Cell-Free Adipose Liquid Extract Combined with Vascularized Lymph Node Transfer for Treating Lymphedema: Implications for Human Therapy

Background: To evaluate whether the application of cell-free adipose liquid extract (ALE) synergistically enhances the therapeutic efficacy of vascularized lymph node transfer (VLNT) by reconstructing the lymphatic-vascular network and restoring the lymph node immune niche in a rat lymphedema model. Methods: A rat hindlimb lymphedema model was established involving popliteal lymph node excision. Animals were randomized into four groups: Control, ALE only, VLNT only, and VLNT combined with ALE (VLNT + ALE). ALE was prepared using a mechanical emulsification and filtration protocol. Edema resolution was monitored by limb circumference. Lymphatic drainage function was visualized via indocyanine green (ICG) lymphography. Histological assessments included Masson's trichrome and immunofluorescence for LYVE-1 (lymphangiogenesis), CD31 (angiogenesis), and MECA-79 (high endothelial venules, HEVs) to evaluate structural and functional regeneration. Results: The VLNT + ALE group achieved the most rapid and significant edema regression compared to VLNT or ALE alone. ICG lymphography revealed that the combined therapy orchestrated the formation of continuous, linear lymphatic channels, effectively eliminating dermal backflow. Histologically, ALE treatment significantly increased the density of both LYVE-1+ lymphatic vessels and CD31+ blood vessels, suggesting a dual-regenerative effect on the microenvironment. ALE directly promoted tube formation in human lymphatic endothelial cells (HLECs), confirming its pro-lymphangiogenic activity in vitro. Crucially, within the transplanted lymph nodes, ALE treatment restored the expression of MECA-79+ HEVs to levels comparable to healthy young nodes. This indicates that ALE not only supports graft survival but also preserves the essential lymphoid microarchitecture required for immune surveillance. Conclusions: Cell-free ALE synergistically enhances the therapeutic efficacy of VLNT by promoting vascular-lymphatic coupling and restoring the functional immune niche of transplanted nodes. As a readily available, safe, and cell-free biologic adjuvant, ALE represents a promising translational strategy to optimize surgical outcomes in secondary lymphedema.