Table 3.

Schematic summary of technical approaches exploited for organotypic multi‐cultures mimicking CRC.

Part I
Co‐culture settings
Matrix	Device (methods)	Cell types	Application	Technology	Ref
ORGANOTYPIC MULTI‐CULTURES
	Tumoroids formed in agarose micro‐molds	HCT116; HIF; human monocytes	Drug response analysis, co‐culture self‐organization	Metabolic activity analyzers; size measurement; IHC; flow cytometry; live imaging	[183]
	Tumoroids, formed in cell‐repellent surface 96‐well microplates, co‐cultured with Immune cells	HT‐29; MS‐5; healthy donors PBMC (pan‐T cells and NK cells)	Immune infiltrate characterization; immunotherapy response analysis	Flow cytometry; IHC	[186]
Collagen I	CRC Spheroids seeded on the top of a blood endothelial cells layer covering collagen I‐embedded CAFs	SW480, SW620; HLFs;patient‐derived CAFs; BECs	Invasion model; drug response analysis	IF; qRT‐PCR; Circular chemo‐repellent‐induced defect (CCID) assay; Intracellular Ca2+ and 12(S)‐HETE assays	[188]
	Spheroids onto confluent monolayers of myofibroblasts, colon epithelial cells, or HUVEC	HT29, LS180, SW948; CCD‐18Co; CCD‐841CoTr; HUVECs	Drug response analysis; ROS detection	ELISA assay; ROS measurement	[184]
Matrigel	Mesenchymal and endothelial cells are co‐cultivated to obtain endothelial tubes (ETs). CRC tumor Spheroids are added after 5 days.	Patient derived‐tumor spheroids; bone marrow‐derived mesenchymal stromal cells (MSCs); HUVECs	Microvascular niche model	Time‐lapse imaging	[189]
Collagen I; Laminin	Matrix droplets, Cancer mass, formed mixing CRC cells with collagen I gel, are embebbed into a stromal compartment (HDFs+HUVECs).	HT29, HCT116; CCD‐841CoN; HDFs; HUVECs	Invasion studies; endothelial networks measurement	IF; optical projection tomography; qRT‐PCR	[187]
Collagen I; Laminin	Matrix droplets, Cancer mass, formed mixing CRC cells with collagen I gel, are embebbed into a stromal compartment (HDFs+HUVECs).	HT29, HCT116; HDFs; HUVECs	Invasion studies; endothelial networks interactions;	IF; qRT‐PCR; Collagen gel density measurement; WB analysis	[190]
Collagen I	ALI, The inner transwell containing tissue and collagen placed into an outer dish containing medium	Patient‐derived normal and tumoral tissue fragments	ALI PDO cultures characterization; drug response analysis	Immunostaining; cell viability assays.	[104]
Part II
Collagen I	ALI, The inner transwell containing tumor tissue and collagen placed into an outer dish containing medium	Mouse and patient‐derived organoids	ALI PDO cultures characterization; immunotherapy response analysis	Exome sequencing; immunostaining; qRT‐PCR; cytotoxicity assays.	[105]
PCL scaffold; collagen I	3D bioprinting, cancer cells, CAFs, and TECs seeded onto sterile scaffolds	HCT116; activated HUVECs; HELFs	Co‐culture characterization, drug response analysis	IF analysis; RNAseq; qRT‐PCR; live/dead assays; in vivo experiments.	[107]
Rat‐derived dECM	CRC cell lines cultured on liver and lung dECM coated dishes	HT‐29, CRC119, SW480, and Caco2	Drug response analysis; histopathological analysis; gene expression profile analysis; metastatic model	Tissue decellularization; proliferation, apoptosis, anoikis and invasion assays; microarray; in vivo experiments.	[112]
Patient‐derived dECM scaffold	dECM injected with myofibrobastas, epithelial and endothelial cells	Human colonic organoids, myofibroblasts and microvascular endothelial cells	CRC progression model	Illumina sequencing; immunostaining;	[109]
Patient‐derived dECM scaffold; Collagen I	dECM injected with cancer cells	HT29, HCT116	Drug response analysis; EMT induction analysis; gene expression profile analysis; metastatic model	Tissue decellularization; IHC; IF; microarray, qRT‐PCR; migration assay; cytotoxicity assay.	[110, 111]
Fibrin gel	CoC, fibroblasts, endothelial and CRC cells injected into each tissue unit self‐organized within an extracellular matrix.	HCT116, SW480; NHLF; human endothelial colony‐forming cell‐derived endothelial cells	Vascularized micro‐tumor characterization; drug response analysis; tumor–stromal interactions.	IF; time lapse imaging; scRNAseq; gene expression profiles	[191]
Fibrin gel	CoC, the mixture of LFs, HUVECs and CRC cells injected into the central channel to form perfusable vassels. NK cells are introduced into the vessels through the side channels.	HT29, SW480, SW620, HCT116, LoVo and SW48; LFs; HUVECs; healthy donors PBMC (NK cells)	Extravasation, migration, immunotherapy response analysis	Time‐lapse imaging; immunocytochemistry	[192]
Matrigel; Collagen I; Collagen IV	CoC; epithelial channel (epithelial and cancerous cells) and endothelial channel separated by a porous membrane. CAFs, can be incorporated into the epithelial channel.	Caco2; HUVECs; HT29; HCT116; patient‐derived organoids and CAFs;	Intravasation; metabolic analysis; invasion;	IF; qRT‐PCR; invasion assays; LC‐MS	[122]
Thiolated hyaluronic acid, thiolated gelatin, and polyethylene glycol diacrylate (PEGDA)‐based hydrogel system	multi‐OoC, primary gut site (intestine epithelial cells and colon carcinoma cells) and secondary liver site in 2 chambers connected in series	HCT116; INT407; HepG2	Metastatic model; stiffness variation; drug response analysis.	IHC; migration‐invasion assays; cell viability assays	[124]
Thiolated hyaluronic acid, thiolated gelatin, and polyethylene glycol diacrylate (PEGDA)‐based hydrogel system	multi‐OoC, the main chamber populated by CRC cells connected with 4 other chambers (1.liver cells; 2.lung cells; 3.endothelial cells; 4.only matrix) by a double branching microfluidic channel.	HCT116; HepG2; A549; HUVECs	Metastatic model	Cell viability assays; fluorescent imaging.	[125]
Collagen I; Fibronectin	multi‐OoC, intestinal, hepatic, tumoral and connective culture chambers interconnected with microchannels on the microfluidic plate.	HCT116; HepaRG; Caco2; TIG121	Drug response analysis	Gene expression analysis; LC‐MS; cell viability assays;	[126]