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Objective: Cultivation of chondrocytes in vitro as chondrospheres or 3D tissue spheroids allows maintaining their chondrogenic phenotype. We hypothesized that chondroblasts derived from human perichondrium could serve as a new more superior cell source for tissue spheroids-based cartilage regeneration. The regenerative potential of tissue spheroids fabricated from chondroblasts derived from human rib's perichondrium has been investigated in vitro. Methods: Human chondrocytes have been isolated from human cartilage and human chondroblasts have been isolated from human rib's perichondrium. Tissue spheroids have been fabricated using ultra-low attachment spheroid microplates (Corning, USA). The diameter of tissue spheroids have been estimated as well as their biomechanical properties ("Microsquisher", CellScale, Toronto, Canada). Regenerative potential of tissue spheroids have been investigated by study of tissue spheroids fusion kinetics, tissue spheroids spreading kinetics and, finally, by morphological study of in vitro regeneration of microdefect in the organ culture of isolated piece of human cartilage. Results: It has been demonstrated that 3D tissue spheroids fabricated from perichondrial cells grow faster and synthesize more extracellular matrix. Also, they fuse, spread and regenerate standard microdefect in cartilage organ culture more efficiently than chondrospheres fabricated from cartilage chondrocytes. Conclusion: Taken together our data strongly suggest that chondrospheres fabricated from chondroblasts isolated from human rib's periochondrium have superior regenerative potential estimated in vitro as compared to chondrospheres fabricated from differentiated chondrocytes isolated from human cartilage. [ABSTRACT FROM AUTHOR]