C-2012: Post-thaw characterization of cord blood-derived mesenchymal stromal cells cryopreserved for up to five years

Abstract

Mesenchymal stromal cells (MSCs) are increasingly used as therapeutic agents for a variety of diseases. Because long-term banking of MSCs is critical for their clinical applications, we sought to determine whether the viability, proliferation potential, capacity for differentiation, migration ability and amenability for genetic manipulation are maintained in MSCs cryopreserved for up to five years. MSCs were established from the mononuclear cell fraction of an umbilical cord blood (CB) sample. Primary adherent MSC monolayers were sub-cultured until passage 12 and aliquots of various cell passages were frozen at −70 °C in the presence of 10% dimethylsulfoxide (Me 2 SO) and 10% fetal bovine serum. MSCs were thawed rapidly at 37 °C, sub-cultured to about 80% confluency, trypsinized, and subjected to trypan blue viability test, seeded in the presence of osteogenic and chondrogenic media, analyzed by flow cytometry for expression of surface markers and by RT-PCR for gene expression of tissue markers, and assessed for migration response. We found that although the viability decreased by up to 20% post-thaw, the proliferation potential of CB-MSCs as measured by population doubling level of 7 was not affected by cryopreservation, nor was their ability to differentiate to osteoblasts (by staining of calcium deposits using Alizarin red) and to chondrocytes (by staining of proteoglycans using Alcian blue). The MSCs retained positive expression for the stromal markers CD73, CD90 and CD105 and negative expression for the hematopoietic markers CD34 and CD45. In addition, they expressed pluripotency genes and early tissue markers for muscle, bone and cartilage. Furthermore, the MSCs retained their migratory response towards the chemokine stromal cell-derived factor, which is up-regulated at sites of tissue injury. In conclusion, CB-derived MSCs cryopreserved for up to 5 years at −70 °C in the presence of 10% Me 2 SO demonstrate high-efficiency recovery of viable, proliferative and functional cells which can be employed for clinical transplantation or manipulated for gene therapy.