Table 2 –

Troubleshooting table

Step	Problem	Possible reason	Solution
Step 1: Thawing and expansion of hiPSC and NHDF	Poor attachment of hiPSC	hiPSC medium wasn’t supplemented with Y-27632	Supplement medium with Y-27632
		Imperfect coating	Plate cells in appropriately coated plates
	Spontaneous differentiation of hiPSC	Cultures were too sparse or too dense	Adjust passage ratio
	hiPSC are proliferating very slowly	hiPSC will only show their normal growth behavior after being passaged several times	Wait for some passages to occur
	NHDFare proliferating very slowly	Cultures were too sparse	Adjust passage ratio
Step 2Avi: Passaging of hiPSC	Many dead cells	EDTA incubation lasted too long	Monitor the cells under a microscope and stop dissociation at the right time
Step 8: Cardiac differentiation	No spontaneously contracting cells on day 12	Poor quality hiPSC culture	See above problems as the problem, and hence solution are likely to be a consequence of problems during the expansion of hiPSC
		Small molecules were not added or wrongly added in the CDM medium	Supplement CDM medium with the right molecules at the right time
		Small molecules might be old	Prepare new stocks of small molecules
Step 18: Fabrication of pillars	Bubbles in PDMS pillars	PDMS was left to sit too long before pouring into mold and therefore was not as viscous; vacuum pump not used to pull bubbles out; centrifuge was not set high enough to push bubbles out.	Pour PDMS immediately after mixing, immediately transfer mold with PDMS to de-bubble in the vacuum chamber (can increase vacuuming time to 30 min); centrifuge PDMS into Pillar formation molds at 400 g for 5 min.
Step 92: Generation of cardiac tissues	Tissues did not form	Fibrinogen, thrombin, or aprotinin stocks may not be fresh. If the tissue did not gel properly from the beginning, it is likely the fibrinogen or thrombin. If the tissue did gel properly but subsequently fell apart, it is likely the aprotinin.	Remake the fibrinogen and thrombin fresh, and test that the two gel when mixed together by combining them in a 5:1 ratio in a petri dish, and confirming that they gel by touching hydrogel with pipette tip. If it is the aprotinin, make a fresh stock. If this does not solve the problem, increase the aprotinin concentration to 6.6 mg/mL or decrease the amount of fibroblasts added to the coculture (be cognizant of the fibroblasts already present in the hiPS-CM differentiation culture).
	Tissues are not; beating well	Dt Cardiac differentiation efficiency may have been too low and/or the cell digestion was too harsh.	Only use hiPS-CM differentiations with an efficiency ^ 80 %. Limit the time in collagenase during digestion. If cells are difficult to dissociate, use the cells a day or two early when they will be easier to digest.
	Medium turninig yellow quickly	Tissue is too metabolically active to be supported by surrounding medium	Ensure tissues are cultured in a large bath of medium. Ensure medium has fatty acids (B-27 supplement) and buffer (bicarbonate)
Step 39 Dxiv: Muscle Strip Myograph System measurements of contractile force	Force not increasing at higher stimulation frequencies	Tissue may not be stretched to allow optimal force generation. Or, organ bath solution may not be being refreshed and thereby has become toxic to the tissue.	Use the Frank-Starling force-length relationship to continuously increase tissue length within the Muscle Strip Myograph System until the force output is stable. Then retry force-frequency response. If the organ-bath solution is bubbling or has not been exchanged recently, replace with fresh solution and ensure it is bubbled properly.
Step 39Fxxi: Imaging of histological sections of adult-like cardiac tissues	High background in immunostained samples	Inadequate wash steps	Increase the time of wash steps (can do 24 hr washes on a shaker at 4 °C) and add gentle detergent (0.01 % Triton-X) to facilitate removal of excess antibody.