In cell-based tissue engineering, fibrin hydrogel can be utilized to produce scaffolds to treat cartilage. However, the optimal fibrin formulation for cartilage regeneration has not yet been studied. This study aimed to find the optimal fibrin formulation and determine whether fibrin optimized with human adipose-derived stem cells (hASCs) increased the in-vivo chondrogenic potential. To find the optimal formulation, fibrin constructs were divided into twelve groups with different ratios of fibrinogen (10, 20, 30, and 50 mg/mL) to thrombin (10, 50, and 100 IU/mL), following which the physical and biological properties of cell-free and cell-embedded fibrin were investigated. The results from cell-free hydrogels showed that increases in the concentrations of fibrinogen and thrombin corresponded to increases in stiffness and initial weight. Moreover, hydrogel degradation was inhibited in high-concentration formulations. In cell-embedded fibrin constructs, the variation of gel formulation did not affect cell viability. However, cell behavior depended on the gel formulation. hASCs within high-concentration fibrinogen formulation maintained a round morphology similar to natural chondrocytes. Variations in thrombin concentration had a lesser effect on cell morphology. In terms of in-vivo cartilage formation, the formulation with 30 mg/mL fibrinogen and 100 IU/mL thrombin showed the highest cartilage formation, as evidenced through collagen type II alpha 1 chain (COL2) and safranin-O, 4 weeks after implantation. The results may lead to optimally designed 3D bio-scaffolds in which we can control both cell survival and chondrogenic potential for cartilage tissue engineering. Scaffolds made with the optimal fibrin formulation can be applied to develop cell therapies with mesenchymal stem cells to treat osteoarthritis. (C) 2019 Published by Elsevier Inc.