Experimental Problem	Troubleshooting Tip
Monolayers fail to form from organoids	1) Optimal culturing conditions for this protocol vary between experimental needs. Human and mouse monolayers derived from organoids should be cultured using diluted basement membrane matrix when using glass or plastic culture dishes. However, rat tail collagen is optimal for monolayer experiments of mouse or human origin that require polyester membrane inserts. 2) Optimal culturing conditions vary depending on the experimental set up. Cultures using single cell suspension are ideally cultured with 3D FGO media whereas intact organoids are cultured with 2D FGO media.
Monolayers derived from aged patients (>55 years) or mice (>18 months) failed to grow and reach confluency	If using stomach tissue collected from aged mice (>18 months) or patients (>55 years) the density of organoids used for the generation of the monolayer should be doubled given that the growth efficiency of organoids is decreased.
Organoids fail to form from glands	It is recommended that fresh (newly purchased if possible) gastrin be used.  Use freshly re-suspended gastrin every 4 months.
Organoids fail to attach and form monolayers	Ensure that efficient removal of basement membrane matrix has been achieved after harvesting organoids for the transfer to culture plates for monolayers.
Cultures derived from aged patients (>55 years) or mice (>18 months) fail	During the digestion and incubation periods, 15 - 30 min of initial incubation followed by 5 min intervals of incubation as necessary is sufficient. It should be noted that patients greater than 55 years of age may have glands that are more easily dissociated and therefore require less digestion. Similarly, FGOs derived from aged patients have displayed slower growth time and increased sensitivity to centrifugation. To obtain the best results, limit any excess centrifugation when working with aged tissue and replace media in a timely manner.