Water permeability parameters of dermal fibroblast employed in tissue engineering in subzero temperatures

Abstract

Fibroblast is a crucial kind of cell in the construction of the tissue engineered dermal equivalent. In order to optimize the cryopreservation protocols of the tissue-engineered dermis, the characteristics of dermal fibroblast in subzero temperatures are required, which include the water permeability of the cell membrane and the apparent activation energy. Using the differential scanning calorimeter (DSC), the volumetric shrinkage during freezing of human dermal fibroblast suspensions was obtained at the cooling rate of 5°C·min⁻¹ in the presence of extracellular ice. To ensure the presence of extracellular ice, a small quantity of ice nucleation bacteria (INA bacteria), pseudomonas syringae was added in the samples. And based on the Karlsson’s model, a nonlinear-least-squares curve fitting technique was implemented to calculate the cryogenic parameters. At the reference temperature T _R(=0°C), the water permeability of membrane L _pg=0.578 μm·min⁻¹·atm⁻¹ and the apparent activation energy, E _LP=308.8 kJ·mol⁻¹. These parameters were then used to simulate water transport of fibroblast during constant cooling at rates between 0.01–50°C · min⁻¹. The simulation results were analyzed to predict the amount of water left in the cell after dehydration and the “optimal cooling rate” for fibroblast cryopreservation. For the dermal fibroblast with DMEM solution, a cooling rate of 4.6°C · min⁻¹ was optimal.