Table 1.

3D printing assisted spheroid assembly

Printing strategy	Target tissue	Spheroid generation method	Materials	Cell type (density)	Spheroid size/spheroidization time (ST)/fusion time (FT)	Feature	Ref
Extrusion-based printing	Breast cancer	Falcon 8 chamber polystyrene vessel	Matrigel,gelatin-alginate, collagen-alginate	MCF10A, MCF10A-NeuN, MDA-MB-231, MCF7	5000 cells/well Size~100 µm ST: MCF10A cells, 8–10 d;  MCF-7, MDA-MB-231, MCF10A-NeuN, 5–6 d FT: N.A.	•High cell viability in mono- and co-culture •More resistant to paclitaxel than individual cells •Size limitation of spheroid •Limited control over spheroid arrangement	[121]
Extrusion-based printing	Breast cancer	8-well chamber slide	Matrigel	MCF10A MDA-MB-231 (10000 cells/well) HUVECs	Size<70 µm ST: MCF10A, 8 d; MDA-MB-231, 5 d FT: N.A.	•MDA-MB-231 migrates out of spheroids in co-culture with HUVECs •Limited control on bioprinted spheroid location and number •Size limitation of spheroid	[120]
Extrusion-based bioprinting	Breast cancer	Agarose molds cast in MicroTissues®3D Petri Dishes®	Thiol-modified HA, unmodified high concentration HA,	ASCs (2500 cells/spheroid) MDA-MB-231	4800 spheroids/mL Size: 228 ± 22 μm ST: 2 d FT: N.A.	•Reduction of the lipid content •Increased fibronectin, collagen I and collagen VI expression •Limited control over spheroid arrangement •Size limitation of spheroid	[122]
Extrusion-based printing (capillary micropipette)	Cardiac tissue	Pellet centrifugation	Collagen type I (Bio-paper)	Cardiac and endothelial cells, VEGF	Size: 300/500 μm ST: A few minutes FT: 70 h	•Synchronously beating after 90 h •Size-consistent spheroids • Weak spheroids	[123]
Extrusion-based printing (capillary micropipette)	Vascular	Pellet centrifugation	Agarose as temporary support	Chinese Hamster Ovary cell, Human umbilical vein smooth muscle cells, Human skin fibroblasts, porcine aortic smooth muscle cells	Size: 300/500 μm ST: 1-2 h FT: 5-7 d	•Size consistent spheroids • Long fusion time •Large quantity of spheroids preparation is time consuming •Non-uniform structure • Weak spheroids	[138]
Multifunctional Fabion 3D bioprinter with the turnstile system	Thyroid gland	Hanging drop	Collagen	Individual thyroid explants and allantoides	Size: thyroid, 388.2 μm±45.3; Allantoide, 493.6 μm±114.3 ST: 18-24 h	•Turnstile allows the deposition of spheroid one at a time •Improved vascularization	[139]
Scaffold-free bioprinter/Regenova/kenzan method	Glioblastoma	96-well U-bottom plates	N.A.	iPSC-derived human neural progenitor cells (40,000 cells/well), U118 human glioma cells (10,000 cells/well)	Size: 500 µm ST: 48 h FT: 3 weeks	•Mechanical damage to the integrity of spheroids •Fixed spacing between needles • High cost	[131]
Aspiration-assisted bioprinting	Post-myocardial infarction (MI) scarring	Ultra-low attachment 96-well round-bottom plates	HA modified with either adamantane (Ad) or β-cyclodextrin (CD)	Human MSCs, Human cardiac fibroblasts Human iPSC-CM	Size: 5000 cells/200 µm 10000 cells/400 µm ST: 96h FT: 4d	•High resolution positioning (~10% spheroid size) •High density micro-tissue • High cell viability	[137]
Aspiration-assisted bioprinting	/	U-bottom 96-well microplate	Fibrin	3T3,mouse mammary carcinoma line 4T1, HUVECs/MSCs, HDF, electrocytes 2500-10,000 cells/well	80-800 µm (~30 s/spheroid) ST: 24h	•~11%with respect to the spheroid size --position accuracy •Non-uniform spheroids printing	[135]
Aspiration-assisted bioprinting	Osteogenic tissues, cartilage	96-well plate	Carbopol, alginate microparticles	Human MSC spheroids	Osteogenic spheroid, 20000 cells/well; chondrogenic spheroids, 50000 cells/well	•Alginate microparticles: ~34% positional accuracy	[136]
Bio-P3 instrument	Tumor	Nonadhesive agarose micro-mold	N.A.	Rat hepatoma (H35), human ovarian granulosa (KGN), human breast cancer (MCF-7) cells	1250 cells/spheroid feature, 25,000–40,000 cells/toroid feature, and 250,000 cells/honeycomb feature ST: spheroid and toroids, 18-24 h; honeycomb, 48 h FT: 48 h	•Allow large microtissue pick-up • Manually operated •Long fabrication time Limited gripper size available •Optical clarity required	[134]
	Hepatoma	Nonadhesive agarose micro-mold	N.A.	HepG2	375 000 cells/honeycomb mold ST: 24 h	•Syringe pump-better flow control •Allow large part pick-up • Long fabrication time •Optical clarity required	[133]
Micro-manipulator	/	Non-adhesive round-bottom 96-well plate	N.A.	NIH/3T3 spheroids	3000 cells/well	•Spheroid size >300 μm	[140]
Microvalve printing	Breast cancer	Culture in Matrigel for 7 days (0.5 million cells/mL)	Elastin-like protein-RGD hydrogels	Human premalignant breast epithelial cells (MCF10ATs) spheroid	~ 50 μm	•Maintained morphology and size	[124]
Laser direct-write	Breast cancer	High-voltage electric field-driven microbead fabrication	Alginate/collagen Alginate/gelatin	MDA-MB231, MCF-7, or mixed MDA-MB-231/ MCF-7 breast cancer cells	Size: 300–400 μm	•Real-time video monitoring • Microbead shifting • Limited resolution	[125]
Acoustic droplet printing	Oral cancer	Hanging-drop method	GelMA	Oral squamous cell carcinoma (OSCC) cell line CAL27 spheroids, CAF	600 cells/spheroid, Size ~ 150 μm	• Nozzle-free • Contact-free • Low cell damage	[126]