Measurement of Spatiotemporal Intracellular Deformation of Cells Adhered to Collagen Matrix During Freezing of Biomaterials

Freezing of biomaterials is adopted in a wide variety of biomedical applications including cryopreservation of native and engineered tissues [1], preservation of biospecimens [2], decellularization of native tissues for scaffold engineering [3,4], and cryosurgery of diseased tissues [5]. Although the objectives of these cryomedicine applications are diverse, it is commonly critical to understand the effects and consequences of freeze/thaw (F/T) on the functionality of tissues and biomaterials. However, the mechanism of the freezing-induced biophysical interactions within tissues and biomaterials is not fully understood yet, and many of the freezing protocols were developed empirically and tissue-type specifically. Quantitative knowledge of freezing-induced biophysical phenomena will be useful to design successful cryomedicine applications. Although post-thaw cell viability [6] has been the primary target of these applications, it has recently been recognized that for tissues, where cells are embedded in a complex three-dimensional extracellular matrix (ECM), other features beyond viability are also important to the functionality of biomaterials. This includes the microstructure of the ECM, state of the cell-matrix adhesion, and the cytoskeletal structure and organization [7–10]. Freeze/thaw of biomaterials has been reported to induce microstructural changes in the extracellular matrix due to interstitial ice formation [8,11,12]. This tissue-level microstructural damage has been investigated using multiphoton-induced autofluorescence and second harmonic generation microscopy [13], magnetic resonance imaging [14], and histological analysis [10,15]. Though successful preservation of microstructures was reported for aortic and pulmonary valves [8] and articular cartilages [16], significant change in tissue functionality, as well as in structural and mechanical properties was also observed in other types of tissues [17–19]. To explain these microstructural changes, it was proposed that they result from complex cell-fluid-matrix interactions during freezing [7,20,21]. These studies were based on a hypothesis that biological tissues can be approximated as a poroelastic material that consists of fibrous collagen ECM saturated with interstitial fluid. These studies suggest that the volumetric expansion associated with the freezing of the interstitial fluid induces complex interactions among the ECM, fluid and cells, and these interactions result in the enlarged pore structure of the ECM and extrusion of the interstitial fluid post-thaw [7,21–23]. A measurement technique was developed [23] to quantify spatiotemporal deformation inside the ET, and these studies found that ECM deformation during freezing can be correlated to the change in ECM microstructure post-thaw [7,20]. Recently, it has been proposed that the cells play an important role in these interactions by providing structural support to the ECM and mitigating the post-thaw structural changes of the ECM. This protective role of the cells is thought to be associated with increased structural strength by cell-matrix adhesion and the strength of the cytoskeleton [24]. This finding is potentially very useful to design cryomedicine applications by (i) providing a new way to preserve functional tissues with a reduced amount of toxic cryoprotective agents and (ii) controlling and modulating the hierarchical porous structures (i.e., ECM and cytoskeleton) mechanistically during freezing, which may enable decellularization with minimal structural change of the ECM during freezing. In order to demonstrate this cell-mediated structural support, it is crucial to quantify the spatiotemporal deformation of the cells attached to the ECM during the freezing-induced complex cell-fluid-matrix interactions. However, it is very difficult to measure these cellular and sub-cellular deformations (i.e., small length scales) within very short time intervals (i.e., small time scales). Currently available deformation measurement techniques have been developed to quantify tissue and ECM-level deformation [23] or to assess traction force distribution on the cell periphery during slow processes, such as matrix remodeling and migration [25,26]. Thus, a new measurement method is necessary that is capable of quantifying the rapid cellular/subcellular deformation. The objective of the present study is to develop a technique to measure the intracellular deformation of cells adhered to a collagen matrix. The technique is then applied to quantify freezing-induced spatiotemporal mechanical loading on the cells adhered to the collagen matrix. As a model tissue, human dermal fibroblasts labeled with fluorescent nanoparticles were seeded on type I collagen matrix and exposed to freezing. During freezing, the movement of intracellular fluorescent nanoparticles was imaged and further analyzed to determine spatiotemporal deformation of the cytoplasm. The results are discussed to establish a mechanistic understanding of freezing-induced biophysical processes around and inside cells embedded within the ECM.