Table 1.

Overview of organoid culture techniques in cancer research.

	Organoid culture techniques
	Submerged Matrigel culture	Air-liquid interface culture	Microfluidic 3D culture
Tissue processing before culture	Tissues are dissociated mechanically or/and enzymatically	Tissue is minced into small fragments	Tissues are dissociated mechanically or/and enzymatically; by filtering 40–100 μm-sized tumor fragments are collected and pelleted in ultra-low-attachment plates
Culture matrix And Culture equipment	Matrigel Dish or plate	Collagen Inner dish, Outer dish (Transwell plates with permeable membrane inserts)	Collagen Microfluidic device
Plating condition	Cells culture underneath medium in mixture with 3D Matrigel	Mixture of tissue fragments and collagen plated on the inner dish with a bottom collagen layer; medium is added into an outer dish that can diffuse into the inner trans-well dish through a permeable membrane; top of collagen layer is exposed to air	Spheroid-collagen mixture is inoculated into central gel region of the device; medium is added into media channels on both sides
Preserved cell types of original tumor tissue in culture	Cancer cells exclusively	Cancer cells, tumor-infiltrating myeloid and lymphoid cells, native immune cells, and stromal cells	Cancer cells, tumor-infiltrating myeloid and lymphoid cells, native immune cells, and stromal cells
Modeling the tumor immune microenvironment	PBMCs, DCs and other immune cells can be added to the culture	Immune cells of tumor tissue are faithfully preserved	Immune cells can be added in the medium; immune cells of tumor tissue are faithfully preserved
Advantages	Organoid expansion is convenient	Cellular complexity and architecture of the tumor tissue are maintained as an intact en bloc unit without reconstitution	Cellular diversity and architecture of the tumor tissue are maintained; small amount of medium/reagents and small number of cells are required; it can be automated; mimicking physiological shear flow
Limitations	Stromal components and immune cells are usually not preserved in tissue processing stage, determining the growth factors and/or inhibitors required to maintain all subclones is the laborious and time-consuming process; does not reflect the native tumor-infiltrating immune cells, lack of immune components hinders immunotherapy assessment; allogeneic cultures will result in high background killing compared with autologous systems	Determining the growth factors and/or inhibitors required to maintain all subclones is the laborious and time-consuming process; necrosis and hypoxic cores of organoids; the immune components decline over time and do not persist beyond ~2 months	Determining the growth factors and/or inhibitors required to maintain all subclones is the laborious and time-consuming process; size limitation; requires specialized equipment; the immune components decline over time
Possible future improvements	Culture duration can be extended; establishing organoid biobanks for model standardization across laboratories; incorporating multiple organoid types into single microchips; using synthetic scaffolds with precise ECM composition that is essential for reproducible research; increase immune cellular complexity by both incorporated into, and preserved in; to overcome to the formation of a necrotic core, and better recapitulation of native TME organoid vascularization and perfusion are needed
References	(33–41)	(42, 43)	(44–47)