Department of Histology
Senescence of Cryopreserved Umbilical Cord Mesenchymal Stem Cells in Platelet Lysate Containing Medium
Jeanne Pawitan1,2, Noviyanti Goei3,4, Isabella K. Liem2,5, Dian Mediana3,4.
1Department of histology, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia; 2Stem Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital - Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia; 3Biomedical Master program, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia; 4Department of Anatomy, Faculty of Medicine Universitas Trisakti, Jakarta, Indonesia; 5Department of Anatomy, Faculty of Medicine Universitas Indonesia, Jakarta, Indonesia
Background: For cell therapy, cells need to be characterized, including their senescent profile. Multiple harvest explant method (MHEM) derived umbilical cord mesenchymal stem cells (UC-MSCs) that were cultured in platelet lysate (PL) containing medium was shown to undergo senescent beginning at passage-10 (P-10), but until P17, the senescent percentage was below 5%. However, for the same cells, there are no senescent data after cryopreservation.
Methods: MHEM derived UC-MSCs were isolated and cultured in PL containing medium as described previously , and cryopreserved in 10% PL, 10% dimethyl sulfoxide (DMSO) containing alpha minimal essential medium (αMEM) with a cell concentration of 500,000 cells/mL. Cryopreservation procedure was done at -20o C for 24 hours, and then moved into liquid N2 tank, vapour phase for one month. Thawed cells were recultured until P-6 (six replicates each, one for reculture and five for staining) in 12-well plate with a seeding density of around 20,000 cells per well. Upon 80-90% confluent, senescent (β galactosidase) staining was done for all passages, random photographs was taken from all stained wells, and senescent percentage was recorded.
Results: No senescent cells were observed at P-2, and senescent cells began to appear at P-3 in all wells and were consistent until P-6, but the percentage was below 5%. Moreover, senescent cells appeared larger compared to nonsenescent cells.
Discussion: Our previous result showed that reculture after cryopreservation in the same method as this research caused enlargement of cell size at P-2 compared to fresh cells (mean value 2674 vs 2056) . A study showed an increase in cell size with increasing passage, which was supposed to be due to senescence , which corroborated our result.
Conclusion: cryopreserved MHEM derived UC-MSCs are at best senescent profile at P-2 .
This study was funded by a grant from the Department of National Education of the Republic of Indonesia (PUSNAS 2015, contract number: 0435/UN2.R12/HKP.05.00/2015).
 Pawitan JA, Liem IK, Budiyanti E, Fasha I, Feroniasanti L, Jamaan T, Sumapradja K. Umbilical cord derived stem cell culture: multiple-harvest explant method. Int J PharmTech Res 2014a;6(4): 1202-8.
 Goei N, Liem IK, Pawitan JA, Mediana D. Effect of Platelet Rich Plasma on Post Cryopreservation Viability, Morphology, and Proliferation of Human Umbilical Cord Stem Cells. Online Journal of Biological Sciences 2015; 15 (2): 42-8.
 Scheers I, Lombard C, Paganelli M, Campard D, Najimi M, Gala J, et al. Human umbilical cord matrix stem cells maintain multilineage differentiation abilities and do not transform during long-term culture. Plosone. 2013; 8(8): 12 pages. DOI: 10.1371.
11:00 - 12:30
|Mesenchymal Stromal Cells||Senescence of Cryopreserved Umbilical Cord Mesenchymal Stem Cells in Platelet Lysate Containing Medium||Room 111-112|