Low back pain is one of the most disabling health problems worldwide and is correlated with intervertebral disc degeneration. Intervertebral disc degeneration is considered to start in the nucleus pulposus concomitant with the transition from notochordal to chondrocyte-like cells. Current treatments for intervertebral disc degeneration only reduce the symptoms or are highly invasive and affected by complications. A minimally invasive procedure aiming for biological and mechanical repair of the degenerated intervertebral disc is desired. We proposed the injection of mesenchymal stem cells in a hydrogel derived from notochordal cell-rich matrix. In this study, we first investigated whether notochordal cell matrix hydrogel could induce chondrogenesis in adipose derived mesenchymal stem cells and bone marrow derived mesenchymal stem cells in vitro. Thereafter, we investigated whether the injection of notochordal cell matrix hydrogel could directly restore the mechanical behavior of degenerated intervertebral discs ex vivo. We showed that mesenchymal stem cells cultured in notochordal cell matrix hydrogel remained viable and produced the two most important extracellular matrix components of the nucleus pulposus: glycosaminoglycans and collagen type II. Based on the higher fibrotic collagen I production by adipose tissue derived mesenchymal stem cells, we recommend the use of bone marrow derived mesenchymal stem cells for future experiments with notochordal cell matrix hydrogel. Furthermore, we showed that notochordal cell matrix hydrogel might restore mechanical behavior of the intervertebral disc upon early degeneration. However, this needs to be confirmed in a study with a higher sample size. An obvious next step towards in vivo application of notochordal cell matrix hydrogel would include ex vivo injection of notochordal cell matrix hydrogel alone or supplemented with mesenchymal stem cells in degenerated intervertebral discs.