Fluorescence imaging case study: 3D cell culture model for drug screening purposes
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3D cell culture model for drug screening purposes C. Roma-Rodrigues, L.R. Raposo, P.V. Baptista and A.R. Fernandes from UCIBIO, DCV, FCT-NOVA, Portugal; written by T.M. van Haaften from CytoSMART Technologies, Netherlands Case study: Researchers from UCIBIO Universidade Nova de Lisboa have used fluorescence live-cell imaging to asses anticancer drug response in a 3D cell model. Spheroid cell culture emerges as a powerful in vitro tool for research into oncology treatment. Although most research regarding cancer biology is based on experiments using in vitro 2-dimensional (2D) cell cultures, this model often lacks the complexity to mimic in vivo structures of the tissue or tumour. By mimicking the 3-dimensional (3D) network of cell-cell interactions and oxygen- and nutrient gradients, tumour spheroids resemble many aspects of the pathophysiologic environment within human tumour tissue [4,5]. “The Lux3 FL allows us to continually monitor our 3D tumour spheroid formation and assess reponse to drugs in 2D or 3D cell models in an easy and user-friendly manner!” Tumour cells are less sensitive to drugs in 3D than in 2D cultures. This effect may be caused by reduced direct access to compounds in the medium or by pathophysiological characteristics of the 3D cellular construct due to hypoxia or differences in cell cycle [5]. The extent to which biological processes are affected, as well as the timing and depth of internalisation are aspects that are essential to take into account in the development of efficient nontoxic anti-tumour drugs. By using continuous live-cell imaging, the CytoSMART Lux3 FL enables to monitor drug internalisation into the tumour spheroid, as well as following biological processes and morphologic changes of the tumour spheroid. This case study provides a proof of concept for the evaluation of anticancer drug response using live-cell imaging. After an 8-day maturation period, the spheroid consisting of the colorectal carcinoma HCT116 cell line was incubated with CellTrace™ CFSE (peak excitation 492 nm; peak emission 517 nm) in phenol-red depleted medium [1], to stain viable cells with green fluorescence. After washing three times with fresh medium, the spheroid was exposed to 10 µM Doxorubicin, a red fluorescent anti-tumour drug. Doxorubicin has emission signal at 590-595 nm upon excitation at 450-480 nm and binds to DNA [3]. Figure 1. HTC116 spheroid after 0h, 12h, 24h and 48h exposure to 10µM Doxorubicin. A) Brighfield channel; B) Red channel (Doxorubicin); C) Green channel (CellTrace™ CFSE); D) Overlay of the three channels. Scale bar represents 200 µm. The absorption of Doxorubicin by the cells in the spheroid and its effect on spheroid integrity and viability was monitored using timelapse imaging (Fig. 1). Brightfield and fluorescen images were captured every 15 minutes for 2 days using the CytoSMART Lux3 FL that was placed inside a 37° 5% CO₂ incubator [6]. The mean intensity of C total fluorescent signal was determined with ImageJ. Research use only. Not intended for diagnosti

Results The initial time point of drug internalisation can be determined with continuous live cell imaging (Fig. 2). The trend of red fluorescence signal indicates that Doxorubicin infused rapidly into the tumour spheroid from t=3h 45min after drug treatment and slowed down after t=6h. At t=1h 45min and t=21h the focus is adjusted resulting in a shift in fluorescence signal. The viability of the tumour cells is monitored by green fluorescence signal. Although the signal naturally fades due to proliferation, the overall exponential reduction in fluorescence signal was an indication of loss...