(249f) Hemoglobin Regulates the Migration of Glioma Cells Along Poly(ε-caprolactone)-Aligned Nanofibers

This work seeks to integrate two disparate technologies and examine their combined effect on U251 glioma cell migration. Aligned fibers have been shown to facilitate cell migration in the direction of fiber alignment while oxygen-carrying solutions improve the metabolism of cells in hypoxic culture. Therefore in this work, U251 aggregate migration on poly(ε-caprolactone) (PCL) aligned fibers was studied in cell culture media supplemented with the O₂ storage and transport protein hemoglobin (Hb) obtained from bovine, earthworm and human sources at concentrations ranging from 0-5 g/L within a cell culture incubator and oxygen tensions ranging from 1-19% O₂. In one set of experiments conducted on tissue culture polystyrene – representing a surface conducive to random cell movement – cell viability and the total number of live cells were compared for individually cultured U251 cells under different culture conditions. To determine the effect of Hb concentration and incubator O₂ tension on Hb oxidation, Hb auto-oxidation rate constants were measured 24 hours after Hb incubation at 37°C. Individual cell migration was also quantified using a wound healing assay up to 24 hours after cell seeding. In another set of experiments, U251 cell aggregates were developed and aggregate dispersion/cell migration quantified for cells cultured on PCL-aligned fibers up to 72 hours after aggregate seeding. The results of this work show that the presence of bovine or earthworm Hb improved individual cell viability at 1% O₂ while human Hb adversely affected cell viability at increasing Hb concentrations and decreasing oxygen levels. It was also observed that Hb auto-oxidation rate constants increased with decreasing O₂ levels, increasing Hb concentrations, and was greatest for bovine Hb and the least for earthworm Hb under identical culture conditions. The presence of cells decreased the rate of Hb auto-oxidation. The wound healing assay showed no differences in individual cell migration under most conditions, except for an increase in cell migration at 0.5 g/L bovine Hb at 5% O₂, and a reduction in cell migration at 1% O₂ in the presence of 5 g/L earthworm Hb. The control data also suggests that decreasing the O₂ tension in the incubator from 5% O₂ to 1% O₂ decreased aggregate dispersion on the PCL aligned fibers. However, the addition of bovine Hb at 5% O₂ significantly improved aggregate dispersion. At 19% O₂ Hb did not impact aggregate dispersion. Also at 1% O₂, aggregate dispersion appeared to increase in the presence of earthworm Hb, but only at the latter time points. Taken together, these results show that Hb-based O₂ carriers can be utilized to improve O₂ availability and the migration of glioma spheroids in vitro.