Table2.

Troubleshooting table.

Step	Problem	Possible reason	Solution
6	Imprecise removal of anterior segment of the eye	Damaging tissue by separately removing cornea and lens using dull scissors	Do not attempt separate removal of cornea and lens. Typically the lens is attached to the anterior segment and it is removed with the anterior segment in one piece. Use sharp scissors.
13	Incomplete removal of choroidal tissues from RPE sheet	Insufficient dispase digestion	Treat with dispase for longer duration. Residual choroidal tissues on the RPE sheet increase the chance of contamination that may lead to abnormal cell growth. Make sure to exclude the choroidal tissues by viewing under a dissection microscope.
20	Abnormal pigmentation of cells	donor related problem	Cell cultures that do not show pigmentation or show hyperpigmentation should be discarded. Use a differernt donor eye.
	Low yield of RPE (<70% confluency)	donor related problem long postmortem interval	Obtain new donor eye with postmortem interval < 24h.
27	Low TER after 1month of culture	contamination overgrowth abnormal pigment	Discard the cells and obtain a new donor eye.
28 a-c	Damage to membrane during removal from Transwell	Mechanical damage from pressure of razor blade	Use fine forceps and new razor blade; apply minimal pressure.
28a	Weak immunopositivity for tight junction proteins	Antibody expired or degraded	Obtain new antibodies.
28a	Poor apical localization of Na/K ATPase	Delay in fixation	Rapid fixation of membrane in paraformaldehyde.
28c	The Transwell support does not section well	Difference in density between Transwell support and plastic embedded membrane	Take special care when ultrathin sectioning to obtain intact sections.