TITLE: U-251MG Spheroid generation using a scaffold based method protocol
AUTHORS: Lara Carroll, Brijesh Tiwari, James Curtin, Janith Wanigasekara

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">3D cell culture is a technique that is used to grow cells in vitro that will mimic an in vivo environment. 3D cell models are a helpful learning tool for researchers to better understand disease mechanisms and to explore different therapeutic properties of drugs. 3D cell cultures can be developed using patient derived cancer cells. Once they have been grown, these 3D cells can be used to screen for small molecule drugs or for genetic modification in for analysis of disease pathways or to predict drug treatments toxicity or efficacy. 3D cell cultures are a big step towards the more ethical testing of drug toxicity and efficacy as they decrease the need to use animals in research as well as providing more reliable results as the cells used are of human physiology.</div><div class = "text-block">Cellusponge are 3D porous hydroxipropylcellulose scaffolds that are designed for use with cells that do not require specific ligands. As well as the standard non-coated cellusponge, there are two more of the same type of scaffold available for use that are made with two different coatings to allow for improved adaptation of different cell types, these are called Cellusponge-Gal and Cellusponge-Col. Cellusponge is a no-coating approach that is intended for use in the development of general soft tissue 3D culture. It has been used as soft matrix for 3D cell culture and 3D tumour model.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dg6gbifip.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dg6kbifip.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dg6qbifip.jpg" /></div><div class = "text-block">We used Bio-Byblos 9mm Cellusponge scaffold plates and adapted protocols published by:</div><div class = "text-block">1. ebersmedical.com. n.d. 3D Cellusponge Cell Seeding Protocol_Multi-well Plate Format Sponge. [online] Available at: <https://ebersmedical.com/files/bbbmd/cellusponge_seeding_protocol.pdf> [Accessed 17 April 2021].</div><div class = "text-block">2. ebersmedical.com. n.d. Cellusponge Scaffolds. [online] Available at: <https://ebersmedical.com/tissue-engineering/scaffolds/cellusponge> [Accessed 17 April 2021]. </div></div>

[STEPS]
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Once U-251MG cell flasks are 70-80% confluent, medium from the flask is aspirated and U-251MG tumor cell monolayers re washed with 1X phosphate buffered saline (PBS; 5ml for a 25cm2 or 4ml for a 75cm2 flask), add 0.25 X Trypsin-EDTA cell dissociation enzyme (2ml for a 25cm2 or 4ml for a 75cm2 flask) and incubate cells at 37°C for 2-5 min.
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Check cell detachment under a microscope and neutralize cell dissociation enzyme with complete growth medium. The 0.25 X Trypsin-EDTA reagent is neutralized using 4 volumes of complete medium.
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Centrifuge U-251 MG single cell suspension at 5000rpm for 5 min, remove supernatant, tap the tube and re-suspend cell pellet in complete growth medium using a pipette. This should yield a single cell suspension without cell clusters.
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Cells with a viability >90% can be used for spheroid generation. Mix 10µl of cells with 10µl of Trypan Blue and count cells using a haemocytometer.
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For a 9mm scaffold, a cell suspension with the cell density of 5000k cells/ml must be obtained.
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Slowly place a cellusponge disk in the middle of each well in a 24-well plate
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Added 100μL of U-251MG cell suspension on top of the sponge for a 9mm disk and incubated it for 3 hours for U-251MG cells. This will remove any air bubbles within the cellusponge.
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After incubation, 500μL of complete media was added to each well in a 24-well plate. Media should be added slowly along the edge of each well, not directly onto the cellusponge.
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The 24-well plate cellusponge was then incubated overnight in an incubator (37°C, 5% CO2, 95% humidity).
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After overnight incubation, the seeded sponge is transferred into a new well plate, the media is replenished and the culture should be continued.
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Tumor spheroid formation was observed within 3 days for U-251MG cell line.