APPNOTE-Monitoring the confluence per cell type in two simultaneous co-cultures
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Monitoring the confluence per cell type in two simultaneous co-cultures Marc van Vijven, Msc 1; Dr. Inge Thijssen-van Loosdregt 1 1 CytoSMART Technologies B.V., Eindhoven, The Netherlands; Introduction Using live-cell imaging, researchers are able to determine not The most common fluorescence live-cell imaging setup only whether, but also when and how certain cellular events is currently a fluorescence microscope, with a stage top occur in culture. The number of read-outs and consequently the incubation box to regulate the culture conditions. However, obtained information from one live-cell imaging experiment can there are practical issues when using a high-end microscope be increased by including one or multiple fluorescent probe(s) for live-cell imaging. The regulation of the culture conditions and corresponding imaging channel(s), besides the standard in the incubation box is more sensitive to variations compared brightfield channel. In co-cultures, fluorescence imaging to a dedicated incubator [8, 9]. This variability may disturb the facilitates distinction of the included cell types and (quantified) cultures and can distort the results [10, 11]. Besides that, images read-outs per cell type, by assigning each cell type a particular are only captured at certain time points, but the microscope fluorescent label [1]. Co-culture setups are commonly applied is unavailable for other users during the entire live imaging in cancer research, since tumors and their environment involve many different cell types: e.g., immune cells aim for elimination The CytoSMART Lux3 FL Duo Kit could overcome these issues. of the cancer cells [2], endothelial cells are stimulated to This system fits in a regular incubator, and therefore enables vascularize a tumor [3], and fibroblasts can either promote or two-channel fluorescence live-cell imaging (green, red) in a inhibit tumor growth [4-6]. A commonly determined read-out constant and optimal culture environment. By connecting in this context is the confluence per cell type, which provides two devices within the same incubator to a single laptop, two insight into interactions between the different cell types; these cultures can be monitored simultaneously and compared can either support each other during the proliferation stage or side-by-side without temporal or inter-sample variation in compete and eliminate each other [1]. How the cells specifically affect each other often relates to the ratio of the various cell types in a tissue environment, where the most abundantly In this proof-of-concept study, we determine the effect of the present cell type is generally dominant, but this depends on the seeding ratio on proliferation of a cancer cell line and fibroblasts specific interaction and the cell types involved [7]. Fundamental in co-culture, using side-by-side fluorescence live-cell imaging. knowledge involving such interactions can be relevant in e.g., The CytoSMART Lux3 FL Duo Kit and corresponding cloud- cancer research, where potential clinical therapies could be based image analysis algorithm for brightfield and fluorescence based on insight in interactions between cancer cells and channel confluence were applied. With these tools, the surrounding other cell types. However, this insight requires an confluence over time of fluorescently labeled MDA-MB-231 cells imaging system that enables fluorescence live-cell imaging of (metastatic breast cancer cell line) and 3T3 cells (fibroblasts) in the co-cultures over time. co-cultures with two different seeding ratios was determined. This provided fundamental insight into interactions between the cell types, which may ultimately be related to tumor growth. Materials and methods tFP602-labeled MDA-MB-231 cells (Innoprot P20317; red fluorescent) and non-fluorescent 3T3 fibroblasts (ATCC® CL- Co-cultures were monitored for 2 days using the CytoSMART 173™) were separately cultured to sub-confluency in DMEM Lux3 FL Duo Kit (37° 5% CO2), with a 1 h interval. At each C; (Gibco) supplemented with 10% FBS (Gibco) and 1% pen-strep time point, the brightfield and red fluorescence channel were (Gibco), under standard culture conditions (37° 5% CO2). CoC; imaged. Confluence per cell type was determined using the cultures of MDA-MB-231 cells and 3T3 fibroblasts were seeded integrated algorithm in the CytoSMART cloud. in 24-well plates at a total density of 50,000 cells per well,

Monitoring the confluence per cell type in two simultaneous co-cultures Results Images from the time-lapse videos of the two seeding ratios, as well as the determined confluence per cell type over time only the 3T3 fibroblasts proliferated, and confluence of the are displayed in Fig. 1. In the 1:1 seeding ratio, both cell types MDA-MB-231 cells did not increase over time. The final ratio of proliferated, but total confluence after 2 days was lower than for the 1:10 condition. For this 1:1 seeding ratio, the final ratio of Figure 1: Side-by-side comparison of confluence per cell type in...

Monitoring the confluence per cell type in two simultaneous co-cultures The confluence quantifications over time indicated competition report specific fibroblasts supporting and accelerating tumor between the MDA-MB-231 cells and the 3T3 cells. However, the growth. These cells are therefore named cancer-associated initial ratio determined whether proliferation for both cell types fibroblasts [6]. Opposite to the inhibitory property of regular was relatively low, or only MDA-MB-231 cells were eliminated. fibroblasts, cancer-associated fibroblasts are more active, and In previous research, it...