Effect of freezing on cell viability and mechanical strength of bioartificial tissues

The effect of freezing on the viability and mechanical strength of bioartificial tissues was determined under a variety of cooling conditions, with the ultimate aim of optimizing the cryopreservation process. Bioartificial tissues (i.e. tissue-equivalents or TEs) were prepared by incubating entrapped human foreskin fibroblasts in collagen gels for a period of 2 weeks. The bioartificial tissues were frozen using a controlled rate freezer at various cooling rates (0.5,2, 5, 20, 40 and > 1000°C/min or slam freezing). The viability (<60 min after thawing) of the fibroblasts in the bioartificial tissue was assessed using the Ethidium Homodimer (dead cells stain red) and Hoechst (live cells stain blue) assay. Uniaxial tension experiments were performed on an MTS Microbionix System (Eden Prairie, MN) to assess the post-thaw mechanical properties (Maximum Stiffness; Ultimate Tensile Stress; and Strain to Failure) of the frozen-thawed bioartificial tissue (≤ 3 hours after thawing). The results suggest that cooling rates of either 2 or 5°C/min are optimal for preserving both the cell viability and mechanical properties of the bioartificial tissues, post-freeze. Bioartificial tissues were also frozen using a directional solidification stage at 5°C/min. The post-thaw viability results are comparable in both the directionally cooled and the controlled rate freezer samples. However, the mechanical properties of the directionally cooled samples are significantly different (with a higher maximum stiffness and a lower strain to failure) than those obtained for samples frozen using a controlled rate freezer. This suggests that the directionality of ice propagation into the sample affects the measured mechanical properties.