Influence of Cryopreservation on the Biological Characteristics of Adult Bone Marrow Mesenchymal Stem Cells.
Authors: Luo Yi , Huang He , Liang Bin , Yu Qin 
Published: April 2020

Abstract
To create a stable system for culture and proliferation of MSCs in vitro and an optimized method to cryopresevation of MSCs. Human MSCs cultured in DMEM with low glucose containing 10% FBS. The P1 passage cells cultured in vitro were cryopreseved with two-step freezing procedure or program-controlled freezing procedure, using a DMEM media containing 5% DMSO and 30% FBS. The cells were resuscitated after 3-month cryopreservation. Growth characteristics of passaged MSCs cryopreserved with various methods was analyzed with MTT. The FACS was performed to examine the expression of cell surface molecules. To observe the multuple differentiate potential of MSCs, we induced the MSCs to differentiate into osteogenic cells, lipocytes or neuron cells.The MSCs acquired in this study were homogenous population and have multiplication potential. Over (2~3)×1010 MSCs would have been obtained from 10-ml bone marrow aspirate at 5th passage. The cells were positive for CD29,CD44,CD166, and negative for CD14,CD34,CD45,HLA-DR. It was showed that the rate of trypan-blue resistance was 79%±3.61%,87.67%±2.52% for two-step freezing procedure and program-controlled freezing procedure, respectively. There was significant difference between the two methods(P=0.027). As cells before cryopreservation, the passage 1, 4 and 8 of MSCs after cryopreservation with the two procedures have same growth kinetics. The proliferation potential of P8 passage was lower than that of P1 and P4 significantly(P&lt;0.01). We compared the growth kinetics of same passage MSCs before or after cryopreservation simultaneously. Human MSCs cultures that were cryopreserved by program-controlled freezing procedure grew at faster rates and generated significantly more cells by the end of 8 day growth period as compared to cultures before cryopreservation and after two-step freezing procedure. It was showed that the proliferation potential of P4 cells on days 5, 6 and P8 cells on days 4 through 8 after programmed cryopreservation was enhanced than that before cryopreservation or after two-step procedure preservation(P&lt;0.05). Percentage of CD29 positive cell was(43.97±7.73)%,(87.87±7.35)% and (77.17±10.7)% for MSCs after two-step freezing procedure, before cryopreservation and after programmed cryopreservation, respectively(P&lt;0.01). As for programmed cryopreservation, percentage of CD29 positive cell was same as that before cryopreservation(P&gt;0.05). CD29, CD44 and CD166 expressed stably in each group 7 days, 3 months and 6 months after programmed cryopreservation. MSCs treated with osteogenic medium formed several scattered nodules and then the nodules were calcipectic which was demonstrated by Von Kossa staining. A larger mount of orange lipocytes were observed after MSCs were treated with lipogenic medium and stained with oil red O. The rate of transduction into lipocyte was no difference between before and after cryopreservation(P=0.669). After induction into neron cell, the cells expressed nestin, NSE and NF-M. No difference were observed between before and after cryopreservation(P=0.423, P=0.787, P=0.299). The method can isolate and culture a homogenous population of cells that have unique growth, phenotype. It is ideal that preserving MSCs with DMEM medium containing 5%DMSO and 30%FBS and programmed cryopreservation.