APPNOTE-Monitoring the confluence ratio per cell type in co-cultures with varying seeding ratios
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Monitoring the confluence ratio per cell type in co-cultures with varying seeding ratios Marc van Vijven, Msc 1; Dr. Inge Thijssen-van Loosdregt 1 1 CytoSMART Technologies B.V., Eindhoven, The Netherlands; Introduction Live-cell imaging enables researchers to determine not only There are also practical issues when using a regular microscope. whether, but also when and how certain cellular events occur Although images are only captured at certain time intervals, in culture. By including one or multiple fluorescent probe(s) the microscope is unavailable for other users during the and corresponding channel(s) – besides a brightfield channel entire live imaging experiment. It is practically infeasible to – the number of read-outs and consequently the obtained optimize microscope use by performing continuous imaging information from one experiment increases. This strategy is experiments during the waiting intervals of the time-lapse for example applied in co-cultures. Each included cell type is experiment. Besides that, only one culture vessel can be then assigned a particular fluorescent label, which facilitates imaged per run. If multiple conditions in separate vessels need distinction of the cell types based on their label and (quantified) to be covered, repeated runs of the same experiment may be read-outs per cell type [1]. Co-cultures are widely used in cancer required, or multiple culture vessels may need to be placed research, since many different cell types play a role in or around under the microscope alternately to be imaged manually. Both tumors: immune cells aim for elimination of the cancer cells strategies are labor-intensive, time-consuming, and increase [2], endothelial cells are stimulated to vascularize a tumor [3], variability in culture conditions and consequently in results. fibroblasts can either promote or inhibit tumor growth [4-6], A system that could overcome these issues is the CytoSMART and many more. A relevant determined read-out in this context Multi Lux3 FL. This is a dedicated system for long-term time- is the confluence per cell type in the co-culture, which provides lapse imaging, enabling optimal use of imaging systems within insight into the cell types either supporting each other during a laboratory. It fits in a regular incubator, and therefore enables proliferation or competing and eliminating each other [1]. These interactions relate to the number of cells of the various (green, red) in a constant and optimal culture environment. By cell types, where the cell type with the largest relative presence connecting four devices within the same incubator to a single is generally dominant, but this depends on the specific laptop, multiple conditions can be monitored simultaneously interaction [7]. Potential clinical cancer therapies could be without variation in temperature and CO2 level. The automated based on fundamental knowledge regarding the interactions cloud-based image analysis enables confluence analysis per between cancer cells and other cell types. However, a proper cell type over time. Consequently, co-culture comparison imaging system for the fluorescence live-cell imaging of the studies can be performed with minimal variability in culture- co-cultures is a prerequisite to gain sufficient insight in the and imaging conditions, and directly available confluence data per imaging channel – and consequently per cell type. In this proof-of-concept study, we demonstrate the applicability of Currently, fluorescence live-cell imaging is predominantly simultaneous fluorescence live-cell imaging to determine the performed using a fluorescence microscope with a stage effect of various seeding ratios on proliferation of a cancer cell top incubation box. However, the regulation of the culture line and fibroblasts in co-culture. The CytoSMART Multi Lux3 conditions in the incubation box – usually 37° and 5% CO2, C FL and corresponding cloud-based image analysis algorithm with the latter regulating the culture medium pH – is more for confluence were used to determine confluence over time sensitive to variations compared to a dedicated incubator of fluorescently labeled HeLa (cervical cancer cell line) and 3T3 [8, 9]. If the cells in a co-culture have different sensitivities to cells (fibroblasts) in co-cultures with various seeding ratios. This temperature and pH [10, 11], the variability in these factors may provided fundamental insight into interactions between the disturb the cultures and distort results. cell types, which may ultimately be related to tumor

Materials and methods tGFP-labeled HeLa cells (Innoprot P20107; green fluorescent) and non-fluorescent 3T3 fibroblasts (ATCC® CL-173™) were separately cultured to sub-confluency in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% pen-strep (Gibco), under standard culture conditions (37°C; 5% CO2). Co-cultures of HeLa cells and 3T3 fibroblasts were seeded in 24-well plates at a total density of 50,000 cells per well, at ratios of 1:1,3:1, 10:1 and 20:1 (HeLa:3T3). The co-cultures were monitored for 90 h using the CytoSMART Multi Lux3 FL (37°C; 5% CO2), making a snapshot every 1 h, with...

Monitoring the confluence ratio per cell type in co-cultures with varying seeding ratios Discussion In co-cultures, fluorescence live-cell imaging can facilitate The confluence quantifications over time indicated that the distinction of the cell types, when each included cell type is HeLa cells eliminated the 3T3 cells at any seeding ratio, although then assigned a particular fluorescent label. In cancer research, the initial ratio affected the time before this elimination was potential therapies could be based on fundamental knowledge initiated. This effect was clearly visible with...

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