Park Seungman, Seawright Angela, Park Sinwook, Craig Dutton J, Grinnell Frederick, Han Bumsoo
School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA.
Department of Aerospace Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA.
J Mech Behav Biomed Mater. 2015 May;45:32-44. doi: 10.1016/j.jmbbm.2015.01.014. Epub 2015 Jan 24.
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.
冷冻保存是组织工程和再生医学的关键支撑技术之一，它能够在不丧失工程组织（ETs）功能的情况下，为其提供可靠的长期储存。然而，设计和开发能够保证解冻后组织功能的冷冻保存方案仍然极具挑战性。冷冻保存面临的主要挑战之一在于难以确定有效且毒性较小的冷冻保护剂（CPAs），以确保解冻后组织的功能。因此，在本研究中，我们检验了一个假设：调节嵌入细胞外基质（ECM）中的细胞的细胞骨架结构，可以减轻冷冻诱导的功能变化，并减少在冷冻保存期间维持ETs功能所需的CPA用量。为了验证这一假设，我们通过将成纤维细胞接种到I型胶原基质中制备了皮肤等效物，从而形成三种不同的细胞骨架结构。这些ETs在有或没有CPAs的情况下，暴露于各种冻融（F/T）条件下。评估了冷冻诱导的细胞-流体-基质相互作用以及ETs随后的功能特性。结果表明，细胞骨架结构和CPA的使用与解冻后功能特性的保存密切相关。随着通过应力纤维形成使细胞骨架结构变强，ET的功能保存得更好。它还降低了保存解冻后功能所需的CPA浓度。然而，如果冷冻诱导的细胞-流体-基质相互作用程度过高，细胞骨架结构会被完全破坏，有益效果变得微乎其微。
