Development of a Robust Gel-Free 3D Culture System for Generating Spheroids from Axolotl Blastema Cells

Regenerative biology is a pivotal field aimed at understanding and harnessing the ability of organisms to restore damaged tissues and organs. The axolotl (Ambystoma mexicanum) is a key model organism renowned for its exceptional regenerative capabilities, particularly in limb regeneration. However, current in vitro research is hampered by the lack of well-defined axolotl blastema cell lines and unreliable primary culture protocols. To address these challenges, we present a novel, robust, gel-free 3D culture system for axolotl blastema cells. This system overcomes limitations of 2D culture methods by enabling the formation of spheroid structures that closely mimic the in vivo environment. Using this protocol, we observed that spheroids derived from axolotl blastema tissues retained key regenerative markers, including PRRX1, and exhibited stable expression of crucial blastema markers through extended culture periods. Our approach facilitates the study of cellular and molecular mechanisms underlying limb regeneration in axolotls and provides a valuable tool for drug testing and regenerative research. The development of this 3D culture system represents a significant advancement in regenerative biology, offering a more consistent and reliable model for exploring the regenerative potential of axolotl cells. ### Competing Interest Statement The authors have declared no competing interest.