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 a 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 ETs 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.

Comparative quasi-static mechanical characterization of fresh and stored porcine trachea specimens

“…Alterations in cell and tissue properties associated with freezing are generally associated with the physical processes involving water such as ice formation or dehydration that can affect cellular integrity and structures such as the extracellular matrix and its associated proteins [13][14][15]. Similar observations were made in a study by Ternifi et al [16] that reported a large drop in the elastic modulus of kidney tissues that were subjected to freezing.…”

Microstructural Parameter-Based Modeling for Transport Properties of Collagen Matrices

“…Since fibrils are connected via branching points, the model yields connections that appear to be in a bent configuration ( Fig. 2(d)) [30]. Thus, the parameter-based model mimics the tortuous collagen fibrils as shown in many SEM images.…”

“…In addition, this study showed that other imaging techniques, such as the SEM, produce more accurate feature size or diameter. Along those lines, we recently measured the fibril diameter of rat tail collagen (monomer-based collagen) as 101.6 nm using SEM [30]. Based on those results, an additional group with 100 nm-sized diameters was included to the parameter-based model to examine the effect of fibril diameter on permeability.…”

“…Recently, it was found that confocal microscopy tends to overestimate the size of an object compared to scanning electron microscopy (SEM) [29]. Thus, we also utilized 100 nm-sized diameter measured by SEM for monomer matrix (type I rat tail collagen) as reported in the other studies [30]. Figure 2(c) shows a close-up image of 3D fibril network with 100 nm diameter obtained from the parameter-based model.…”

Freezing does not alter multiscale tendon mechanics and damage mechanisms in tension

“…While the formation of ice crystals may alter collagen integrity, this alteration has only been observed in engineered biomaterials. 19,20 This suggests that native tendon and ligament are unaffected by the ice crystals, especially when carefully frozen. We preserved fascicles by freezing the whole rat tail with its skin intact without freezing the isolated individual fascicles, and placing fascicles in plastic bags, wrapped in PBS-soaked gauge.…”