Spatiotemporal Measurement of Freezing-Induced Deformation of Engineered Tissues

Abstract

In order to cryopreserve functional engineered tissues (ETs), the microstructure of the extracellular matrix (ECM) should be maintained as well as the cellular viability since the functionality is closely related to the ECM microstructure. Since the post-thaw ECM microstructure is determined by the deformation of ETs during cryopreservation, freezing-induced deformation of ETs was measured with a newly developed quantum dot (QD)-mediated cell image deformetry system using dermal equivalents as a model tissue. The dermal equivalents were constructed by seeding QD-labeled fibroblasts in type I collagen matrices. After 24 hour incubation, the ETs were directionally frozen by exposing them to a spatial temperature gradient (from 4 °C to −20 °C over a distance of 6 mm). While being frozen, the ETs were consecutively imaged, and consecutive pairs of these images were two-dimensionally cross-correlated to determine the local deformation during freezing. The results showed that freezing induced the deformation of ET, and its magnitude varied with both time and location. The maximum local dilatation was 0.006 s −1 and was always observed at the phase change interface. Due to this local expansion, the unfrozen region in front of the freezing interface experienced compression. This expansion-compression pattern was observed throughout the freezing process. In the unfrozen region, the deformation rate gradually decreased away from the freezing interface. After freezing/thawing, the ET experienced an approximately 28% decrease in thickness and 8% loss in weight. These results indicate that freezing-induced deformation caused the transport of interstitial fluid and the interstitial fluid was extruded. In summary, the results suggest that complex cell-fluid-matrix interactions occur within ETs during freezing, and these interactions determine the post-thaw ECM microstructure and eventual post-thaw tissue functionality.

Percutaneous Tumor Ablation

“…We think that the Co-CACH intraprocedural targeting efficacy is the essence of safety. To us, the ice margin assists with the deposition of the selected LMW free drugs mixture and with a marginal fluid flow to which factors such as a peripheral increased hydraulic conductivity, an augmented extracellular fluid fraction triggered by the freezing, the volume of injectate, 12,13,31 and a more central fluid-impervious hard ice 6,16,32 contribute. The slush ice region whose outer rim is CT imageable would act as a tunable fluid-channeling network that can be switched on and off with time-adjusted freeze and thaw sequences.…”

“…9,10 However, it is not known which timing, route of administration, formulation, or dosing for the associated cytotoxic drugs is best for improving the effectiveness and predictability of the cryoablative ice margin. [11][12][13] Many studies use drugs formulated with vehicles such as a matrix, capsule, or nanoparticle that facilitate tumor targeting, drug residence, slow release rate, and intracellular uptake. Promising combination therapy associated with nanoparticleencapsulated doxorubicin and CA 14 demonstrate enhanced eradication of cancer stem cells (CSTs) in vitro.…”

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

“…The cells were then trypsinized and used to prepare the engineered tissues. The cells were labeled with another smaller size particle, 20 nm diameter quantum dots (Qtracker 655, Invitrogen, Carlsbad, CA), according to the protocol described earlier [23].…”

“…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][22][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].…”

“…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.…”