Document Type : Original Article
Authors
1
Cellular and Molecular Biology Research Center, Babol University of Medical Sciences, Babol, Iran
2
Department of Anesthesiology, Roohani Hospital, Babol University of Medical Sciences, Babol, Iran
3
Cellular and Molecular Biology Research Center, Babol University of Medical Sciences, Babol, Iran.
4
Department of Pharmacology & Physiology, School of Medicine, Babol University of Medical Sciences, Babol, Iran.
5
Student Research Committee, Babol university of medical Sciences, Babol, Iran
6
Department of Pharmacology & Physiology, School of Medicine, Babol University of Medical Sciences, Babol, Iran
Abstract
Cryopreservation is the method of choice for long term storage of human PBMCs. This study was designed to
compare the different combinations of variables affecting the cryopreservation of PBMCs samples.
The viability of PBMCs separated from 2×5 ml peripheral blood samples obtained from 16 healthy adult
volunteers, were measured using trypan blue dye exclusion method just before freezing with different
concentrations of DMSO (10, 15, and 20%) and FBS (40 or 70%) at two different temperatures (either 4oC or
25oC). Then after 2 weeks the cells were thawed and the viability was measured again. Also the PBMCs
response to PHA was measured after 48 h using MTT assay. The effects of the different variables were
calculated and compared among the groups.
A total of 192 PBMCs cryotubes made from blood samples of 16 volunteers were tested. The viability of the
cells obtained by the two centrifugation procedure was the same (both more than 99%). The concentration of the
FBS (40 vs 70%) did not show to have significant effects on either cells viability or response to PHA. On the
other hand 20% DMSO concentration and freezing temperature at 25oC decreased both cells.
Based on the obtained results, it is recommended to centrifuge the PBMCs under higher revolt speed at shorter
time (700 g for 20 minutes) and decrease the FBS concentration to 40%. The DMSO concentration should be
kept at 10-15% and the freezing medium be cooled down to 4oC.
Keywords