Prevention of apoptosis as a possible mechanism behind improved cryoprotection of hematopoietic cells by catalase and trehalose.
BACKGROUND
Our previous in vitro work has shown the usefulness of membrane stabilizers and antioxidants as additives in conventional freezing medium to freeze mouse and human hematopoietic cells. The present work was carried out using murine model to test the in vivo engraftment ability of mouse bone marrow frozen with (test cells) or without (control cells) addition of a combination of trehalose and catalase in the medium containing 10% dimethyl sulfoxide (DMSO).


METHODS
Viability, nucleated cell recovery, and progenitor content of revived cells were measured. Freezing efficacy was tested by in vivo assays like colony forming unit-spleen (CFU-S), pre-CFU-S, and short-term engraftment of frozen marrow in irradiated mice. Long-term engraftment ability of frozen marrow was assessed using a Ly5.1-Ly 5.2 chimera model. Levels of apoptosis and reactive oxygen species (ROS) generated in revived cells were estimated. The former by Annnexin V, TUNEL, and DNA laddering and the latter by DCFH-DA probe.


RESULTS
Our results show that the combination of catalase and trehalose with 10% DMSO improves freezing efficacy not only in terms of viability, cell recovery, and progenitor content but also by in vivo assays like CFU-S, pre-CFU-S, and short- and long-term engraftment. Both the level of apoptosis and ROS generation were considerably reduced in test set as compared to control set.


CONCLUSIONS
We conclude that inclusion of a combination of trehalose and catalase in conventional freezing medium leads to enhanced engraftment potential of cryopreserved mouse bone marrow cells probably by preventing apoptotic cell death. Our observation using animal model may have significant clinical implications.
Sasnoor
Lalita M
LM
National Centre for Cell Science, Ganeshkhind, Pune, India.
Kale
Vaijayanti P
VP
Limaye
Lalita S
LS
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001854:Bone Marrow Cells / Q000378:metabolism / Q000502:physiology*; D002374:Catalase / Q000494:pharmacology*; D002470:Cell Survival; D003114:Colony-Forming Units Assay; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004338:Drug Combinations; D005260:Female; D005609:Free Radicals / Q000378:metabolism; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism / Q000502:physiology*; D008297:Male; D051379:Mice; D014199:Trehalose / Q000494:pharmacology*
D003451:Cryoprotective Agents; D004338:Drug Combinations; D005609:Free Radicals; D014199:Trehalose; D002374:Catalase