title: Preservation of tissue microstructure and functionality during freezing by modulation of cytoskeletal structure.
abstract: Cryopreservation is one of the key enabling technologies for tissue engineering and regenerative medicine, which can provide reliable long-term storage of engineered tissues (ETs) without losing their functionality. However, it is still extremely difficult to design and develop cryopreservation protocols guaranteeing the post-thaw tissue functionality. One of the major challenges in cryopreservation is associated with the difficulty of identifying effective and less toxic cryoprotective agents (CPAs) to guarantee the post-thaw tissue functionality. In this study, thus, a hypothesis was tested that the modulation of the cytoskeletal structure of cells embedded in the extracellular matrix (ECM) can mitigate the freezing-induced changes of the functionality and can reduce the amount of CPA necessary to preserve the functionality of ETs during cryopreservation. In order to test this hypothesis, we prepared dermal equivalents by seeding fibroblasts in type I collagen matrices resulting in three different cytoskeletal structures. These ETs were exposed to various freeze/thaw (F/T) conditions with and without CPAs. The freezing-induced cell-fluid-matrix interactions and subsequent functional properties of the ETs were assessed. The results showed that the cytoskeletal structure and the use of CPA were strongly correlated to the preservation of the post-thaw functional properties. As the cytoskeletal structure became stronger via stress fiber formation, the ET's functionality was preserved better. It also reduced the necessary CPA concentration to preserve the post-thaw functionality. However, if the extent of the freezing-induced cell-fluid-matrix interaction was too excessive, the cytoskeletal structure was completely destroyed and the beneficial effects became minimal.
authors: School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA.
authors: Seungman
authors: S
authors: Park
authors: School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA.
authors: Angela
authors: A
authors: Seawright
authors: School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA.
authors: Sinwook
authors: S
authors: Park
authors: Department of Aerospace Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
authors: J
authors: J
authors: Craig Dutton
authors: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA.
authors: Frederick
authors: F
authors: Grinnell
authors: School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA; Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA; Birck Nanotechnology Center, Purdue University, West Lafayette, IN, USA. Electronic address: bumsoo@purdue.edu.
authors: Bumsoo
authors: B
authors: Han
mesh_terms: D001696:Biomechanical Phenomena; D002470:Cell Survival; D024042:Collagen Type I / Q000378:metabolism; D015925:Cryopreservation; D003599:Cytoskeleton / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D022502:Stress Fibers / Q000378:metabolism; D023822:Tissue Engineering
chemical_list: D024042:Collagen Type I
keywords: Cryoprotective agent (CPA); Cytoskeletal structure; Dermal equivalents; Viscoelastic properties