Teo Ka Yaw, Dutton J Craig, Han Bumsoo
Department of Mechanical and Aerospace Engineering, University of Texas at Arlington, Arlington, TX 76019, USA.
J Biomech Eng. 2010 Mar;132(3):031003. doi: 10.1115/1.4000875.
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 h incubation, the ETs were directionally frozen by exposing them to a spatial temperature gradient (from 4 degrees C to -20 degrees 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.
为了冷冻保存功能性工程组织（ETs），细胞外基质（ECM）的微观结构应得以维持，细胞活力也应保持，因为其功能与ECM微观结构密切相关。由于解冻后ECM的微观结构由冷冻保存过程中ETs的变形决定，因此使用新开发的量子点（QD）介导的细胞图像变形测量系统，以真皮替代物作为模型组织，测量了ETs的冷冻诱导变形。通过将QD标记的成纤维细胞接种到I型胶原基质中构建真皮替代物。孵育24小时后，通过将ETs暴露于空间温度梯度（在6毫米的距离内从4℃降至-20℃）进行定向冷冻。在冷冻过程中，对ETs进行连续成像，并对这些图像的连续对进行二维互相关，以确定冷冻过程中的局部变形。结果表明，冷冻诱导了ET的变形，其大小随时间和位置而变化。最大局部膨胀率为0.006 s(-1)，且总是在相变界面处观察到。由于这种局部膨胀，冷冻界面之前的未冷冻区域受到压缩。在整个冷冻过程中都观察到了这种膨胀-压缩模式。在未冷冻区域，变形率从冷冻界面逐渐降低。冷冻/解冻后，ET的厚度大约减少了28%，重量损失了8%。这些结果表明，冷冻诱导的变形导致了间质液的运输，并且间质液被挤出。总之，结果表明在冷冻过程中ETs内发生了复杂的细胞-流体-基质相互作用，这些相互作用决定了解冻后ECM的微观结构和最终解冻后组织的功能。
