Transfection of Bone Marrow Cells In Vitro or In Vivo Prior to Cryopreservation with Manganese Superoxide Dismutase (MnSOD-PL) Protects Frozen Cells from Ionizing Irradiation.

Overexpression of MnSOD in IL-3 dependent murine hematopoietic progenitor cell line 32D cl 3 protects against apoptosis induced by ionizing irradiation, TNF-α, IL-3 withdrawal or other inducers of cytotoxic stress. Cryopreservaton of bone marrow is routinely used in clinical marrow transplantation with the necessary cytotoxic loss of a fraction of cells from the stress of thawing. To determine if overexpression of MnSOD protected marrow from the stress of thawing and also whether frozen cells resisted ionizing irradiation, 32D cl 3 and subclone 2C6 stably transfected and overexpressing MnSOD were tested. Exponentially growing cells 2C6 cells demonstrate increased irradiation survival with an increased shoulder on the survival curve n = 4.95 ± 0.1 compared to 32D cl 3 n = 2.77 ± 0.1 (p = 0.011), with no significant change in the Do = 2.11 ± 0.08 vs. Do = 2.36 ± 0.10 Gy. Cells from 32D cl 3 and 2C6 were frozen in liquid nitrogen in 50% DMSO and stored at a −140°C for a minimum of 24 hrs and irradiated to doses ranging from 0 to 800 cGy while frozen. The cells were thawed 24 hr later, plated in methylcellulose and colonies of greater than 50 cells counted seven days later. The data was analyzed using linear quadratic and single-hit, multi-target models. Cells from 2C6 were more resistant to thawing with survival of 24.3 ± 0.6% compared to 20.9 ± 0.8% for 32D cl 3 (p = 0.018) and to irradiation as shown by an increase in the Do of 2.036 ± 0.225 compared to 1.455 ± 0.149 Gy for frozen 32D cl 3 cells (p= 0.0287). The frozen 2C6 cells resisted the stress of thawing with an increased plating efficiency compared to the frozen 32D cl 3 cells (121.4 ± 3.2 colonies for 2C6 cells and 104.3 ± 3.9 colonies for 32D cl 3 per 500 cells plated, p=0.0018). To determine whether fresh bone marrow transfected in vivo, or in vitro, then frozen and irradiated, also was protected, C57BL/6NHsd mice were injected with MnSOD-PL (100 ug plasmid DNA in 100 μl). Twenty-four hours later, bone marrow was isolated from MnSOD-PL injected mice as well as control mice (a subpopulation transfected with MnSOD-PL in vitro) and frozen in aliquots of 1 × 10⁶ cells. The cells were irradiated while frozen to doses ranging from 0 to 800 cGy, plated in methylcellulose and colonies of greater than 50 cells counted 7 days later. The colonies were divided between nonadherent and adherent in the methylcellulose. The Do was calculated for the adherent colonies, nonadherent colonies and total colonies. In all three cases the Do for the bone marrow treated with MnSOD-PL in vitro or in vivo was higher than the Do of the control bone marrow. The percent HA-epitope tagged total transfected cells was 22.1 ± 3.7%. The data indicates that MnSOD overexpression protects frozen bone marrow perhaps important to increasing preservation efficiency.