U-251MG Spheroid Generation Using Hanging Drop Method Protocol v1

The use of 3D cell culture has been a major step in developing cellular models that can mimic physiological tissues. Traditional 2D cell cultures are often unable to accurately represent the cellular functions and responses that are present in tissues, as a result, research findings based on 2D cultures tend to be skewed with limited predictive capability. 3D cell cultures can be grown from cells obtained from cancer organoids in patients. These models are useful for understanding disease mechanisms and exploring drug therapeutics in areas such as toxicity and efficacy. In order to gather more physiologically relevant data, a variety of 3D cell culture techniques have been developed to mimic the in vivo characteristics of physiological tissues. This protocol describes in vitro generation of U-251MG spheroids using the hanging drop method. Advantages of using hanging drop plate method are, able to produce uniform size spheroids, low cost, comfortable to handling and suitable for short term culture. The main downside of this method is medium change, different drug treatment at different time points are impossible and labor intensive. This method uses the Perfecta3D hanging drop plate, a novel cell culture device that simplifies the process of spheroid formation, testing and analysis. Rather than having to invert the plates which often results in spillage or detachment, these plates are designed to create hanging drops using a plateau structure at the bottom of the plate. We used HDP1096 Perfecta3D® 96-well hanging drop plates for tumorsphere construction from 3D Biomatrix and adapted protocols published by: Hikage, F., Atkins, S., Kahana, A., Smith, T. and Chun, T., 2019. HIF2A–LOX Pathway Promotes Fibrotic Tissue Remodeling in Thyroid-Associated Orbitopathy. Endocrinology, [online] 160(1), p.3. Available at: . Moraes, G., Wink, M., Klamt, F., Silva, A. and Fernandes, M., 2020. Simplified low-cost methodology to establish, histologically process and analyze three-dimensional cancer cell spheroid arrays. European Journal of Cell Biology, [online] 99(5), pp.2-3. Available at: . sigmaaldrich.com. n.d. HDP1096 Perfecta3D® 96-Well Hanging Drop Plates Protocol. [online] Available at: .