Cooling of Cells and Organs Confers Extensive DNA Strand Breaks Through Oxidative Stress and ATP Depletion

Cell Transplant. 2022 Jan-Dec:31:9636897221108705. doi: 10.1177/09636897221108705.


Cooling at 4°C is routinely used to lower metabolism and preserve cell and tissue integrity in laboratory and clinical settings, including organ transplantation. However, cooling and rewarming produce cell damage, attributed primarily to a burst of reactive oxygen species (ROS) upon rewarming. While DNA represents a highly vulnerable target of ROS, it is unknown whether cooling and/or rewarming produces DNA damage. Here, we show that cooling alone suffices to produce extensive DNA damage in cultured primary cells and cell lines, including double-strand breaks (DSBs), as shown by comet assay and pulsed-field gel electrophoresis. Cooling-induced DSB formation is time- and temperature-dependent and coincides with an excess production of ROS, rather than a decrease in ATP levels. Immunohistochemistry confirmed that DNA damage activates the DNA damage response marked by the formation of nuclear foci of proteins involved in DSB repair, γ-H2Ax, and 53BP1. Subsequent rewarming for 24 h fails to recover ATP levels and only marginally lowers DSB amounts and nuclear foci. Precluding ROS formation by dopamine and the hydroxychromanol, Sul-121, dose-dependently reduces DSBs. Finally, a standard clinical kidney transplant procedure, using cold static storage in UW preservation solution up to 24 h in porcine kidney, lowered ATP, increased ROS, and produced increasing amounts of DSBs with recruitment of 53BP1. Given that DNA repair is erroneous by nature, cooling-inflicted DNA damage may affect cell survival, proliferation, and genomic stability, significantly impacting cellular and organ function, with relevance in stem cell and transplantation procedures.

Keywords: 53BP1; DNA strand breaks; cooling; reactive oxygen species; transplantation; γ-H2Ax.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • DNA / metabolism
  • DNA Damage*
  • Histones* / metabolism
  • Oxidative Stress
  • Reactive Oxygen Species / metabolism
  • Swine


  • Histones
  • Reactive Oxygen Species
  • Adenosine Triphosphate
  • DNA