Research Papers

Role of Cells in Freezing-Induced Cell-Fluid-Matrix Interactions Within Engineered Tissues

[+] Author and Article Information
Altug Ozcelikkale

School of Mechanical Engineering,
Purdue University,
West Lafayette, IN 47907

Craig Dutton

Department of Aerospace Engineering,
University of Illinois at Urbana-Champaign,
Urbana, IL 61801

Bumsoo Han

School of Mechanical Engineering,
Purdue University,
West Lafayette, IN 47907;
Weldon School of Biomedical Engineering,
Purdue University,
West Lafayette, IN 47907
e-mail: bumsoo@purdue.edu

1Angela Seawright and Altug Ozcelikkale have equally contributed to the present work.

2Corresponding author. Present address: 585 Purdue Mall, West Lafayette, IN 47906.

Contributed by the Bioengineering Division of ASME for publication in the JOURNAL OF BIOMECHANICAL ENGINEERING. Manuscript received June 19, 2012; final manuscript received April 29, 2013; accepted manuscript posted May 16, 2013; published online July 10, 2013. Assoc. Editor: John Bischof.

J Biomech Eng 135(9), 091001 (Jul 10, 2013) (12 pages) Paper No: BIO-12-1240; doi: 10.1115/1.4024571 History: Received June 19, 2012; Revised April 29, 2013; Accepted May 16, 2013

During cryopreservation, ice forms in the extracellular space resulting in freezing-induced deformation of the tissue, which can be detrimental to the extracellular matrix (ECM) microstructure. Meanwhile, cells dehydrate through an osmotically driven process as the intracellular water is transported to the extracellular space, increasing the volume of fluid for freezing. Therefore, this study examines the effects of cellular presence on tissue deformation and investigates the significance of intracellular water transport and cell-ECM interactions in freezing-induced cell-fluid-matrix interactions. Freezing-induced deformation characteristics were examined through cell image deformetry (CID) measurements of collagenous engineered tissues embedded with different concentrations of MCF7 breast cancer cells versus microspheres as their osmotically inactive counterparts. Additionally, the development of a biophysical model relates the freezing-induced expansion of the tissue due to the cellular water transport and the extracellular freezing thermodynamics for further verification. The magnitude of the freezing-induced dilatation was found to be not affected by the cellular water transport for the cell concentrations considered; however, the deformation patterns for different cell concentrations were different suggesting that cell-matrix interactions may have an effect. It was, therefore, determined that intracellular water transport during freezing was insignificant at the current experimental cell concentrations; however, it may be significant at concentrations similar to native tissue. Finally, the cell-matrix interactions provided mechanical support on the ECM to minimize the expansion regions in the tissues during freezing.

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Fig. 1

MCF7 cells (left column, (a) and (c)) and Fluoresbrite® YG Microspheres (right column (b) and (d)) embedded in engineered tissues. Images (a) (MCF7 cells) and (b) (microspheres) are imaged with the bright field of a confocal microscope (Olympus IX71). Images (c) and (d) are fluorescent images, the MCF7 embedded QDs, (c), imaged with the TRITC filter and the microspheres, (d), imaged with the FITC filter.

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Fig. 2

Schematic illustration of the representative elementary volume of an engineered tissue. States (I) and (II) correspond to before and during extracellular freezing. The ET is composed of cellular (c) and extracellular (ec) compartments. Each compartment has both solid (s) and fluid (fl) components. As ice forms in the extracellular space, cells dehydrate and make more water available for extracellular freezing. In the meantime, the extracellular space expands increasing the volume of the REV.

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Fig. 3

Dilatation (n ≥ 3) of normal MCF7 concentration (a) and microsphere ETs (b) at X(t) = 2000 μm

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Fig. 4

The y-averaged deformation (top row) and dilatation rates (bottom row) at X(t) = 2000 μm and 4000 μm for (a) and (c) the normal MCF7 cell concentration and (b) and (d) the microspheres

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Fig. 5

Dilatation (n ≥ 3) of doubled MCF7 concentration (a) and quadrupled MCF7 concentration ETs (b) at X(t) = 2000 μm

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Fig. 6

The deformation (top row) and dilatation rates (bottom row) at X(t) = 2000 μm and 4000 μm averaged along the y-axis for (a) and (c) the doubled MCF7 cell concentration and (b) and (d) the quadrupled MCF7 cell concentration

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Fig. 7

Cellular water transport during freezing. (a) Representative cryomicroscopy images of MCF7 cells dehydrating during freezing with the cooling rate of 10  °C/min. The locations of cells are indicated by dark arrows. Scale bar is 20 μm (b) change of cell volume with decreasing temperature for the three cooling rates. The error bars stand for the standard error of the mean. Solid lines are model predictions with the optimal membrane permeability parameters.

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Fig. 8

Extracellular freezing thermodynamics of engineered tissues. Rate of latent heat release (solid line) and the frozen fraction calculated by cumulative integration of the rate of latent heat release with respect to time (dashed line).

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Fig. 10

(a) The variation of estimated freezing-induced dilatation with decreasing temperature for different concentrations of cells and microspheres. Solid lines correspond to ETs with cells while dashed lines correspond to ETs with microspheres. (b) The variation of relative difference of freezing-induced dilatation between the cells versus microsphere ETs with decreasing temperature. Unlike the actual experimental settings, this analysis assumes the sphere diameter to be the same as the diameter of the MCF7 cells in order to isolate the effects of cellular water transport from differential size effects.

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Fig. 9

(a) Temperature measurements obtained at different axial distances from the cold terminal. The underlying lines are polynomial curve fits that were used in the analysis for the estimation of freezing-induced dilatation (Pearson coefficient of correlation R2 > 0.99 for each case). The temporal trends of (b) cellular water transport, (c) extracellular ice formation, and (d) estimated freezing-induced dilatation at two axial locations, x = 2000 μm and x = 4000 μm.



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