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

Abstract

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(®); 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 ± 1.4 × 10(6)/g, which was significantly higher than that obtained without Cellamigo (0.66 ± 0.53 × 10(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.

Comparison between isolation protocols highlights intrinsic variability of human umbilical cord mesenchymal cells

“…Issues such as source of donor tissue, donor age, environmental background, and isolation methods have been described to impact upon the overall quality of these cells (Badraiq et al 2014;Mori et al 2014;Swamynathan et al 2014). However, individual variability is still an unanswered issue, which we sought to address studying UC-MSCs.…”

Isolation, cultivation, and characterization of human mesenchymal stem cells

“…With 10–15 × 10 3 MSC/cm , human WJ represents a valuable source for MSC isolation, which usually starts by removing blood and vessels. The cord is cut into pieces, which are then processed by different methods including explant cultures , and enzymatic digestion . Alternative methods without removal of vessels have also been described .…”

Research Advancements in Porcine Derived Mesenchymal Stem Cells

“…Recently, Mori and colleagues have demonstrated an improved explant isolation method using cellamigo- an autoclavable stainless steel mesh over minced human umbilical cord pieces to expose more tissue area in contact with plastic surface. Interestingly, more number of cells were isolated with attainment of more confluence within short duration as compared to MSCs isolated by conventional ex-plant method [52], this method can also be utilized for isolation of pMSCs. Other methods may include use of tissue specific digestion enzymes under low temperature conditions followed by mechanical agitation at regular intervals of time instead of longer enzymatic treatment at higher temperatures which causes over digestion of cells and decrease their potency.…”