doi:10.1007/s12250-016-3889-z

Cell characteristics/ function	2D	3D	References
Cell shape	Single layer	Multiple layers	Edmondson et al., 2014
Morphology	Sheet-like flat and stretched cells in monolayer	From aggregate/spheroid structures	Edmondson et al., 2014
Polarity	Partial polarization	More accurate depiction of cell polarization	Antoni et al., 2015
Stiffness	High stiffness	Low stiffness	Baker and Chen, 2012
Migration	Only one mechanism	Diverse cell migration strategies	Pampaloni et al., 2007 Petrie and Yamada, 2012
Adhesions	Represent exaggerated stages of dynamic in vivo	Generate adhesions comparable with 3D adhesion in vivo	Cukierman et al., 2002
Proliferation	Tumor cells grown in monolayer faster than in 3D spheroids	Similar to the situation in vivo	Antoni et al., 2015
Gene expression/ protein expression	Often display differential gene/protein levels compared with in vivo models	Gene and protein expression in vivo to be present in 3D models	Ravi et al., 2015 Ghosh et al., 2005
Drug sensitivity	Cells are more sensitive to drugs in contrast to 3D cells	Cells are more resistant to anticancer drugs compared with 2D cells	Loessner et al., 2010 Karlsson et al., 2012
Cell-cell interaction	Limited	In vivo-like	Li and Cui., 2014

Table 1. The differences of biological function and cellular characteristics in 2D and 3D systems

Strengths	References
Cells cultured in 3D system can represent a more physiological microenvironment.	Vinci et al., 2012
As compared with 2D cultures, 3D cell cultures more accurately simulate normal	Antoni et al., 2015
cell morphology, proliferation, migration, cell-cell and cell-ECM interactions.	Edmondson et al., 2014
3D cell culture is flexible, cost effective and controllable, as well as a high-throughput platform.	Nickerson et al., 2007
Several 3D models can monitor and control physiological conditions: temperature,	Murakami et al., 2008; Li and Cui., 2014;
pH, oxygen concentration, metabolites and growth factors.	Worthington et al., 2015
Limitations	References
In vivo complex and physiological microenvironment not to be replicated.	Friedl et al., 2012; van Duinen et al., 2015
Poor reproducibility for some biomimetic scaffolds.	Antoni et al., 2015
Some available 3D models to be more time and expensive.	Vinci et al., 2012
Quality of imaging interfering with 3D scaffold size, material transparency and microscope depth.	Antoni et al., 2015

Table 2. Strengths and limitations of 3D cell culture models

Methods	Advantages	Disadvantages	References
MCS cultures	High-throughput assay Best suited for cancer research Reproduces tissue-like organization Co-culture possible	High shear stress Size of spheroid limiting Limited flexibility	Achilli et al., 2012 Asthana and Kisaalita., 2012
3D cell culture using RWV	Low fluid-shear environment Inherent flexibility Easy manipulation of culture conditions Randomized gravitational vectors	Potential length of time Expensive	Barrila et al., 2010 Hjelm et al., 2010 Unsworth et al., 1998 Hammond and Hammond, 2001
Organotypic epithelial raft cultures	Flexibility Suit studying epitheliotropic or fastidious viruses	Expensive Time consuming and laborious	Ozbun and Patterson, 2014 Chow, 2015 Andrei et al., 2010
Scaffold/matrix- based culture	Quiet incorporates growth factors Good extracellular support Easy to set up Available for co-cultures	Expensive Limited in removing cells	Kim, 2005 Breslin and O'Driscoll, 2013
Microfluidic 3D cell culture	The ability to co-culture cells in a spatially controlled manner Generation of and control over (signaling) gradients The integration of perfusion/flow	Expensive special equipment Requirement for high sensitive analytical methods Difficulty to maintain long term flow stability The limited size and low number of cells	van Duinen et al., 2015 Breslin and O'Driscoll, 2013 Li and Cui, 2014
3D perfusion cell culture	Overcomes diffusional limitations Controllable shear stimulates cell functions Can control physiological chemostatic conditions Generates gradients of oxygen, growth factors	High cost	Li and Cui, 2014
3D cell culture by magnetic levitation	Does not induce an inflammatory response by the cultured cells Nontoxic Suitable for co-culture Simple, flexible and effective	Expensive	Souza et al., 2010 Tseng et al., 2013

Table 3. The advantages and disadvantages of different 3D culture models used for human virology

3D models	Devices, scaffolds, technical demands	References
MCS cultures	Microfluidics Hanging drop Pellet culture Spinner flask culture Liquid overlay culture Rotating-wall-vessel bioreactors	Lin and Chang, 2008 Kelm et al., 2003 Achilli et al., 2012
3D cell culture using rwv	Rotating-wall-vessel bioreactors	Antoni et al., 2015
Organotypic epithelial raft cultures	The dermal equivalent is composed of natural dermal elements (collagen matrix with fibroblasts) or a synthetic dermal matrix maintained on a rigid support	Fang et al., 2006 Andrei et al., 2010
Scaffold/matrix- based culture	Natural polymers: such as hydrogel, collagen, Matrigel, laminin, gelatin, hyaluronate, chitosan Synthetic polymers: such as polycaprolactone, polyethylene glycol, polyurethanes and polyanhydrides	Lee et al., 2008 Haycock, 2010
Microfluidic 3D cell culture	Cell patterning inside a hydrogel, exploiting the microfluidic properties and differences in viscosity and pressure 96 microfluidic culture chambers integrated underneath a microtiter plate Microfluidic hanging drop network Dynamically perfused chip-based bioreactor platform	Bischel et al., 2013 Frey et al., 2014 Atac et al., 2013 Trietsch et al., 2013 van Duinen et al., 2015
3D perfusion cell culture	Stirred-suspension culture reactors Rotating-wall-vessel bioreactors Hollow fiber bioreactors Direct perfusion bioreactors	Jasmund and Bader, 2002 Martin and Vermette, 2005 Morin et al., 2003 Zhao and Ma, 2005 Li and Cui, 2014
3D cell culture by magnetic levitation	Consisting of gold nanoparticles, magnetic iron oxide nanoparticles and filamentous bacteriophage	Souza et al., 2010 Haisler et al., 2013

Table 4. Devices, scaffolds and technical demands for different 3D cell culture

Viruses	3D models	Cell types	Application in virology	References
hpv	Organotypic raft cultures	Primary human keratinocytes	HPV-cancer/other virus interaction HPV propagation and infection HPV life cycle Evaluating the efficacy of HPV therapy	Mole et al., 2009 Fang et al., 2006 Andrei et al., 2010
hiv	Organotypic raft cultures	Gingival keratinocytes Primary human keratinocytes	Effect of antiretroviral drugs on primary gingival epithelium Evaluation of multi-targeted drugs	Israr et al., 2010 Balzarini et al., 2013
hcv	3D/RFB 3D/RWV Scaffold-based 3D cultures	FLC4 cells Huh-7 cells HuS-E/2 cell/ Huh-7.5 cells	HCV replication and infection The life cycle of HCV Evaluation of anti-HCV drugs	Murakami et al., 2008 Sainz et al., 2009 Aly et al., 2009
hev	RWV bioreactors	PLC/PRF/5 cells	The viability of virions HEV replication	Berto et al., 2013
hsv	Organotypic raft cultures	Immortalized HaCaT keratin-ocytes Human keratinocytes Primary human keratinocytes	HSV-1 infection, replication and spread Study of antiviral agents	Hukkanen, 1999 Visalli et al., 1997 Balzarini et al., 2013
vzv	Organotypic raft cultures Tissue-like assemblies models using RWV bioreactor	Primary human keratinocytes Human neural progenitor cells	Evaluation of antiviral compounds VZV infection	Andrei et al., 2005 Goodwin et al., 2013
AdV	Multicellular spheroid model Organotypic raft cultures 3D organoids	Primary human keratinocytes HEK-293 cells	Adenovirus mutants Adenovirus vectors	Noya et al., 2003 Wang et al., 2014
NoV	RWV bioreactors	Int-407 cells Caco-2 cells	Virus replication	Straub et al., 2007 Straub et al., 2011

Table 5. Studies of human viruses using 3D cell culture models