs are very rare, and thus conventional microscopy could hardly identify the dividing CTCs from the samples containing a number of blood cells. In this study, we propose to use a wide-field imaging system for investigation of CTC cultivation. The imaging system is composed of a light emitting diode (LED, peak wavelength: 465 nm) and a complementary metal oxide semiconductor (CMOS) imaging sensor by which an area of 6.55 × 4.92 mm2can be visualized in one shot. A cell culture dish is placed on the CMOS image sensor, and images are acquired under the LED illumination without using any lens systems (referred to as “lenss-less imaging”). The CMOS image sensor allows to develop a space-saving and inexpensive platform, so that whole system can be introduced in a general incubator, and real-time monitoring of cell growth is possible. Using this lens-less imaging system, we attempted to observe tumor cell proliferation and distinguish tumor cells from blood cells. HeLa cells were used as model tumor cells. A culture dish containing HeLa cells in 2 ml of the high glucose Dulbecco's modified Eagle's medium (DMEM, 5,000 cells/ml) was placed on the lens-less imaging system, and incubated for 18 hours at 37°C with 5% CO2. Images were captured every 15 min. For comparison, microscopic observation was also performed. As a result, HeLa cells were recorded as a spherical patterns with high intensity by a CMOS image sensor. When divided, HeLa cells were once illustrated as a dark patterns, and then two daughter cells gradually appeared with increasing the intensity. It is well known that adhesive cells temporarily go through a non-adhesive state before their division process. Indeed, we confirmed that the spherical patterns with high intensity represents the HeLa cells adhere on the bottom of the dishes, and the subsequent dark patterns do the cells in the non-adhesive state by microscopic observation. This data indicates that the lens-less imaging system can detect individual cell division events. Taking advantage of the wide field of view, it was possible to monitor the cell division events of several hundreds of cells simultaneously. Recovery of CTCs from blood samples is the first step for the cultivation and further analysis, however it is still hard to avoid blood cell contamination despite the development of sophisticated technology for CTC separation and enrichment nowadays. Consequently, it is essential to differentiate CTCs and the contaminated blood cells, otherwise accurate evaluation of CTC proliferation is impossible. To test whether tumor cells and blood cells were distinguishable based on lens-less imaging, we compared the lens-less images of HeLa cells, red blood cells and JM cells (model white blood cells). A number of differentiation parameters were extracted from the images, and computed for cluster analysis. As a result, it was successfully demonstrated to discriminate HeLa cells, red blood cells and JM cells without any labeling process, suggesting the development of a simple, inexpensive and rapid cell typing system. In summary, the lens-less imaging system could monitor cell division events of HeLa cells in a wide field of view, and differentiate HeLa cells and blood cells with high accuracy. We believe that this system could be a powerful tool to monitor the proliferation of CTCs recovered from patient blood samples.