Table 2.

Two-in-one system for hPSCs expansion and cardiomyocytes (CMs) differentiation.

hPSCs Culture Format	Characteristics	Culture Vessel	Medium for hPSCs Expansion	Yield	Culture Medium and Format for CMs Differentiation	Efficiency of CMs Differentiation	Advantage	Disadvantage	References
Monolayer culture	Use Matrigel-coated multilayer culture plates with active gas ventilation to maintain CO2 stable in all culture plates for culture hiPSCs. Metabolic purification for CMs using lactate-supplemented media.	4-layer or 10-layer of 632 cm2 culture plates	Modified Stem Fit medium: Stem Fit medium (Ajinomoto, Japan) with amount of most ingredients increased for large-scale culture. Dissociation by Accutase.	Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer and 1.7 × 109 hiPSCs in 10-layer culture plates	RPMI + B27 minus insulin for 7 days, MEMα + 5% FBS for later stage up to Day 12. Inducers CHI99021 and BMP4.	66–87%. After selection, 99% cTnT was obtained. 6.2–7.0 × 108 cells (4-layer) and 1.5–2.8 × 109 cells (10-layer).	High efficiency in generation of pure hiPSCs-CMs since all cells are evenly exposed to purification medium.	Difficult scalability. Difficult cell harvesting.	Tohyama et al., 2017 [78]
Monolayer culture	Perform co-differentiation to CMs and endothelial cells (ECs) from hPSCs in 2D model then forming 3D cardiac microtissue (MT) by mixing CMs and ECs.	N/A	E8/ Vitronectin coat. Dissociation by EDTA 0.5Mm.	N/A	2D monolayer. V-bottom 96 well microplates for microtissue formation. BPEL medium + BMP4 + Activin-A + CHIR99021. Wnt inhibitor XAV939 from Day 3 for CMs and VEGF for ECs differentiation.	Enrichment by VCAM1+ antibody increased CMs from 63% to 85%. The ratio of 85% CMs and 15% ECs presented a good microtissue organization.	Approach by co-differentiation to get cardiac identity-endothelial cells prior to microtissue formation helps promote CMs maturation	This protocol includes several steps and is only applicable for laboratory scale.	Giacomelli et al., 2017 [84]
Microcarriers	Expansion of hESC followed by CMs differentiation in a homogenous process	Ultra low attachment T-25 flask with rocker culture	mTeSR1/ Matrigel coat. Clump dissociation by dispase.	Seeding of 2 × 105 cells/mL yielded 3.74 × 106 cells/mL after 7 days	Microcarrier aggregates. RPMI+B27 minus insulin with CHIR and IWP2.	Yield 2.45 × 106 CM/mL with 65.73% expression of cTnT after 12 days differentiation	Integrate hPSCs expansion and CMs differentiation in a continuous process	CMs were separated from microcarrier by enzymatic dissociation and filter through 40 µm cell strainer	Ting et al., 2014 [103]
Microcarriers	hPSC expansion, differentiation, and purification using microcarriers	500 mL controlled bioreactor	N/A	3.66 × 106 cells/mL after 7 days culture (18-fold increase)	CMs induction medium: N/A. CMs purification was developed using lactate- supplemented medium.	1.33 × 106 CMs/mL with 83.1% expression of cTnT after 23 days culture and purification	A high yield of CMs can be obtained using a large volume bioreactor	Removal of microcarriers before further applying CMs for transplantation, drug screening and disease modeling	Steve Oh et al., 2017 [91]
Carrier-free cell aggregates (3D sphere)	Two methods to form aggregates Batch-feeding: stirring- Controlled aggregates were formed for 48 h. Cyclic perfusion feeding: 24 h after inoculation, stirring was paused for 10 min every 2 h to let cell aggregate.	Static 12-well plate. 125-mL Erlenmey-er (working volume 20 mL). Bioreactor 100 mL.	mTeSR1 with Y-27632. Single cell inoculation by Accutase	60 × 106 cells in 100 mL bioreactor	Matrix-independent aggregates (3D sphere). RPMI+B27 minus insulin with CHIR and IWP2.	In bioreactor, efficiencies are 85%, 54%, 68% (n = 3) after 10 days of differentiation Erlenmeyer ~ 60.4%	Applying carrier-free cell aggregates facilitates cell harvesting compared to microcarriers	Maintenance of cell aggregates required ROCK inhibitor during culture process may change hPSCs metabolome profile.	Kempf et al., 2014 [94]
Carrier-free cell aggregates (3D sphere)	The culture process is defined and standardized in compliance with GMP regulations.	6-well plate; 125, 500, and 1000 mL spinner flasks	StemPro medium with 40 ng/mL bFGF and ROCK inhibitor. Single cell inoculation by Accutase.	Seeding at 2.5 × 105 cells/mL yielded 1 × 106 cells/mL after 3 days	Matrix-independent aggregates (3D sphere). RPMI+B27 minus insulin with CHIR and IWP2.	>90% CM purity after 25 days of differentiation Yield 1.5 to 2 × 109 CM/L	Integrate hPSCs expansion and CMs differentiation in a continuous suspension culture. Suspension culture eliminates the need for coating.	Maintenance of cell aggregates required ROCK inhibitor during culture process may change hPSCs metabolome profile.	Chen et al., 2012;Chen et al., 2015 [95]
Hydrogel	hiPSCs were encapsulated in PEG-fibrinogen hydrogels and differentiated into CMs continuously	Prepare PDMS mold on acrylated glass, put in 6-well plate	mTeSR1 with ROCK inhibitor supplemented for the first 24 h	Cells were seeded at 5.5 × 105 hiPSCs per tissue	3D engineered cardiac tissues (micro island). RPMI+B27 minus insulin with CHIR and IWP2.	75% cTnT positive (day 20). Maturation structural of CMs including tubules can be obtained.	Cardiac tissue can be formed in single seeding step. CMs cultured in 3D hydrogel can be sustained longer for maturity.		Kerscher et al., 2016 [102]