TABLE 1.

Overview of tumour organoid culture systems in cancer research

	Tumour organoid culture approach
	Submerged Matrigel culture	Microfluidic 3D culture	ALI culture
Samples	Tumour tissues
Tissue processing before 3D culture	Tissues are dissociated enzymatically and physically	Tissues are dissociated physically and enzymatically; collect 40‐100 μm‐sized spheroid fractions, subsequently maintained in ultra‐low‐attachment plates	Tissues are physically minced into fragments
Culture apparatus	Plate or dish	3D microfluidic culture device	Inner dish, Outer dish, (Transwell plates)
Matrix	Matrigel	Collagen	Collagen
Culture condition	Cell‐Matrigel mixture is plated; medium is added over Matrigel	Spheroid‐collagen mixture is injected into central gel region of device; medium is added into media channels on both sides	Minced tumour tissue fragments are embedded in collagen and plated on bottom collagen layer; medium is added into an outer dish; top of collagen layer is exposed to air
Retained cell types of native tumour tissue in culture	Tumour cells exclusively	Tumour cells, tumour‐infiltrating myeloid and lymphoid cells	Neoplastic cells, native immune cells and stromal fibroblasts
Immune TME	PBMCs, primary leukocytes, TAMs, and DCs can be added in medium	Immune cells can be added in medium; immune TME of primary tissue is faithfully reconstituted	Immune TME of primary tissue is faithfully maintained
Benefits	Easy to enrich and expand	Requires small number of cells and small amount of medium and reagents to test	Preserves diverse immune cells and fibroblasts in TME
Limitations	Lack of non‐neoplastic components	Requires specialized equipment; size limitation; does not reflect recruitment of circulating immune cells into tumour	Creation of uniformly sized organoids; does not reflect recruitment of circulating immune cells into tumour
Refs	43‐46, 50, 52, 54, 77, 79	55, 56, 59	28, 61