Recently remarkable progress has been made in the understanding of lymphangiogenesis due to the availability of specific biomarkers. A sentinel lymph node (SLN) biopsy has already been established as a common surgical procedure, and the clinical usefulness of an SLN biopsy has been confirmed in patients with various types of cancer. In this study, a novel method has been successfully developed for the isolation of anatomically defined lymphatic endothelial cells (LECs) from human sentinel lymphatic channels during an SLN biopsy in breast cancer patients by the use of collagenase II digestion. The isolated cells were cultured in EGM-2MV media with 10% fetal bovine serum (FBS) under hypoxic conditions in an atmosphere of 5% O2, 5% CO2 and 90% N2 at 37degrees C. The cultured cells exhibited a monolayer with a cobblestone appearance. Immunofluorescence analysis using selective lymphangiogenesis markers showed expression of vascular endothelial growth factor receptor 3 (VEGFR-3), prospero homeobox 1 (Prox-1) and Podoplanin. Newly established LECs showed expressions of vascular endothelial growth factor (VEGF) family members except VEGF-D and corresponding VEGF receptors by the use of conventional RT-PCR. Treatment with VEGF-C156S (500 ng/mL) apparently induced phosphorylation of the protein kinase Akt, MAPK and JNK in human isolated LECs as determined by Western blot analysis. Peak induction of Akt, MAPK and JNK occurred at 10 to 15 minutes after incubation of the isolated LECs with VEGF-C156S. The use of the MTS cell proliferation assay showed a significant increase in the growth of human lymphatic endothelial cells with VEGF-C156S treatment. The effects of VEGF-C156S (500 ng/mL) on proliferation activity was significantly with 3% FBS condition alone (MTS score: 0.54+/-0.0104, n=3) and a 3% FBS+VEGF-C156S (MTS score: 0.572+/-0.0061, n=3) condition. The isolated and enzymatic digested method adopted for the culture of human LECs is simple and useful for the investigation of the cellular, molecular and genomic properties of LECs.