An animal-free bioprocess to synthesize 3D human matrix scaffolds using mesenchymal stromal cells

Basement membrane is a specialized extracellular matrix (ECM) that compartmentalizes epithelial and endothelial tissues and provides essential structural and signaling cues for tissue organization. While fibrillar collagens (Col) of the interstitial matrix (e.g., Col I and Col III) are widely used in tissue modeling, the networking collagens that scaffold basement membrane, including human Col IV and Col VI, remain difficult to access. Commercial basement membrane surrogates such as Matrigel(R) are derived from murine tumors and are ill-defined, dilute, variable, and incompatible with animal-free biomanufacturing. Thus, there is a critical need for human-derived basement membrane matrices that are free of xenogenic contaminants and do not rely on animal agriculture. Here, we investigated whether human mesenchymal stromal cells (MSCs) could serve as a platform to produce self-assembling basement membrane components under chemically defined, xeno-free conditions. MSCs from placental, umbilical cord, bone marrow, and adipose tissues were cultured as three-dimensional spheroids and adherent multilayered sheets. Confocal imaging of whole-mount, decellularized spheroid matrices revealed complex networks of fibronectin and Col IV with topological and organizational features characteristic of native basement membrane. Perinatal MSCs produced distinct matrix architectures consisting of apical fibronectin sheets underlaid by continuous Col IV networks. Time-resolved imaging of umbilical cord MSC-derived matrix sheets demonstrated a reproducible sequence of basement membrane assembly that parallels developmental tissue organization. Together, these findings demonstrate that human MSCs, cultured entirely without animal-derived components, can synthesize functional basement membrane proteins that self-assemble into ordered, tissue-like scaffolds. This work establishes MSCs as a scalable, sustainable, and cruelty-free platform for the manufacturing of human basement membrane matrices for bioengineering and regenerative medicine applications.