Organoid_culture_Grid_6well_Amin_Kelley_Pasca

thingiverse

For details on this protocol and its use in human stem cell derived organoid culture, please see the methods section from our preprint: https://www.biorxiv.org/content/10.1101/2023.05.31.541819v1 >> If you use this grid or any modified versions of this grid for a research publication, please cite the above preprint (or the peer-reviewed manuscript when it becomes available). >> The concept for the grid came from Neal Amin, the initial grid was designed by Tommy Li with input from Neal Amin, and the final grid design provided here was designed by Neal Amin. This 3D grid inserts into a standard format 6-well culture plate. It can be printed with PLA, which slowly degrades into lactic acid and is safe for cell culture. The wings around the outside get compressed during insertion into the plate, securing it tightly. Gaps are placed on some lines to allow for slight compression of the grid - this is important since 3D printing is slightly inaccurate each time, and the compression gaps allow the grid to fit even if there are printing imperfections. The height is 6mm but can be easily adjusted. There are small diffusion ports along the bottom that allow for fluids to mix but cultured organoids will not be able to move between them. Over time (1-2d), the grids may slightly push against the plastic of the 6 well culture plate and it may become less secure, or even become dislodged. For this reason, silicone glue can be applied along the outside edges of the grid before insertion into the 6 well culture plate. The silicone is inert and will cure within hours to a day. The silicone adhered grids will stay attached to the 6 well plate for at least months. After securing the grids within a 6 well plate, the grids can be sterilized with 70% ethanol and coated with a hydrophobic solution of polyHEMA to prevent organoids from adhering to the PLA grid. After this sterilization and coating steps, the grids can be further UV sterilized in a standard tissue culture incubator. Finally, the grids can be washed with PBS, culture media can be added (roughly 6mL), and individual organoids can be placed in each well of the grid for long term culture. Media aspiration can be done from the aspiration area. Media can be added in this area as well.