Improved Explant Method to Isolate Umbilical Cord-Derived Mesenchymal Stem Cells and Their Immunosuppressive Properties

摘要

The umbilical cord (UC) has become one of the major sources of mesenchymal stem cells (MSCs). The common explant method of isolating UC-derived MSCs (UC-MSCs) involves mincing the UCs into small fragments, which are then attached to a culture dish bottom from which the MSCs migrate. However, the fragments frequently float up from the bottom of the dish, thereby reducing the cell recovery rate. To overcome this problem, we demonstrate an improved explant method for UC-MSC isolation, which involves the use of a stainless steel mesh (Cellamigo((R)); Tsubakimoto Chain Co.), to protect the tissue from floating after the minced fragments are aligned at regular intervals in culture dishes. The culture medium was refreshed every 3 days and the adherent cells and tissue fragments were harvested using trypsin. The number of UC-MSCs isolated from 1 g of UC using the explant method with Cellamigo was 2.9 +/- 1.4x10(6)/g, which was significantly higher than that obtained without Cellamigo (0.66 +/- 0.53x10(6)/g) (n=6, p<0.01) when cells reached 80-90% confluence. In addition, the processing and incubation time required to reach 80-90% confluence was reduced in the improved explant method compared with the conventional method. The UC-MSCs isolated using the improved method were positive for CD105, CD73, CD90, and HLA class I expression and negative for CD45 and HLA class II expression. The isolated UC-MSCs efficiently inhibited the responder T cells induced by allogeneic dendritic cells in a mixed lymphocyte reaction. Conclusively, we demonstrated that the use of Cellamigo improves the explant method for isolating UC-MSCs.