Abstract
Aims: This study aimed to expand a previous pilot project to verify the effectiveness of a cryopreservation medium using a combination of high molecular weight (HMW) hyaluronic acid (HA) (1.5 MDa) and dimethyl sulfoxide (DMSO) as a cryoprotective agents (CPAs) in cryopreservation of dental pulp stem cells (DPSCs). The combination with HMW HA was used to reduce the concentration of the cytotoxic DMSO further. Conclusion: We extended the pilot project to continue in reducing the DMSO concentration used in the cryopreservation medium. We analysed six new combinations of DMSO and HMW HA classified according reducing DMSO concentration:
1) 5 % DMSO +
0.1 % HA,
2) 5 % DMSO +
0.2 % HA,
3) 4 % DMSO +
0.1 % HA,
4) 4 % DMSO +
0.2 % HA,
5) 3 % DMSO +
0.1 %,
6) 3 % DMSO +
0.2 % HA. According to the rate of the freezing process, the ice crystals are formed intracellularly or extracellularly. Low molecular weight (LMW) CPAs can penetrate the stem cells and provide intracellular protection against ice crystal formation. Oppositely, HMW-CPAs stay extracellularly. The balance in concentration of LMW and HMW CPAs has to be determined for the most effective stem cell protection during cryopreservation. LMW CPAs protect cells from intracellular osmotic stress, and HMW CPAs stabilize the cell membrane and protect stem cells from the formation of extracellular ice crystals. Therefore, the optimal combination of these two cryoprotectants effectively preserve cells from cryoinjury. We tested the gradually reducing DMSO concentration (5%, 4% a 3%) combined with the
0.1 and
0.2 % hyaluronic acid. The average viability of all groups of cells was over 90 % after thawing and 14-day cultivation. All cells expressed CD markers for mesenchymal stem cells. Expressions of CD73, CD 90, CD146 remained the same after cryopreservation. The cryopreserved cells remained highly proliferative active. Moreover, we observed an increased expression of CD49f. CD49f is a part of an integrin family, membrane receptors, which bond with extracellular matrix components or other cells. The bond contributes to cell adhesion, migration, and, therefore, tissue regeneration. CD49f is a "stemness" marker because its higher expression leads to higher proliferation and self-renewal ability and differentiation potential of DPCSs. In our pilot project, DPSCs cryopreserved using 10 % DMSO (control group) had the viability of
93.8 % +-
1.0 % after thawing and one-week cultivation. The average viability of all groups of cells from this project was
93.0 % +-
0.6 % after seven-day cultivation. Even the cells cryopreserved using 3 % DMSO, and HA had a similar viability during cultivation as the control group.