Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors

Table 1 Overview of current in vitro cell culture systems for production of EBs and other cell types

Cell culture methods	Benefit						Detriment						Propose	Yield	Note	Ref.
	a	b	c	d	e	f	g	h	i	j	k	l
1 Hanging drop method	x							x	x	x			Differentiation into three germ layers	ND	Using mES cells	[26]
2 Static suspension culture					x		x						Differentiation into three germ layers and neural lineage	ND	Using mES cells	[30,31]
3 Entrapment of ES cells (methylcellulose)	x							x	x	x			Differentiation into hematopoietic lineage	ND	Using mES cells	[26]
4 Multiwell/ microfabrication
4.1 Round bottomed, low attachment, 96 well plate	x				x			x					Differentiation into cardiac and neural lineage	94% of wells have a single EB with diameter of 415 microns	Using polyvinyl carbonate PCR plate without coating reagents	[37]
4.2 Low adherence, 96 well plate coated with MPC or CS	x				x			x					Differentiation into cardiac lineage	EB formed MPC and CS was increased cardiac differentiation	Using mES cells	[38]
4.3 Round bottomed, low attachment, 96 well plate	x				x			x					Differentiation into hematopoietic lineage	Single EBs were achieved from PC surface but not from PS surface	Comparison of EB formation derived various type of 96 well plate; PS and PS coated with MPC	[39]
4.4 Round bottomed, low attachment, 96 well plate polyvinyl carbonate PCR plate	x				x			x					Differentiation into cardiac lineage	Single EB achieved from PS coated with MPC was near 100%	Comparison of EB formation derived various type of 96 well plate; PS and PS coated with MPC	[40]
4.5 Round bottomed, low attachment, 96 well plate	x				x			x					Differentiation into hematopoietic lineage	Differentiation was achieved with blood cells formed in 90% of EBs	Force aggregation by using centrifugation; Using hES cells	[41]
4.6 V bottomed, 96 well plate	x				x			x					Differentiation into cardiac lineage	> 90% EB formation was achieved from this method	Force aggregation by using centrifugation; Using hES cells	[43]
5 Bioreactor
5.1 A 2-L controlled spinner flask		x	x		x	x	x				x	x	Differentiation into cardiac lineage	4.6 × 109 of cardiomyocytes were produced in a single run	Using MHC-neo ES cells	[53]
5.2 Stirred		x	x	x	x	x	x				x	x	Expansion and differentiation into three germ layers	ES cells went through 13 passages over the same 28 d exhibiting higher pluripotency	Comparison of stirred and static suspension culture	[48]
5.3 Stirred		x	x		x	x	x				x	x	Differentiation into vascular lineage	ND	ND	[28]
5.4 Stirred		x	x	x	x	x	x				x	x	Expansion and differentiation into neural lineage	10 fold increase towards neural differentiation	Using hEC cells	[54]
5.5 Stirred		x	x	x	x	x	x				x	x	Expansion and differentiation into osteogenic lineage	10 fold of calcium per total grams of protein increase over the control culture	Comparison of stirred and static suspension culture; Transplantation	[75]
5.6 Stirred		x	x		x	x	x				x	x	Differentiation into hepatic lineage	No significant difference in the specific albumin productivity of EB derived from different groups	Comparison of stirred suspension culture and hanging drop	[81]
5.7 Stirred + encapsulation (HA and dextran)	x	x	x	x	x	x					x	x	Expansion and differentiation into three germ layers	Dextran can induce EB formation from ES cells	Using mES cells	[31]
5.8 Stirred + encapsulation (agarose) + perfusion	x	x	x		x	x					x	x	Differentiation into cardiac lineage	The cardiomyocytes production in encapsulated culture was higher than without encapsulation	Using MHC-neo ES cells; Comparison of O2 tension	[72]
5.9 Two type of stirred, STLV and static suspension culture		x	x		x		x				x	x	Differentiation into cardiac lineage	EB formed GBI resulted in high EB yield with homogenous in size	Comparison of hydrodynamic condition (shear force)	[65]
5.10 RCCS (STLV and HARV)	x	x	x		x	x					x		Differentiation into three germ layers	3 fold enhancement in generation of EBs compared to static culture	Comparison of different type of bioreactors and suspension culture; Using hES cells	[23]
5.11 STLV	x	x	x		x	x					x		Differentiation into cardiac lineage	> 90% of the NTEBs generated beating area	Comparison of STLV and static suspension	[62]
5.12 HARV+ encapsulation (alginate)	x	x	x		x	x					x		Differentiation into osteogenic lineage	ND	Using mES cells	[70]
5.13 HARV+ encapsulation (alginate) + biograss	x	x	x		x	x					x		Differentiation into osteogenic lineage	ND	Using 70s bioglass	[71]
5.14 Rotary suspension culture using an orbital rotary shaker		x	x		x	x	x					x	Differentiation into three germ layers	20-fold enhancement in the number of cells incorporated into primitive EBs in rotary vs static conditions was detected in the first 12 h	Comparison of rotation, static suspension and hanging drop	[64]
5.15 Orbital shaker + microsphere fibrification	x	x	x	x	x	x						x	Differentiation into three germ layers	Degradable PLGA microspheres releasing RA were incorporated within EBs and induced cystic formation earlier than in non microspheres	Degradable PLGA microspheres releasing RA were incorporated within EBs and induced cystic formation	[68]
5.16 Perfused and dialyzed STLV	x	x	x	x	x	x							Differentiation into neural lineage	Perfused STLV can decrease in expression of markers of undifferentiated stage and increase in expression of markers of differentiation, specifically focusing on the neural lineage	Comparison of perfused and dialyzed STLV, perfused STLV, non-perfused STLV and suspension culture	[73]

a: Homogeneity of EB; b: Scalable production of EB; c: Controlled monitoring; d: Integrated single step of culture (expansion and differentiation); e: Easy to manage; f: Flexible culture cells; g: Heterogeneity of EB; h: Small scale production of EB; i: Labor-intensive procedure; j: Difficult to manage; k: Requires a lot of medium; l: Shear force; ND: No available data; EB: Embryoid body; MPC: Methacryloyloxyethyl phosphorylcholine; ES: Embryonic stem; hES: Human embryonic stem cells; mES: Mouse embryonic stem cells; PCR: Polymerase chain reaction; MPC plate: 96-well polystyrene plate coated with 2-methacryloyloxyethyl phosphorylcholine; PS plate: Polystyrene plate; CS plate: A polystyrene plate coated with a type of glycosaminoglycan; HD: Hanging drop; hEC: Human embryonic carcinoma stem cells; MHC-neo: Myosin heavy chain-neomycin resistance; O₂: Oxygen; RCCS: Rotating cell culture system; HARV: A high aspect rotating vessel; STLV: A slow turning lateral vessel; NTEB: EB derived from nuclear transfer ES; PLGA: Poly(lactic-co-glycolic acid)/poly (L-lactic acid).