Box 1.

Overview of some important parameters when developing a new OoC device

ADVANTAGES	LIMITATIONS
HUMAN TISSUE SOURCE
Embryonic stem cells (ESCs) a , b
Unlimited differentiation potential  More consistent phenotype  Easier to obtain and last longer in culture  Potential to recreate multiple organ-like structures	Ethically controversial (they derived from human embryos) Difficult to create large numbers of genetically diverse cell lines Variability in efficiency of differentiation protocols Difficult to differentiate into distinct, mature cell phenotypes Low efficiency in generating neuronal subtypes Lack of native 3D tissue structure High time and cost when designing OoC devices
Human induced pluripotent stem cells (hiPSCs) a , b , c , d , e , f
No ethical concerns (they derive from adult tissue)  Defined disease phenotypes  Ideal and unlimited source of cells  Patient-specific  Possibility to expand and differentiate into multiple lineages  Genetic homogeneity  Ideal for target-specific drug development  Low preclinical research time	Low efficiency in generating specific neuronal subtypes Lack of native 3D tissue structure High time and cost associated when designing OoC devices Difficult to develop and achieve complete maturation Lack of robust protocols for their differentiation and maturation Availability of patient-specific human cells Limitation in accurate mimicking of human organs Limitation in reproducing cell-cell interactions
Tissue biopsies b , f , g
Derived directly from adult tissue  Maintaining some of the natural ECM and 3D tissue structures	Do not survive more than 48 h Lack of cell proliferation and of human tissue sources
Cell lines a , b , f , h
Widely available and facile handling  Easy to culture and economical  High proliferation under simple culture conditions  Useful in optimizing parameters during OoC development	Lack of natural extracellular matrix Lack the patient-specificity Not accurately recapitulate tissue function Lack the phenotypic function characteristic of the organ they intend to represent
FLOW MANIPULATION
Microfluidic systems f , h , I , j , k , l , m
High reproducibility and sophisticated fluid manipulation  Ideal in mimicking the dynamic cellular environment  Able to sustain complex microfluidic gradients for long time  Can replicate the complexity and interconnectivity of real organs  High throughput and low reagent consumption  Spatial control of liquid composition at subcellular resolution	Presence of air bubbles Laminar flow only produces relative slow diffuse mixing Difficulty in fluid handling
MATERIALS: BIOCOMPATIBLE POLYMERS
Polydimethylsiloxane (PDMS) e , f , h
Transparent and excellent flexibility  Biocompatibility, oxygen permeability and low cytotoxicity  Low cost and easy of processing	Drug adsorption and highly hydrophobic Not degradable Not scalable, due to its softness and elasticity
Poly(methyl methacrylate) (PMMA) n , o , p
Reduce drug, protein or small molecule absorption/adsorption  Can improve the robustness of the OoC during long operations  Low cost, easy to fabricate and manipulate  Low auto-fluorescence and excellent transparency	Affected by important solvents used in microfabrication and sterilization Barely permeable to gas
Polycarbonate (PC) n , p
Transparent and Low cost  High heat resistance and high stiffness and strength	Barely permeable to gas Poor resistance to certain organic solvents
FABRICATION TECHNIQUES
Photolithography e , h , q
Cells can be cultured directly on the patterned materials  Hydrogels can be incorporated, to promote cell seeding and include a physiological ECM environment	Pattern resolution is limited by the light diffraction Expensive and time-consuming Not possible the direct insertion of specific materials (e.g. ECM)
3D printing h , p , r
Cells can be printed continuously and accurately  Controllable resolution, high printing speed, rapid technique and low material costs  Can incorporate proliferation and differentiation cues  Versatile technique able to reproduce 3D geometry  Able to integrate mechanical and electrical sensors	Sometimes, slow printing speeds, not useful for larger tissues or organ printing Low spatial resolution and cellular perturbation Cross-linking: potentially cytotoxic factors, High viscosity of some biomaterials Multiple treatment session with limited micro size precision
Microcontact printing s
Low cost and rapid prototyping	Difficulty in controlling the ink and the surface robustness
Laser-based patterning s
Cells and any particles can be manipulated	Large instrumentation, complex setup
Injection moulding g
Mass production  Low cycle time and highly automated	Restricted to thermoplastic High costs for moulds and complex moulding equipment
Casting u
Process, equipment setup and replication accuracy	Long process time (e.g. labour and lab costs)
CHIP DESIGN
2D system v , w
Study of cell behaviour using simple technologies  Universally known and several protocols available  Simple realization and low cost	Does not adequately represent the natural 3D environment Does not properly reproduce in vivo conditions
3D system v , w
3D architecture very close to in vivo model	Very complex and expensive to build and to control

^a Ronaldson-Bouchard and Vunjak-Novakovic, 2018;

^b Wnorowski et al., 2019;

^c Takahashi et al., 2007 b;

^d Burridge et al., 2016;

^e Cavero et al., 2019;

^f Jodat et al., 2018;

^g Luni et al., 2014;

^h Ahadian et al., 2018;

ⁱ Andersson et al., 2004;

^j Dittrich and Manz, 2006;

^k Kang et al., 2008;

^l Velve-Casquillas et al., 2010;

^m Sivagnanam and Gijs, 2013;

ⁿ Ren et al., 2013;

^o Gencturk et al., 2017;

^p Rodrigues et al., 2017;

^q Chapanian and Amsden, 2010;

^r Nahmias et al., 2005;

^s Martinez-Rivas et al., 2017;

^t Fiorini and Chiu, 2005;

^u Becker and Gärtner, 2008;

^v Osaki et al., 2018b;

^w Coluccio et al., 2019.