title: Comparative cellular and molecular analyses of pooled bone marrow multipotent mesenchymal stromal cells during continuous passaging and after successive cryopreservation.
abstract: The clinical application of human bone marrow derived multipotent mesenchymal stromal cells (MSC) requires expansion, cryopreservation, and transportation from the laboratory to the site of cell implantation. The cryopreservation and thawing process of MSCs may have important effects on the viability, growth characteristics and functionality of these cells both in vitro and in vivo. More importantly, MSCs after two rounds of cryopreservation have not been as well characterized as fresh MSCs from the transplantation perspective. The objective of this study was to determine if the effect of successive cryopreservation of pooled MSCs during the exponential growth phase could impair their morphology, phenotype, gene expression, and differentiation capabilities. MSCs cryopreserved at passage 3 (cell bank) were thawed and expanded up to passage 4 and cryopreserved for the second time. These cells (passive) were then thawed and cultured up to passage 6, and, at each passage MSCs were characterized. As control, pooled passage 3 cells (active) after one round of cryopreservation were taken all the way to passage 6 without cryopreservation. We determined the growth rate of MSCs for both culture conditions in terms of population doubling number (PDN) and population doubling time (PDT). Gene expression profiles for pluripotency markers and tissue specific markers corresponding to neuroectoderm, mesoderm and endoderm lineages were also analyzed for active and passive cultures of MSC. The results show that in both culture conditions, MSCs exhibited similar growth properties, phenotypes and gene expression patterns as well as similar differentiation potential to osteo-, chondro-, and adipo-lineages in vitro. To conclude, it appears that successive or multiple rounds of cryopreservation of MSCs did not alter the fundamental characteristics of these cells and may be used for clinical therapy.
authors.affiliation: Stempeutics Research Malaysia Sdn. Bhd., Technology Park Malaysia, 57000 Kuala Lumpur, Malaysia, India.
authors.forename: Murali Krishna
authors.initials: MK
authors.lastname: Mamidi
authors.forename: Kavitha Ganesan
authors.initials: KG
authors.lastname: Nathan
authors.forename: Gurbind
authors.initials: G
authors.lastname: Singh
authors.forename: Saratha Thevi
authors.initials: ST
authors.lastname: Thrichelvam
authors.forename: Nurul Ain Nasim
authors.initials: NA
authors.lastname: Mohd Yusof
authors.forename: Noor Atiqah
authors.initials: NA
authors.lastname: Fakharuzi
authors.forename: Zubaidah
authors.initials: Z
authors.lastname: Zakaria
authors.forename: Ramesh
authors.initials: R
authors.lastname: Bhonde
authors.forename: Anjan Kumar
authors.initials: AK
authors.lastname: Das
authors.forename: Anish Sen
authors.initials: AS
authors.lastname: Majumdar
mesh_terms: D015415:Biomarkers; D001853:Bone Marrow / Q000378:metabolism* / Q000502:physiology; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D019070:Cell Lineage; D049109:Cell Proliferation; D002470:Cell Survival; D016922:Cellular Senescence; D015925:Cryopreservation / Q000379:methods*; D003470:Culture Media / Q000378:metabolism; D006801:Humans; D016130:Immunophenotyping; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D010641:Phenotype; D013696:Temperature; D013997:Time Factors; D059467:Transcriptome
chemical_list: D015415:Biomarkers; D003470:Culture Media