Table 1.

iPSC-CM maturation methods and characteristics

Maturation method	Approach	Maturation duration	Mature CM features	Ref.
Physical	Prolonged culture time	360 days	(Compared to day 30) Larger cell size Longer sarcomere Lower beating rate Less dual MCL2α/v expression More aligned sarcomere	[ 45 , 139 ]
Metabolic (hormone treatment)	Media +Triiodothyronine (T3) +Glucocorticoid +Dexamethasone	1–2 weeks	Larger cell size Less circularity Up-regulation of α-MHC, SERCA2a Down-regulation of β-MHC Decrease in proliferation Improved contractile force and kinetics Faster calcium transience Increased mitochondrial respiration	[ 39 , 90 , 49 , 91 ]
Metabolic (glucose replacement)	Media Remove glucose +palmitate +oleic acid +carnitine	3–20 days	Larger cell size Less circularity Longer sarcomere Increase in mitochondria number and respiration Improved contractile force and kinetics Upstroke velocity and membrane capacitance	[ 95 , 48 , 101 ]
Physical (substrate modification)	Culture environment Increased substrate stiffness via polydimethylsiloxane (PDMS) or polyacrylic acid (PAA) (6∼10 kPa)	3–9 days	Modulates sarcomere tension and contractility Increased force generation More rectangular shape	[ 69 , 102 , 103 ]
Physical (electrical stimulation)	Continuous electrical stimulation (6.6 V/cm, 1 Hz, 2 ms duration)	4 days	Upregulation: HCN1, MLC2V, SCN5A, SERCA, Kv4.3, GATA4 Increase in TNNT+ cells Cell elongation Ventricular-like AP Increase AP duration Improved calcium handling Coupled with mechanical stimulation via photoactivated PDMS: increased surface N-cadherin signalling, sarcomere shortening	[ 104 , 106 ]
Biochemical/physical	Culture environment CM-FB/MSC co-culture and static/cyclic stretching	2 weeks	Increased contraction velocity Greater twitch amplitude Increased tensile modulus More mature sarcomere structure Connexin 43 clustering N-cadherin surface localization Decreased proliferation potential Better MYL2/7 and MYH7/6 Up-regulation: MLC2, NPPA, CACNA1C, SERCA2 Stretching further increased findings, particularly cyclic	[ 107 ]
Metabolic/biochemical/physical	Media +triiodothyronine, dexamethasone, (TDI) in a microtissue format	7 days	Improved cellular alignment Increased sarcomere length Increased MYH7, ATP2A2, SLC8A1, PLN, PPARG, ADRB1, CHRM2, p-AKT Decreased MYH6, TNNI1 Increased contractile force Increased time to peak Increased relaxation time	[ 109 ]
Metabolic/biochemical	Media and culture environment using EHT format	3–5 weeks	Increased fatty acid oxidation Increased tetrodotoxin sensitivity Enhanced aerobic respiration Sarcomere-arrayed mitochondria Improved calcium handling Higher IK1 density More negative diastolic membrane potential	[ 97 ]