Water transport and IIF parameters for a connective tissue equivalent

Understanding the biophysical processes that govern freezing injury of a tissue equivalent (TE) is an important step in characterizing and improving the cryopreservation of these systems. TEs were formed by entrapping human dermal fibroblasts (HDFs) in collagen or in fibrin gels. Freezing studies were conducted using a Linkam cryostage fitted to an optical microscope allowing observation of the TEs cooled under controlled rates between 5 and 130°C/min. Typically, freezing of cellular systems results in two biophysical processes that are both dependent on the cooling rate: dehydration and/or intracellular ice formation (IIF). Both these processes can potentially be destructive to cells. In this study, the biophysics of freezing cells in collagen and fibrin TEs have been quantified and compared to freezing cells in suspension. Experimental data were fitted in numerical models to extract parameters that governed water permeability, E_Lp and L_pg, and intracellular ice nucleation, Ω_o and κ_o. Results indicate that major differences exist between freezing HDFs in suspension and in a tissue equivalent. During freezing, 55% of the HDFs in suspension formed IIF as compared to 100% of HDFs forming IIF in collagen and fibrin TE at a cooling rate of 130°C/min. Also, both the water permeability and the IIF parameters were determined to be higher for HDFs in TEs as compared to cell suspensions. Between the TEs, HDFs in fibrin TE exhibited higher values for the biophysical parameters as compared to HDFs in collagen TE. The observed biophysics seems to indicate that cell-cell and cell-matrix interactions play a major role in ice propagation in TEs.