Organoids are novel in vitro models to study intercellular crosstalk between the different types of cells in the disease pathophysiology. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), we developed scaffold-free multi-cellular 3D cholangiocyte organoids (3D-CHO) using 'primary' liver cells derived from normal and PSC patients. Human liver samples from healthy donors and PSC patients were used to isolate 'primary' cholangiocytes (EPCAM+/CK-19+), liver endothelial cells (LECs, CD31+), and hepatic stellate cells (HSCs, CD31-/CD68-/ Desmin+/Vitamin A+). 3D-CHOs were formed using cholangiocytes, HSCs, and LECs, and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence, qRT-PCR, and transmission electron microscopy. Transcription profiles for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, SCTR), fibrosis (ACTA2, COL1A1, DESMIN, TGFβ1), angiogenesis (PECAM, VEGF, CDH5, vWF), and inflammation (IL-6, TNF-α) confirmed PSC phenotypes of 3D-CHOs. Since cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal-immunofluorescence demonstrated that the neurokinin-1 receptor (NK-1R) was localized within CK-19+-cholangiocytes and desmin+-HSCs. Moreover, 3D-CHOs from PSC patients confirmed PSC phenotypes with upregulated NK-1R, tachykinin precursor 1, and downregulated membrane metalloendopeptidase. Scaffold-free multi-cellular 3D-CHOs showed superiority as an in vitro model in mimicking PSC in vivo phenotypes compared to 2D cell culture, which can be used in PSC disease-related research.