An innovative minimally invasive method to obtain bonafide elastic cartilage mesenchymal stem cells in xeno-free conditions: a translational perspective

Microtia and related auricular malformations remain among the most challenging problems in reconstructive surgery. Current approaches, including costal cartilage harvesting and auricular chondrocyte progenitor expansion, are constrained by invasiveness, donor-site morbidity, and prolonged culture times. This study aimed to establish a minimally invasive, xeno -free strategy for isolating and expanding mesenchymal stem cells (MSC) from auricular elastic cartilage (MSC-EC) in healthy adults. A tailored mini-biopsy technique was designed to obtain auricular elastic cartilage. Tissue fragments were semi-digested and cultured in StemMACS™ MSC Xeno -free medium, enabling massive cell expansion. Immunophenotypic profiling and CFSE proliferative assays were performed by flow cytometry. Multipotent differentiation into chondrogenic, osteogenic, and adipogenic lineages was assessed in 2D and their capacity to regenerate bona fide elastic-cartilage was evaluated in 3D micro-masses, using histological, cytochemical, immunofluorescence staining and light-sheet fluorescence microscopy. MSC-EC exhibited a canonical mesenchymal phenotype, uniformly expressing HLA-ABC, CD73, CD90, CD105 and CD13, whilst remaining completely devoid of hematopoietic and endothelial lineages (CD45, CD31, CD34, HLA-DRA and CD14), thereby strictly satisfying the International Society for Cell & Gene Therapy (ISCT) criteria. Functionally, CFSE kinetics revealed a high proliferative capacity, paired with robust tri-lineage differentiation potential. Crucially, when transitioned into 3D micro-mass cultures, MSC-EC demonstrated a structurally superior chondrogenic capacity compared to bone marrow-derived MSC. Rather than forming unorganised cellular aggregates, MSC-EC autonomously orchestrated the developmental histogenesis of bona fide elastic cartilage. This mimetic morphology was reached by the precise architectural assembly of a functional extracellular matrix meshwork rich in collagen-II, sulphated glycosaminoglycans and peripheral elastin, seamlessly generating classical lacunae arrangements and chondrons. These biomimetic constructs established a highly organised spatial compartmentalisation. Adult auricular elastic cartilage obtained through our surgical method is a clinically accessible and well-tolerated source of bona fide MSC. The robust proliferative and differentiation capacities of MSC-EC underscore their translational promise for auricular reconstruction and wider osteo- and articular regenerative applications. This study was reviewed and approved (approval number CIE/1719/10/2024) on 21 December 2024.