Hypoxia Inducible Factor alpha (HIF-1α) has been described as an important regulator of cellular oxygen homeostasis. The activation of the HIF-1α transcription factor in response to reduced oxygen availability is an important mechanism used by cells under hypoxic conditions that ensures the maintenance of their essential biological processes. Besides, it was demonstrated that the collection and manipulation of hematopoietic stem cells from mice bone marrow and human umbilical cord in a hypoxic environment seems to promote better transplantation efficacy. Different tools that measure cell viability or proliferation capacity in cryopreserved Peripheral Blood Stem Cells (PBSC) have been used to monitor cell quality and probability of engraftment however none of the current protocols have considered the existence of a hypoxic environment under these circumstances. For these reasons, we investigated whether HIF-1α has any influence on pivotal cellular viability assays of long-term storage PBSC samples.

PBSC were cryopreserved using a mixture of dimethylsulfoxide, 6% hydroxyethyl starch and 4% Human Serum Albumin (HAS) as cryoprotectant giving a final concentration of 5% DMSO without controlled rate freezing and kept stored in a mechanical freezer at -84 degrees Celsius (°C). Cryopreserved samples were divided into Group A (50‒200×106/mL, n = 91), Group B (> 200‒400×106/mL, n = 89) and Group C (> 400×106/mL, n = 83) to also investigate the influence of cellular concentration. Determination of viable cells, clonogenicity, CD34+ recovery, cytokines expression in T lymphocytes, HIF-1α mRNA and HIF-1α protein levels were tested in thawed samples cryopreserved for 3, 12, 24 and 36 months from all three groups.

Viable cells, CFU-GM, CD34+ recovery counts and CD4+ T lymphocytes intracellular TNF-α, INF-γ, IL-4 and IL-10 expression were similar without the influence of cell concentration or cryopreservation period. HIF-1α mRNA and HIF-1α protein levels rose identically among the different cryopreservation periods and had similar values to those observed in control cells exposed to hypoxic conditions; also, no differences were observed among the three groups. To assess the influence of HIF-1α on cell viability tests we analyzed twenty-three new PBSC samples with varying cell concentrations frozen for 3-months in which a HIF blocker (named YC-1) was added to the cryoprotectant solution; interestingly a significant decrease in the number of viable cells, proliferative capacity, cytokine expression and HIF-1α protein was observed.

Our results agree with others previously reported which showed that reducing the effects of oxidative stress increases the number and function of hematopoietic stem cells.

We therefore suggest that maintenance of cell viability during cryopreservation of PBSC appears to be associated with increased levels of HIF-1α regardless of cell concentration or cryopreservation length.