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

Abstract

Regenerative biology seeks to uncover the principles enabling organisms to restore complex tissues and organs. The axolotl (Ambystoma mexicanum), a salamander with unparalleled limb regenerative capacity, remains a premier model system in this field. However, in vitro studies have been limited by the absence of reliable culture platforms for maintaining blastema cell identity and functionality. Here, we present a robust, gel-free 3D culture protocol that enables the formation of spheroids from axolotl blastema cells under fully defined, serum-free conditions. These spheroids preserve the expression of key regenerative markers such as Prrx1, Msx2, and CTGF for at least 10 days in culture, and exhibit cellular stability supported by antioxidant and amino acid supplementation. Importantly, spheroids cultured for 10 days were capable of initiating extra digit formation when transplanted into limb bud after amputation. With unique advantage of fully defined incubation conditions, this technique adds a powerful in vitro platform to existing experimental models of axolotl limb regeneration.