Auricular reconstruction in children with microtia is one of the more complex procedures in plastic surgery. Obtaining sufficient native material to build an ear requires harvesting large fragments of rib cartilage in children. Herein, we investigated how to optimize autologous chondrocyte isolation, expansion and re-implantation using polyglycolic acid (PGA) scaffolds for generating enough cartilage to recapitulate a whole ear starting from a small ear biopsy. Ear chondrocytes isolated from human microtia subjects grew slower than microtia rib or healthy ear chondrocytes and displayed a phenotypic shift due to the passage number. Rabbit ear chondrocytes co-cultured with mesenchymal stem cells (MSC) at a 50 : 50 ratio recapitulated the cartilage biological properties in vitro. However, PGA scaffolds with different proportions of rabbit chondrocytes and MSC did not grow substantially in two months when subcutaneously implanted in immunosuppressed mice. In contrast, rabbit chondrocyte-seeded PGA scaffolds implanted in immunocompetent rabbits formed a cartilage 10 times larger than the original PGA scaffold. This cartilage mimicked the biofunctional and mechanical properties of an ear cartilage. These results indicate that autologous chondrocyte-seeded PGA scaffolds fabricated following our optimized procedure have immense potential as a solution for obtaining enough cartilage for auricular reconstruction and opens new avenues to redefine autologous cartilage replacement.