Table 2.

Troubleshooting Guide for Aggregate Growth

Problem	Possible cause	Solution
Aggregate loss or cell death	Aggregate size is suboptimal (too small or too large)	Re-plate cells using gentler technique (to reduce cell death) or check attachment conditions (to reduce cell loss; e.g., check Matrigel batch, cell density)
Poor recovery of aggregates post-thaw	Aggregate number per vial is suboptimal (too few or too many)	Re-check optimal thaw ratio
	Aggregates lost at handling	Re-plate cells using gentler technique (to reduce cell death) or check attachment conditions (to reduce cell loss, e.g., check Matrigel batch, cell density)
Too many aggregates post-thaw or split	Too many aggregates plated	Plate fewer aggregates at next split (and monitor for faster expansion and increased risk of spontaneous differentiation)
Sparse aggregates post-thaw or split	Too few aggregates plated	Increase density at initial plating. Split cells at 5-6 days post-split and use a lower split ratio (1:2-1:4) to reset and recover.
Excess spontaneous differentiation	Rough handling, suboptimal aggregate density, skipped medium changes	hESCs with spontaneous differentiation are more adherent than normal aggregates and can be removed at passage by shortening the ReLeSR exposure time. This will sacrifice some healthy aggregates (also remaining attached to the plate) but removes areas of differentiation from the passage.