We compared the morphological and functional characteristics of cultured unfrozen hepatic stellate cells (HSCs) and cryopreserved HSCs obtained from human livers. We used liver tissues obtained by surgical resection from patients with metastatic liver cancer or with hepatocellular carcinoma. HSCs were isolated and allowed to spread in culture. Comparison of morphological and functional features between the unfrozen HSCs and cryopreserved HSCs was performed at each passage using the following techniques: light microscopy, immunohistochemistry, cell growth curve, metallothionein (MTT) assay, and PI staining, Western blot, real-time polymerase chain reaction (PCR), and gene expression analysis using microarrays. The purity of HSCs was more than 90% in all passages. alpha-Smooth muscle actin (SMA-)positive HSCs gradually increased in successive passages, and the positive cell rate and rate of increase in cell number were similar in both groups. Expression of platelet-derived growth factor (PDGF) receptor, transforming growth factor (TGF)-beta receptor, and alpha-SMA mRNAs and protein was similar during each passage in the two groups. Gene expression was nearly identical at each passage in unfrozen and frozen/thawed samples obtained from the same patient. In conclusion, an adequate protocol for the cryopreservation of human primary cultured HSCs could be established.