Primary Cell‐Based Lymphatic Spheroids in Synthetic PEG Hydrogels: A Scalable Model of Lymphatic Sprouting

ABSTRACT

Lymphatic vessels (LVs) are an essential component of the lymphatic system, which serves as the main route of immune cell trafficking and maintains tissue fluid homeostasis. LVs form via lymphangiogenesis during embryonic development and remain mostly quiescent in adults, reactivating in specific contexts such as inflammation. While vascular endothelial growth factor C (VEGF‐C) signaling through VEGFR‐3 is a well‐established driver of lymphangiogenesis, the influence of supporting cells and microenvironmental mechanics is less understood. Although animal models have been indispensable for lymphatic research, tissue‐engineered systems offer reproducibility, tunable microenvironments, and the integration of primary cells, enabling precise mechanistic studies and translational applications. We developed reproducible spheroid models to recapitulate lymphatic sprouting in vitro. Spheroids composed of primary rat lymphatic endothelial cells (LECs) and rat lymphatic muscle cells (LMCs) were coated with laminin and collagen IV to mimic collecting LV architecture. When embedded in 10% polyethylene glycol (PEG) hydrogels, these spheroids formed robust lymphatic networks, and VEGF‐C supplementation enhanced cell viability and sprouting. To improve translational relevance, we co‐cultured primary human LECs with fibroblasts to generate human spheroids, which exhibited similar sprouting patterns. Our spheroid‐based platforms capture essential cellular and mechanical features of lymphatic sprouting, providing a bottom‐up approach to reconstructing LV formation in vitro. By incorporating primary human cells, these models may be adapted for patient‐specific disease modeling and therapeutic screening, offering a versatile and translationally relevant system to advance lymphatic research.