The effects of hypothermic storage on passaged chondrocyte viability and redifferentiation potential

Autologous chondrocyte implantation (ACI) is a cell-based therapy used to treat focal articular cartilage defects. In ACI, chondrocytes from nonload-bearing healthy cartilage regions are isolated and sent to a cell manufacturing laboratory, where they are expanded for cell number in monolayer culture. The expanded cells are then transported back to the clinic for reimplantation into the defect site. The storage and transport conditions from the cell manufacturing facility to the clinic implantation may influence reparative potential. However, the impact of acute hypothermic storage of passaged chondrocyte, which is thought to preserve cell viability, is not fully known. Here, we tested the hypothesis that acute hypothermic storage negatively impacts passaged chondrocyte viability and reduces the capacity for redifferentiation. Passaged chondrocytes were stored either in monolayer culture or in suspension at 36°C, 19°C or 8°C. In monolayer culture, hypothermic temperatures preserved cell viability, promoted cell rounding, reduced proliferation, depolymerized filamentous actin and reduced the mRNA levels of specific matrix molecules compared to 36°C. The effects of hypothermia were context-dependent. Exposure of passaged cells in suspension to hypothermia promoted cell viability and reduced cell aggregation. At 8°C, cells in suspension enhanced expression of specific matrix molecule mRNA levels. Subsequently, when cells in suspension at 8°C were seeded in three-dimensional within agarose molds, aggrecan expression was enhanced and formed thicker tissues. Therefore, in contrast to our hypothesis, we found that hypothermic storage did not have a negative impact; when stored for 1 day in suspension, it had lasting effects on matrix deposition. The storage of passaged chondrocytes under hypothermic conditions may be beneficial for ACI, warranting further investigations of cell hypothermic storage for in vivo repair.