Wharton’s Jelly (WJ) tissue is a promising biomaterial, for tissue engineering applications. However, its preservation over a long period in order to be readily available needs further optimization. A possible solution could be the vitrification and storage of WJ tissue at low temperatures. The aim of this study is to evaluate the effect of low temperature in the WJ tissue, which was stored at -196 °C, either with the vitrification or conventional cryopreservation methods. WJ tissues were isolated from human umbilical cords, cryopreserved with the above methods and remained for 1 year at -196 °C. Histological analysis of tissue’s extracellular matrix (ECM), isolation, and characterization of mesenchymal stromal cells (MSCs) were performed. Histological analysis revealed the presence of ECM components such as collagen, sulfated glycosaminoglycans (sGAGs), and the presence of cell nuclei only in vitrified samples. Furthermore, MSCs were isolated and expanded successfully from vitrified WJ tissues, whereas a few viable cells were obtained from conventionally cryopreserved tissues that were not further expanded. In conclusion, this study indicated the proper preservation of vitrified WJ tissues after 1 year of storage, which eventually could be used in tissue engineering and regenerative medicine approaches.