Abstract
Retinoblastoma (RB) is a primary intraocular malignancy in childhood. Relapses may develop and cause secondary cancers during later development. This study was set up to identify optimal cell culture conditions for RB cell growth in vitro and to optimize tumor growth in an in vivo model. RB cell lines (Y79 and WERI-Rb1) were cultivated under three different in vitro conditions and apoptosis, proliferation and cell growth, as well as expression profiles of two epithelial-mesenchymal transition (EMT) markers, were analyzed. EMT gene expression profiles were not generally changed, whereas apoptosis levels, tumor cell proliferation, and in vitro growth were significantly influenced by different cell culture conditions. In order to optimize the time-limited chick chorioallantoic membrane (CAM) assay, we investigated two different time points of tumor cell inoculation (embryonic development day EDD8 and EDD10) as well as three different cell concentrations. We showed that inoculation at EDD8 led to decreased tumor formation and chicken viability, whereas different cell concentrations did not change size and weight of developing tumors. Our findings demonstrate that medium conditions in vitro as well as the starting point for CAM inoculation in ovo significantly influence the experimental outcome of investigations using RB cell lines.
Highlights
Retinoblastoma (RB) is the most common malignant pediatric intraocular tumor [1], representing 2.5–4% of all pediatric cancers, and approximately 9000 new cases develop each year worldwide [1,2]
While culture conditions seemed to have no influence on the endogenous apoptosis rate of Y79 cells (Figure 2A), cell death levels were significantly increased in WERI-Rb1 cells cultivated in Dulbecco’s modified Eagle’s medium (DMEM) and RPMI media and 5% CO2 compared to cells grown in RB medium and 10% CO2 (Figure 2D)
DMEM and RPMI media conditions significantly decreased the proliferation rate of Y79 cells compared to RB medium conditions (Figure 2B), whereas RPMI medium conditions seemed to have a positive effect on WERI-Rb1 proliferation compared to RB and DMEM media conditions (Figure 2E)
Summary
Retinoblastoma (RB) is the most common malignant pediatric intraocular tumor [1], representing 2.5–4% of all pediatric cancers, and approximately 9000 new cases develop each year worldwide [1,2]. The total survival rate is high with a good eye-salvage rate (95%); saving vision, fails in advanced cases [3]. Untreated tumors expand and may extend beyond the eye causing metastatic spread [2]. Different cultivation protocols for the same RB cell lines have been published so far [3,5–8]. It is wellknown that varying cell culture conditions, e.g., using different media and/or supplements as well as different CO2 concentrations, have a huge impact on the tumor cell growth, viability and gene expression, e.g., of epithelial-mesenchymal transition (EMT) markers. It is comprehensible that experiments using the same RB cell lines are still not verifiable across different labs as they are carried out using different cultivation protocols
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