TITLE: U-251MG Spheroid Generation Using Hanging Drop Method Protocol
AUTHORS: Lara Carroll, Brijesh Tiwari, James Curtin, Janith Wanigasekara

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><div class = "text-block"><span>In order to gather more physiologically relevant data, a variety of 3D cell culture techniques have been developed to mimic the </span><span style = "font-style:italic;">in vivo </span><span>characteristics of physiological tissues. This protocol describes </span><span style = "font-style:italic;">in vitro </span><span>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. </span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dgagbifip.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dgakbifip.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dgaqbifip.jpg" /></div><div class = "text-block">We used HDP1096 Perfecta3D® 96-well hanging drop plates for tumorsphere construction from 3D Biomatrix and adapted protocols published by: </div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span>Hikage, F., Atkins, S., Kahana, A., Smith, T. and Chun, T., 2019. HIF2A–LOX Pathway Promotes Fibrotic Tissue Remodeling in Thyroid-Associated Orbitopathy. </span><span style = "font-style:italic;">Endocrinology</span><span>, [online] 160(1), p.3. Available at: <https://academic.oup.com/endo/article/160/1/20/5150427>.</span></li><li style = "counter-reset:ol0;"><span>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. </span><span style = "font-style:italic;">European Journal of Cell Biology</span><span>, [online] 99(5), pp.2-3. Available at: <https://www.sciencedirect.com/science/article/pii/S0171933520300340>. </span></li><li style = "counter-reset:ol0;"><span>sigmaaldrich.com. n.d. </span><span style = "font-style:italic;">HDP1096 Perfecta3D® 96-Well Hanging Drop Plates Protocol</span><span>. [online] Available at: <https://www.sigmaaldrich.com/technical-documents/protocols/biology/perfecta3d-hdp1096-media-exchange-protocol.html>.</span></li></ol></div></div>

[STEPS]
?. U-251 MG cells are cultured in 100mm or 150mm dishes until reaching approximately 80-90% confluency and washed with 1X PBS solution.
?. Dissociate U-251 MG cells by incubating for 4 minutes at 37°C with Trypsin-EDTA solution (0.25%w/v) and inactivate the trypsin by re-suspending in the full growth medium pre-warmed in a 37°C water bath.
?. Centrifuge U-251 MG cell suspension for 5 minutes at 300g, re-suspend the cell-pellet in 1ml of full growth medium and pipette cell suspension up and down thoroughly to obtain a uniform single cell suspension.
?. With a pipette, add sterile, room temperature PBS to the reservoirs located on the peripheral rim of the plate and tray which are divided into sections. Add 2ml per plate reservoir section and 1ml per tray reservoir section. This prevents the hanging drop from drying out during incubation. Note: Take care to not tilt the plate if filling with water as this may result in spillage and contamination of hanging drops.
?. Prepare a 10ml stock solution of U-251MG cell suspension in full growth medium at a concentration of 2.5x105 cells/mL in order to achieve 5000 U-251MG cells per 20μl hanging drop. (Each hanging drop well is able to hold 20-50μl cell suspension and any volume above 50μl will result in drop instability). Mix the cell suspension thoroughly by gently pipetting up and down using a 1000μl pipette to ensure all cells are fully suspended within the media.
?. Hanging drops can be formed by carefully pipetting 20-50μl of cell suspension into the centre of each well from the top side of the plate. Use a multi-channel pipette but only add 2-4 drops at once as multiple seeding can affect the accuracy and placement of the drops. Hanging drops should be formed on and confined to the bottom of the plate.
?. Place the lid on the plate and insert the assembly into a humidified cell culture incubator at 37°C and 5% CO2. After a couple of hours, individual cells should begin to aggregate and form into spheroids. Time taken for spheroid formation differs between cell types but is typically a minimum of 4 days. U-251MG tumorspheres will form within 4 days and formation can be confirmed visually and monitored daily using a bright-field microscope.NOTE: Extra care must be taken not only when transporting the plates to/from the incubators, but when opening and closing the incubator in general as excessive movement can cause the plates to shift and the spheroids to either fall or form incorrectly.
?. Cell culture media should be exchanged on a daily basis to provide enough nutrients for cells and to prevent osmolality shift of the media. Add 5μl of fresh media back into the hanging drops by placing the end of the pipette tips in the neck region of the access holes/wells and slowly dispense fresh media into the access holes.
?. Once formed, tumorspheres can be transferred from the hanging drop plate to low attachment plates/pre-coated wells in the dish by adding 50μl of fresh media into each hole.