Biophysics of freezing of tissue equivalents

Understanding the biophysical processes which govern injury during freezing of a tissue equivalent (TE) is an important step in understanding and improving cryopreservation in these systems. TEs were formed by entrapping human dermal fibroblasts (HDFs) in collagen or in fibrin gel. Freezing studies were performed using a Linkam cryostage fitted to an optical microscope allowing observation of TE cooling under controlled rates between 5-130°C/min, Typically, freezing results in two biophysical processes inversely dependent on cooling rate: dehydration or intracellular ice formation (IIF). Both of these processes can be lethal to cells. In this study we have measured these biophysical processes and fit the behavior to models so as to extract parameters that govern water permeability, E _Lp and L _pg, and parameters that govern intracellular ice nucleation, Ωo and κo. Biophysical behavior was also found to depend on cell-cell and cell-matrix interactions. For example, HDFs in suspension show 55% IIF whereas HDFs in collagen and in fibrin TE 100% IIF at 130 C/min cooling rate. Further differences were noted and quantified by obtaining biophysical parameters for HDF cells in suspension vs. entrapped in collagen or fibrin gels.