A Rapid, Simple, and Reproducible Method for the Isolation of Mesenchymal Stromal Cells from Wharton's Jelly Without Enzymatic Treatment

The co-infusion of mesenchymal stromal cells (MSCs) with hematopoietic stem cells could improve the hematopoietic engraftment after cord blood transplant. Adult bone marrow is the major source of MSCs for cell therapy. However, bone marrow aspiration involves an invasive procedure and, in the case of a cord blood transplant, requires the use of a third party. The umbilical cord matrix, called Wharton's jelly (WJ), was previously shown to be a valuable source of MSCs. However, the process of cell separation is not standardized and needs to be optimized. In this study, we focused on the efficiency of the isolation procedure and expansion of cells from WJ MSCs isolated from human full-term umbilical cords. MSCs were isolated from the WJ without enzyme digestion or dissection. The procedure was based only on the plastic adhesion capacities of MSCs. Briefly, umbilical cord segments of 5-10 cm were cut longitudinally and plated with the WJ onto a plastic surface for 5 days in an appropriate culture medium. After removing the cord segment, the culture was pursued until subconfluency. The number of cells and their phenotypes, clonogenic capacities, differentiation capacities, immunomodulation, and hematopoietic supportive functions were evaluated. Using this method, we were able to isolate MSCs from all human umbilical cords analyzed (n=50). We obtained a mean of 1.4 x 10(8) cells at the second passage and >7 x 10(9) cells at the third. The expanded cells expressed characteristic markers and presented typical functional properties of MSCs such as differentiation capacities, immunologic properties, and hematopoietic supportive functions. In conclusion, we have established a simple, rapid, and reproducible protocol to isolate abundant MSCs from short segments of umbilical cords.