Quality and safety of cryopreserved human mesenchymal stem cell(MSC) during serial subculture

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Objectives:1) To examine surface marker profile of human bone marrow-derived mesenchymal stem cells (MSCs) during the two subculture processes; during the primary culture and subculture (the 2nd passage) and during cryopreservation and subculture (the 3rd passage).2) To examine the safety of cryopreserved MSC.Methods:1) The expression of the stem cell markers, CD105, CD133, CD166 and CD271, was analyzed at the end of each individual passage.2) The expression level of CD271 at each time point was compared. The viability of MSC during osteogenic differentiation was examined in vitro.3) In vitro osteogenic potential of MSCs was compared in the presence or absence of bone marrow washing process.4) Chromosome analysis was performed using the cryopreserved MSCs by G-banding.Results :1) MSCs were 100% positive for CD105, CD133 and CD166 antigens at the 2nd and 3rd passages.2) The expression of CD271 (+) and CD271 (-) cells was similar at either passage, without difference in the ratio of CD271 (+)/CD271 (-) cells.3) Both CD271 (+) and CD271 (-) cells preserved their multilineage differentiation potential, even after the primary culture or even after cryopreservation.4) Viability of osteocytes was significantly higher for CD271(+)cells at the 2nd passage and for CD271 (-) at the 3rd passage.5) Washing process of the bone marrow increased significantly in vitro viability of osteocytes.6) G-banding analysis showed no chromosomal abnormality at each passage after primary culture and after cryopreservation (5, 7 and 10 years). However, there was a decline in cell viability for the MSCs that had been cryopreserved for 5, 7 and 10 years.Conclusion :Osteogenic potential of CD271 (+) MSCs decreased after cryopreservation. G-band chromosome analysis revealed no cryopreservation-related abnormality. Washing of the bone marrow before primary culture may be an appropriate step for preparation of MSC.