Date of Award
Master of Science in Engineering (MSE)
Committee Member 1
Committee Member 2
Long term preservation of functional engineered tissues can significantly advance tissue engineering industry and regenerative medicine. Several preservation techniques have been proposed and investigated for this purpose, and cryopreservation is a leading candidate. While tissues are cryopreserved, ice forms in both the extracellular and intracellular spaces and causes freezing-induced spatiotemporal deformation of the tissue. During this process the cells undergo dehydration by the freezing-induced osmotic pressure difference and mechanical deformation, transmitted through cell-extracellular matrix adhesions. However, the significance and interaction of these cellular level transport and mechanics processes are not well understood. Therefore, this study aims to establish mechanistic understanding of these interactions so that cryopreservation of functional engineered tissues can be achieved. First, the relative significance of both processes was quantified. Two groups of tissue constructs were created- i) engineered tissue (ET) with MCF7 human breast cancer cells, and ii) ET with osmotically inactive fluorescence microparticles. Tissue deformations were measure through cell image deformetry (CID). It was found that intracellular water transport, i.e., dehydration, during freezing was insignificant at the experimental cell concentrations examined in this study.
Second, based on this result, a hypothesis was tested that modulation of cytoskeletal structure can minimize the freezing-induced deformation and improve the cryopreservation outcome. As model ETs, dermal equivalents were created with three different cytoskeletal and cell-matrix adhesion configurations were created. After exposing these dermal equivalents to freeze/thaw (F/T), the post-thaw actin structures and cellular viability were examined as well as tissue deformation measured using CID. Additionally, the effects of cytoplasmic properties on the mechanical behavior of a cell embedded in a 3D matrix were analyzed using a biphasic model to estimate the stress distributions that develop within the cell during tissue deformation. Stress fibers formed due to the different cell configurations resulted in better actin structure preservation at a higher tissue deformation. Additionally, the stress was estimated to be highest at the localized adhesion sites at the end of the stress fibers, which may be the cause of low membrane integrity of the stress fiber induced configurations. This may indicate that if the cytoplasmic strength increased and the tissue deformation decreased (with the presence of a cryoprotective agent) the tissue and cellular functionality may be better preserved.
Seawright, Angela Christine, "Modulation of Cell-Matrix Interaction for Cryopreservation of Engineered Tissue" (2013). Open Access Theses. 74.