Water transport and IIF parameters for a connective tissue equivalent

Area of Science:

• Biophysics
• Cryobiology
• Tissue Engineering

Background:

• Cryopreservation is crucial for preserving biological tissues and cells.
• Understanding freezing injury mechanisms in tissue equivalents (TEs) is vital for improving cryopreservation techniques.
• TEs, composed of cells within a matrix, present unique freezing dynamics compared to cell suspensions.

Purpose of the Study:

• To quantify and compare the biophysical processes governing freezing injury in human dermal fibroblasts (HDFs) within collagen and fibrin TEs versus cell suspensions.
• To investigate the influence of cooling rate on dehydration and intracellular ice formation (IIF) in HDFs within different TEs.
• To determine key biophysical parameters, including water permeability and intracellular ice nucleation, for HDFs in various cryopreservation contexts.

Main Methods:

• Human dermal fibroblasts (HDFs) were encapsulated in collagen or fibrin gels to form TEs.
• Freezing studies were conducted using controlled cooling rates (5-130°C/min) on a Linkam cryostage equipped with an optical microscope.
• Experimental data were fitted to numerical models to extract biophysical parameters related to water permeability and intracellular ice nucleation.

Main Results:

• At 130°C/min, 100% of HDFs in both collagen and fibrin TEs underwent intracellular ice formation (IIF), compared to 55% in suspension.
• Water permeability and IIF parameters were significantly higher for HDFs within TEs than in cell suspensions.
• HDFs in fibrin TEs showed higher biophysical parameter values than those in collagen TEs, suggesting matrix-specific effects.

Conclusions:

• Cell-cell and cell-matrix interactions within TEs significantly influence freezing injury, promoting IIF and altering water transport compared to suspensions.
• Fibrin TEs exhibit distinct biophysical responses to freezing compared to collagen TEs, highlighting the importance of matrix composition.
• These findings provide critical insights into the biophysics of freezing cells within engineered tissues, informing strategies for enhanced cryopreservation.