High yield of human umbilical cord-derived mesenchymal stromal cells in an animal component-free culture medium

Abstract

Background & Aim Mesenchymal stromal cells (MSCs) can be isolated from adult and new born tissue sources. Unlike adult bone marrow, the collection of human umbilical cord (UC) tissue offers the advantage of being noninvasive. MSCs isolated from UC tissues are also not negatively impacted by advanced age and certain diseases, which serve their banking for future clinical applications. We have optimized MesenCult™-ACF Plus (MACFP), an improved animal component-free (ACF) culture medium for the efficient derivation and expansion of MSCs from UC tissue. Methods, Results & Conclusion Wharton's Jelly was dissected from the UC, cut into 1-2 mm3 fragments and placed into individual wells of a 24-well pre-coated tissue culture plate in UC-MesenCult™-ACF Plus (UC-MACFP) medium supplemented with an optimized cytokine cocktail, or in control medium containing fetal bovine serum (FBS). MSCs migrated from the explanted fragments in 10-14 days and were replated at 1.5–3.0 × 103 cells/cm2 for long-term expansion in each medium. The proliferative potential of UC-derived MSCs was measured by counting cells at each passage (P) up to P6 in both media and their phenotype was assessed. MSC differentiation potential was examined by culturing these cells in adipogenic and osteogenic differentiation media. The average yield of MSCs from UC was higher in UC-MACFP than in FBS medium (9.9±3.4 × 105 vs. 4.4±1.8 × 105 cells/g [mean ± SEM]; n=11 and 5, respectively). Cell outgrowth was further increased to 2.6±1.0 × 106 cells/g (n=9) with addition of 2.5% human AB serum to UC-MACFP medium. The average fold-expansion of UC-MSCs at each subculture over 6 passages was comparable in UC-MACFP (13.5 ± 0.9; n=13) and in FBS media (12.8±0.6; n=6). The phenotype of MSCs was similar in both UC-MACFP and FBS media, with >95% of cells positively expressing CD105, CD73 and CD90, and