Abstract: 【Abstract】 Objective To establish a complete system for the isolation, purification, identification, and culture of Kaposiform hemangioendothelioma-derived endothelial cells （KHE-ECs）, to analyze the phenotype of KHE-ECs, and to explore the possibility of establishing a KHE-EC bank. Methods A novel digestion solution for KHE tumors （patent number: CN202410500224.2） was formulated using collection fluid, LiberaseTM and dispase stock solutions, and was used to process tumor tissues to obtain cells. High-purity KHE-ECs were purified using CD31+ immunomagnetic beads. The EGM-2 complete medium containing 10% fetal bovine serum and 2% penicillin-streptomycin solution was employed for cell culture. To verify the characteristics of KHE-ECs, immunofluorescence assay was conducted to determine the expression of endothelial cell-specific markers CD31 and CD34, KHE disease markers podoplanin （D2-40）, prospero-related homeobox 1 （Prox-1）, and lymphatic vessel endothelial hyaluronan receptor 1 （LYVE1）, as well as an infantile hemangioma-specific diagnostic marker glucose transporter 1 （GLUT-1）. Human umbilical vein endothelial cells （HUVECs） served as controls for the phenotype analysis of KHE-ECs, including cell viability, cytoskeleton, proliferation, migration, invasion, tube formation, and sprouting ability. Results Primary cells were successfully isolated from KHE tumor tissues, and high-purity KHE-ECs were obtained by using CD31+ immunomagnetic beads. The cells exhibited typical spindle-shaped morphology and an adherent growth pattern. Immunofluorescence assay showed that KHE-ECs expressed CD31, CD34, D2-40, Prox-1, and LYVE1, but did not express GLUT-1. There were significant differences in cell morphology, cell viability, and cytoskeletal structures between KHE-ECs and HUVECs. Additionally, the KHE-EC group showed significantly increased percentages of proliferative cells （29.1% ± 2.5%）, numbers of migratory cells （114.3 ± 9.4） and invasive cells （110.0 ± 6.1）, tube length （32 121.0 ± 892.0 μm）, and number of sprouting cells （25.0 ± 3.6） compared with the HUVEC group （13.0% ± 2.2%, 38.0 ± 3.6, 35.3 ± 2.3, 25 345.0 ± 448.1 μm, 5.0 ± 1.0, respectively, all P ≤ 0.001）. Conclusion An innovative digestion solution specifically for KHE tumors was formulated for the first time, and high-purity and well-growing KHE-EC strains were successfully isolated and purified by using the novel digestion solution in combination with CD31+ immunomagnetic beads, providing a stable and reliable cell source for subsequent experimental studies on KHE and laying the foundation for establishing a KHE-EC bank.

Key words: Hemangioendothelioma, Endothelial cells, Primary cell culture, Kaposiform hemangio-endothelioma, Digestive fluid