Cell culture and gamma irradiation

Primary human adipose-derived mesenchymal stem cells (hADSCs) were acquired from ATCC (ATCC PCS-500-011, Manassas, VA) and cultured in high glucose Dulbecco’s Modified Eagle Medium (Corning; Corning, NY) with 10% Fetal Bovine Serum (Atlanta Biologicals; Flowery Branch, GA) and 1% penicillin-streptomycin (HyClone; Logan, Utah). These hADSCs were selected as they are known to differentiate along several lineages and they are often used for regenerative medicine or tissue engineering applications in the literature. One day prior radiation exposure, cells were passaged and counted using a Scepter^™ (EMD Millipore; Darmstadt, Germany) automated cell counter to ensure the proper cell density was plated for each experiment. Depending on the experiments being performed during that trip, cells were either plated in T25, 6- or 24-well plates.

During travel from the facilities at Clemson University to the radiation facility at Wake Forest School of Medicine (~4 hours), cell culture systems were kept in a portable incubator capable of maintaining proper temperature. Upon arrival, all cell culture systems were placed in a standard incubator (37°C, 5% CO₂) for 1 hour to re-acclimate the cells and media. After this period, cells were irradiated with a custom Cs¹³⁷ gamma irradiator for a total dose of 2Gy and either a lower dose rate (LDR; 1.40Gy/min) or a higher dose rate (HDR; 7.31Gy/min), then placed back in the portable incubator for travel back to Clemson University. Control group cells traveled to the radiation facility but received 0Gy of radiation. Note that the low dose rate for this study is much higher than those from some applications, such as brachytherapy. It is comparable to the low dose rates used in gamma knife.