Cartilage tissue engineering (TE) is a promising osteoarthritis therapy whereby cellseeded constructs are generated in vitro for use in restoring degenerated cartilage in patients. While cartilage TE technology has exhibited growing clinical success, it continues to be encumbered by the utilization of high cost and laborious protocols, such as the need for frequent replenishment of culture media (every other day) during the duration of standard in vitro cultivation phases (2-8 weeks). This constitutes a significant time/cost burden for researchers and clinical technicians. Interestingly, the adoption of this convention is based on traditional cell culture protocols, rather than on a fundamental understanding of the stability of culture media constituents in current cartilage TE culture systems, leading one to consider that current TE replenishment protocols may be far from optimized. In the current study, we hypothesize that larger media volumes can be used to:​ 1) mitigate the depletion of constituents and accumulation of waste products in tissue constructs over time and accordingly, 2) reduce the media replenishment frequency required to generate engineered cartilage with functional mechanical properties and composition.

Bovine chondrocyte-seeded agarose constructs (Ø4mm×2mm) were cultivated for 7 weeks in chondrogenic media of increasing cumulative media volumes (3mL, 6mL, 9mL, 18mL, and 54mL) and replenishment frequencies, including the conventionally utilized thrice-weekly and lowered frequencies of weekly, biweekly, and replenishment-free. The stability of influential media constituents (glucose, ascorbic acid, insulin), waste product accumulation (assessed via pH), and the properties of constructs were monitored over time. Results demonstrated that concentrations of growth-promoting media constituents and pH decreased over culture duration but this decrease can be mitigated by the use of larger replenished media volumes. For all replenishment frequencies, tissue construct mechanical properties and sulfate glycosaminoglycan (sGAG) content generally increased with replenished media volumes. For weekly, biweekly, and replenishment-free frequencies, the generation of constructs with native properties required the higher replenished media volumes per replenishment but did not require the use of higher cumulative media volumes. These results suggest that functional engineered cartilage can be generated with lower media replenishment frequencies or replenishment-free conditions. These protocols may be adopted in clinical and research-grade TE platforms to reduce labor costs and contamination risk.