TABLE 2.

Advanced 3D cell culturing technique comparison.

	Function	Preparation	Advantages and Applications
Hydrogel-based support	1.ECM can be replicated (Antoni et al., 2015) 2.Can be loaded with biological fluids and water (Antoni et al., 2015) 3.Can osmoregulate (Godugu et al., 2013)	1.Chemical crosslinking, free radical polymerization, irradiation crosslinking, and physical crosslinking via polyelectric complexation, hydrogen bonding, and hydrophobic association (Godugu et al., 2013)	1.Smart hydrogels can respond to environmental stimuli such as changes in temperature, pH, ionic strength, radiation, metal, electric field and more (Godugu et al., 2013) 2.Intestinal flow and diffusive transport (Langhans, 2018) 3.Act as drug storehouses, tissue barriers, and a bioactive moieties delivery system that stimulates the natural reparative process (Torkian et al., 2004; El-Sherbiny and Yacoub, 2013)
Polymeric hard material based support	1.The scaffold is used to replicate the in vivo ECM since cells can attach and form 3D cultures (Dhandayuthapani et al., 2011)	1.The cells are matured on the scaffold to model tumors or tissue (Shantha and Harding, 2003) 2.The cells are then cut to a diameter that fits inside a given test vessel (Hoffman, 2001)	1.The cell treatment procedures are very similar to 2D cell culture (Hoffman, 2001) 2.Very reproducible (Costa et al., 2016) 3.Tumoroids grown using patient samples show promising signs for drug screening and drug development (Peppas et al., 2000) 4.Tissue regeneration in bone, ligaments, cartilage, skeletal and vascular muscle, and central nervous system tissue (Haycock, 2011)
Hydrophilic glass fiber	1.Model the ECM (Cushing and Anseth, 2007) 2.Can be used in migration, invasion, chemo-invasion, and angiogenesis assays (Cushing and Anseth, 2007)	1.Commonly performed using the SeedEZTM lab device by Lena Biosciences 2.3D cell cultures will be more consistent in shape, spread, thickness, and cell distribution in the X, Y, and Z dimensions (Cushing and Anseth, 2007)	1.Can perform spot culture experiments, mixed cell cultures, sol-state gel suspension experiments, non-contact and contact co-culture methods via the three-dimensional feeder layer technique, stack and culture experiments, and side-by-side cultures (Cushing and Anseth, 2007) 2.Cells may be primary cells, secondary cells, and cell lines of various origins and sources (Cushing and Anseth, 2007) 3.Can culture advanced 3D tumor models for long durations of time in vitro (Cushing and Anseth, 2007)
Magnetic levitation	1.The magnetic forces allow cell aggregation while inducing ECM synthesis (Godugu et al., 2013) 2.Promotes cell-cell interaction (Godugu et al., 2013)	1.Created by loading the cells with magnetic nanoparticles and then are exposed to an external magnetic field that causes cells to aggregate into a spheroid (Souza et al., 2010; Talukdar and Kundu, 2012)	1.Does not require a specific medium (Talukdar and Kundu, 2012) 2.Works with normal 2D cell culture techniques (Talukdar and Kundu, 2012) 3.Works with a wide range of cell types (Souza et al., 2010) 4.Not just limited to 96 well-plates (Adine et al., 2018) 5.Takes about 16 h for spheroids to form (Talukdar and Kundu, 2012) 6.Can form a 3D culture without the use of an artificial protein substrate (Talukdar and Kundu, 2012) 7.Can synthesize ECM while forming (Talukdar and Kundu, 2012)
Spheroid microplates with ultra-low attachment coating	1.The ultra-low attachment coating reduces cell adherence to promote spheroid formation (Dhandayuthapani et al., 2011) .	1.Typically made out of polystyrene and treated with hydrophilic or hydrophobic coatings or made with natural polymers such as agarose (Haisler et al., 2015) 2.The v-shaped bottomed wells promote consistent spheroid formation in all the wells (Dhandayuthapani et al., 2011)	1.Transfer of spheroids to a new plate is often unnecessary due to the large volume 96- or 384-well plates (Haisler et al., 2015; Imamura et al., 2015) 2.The spheroids of human breast cancer cells mimicked characteristics in vivo such as hypoxia, dormancy, anti-apoptotic features, and drug resistance in one study (Coleman et al., 2007) 3.3D neurospheres have proven useful in studying growth kinetics and drug toxicity (Imamura et al., 2015)