Cell-based transplantation therapies, such as autologous chondrocyte implantation (ACI), are used to treat focal cartilage defects caused by trauma or degeneration. In ACI, chondrocytes are isolated from non-load-bearing regions of healthy cartilage regions and then sent to a cell manufacturing laboratory, where they are expanded for cell number in monolayer culture. Once a large number of cells are obtained, they are transported to the clinic for reimplantation into the defect site. The storage and transport conditions from cell manufacturing to implantation may be a critical time that could influence cell viability and redifferentiation potential. Although hypothermic storage at sub-physiological temperatures is commonly used to preserve cell viability, long-term storage of cartilage under hypothermic conditions can impair chondrocyte viability and function. However, the impact of short-term, acute hypothermic storage on passaged chondrocytes remains largely unknown. 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, 19 or 8°C. In monolayer culture, hypothermic temperatures preserved cell viability with no difference compared to storage at 36°C for up to three days. Additionally, hypothermic temperatures promoted cell rounding, reduced proliferative capacity, depolymerized filamentous actin, and led to a slight reduction in the mRNA levels of specific matrix molecules compared to 36°C. Intriguingly, the effects of hypothermia were context-dependent. Exposure of passaged cells in suspension to hypothermia promoted the maintenance of cell viability and reduced aggregation compared to 36°C. When stored at 8°C in suspension, passaged cells exhibited enhanced expression of specific matrix molecule mRNA levels compared to cells at 36°C in suspension. Subsequently, when passaged cells in suspension at 8°C were seeded in 3D within adherent agarose molds, there was an increase in aggrecan expression 10 days after seeding. The tissues formed by cells stored in suspension at 9°C were thicker and stained more intensely for aggrecan. 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 repair.