Table 1.

Overview of Ex Vivo Tumor Organoid Culture Systems Modeling the Tumor Immune Microenvironment

Feature	Method
	Submerged Matrigel culture	Microfluidic 3D culture	ALI culture
Source	Patient-derived and mouse-derived tumor specimens
Tissue processing before culture	Tissues are dissociated physically and enzymatically (e.g., collagenase, dispase, and trypsin)	Tissues are dissociated physically and enzymatically (collagenase); samples are passed over filters to collect 40–100 μm-sized spheroid fractions, subsequently maintained in ultra-low-attachment plates	Tissues are physically minced into fragments
Matrix	Matrigel	Collagen	Collagen
Culture instrument	Any size of plate or dish	3D microfluidic culture device	Diverse cell culture inserts and dishes or plates, including multiwells
Plating procedure	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 tumor 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
Cell types of components retained in culture	Tumor cells exclusively; difficult to maintain stromal components long-term	Tumor cells, tumor-infiltrating lymphoid and myeloid cells, including DCs, MDSCs, and TAMs; determined by flow cytometry	Tumor cells, native immune cells (T and B cells, myeloid cells, macrophages, and NK cells) and stromal fibroblasts; determined by flow cytometry, single cell RNA-seq, and immunofluorescence
Culture period	Long-term culture to maintain and expand tumor organoids; short-term reconstitutive co-culture with different types of immune cell	Short-term culture; long-term culture is not reported	Tumor cells can propagate long-term; immune cells and fibroblasts in both human and mouse organoids decline over a 1–2-month period
Advantages	Easy to enrich and expand tumor organoids; can recapitulate genetic and morphological alterations of original tumor; potential recapitulation of clinical responses to chemotherapy and/or radiation	Requires small number of cells and small amount of medium and reagents to test; preserves multiple different types of cell in TME; enables study of tumor-immune interactions	Recapitulates genetic and morphological alterations of original tumor; preserves diverse immune cells and fibroblasts in TME; enables study of tumor–immune interactions
Limitations	Lack of native immune and stromal components; exogenously added TME only	Size limitation; requires specialized equipment; restricted to native tumor-infiltrating immune cells; does not reflect recruitment of circulating immune cells into tumor	Creation of uniformly sized organoids; restricted to native tumor-infiltrating immune cells; does not reflect recruitment of circulating immune cells into tumor
Co-culture system to reconstitute immune TME	Organoids can be co-cultured with PBMCs, primary leukocytes, TAMs, and DCs that are added to medium	Immune cells (Jurkat cells) can be added in medium to assess T cell infiltration into organotypic tumor spheroids; immune TME of primary tissue is faithfully reconstituted	Immune TME of primary tissue is faithfully reconstituted
Potential of immune cells in culture	Co-culture of autologous PDOs and PBMCs enriches tumor-reactive T cells, which can be used to assess efficiency of T-cell mediated cytotoxicity; enables assessment of tumor organoid killing by co-culture with TILs and CAR cells	Recapitulates response to anti-PD-1 antibody; useful culture system to test therapeutic combinations to enhance response to PD-1 response; secreted cytokine profiling	Recapitulate functional T cell activation and tumor-killing responses to anti-PD-1 and anti-PD-L1 antibodies; TCR repertoire highly conserved between TILs of original tumor and ALI PDOs
Refs	[37–40,50,52,53,72,92]	[43,44,55]	[36,46]