Table 3:

Main characteristics of cell-based approaches that are used for validation of molecular networks

Cell-based approaches
In vitro cell models	• immortalized cell lines  • primary cells isolated from different human tissues  • stem cells  • induced pluripotent stem cells (iPS)
Types of cell culture systems
2D	Cells are growing in a polystyrene rigid plastic surface (Petri dishes, flasks)
3D	Cells are growing as spheroids, in suspension, or organoids surrounded by ECM, or on scaffolds
3D cell culture systems
Spheroids	Liquid overlay culture (LOC)	Hanging drop	Microtechnology	Bioreactor
	Spheroids are placed on a thin layer of matrix composed by a mixture of matrigel with specific growth factors depending on the cell type.	Formation of spheroids in a small volume of cell suspension as a droplet.	Cell suspension is placed on 20 μm to 500 μm micro chambers with various shapes: honeycomb, round, or square(Karp et al., 2007)	Dynamic cell culture condition created by continuous stirring in appropriate glass containers for massive spheroid production(Ou & Hosseinkhani, 2014)
Organoids	Examples of organoids and use:  • human epithelial cells, breast cancer cells, acini-like structures that secreted milk proteins (Emerman, Enami, Pitelka, & Nandi, 1977)  • exocrine pancreas cells used as a platform to study pancreatitis and pancreatic cancer (Huang et al., 2015)  • human derived organoids can be altered genetically, by the genome editing CRISPR/Cas9 technology, to study the role of genes in molecular pathways of interest (Fujii, Matano, Nanki, & Sato, 2015)
Organ-Chips	• Human breathing lung alveolus chip, blood-brain barrier, kidney, skin, placenta, intestine, etc. (Huh et al., 2010; Kasendra et al., 2018; Sances et al., 2018)
Scaffolds	Mineral	Polymeric custom shapes Hydrogel	De-cellularized tissues and organs	3D bioprinting
	Use for bone, dental, craniofacial and axon regeneration: -calcium phosphates composite scaffolds -graphene-based nanocomposites	Use for reconstruction of bone, cartilage, muscle tissues: -biodegradable/bioabsorbable synthetic polymers (PCL, PLA, PEG) -biopolymers (collagen, gelatin, hyaluronic acid, chitosan)	Use for engineering blood vessels and vascularized tissues grafts: -urinary bladder -vagina -liver -heart valves -tendons	Use to create tissue/organ by three bio-printing techniques: -Inkjet -laser-assisted -extrusion

PCL: poly-e-caprolactone, PLA: poly lactic acid, PEG: polyethylene glycol