Cell therapies present a feasible option for the treatment of degenerated cartilaginous and intervertebral disc (IVD) tissues. Microenvironments of these tissues are specific and often differ from the microenvironment of cells that, could be potentially used for therapy, e.g. human adipose-derived stem cells (hASC). To ensure safe and efficient implantation of hASC, it is important to evaluate how microenvironmental conditions at the site of implantation affect the implanted cells. This study has demonstrated that cartilaginous tissue-specific osmolarities ranging from 400-600 mOsm/L affected hASC in a dose- and time-dependent fashion in comparison to 300 mOsm/L. Increased osmolarities resulted in transient (nuclear DNA and actin reorganisation) and non-transient, long-term morphological changes (vesicle formation, increase in cell area, and culture morphology), as well as reduced proliferation in monolayer cultures. Increased osmolarities diminished acid proteoglycan production and compactness of chondrogenically induced pellet cultures, indicating decreased chondrogenic potential. Viability of hASC was strongly dependent on the type of culture, with hASC in monolayer culture being more tolerant to increased osmolarity compared to hASC in suspension, alginate-agarose hydrogel, and pellet cultures, thus emphasizing the importance of choosing relevant in vitro conditions according to the specifics of clinical application.