Table 5:

Troubleshooting guide

Step	Problem	Possible reason	Solution
10	Differentiating ESC colonies	Suboptimal culture medium and culture of ESC	Prepare fresh N2B27 2iLIF medium and store at 4°C for only up to 10 days.
			Passage ESCs before they reach 80% confluency. Individual colonies should not be touching.
29	There are clumps of ESCs, making it difficult to accurately count the cell density	Insufficient trypsinisation and pipetting to break up ESC colonies	Extend trypsinisation.
			Pipette up and down for more times after stopping trypsinisation.
39	Some microwells of AggreWell are empty after seeding of ESCs	There are bubbles remained in microwells before cell seeding	Check for any remaining bubbles in microwells. Repeat centrifugation of AggreWell plate with anti-adherence rinsing solution until there are no bubbles.
		Insufficient treatment of AggreWell with anti-adherence rinsing solution	Ensure anti-adherence rinsing solution is left in the AggreWell for at least 30 min.
	Unequal distribution of cells across microwells	Cell suspension is non-homogenous and not distributed across the AggreWell	Resuspend final cell pellet with sufficient pipetting and add the suspension of cell mixture drop by drop across the area of the well.
		AggreWell plate is not balanced during centrifugation	Ensure AggreWell plate is balanced precisely during centrifugation.
40–42	Cell aggregates are dislodged from microwells	Medium change is too vigorous	Remove medium very slowly and add medium slowly along the side of AggreWell.
		Improper handling of the AggreWell plate	Move the AggreWell plate carefully and avoid dragging of the plate. Lift the lid gently with one hand holding the bottom of the plate.
49	EiTiX embryos do not form or low yield of EiTiX embryos	Missing one or more cell type(s) in the cell seeding mixture or miscounting of cell density for seeding	Ensure all three cell types have been added. Clumps of ESC should not be observed when counting on a haemocytometer (see troubleshooting for Step 29).
		ESCs are differentiating in culture	Ensure ESC colonies are round and have a defined boundary (see troubleshooting for Step 10). Thaw a new vial of ESCs if necessary.
		ESCs have been cultured for too long	Thaw a new vial of ESCs with lower passage number and avoid culturing ESCs for more than 10 passages. If possible, avoid using ESCs with passage number over 30.
		Contamination with mycoplasma or other microorganisms	Perform mycoplasma testing every 2 weeks and look for signs of contamination.
	EiTiX embryos are damaged or disintegrated	EiTiX embryos are dislodged from microwells too vigorously or transferred too vigorously	Pipette up and down gently to dislodge EiTiX embryos from AggreWell and only pipette along the boundary of the well.
			Cut a wider bore opening of the P1000 pipette tip.
56	EiTiX embryos fuse together	Overcrowding of EiTiX embryos	Gently shake the plate containing selected Day 4 EiTiX embryos to distribute them.
			Avoid culturing more than 50 EiTiX embryos in a 6-well.
56, 61, 62, 67	Low efficiency of EiTiX embryos progressing to the next day	EiTiX embryos are left at RT for too long or exposed to excessive temperature fluctuation	Perform selection of EiTiX embryos and medium change as quickly as possible. If possible, designate an incubator only for EiTiX embryo culture and minimise opening of incubator.
		Suboptimal peri-implantation culture medium	Test the batch of FBS.
			Make new stock solutions of β-estradiol, progesterone and NAC. Store at −20°C for up to 1 month and avoid repeated freezing and thawing.
		Suboptimal rat serum in post-implantation culture medium	Produce rat serum in-house if possible.
			Validate the quality of the batch of rat serum by using it to culture natural mouse embryos.
			Remove any blood clotting proteins appearing on top after centrifuging at 600g for 40 min at 4°C.
			Do not refreeze heat-inactivated rat serum and use thawed aliquots within 2 days.
	Contamination of culture medium	Exposure of culture medium to microorganisms	Filter post-implantation medium.
			Minimise opening of culture plate and use sterile filtered pipette tips.
93	MEFs do not attach to the plate	MEFs did not survive thawing	Ensure steps involved in the thawing of MEFs are performed quickly.
	MEFs do not extend processes to cover the well	Unhealthy MEFs	Purchase MEFs of good quality.
102	TSCs are differentiating	The medium is not well conditioned by MEFs	Ensure that MEFs are healthy and have not been in culture for more than 2 weeks.
			Ensure to condition the MEFs well for at least 6 hours before plating TSCs.
		Suboptimal heparin and/or Fgf4	Prepare fresh batch of heparin and/or Fgf4.
110	TSCs are not dissociating properly	Insufficient washing of well with PBS before the addition of trypsin-EDTA, insufficient trituration	Ensure to wash TSCs sufficiently with PBS before adding trypsin-EDTA.
			Triturate TSCs well to break down any cell clumps.