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TECHNICAL PAPERS: Cell

# Cellular Response of Adipose Derived Passage-4 Adult Stem Cells to Freezing Stress

[+] Author and Article Information
Ram V. Devireddy1

Bioengineering Laboratory, Department of Mechanical Engineering, Louisiana State University, Baton Rouge, LA, USA

Sreedhar Thirumala

Bioengineering Laboratory, Department of Mechanical Engineering, Louisiana State University, Baton Rouge, LA, USA

Jeffrey M. Gimble

Stem Cell Laboratory, Pennington Biomedical Research Center, Louisiana State University System, Baton Rouge, LA, USA

1

Author to whom correspondence should be addressed. Tel: 1-225-578-5891; fax: 1-225-578-5924; e-mail: devireddy@me.lsu.edu

J Biomech Eng 127(7), 1081-1086 (Jul 13, 2005) (6 pages) doi:10.1115/1.2073673 History: Received February 20, 2005; Revised June 14, 2005; Accepted July 13, 2005

## Abstract

A differential scanning calorimeter technique was used to generate experimental data for volumetric shrinkage during cooling at $20°C∕min$ in adipose derived adult stem cells (ASCs) in the presence and absence of cryoprotective agents (CPAs). By fitting a model of water transport to the experimentally determined volumetric shrinkage data, the membrane permeability parameters of ASCs were obtained. For passage-4 (P4) ASCs, the reference hydraulic conductivity $Lpg$ and the value of the apparent activation energy $ELp$ were determined to be $1.2×10−13m3∕Ns$ and $177.8kJ∕mole$, respectively. We found that the addition of either glycerol or dimethylsulfoxide (DMSO) significantly decreased the value of the reference hydraulic conductivity $Lpg(cpa)$ and the value of the apparent activation energy $ELp(cpa)$ in P4 ASCs. The values of $Lpg(cpa)$ in the presence of glycerol and DMSO were determined as $0.39×10−13$ and $0.50×10−13m3∕Ns$, respectively, while the corresponding values of $ELp(cpa)$ were 51.0 and $61.5kJ∕mole$. Numerical simulations of water transport were then performed under a variety of cooling rates $(5–100°C∕min)$ using the experimentally determined membrane permeability parameters. And finally, the simulation results were analyzed to predict the optimal rates of freezing P4 adipose derived cells in the presence and absence of CPAs.

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## Figures

Figure 1

Volumetric response of P4 ASCs as a function of subzero temperatures obtained using the DSC technique in the presence of extracellular ice (A), in the presence of extracellular ice and glycerol (B), and in the presence of extracellular ice and DMSO (C). The filled circles represent the experimentally obtained water transport (volumetric shrinkage) at a cooling rate of 20°C∕min. The model-simulated dynamic cooling response at 20°C∕min is shown as a solid line and was obtained by using the best-fit membrane permeability parameters (Lpg and ELp or Lpg[cpa] and ELp[cpa]) (Table 1) in the water transport equation (Eqs. 1,2). The model-simulated equilibrium cooling response obtained is shown as a dotted line in all the figures. The nondimensional volume is plotted along the y axis and the subzero temperatures are shown along the x axis. The error bars represent the standard deviation for the mean values of six separate DSC experiments (n=6).

Figure 2

Volumetric response of adipose derived (P4) cells at various cooling rates as a function of subzero temperatures using the best-fit membrane permeability parameters (shown in Table 1). The changes in the normalized cell volume (V∕Vo) are shown as a function of temperature for different cooling rates in P4 ASC suspensions without CPA (A), with 10% glycerol (B), and with 10% DMSO (C). In all the figures, the water transport curves (solid lines) represent the model-simulated response for different cooling rates (from left to right: 5, 10, 20, 40, 60, 80, and 100°C∕min). The subzero temperatures are shown along the x axis while the nondimensional volume is plotted along the y axis.

Figure 3

Contour plots of the goodness of fit parameter R2(=0.98) for water transport response in P4 ASCs in medium with no CPAs, in medium with 10% glycerol, and in medium with 10% DMSO. The enclosed region corresponds to the range of parameters that best fit the water transport data at a cooling rate of 20°C∕min with R2=0.98. Note that the best-fit parameters (Table 1;Vb=0.6Vo) are represented within the contours by a “#” (absence of CPA), “&” (10% glycerol), and “*” (with 10% DMSO). The membrane permeability at 0°C, Lpg (or Lpg[cpa]) (m3∕Ns), is plotted on the y axis while the apparent activation energy of the membrane, ELp (ELp[cpa]) (kJ/mol) is shown on the x axis.

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