Successful improvement of cryopreservation protocols for cells in suspension requires knowledge of how such cells respond to the biophysical stresses of freezing (intracellular ice formation, water transport) while in the presence of a cryoprotective agent (CPA). This work investigates the biophysical water transport response in a clinically important cell type—isolated hepatocytes—during freezing in the presence of dimethylsulfoxide (DMSO). Sprague-Dawley rat liver hepatocytes were frozen in Williams E media supplemented with 0, 1, and 2 M DMSO, at rates of 5, 10, and 50°C/min. The water transport was measured by cell volumetric changes as assessed by cryomicroscopy and image analysis. Assuming that water is the only species transported under these conditions, a water transport model of the form dV/dT = f(Lpg([CPA]), ELp([CPA]), T(t)) was curve-fit to the experimental data to obtain the biophysical parameters of water transport—the reference hydraulic permeability (Lpg) and activation energy of water transport (ELp)—for each DMSO concentration. These parameters were estimated two ways: (1) by curve-fitting the model to the average volume of the pooled cell data, and (2) by curve-fitting individual cell volume data and averaging the resulting parameters. The experimental data showed that less dehydration occurs during freezing at a given rate in the presence of DMSO at temperatures between 0 and −10°C. However, dehydration was able to continue at lower temperatures (<−10°C) in the presence of DMSO. The values of Lpg and ELp obtained using the individual cell volume data both decreased from their non-CPA values—4.33 × 10−13 m3/N-s (2.69 μm/min-atm) and 317 kJ/mol (75.9 kcal/mol), respectively—to 0.873 × 10−13 m3/N-s (0.542 μm/min-atm) and 137 kJ/mol (32.8 kcal/mol), respectively, in 1 M DMSO and 0.715 × 10−13 m3/N-s (0.444 μm/min-atm) and 107 kJ/mol (25.7 kcal/mol), respectively, in 2 M DMSO. The trends in the pooled volume values for Lpg and ELp were very similar, but the overall fit was considered worse than for the individual volume parameters. A unique way of presenting the curve-fitting results supports a clear trend of reduction of both biophysical parameters in the presence of DMSO, and no clear trend in cooling rate dependence of the biophysical parameters. In addition, these results suggest that close proximity of the experimental cell volume data to the equilibrium volume curve may significantly reduce the efficiency of the curve-fitting process.

1.
Aggarwal
S. J.
,
Diller
K. R.
, and
Baxter
C. R.
,
1988
, “
Hydraulic permeability and activation energy of human keratinocytes at subzero temperatures
,”
Cryobiology
, Vol.
25
, pp.
203
211
.
2.
Bevington, P. R., and Robinson, D. K., 1992, Data Reduction and Error Analysis for the Physical Sciences, McGraw-Hill, New York.
3.
Bischof
J. C.
,
Ryan
C. M.
,
Tompkins
R. G.
,
Yarmush
M. L.
, and
Toner
M.
,
1997
, “
Ice formation in isolated human hepatocytes and tissue of human liver
,”
ASAIO J.
, Vol.
43
, pp.
271
278
.
4.
Borel Rinkes
I. H.
,
Toner
M.
,
Ezzell
R. M.
,
Tompkins
R. G.
, and
Yarmush
M. L.
,
1992
a, “
Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration
,”
Cryobiology
, Vol.
29
, pp.
443
453
.
5.
Borel Rinkes
I. H.
,
Toner
M.
,
Sheeha
S. J.
,
Tompkins
R. G.
, and
Yarmush
M. L.
,
1992
b, “
Long-term functional recovery of hepatocytes after cryopreservation in a three-dimensional culture configuration
,”
Cell Transp.
, Vol.
1
, pp.
281
292
.
6.
Borel Rinkes
I. H.
,
Toner
M.
,
Tompkins
R. G.
, and
Yarmush
M. L.
,
1994
, “
An extracorporeal microscopy perfusion chamber for on-line studies of environmental effects on cultured hepatocytes
,”
ASME JOURNAL OF BIOMECHANICAL ENGINEERING
, Vol.
116
, pp.
135
139
.
7.
Chesne´
C.
, and
Guillouzo
A.
,
1988
, “
Cryopreservation of isolated rat hepatocytes: a critical evaluation of freezing and thawing conditions
,”
Cryobiology
, Vol.
25
, pp.
323
330
.
8.
Chesne´
C.
,
Guyomard
C.
,
Fautrel
A.
,
Poullain
M. G.
,
Fremond
B.
,
De Jong
H.
, and
Guillouzo
A.
,
1993
, “
Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation
,”
Hepatology
, Vol.
18
, pp.
406
414
.
9.
Cosman
M. D.
,
Toner
M.
,
Kandel
J.
, and
Cravalho
E. G.
,
1989
, “
An integrated cryomicroscopy system
,”
Cryo-Lett.
, Vol.
10
, pp.
17
38
.
10.
Darr, T. B., and Hubol, A., 1995, “Investigation of subzero water transport properties for isolated hepatocytes and hepatocytes cultured in spheroids,” ASME BED- Vol. 29, pp. 269–270.
11.
Darr
T. B.
, and
Hubel
A.
,
1997
, “
Freezing characteristics of isolated pig and human hepatocytes
,”
Cell Transp.
, Vol.
6
, pp.
173
183
.
12.
De Loecker
P.
,
Fuller
B. J.
,
Koptelov
V. A.
, and
De Loecker
W.
,
1993
, “
Metabolic activity of freshly prepared and cryopreserved hepatocytes in monolayer culture
,”
Cryobiology
, Vol.
30
, pp.
12
18
.
13.
De Loecker
R.
,
Fuller
B. J.
,
Gruwez
J.
, and
De Loecker
W.
,
1990
, “
The effects of cryopreservation on membrane integrity, membrane transport and protein synthesis in rat hepatocytes
,”
Cryobiology
, Vol.
27
, pp.
143
152
.
14.
Diller
K. R.
, and
Bradley
D. A.
,
1984
, “
Measurement of the water permeability of single human granulocytes on a microscopic stopped-flow mixing system
,”
ASME JOURNAL OF BIOMECHANICAL ENGINEERING
, Vol.
106
, pp.
384
393
.
15.
Diller
K. R.
, and
Lynch
M. E.
,
1983
, “
An irreversible thermodynamic analysis of cell freezing in the presence of membrane permeable additives. I. Numerical model and transient cell volume data
,”
Cryo-Lett.
, Vol.
4
, pp.
295
308
.
16.
Diller
K. R.
, and
Lynch
M. E.
,
1984
, “
An irreversible thermodynamic analysis of cell freezing in the presence of membrane permeable additives. II. Transient electrolyte and additive concentrations
,”
Cryo-Lett.
, Vol.
5
, pp.
117
130
.
17.
Harris
C. L.
,
Toner
M.
,
Hubel
A.
,
Cravalho
E. G.
,
Yarmush
M. L.
, and
Tompkins
R. G.
,
1991
, “
Cryopreservation of isolated hepatocytes: intracellular ice formation under various chemical and physical conditions
,”
Cryobiology
, Vol.
28
, pp.
436
444
.
18.
Hubel, A., and Darr, T. B., 1995, “Cryobiophysical parameters for freshly isolated human hepatocytes and G2/G3A cells,” ASME HTD- Vol. 322, pp. 73–74.
19.
Hubel
A.
,
Toner
M.
,
Cravalho
E. G.
,
Yarmush
M. L.
, and
Tompkins
R. G.
,
1991
, “
Intracellular ice formation during the freezing of hepatocytes cultured in a double collagen gel
,”
Biotech. Prog.
, Vol.
7
, pp.
554
559
.
20.
Karlsson
J. O.
,
Cravalho
E. G.
,
Borel
R. I.
,
Tompkins
R. G.
,
Yarmush
M. L.
, and
Toner
M.
,
1993
, “
Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide
,”
Biophys. J.
, Vol.
65
, pp.
2524
2536
.
21.
Karlsson
J. O.
,
Eroglu
A.
,
Toth
T. L.
,
Cravalho
E. G.
, and
Toner
M.
,
1996
, “
Fertilization and development of mouse oocytes cryopreserved using a theoretically optimized protocol
,”
Human Reprod.
, Vol.
11
, pp.
1296
1305
.
22.
Kedem
O.
, and
Katchalsky
A.
,
1958
, “
Thermodynamic analysis of the permeability of biological membranes to non-electrolytes
,”
Biochim. Biophys. Acta
, Vol.
27
, pp.
229
246
.
23.
Leibo
S. P.
,
1980
, “
Water permeability and its activation energy of fertilized and unfertilized mouse ova
,”
J. Memb. Biol.
, Vol.
53
, pp.
179
188
.
24.
Levin
R. L.
,
Cravalho
E. G.
, and
Huggins
C. E.
,
1976
, “
A membrane model describing the effect of temperature on the water conductivity of erythrocyte membranes at subzero temperatures
,”
Cryobiology
, Vol.
13
, pp.
415
429
.
25.
Loretz
L. J.
,
Li
A. P.
,
Flye
M. W.
, and
Wilson
A. G.
,
1989
, “
Optimization of cryopreservation procedures for rat and human hepatocytes
,”
Xenobio.
, Vol.
19
, pp.
489
498
.
26.
Mazur
P.
,
1963
, “
Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing
,”
J. Gen. Physiol
, Vol.
47
, pp.
347
369
.
27.
Mazur
P.
,
1984
, “
Freezing of living cells: mechanisms and implications. [Review]
,”
Am. J. Physiol
, Vol.
247
, pp.
C125–C142
C125–C142
.
28.
Mazur
P.
,
1990
, “
Equilibrium, quasi-equilibrium, and nonequilibrium freezing of mammalian embryos. [Review]
,”
Cell Biophys.
, Vol.
17
, pp.
53
92
.
29.
McCaa
C.
,
Diller
K. R.
,
Aggarwal
S. J.
, and
Takahashi
T.
,
1991
, “
Cryomicroscopic determination of the membrane osmotic properties of human monocytes at subfreezing temperatures
,”
Cryobiology
, Vol.
28
, pp.
391
399
.
30.
McGrath, J. J., 1988, “Membrane transport properties,” in: Low Temperature Biotechnology: Emerging Applications and Engineering Contributions, J. J. McGrath and K. R. Diller, eds., ASME, New York, pp. 273–330.
31.
McGrath, J. J., Gao, D. Y., Tao, J., Benson, C., Critser, E. S., and Critser, J. K., 1992, “Coupled transport across the murine oocyte plasma membrane: water and cryoprotective agents,” in: Topics in Heat Transfer, M. Toner, M. I. Flick, B. M. Webb, et al., eds., ASME HTD-Vol. 206–2, New York, pp. 1–14.
32.
Rule
G. S.
,
Law
P.
,
Kruuv
J.
, and
Lepock
J. R.
,
1980
, “
Water permeability of mammalian cells as a function of temperature in the presence of dimethylsulphoxide: correlation with the state of the membrane lipids
,”
J. Cell. Physiol.
, Vol.
103
, pp.
407
416
.
33.
Toner
M.
,
Cravalho
E. G.
, and
Armant
D. R.
,
1990
, “
Water transport and estimated transmembrane potential during freezing of mouse oocytes
,”
J. Memb. Biol.
, Vol.
115
, pp.
261
272
.
34.
Toner
M.
,
Tompkins
R. G.
,
Cravalho
E. G.
, and
Yarmush
M. L.
,
1992
, “
Transport phenomena during freezing of isolated hepatocytes
,”
AIChE J.
, Vol.
38
, pp.
1512
1522
.
35.
Toner
M.
,
Cravalho
E. G.
, and
Karel
M.
,
1993
a, “
Cellular response of mouse oocytes to freezing stress: prediction of intracellular ice formation
,”
ASME JOURNAL OF BIOMECHANICAL ENGINEERING
, Vol.
115
, pp.
169
174
.
36.
Toner
M.
,
Cravalho
E. G.
,
Stachccki
J.
,
Fitzgerald
T.
,
Tompkins
R. G.
,
Yarmush
M. L.
, and
Armant
D. R.
,
1993
b, “
Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential
,”
Biophys. J.
, Vol.
64
, pp.
1908
1921
.
37.
Yarmush
M. L.
,
Toner
M.
,
Dunn
J. C.
,
Rotem
A.
,
Hubel
A.
, and
Tompkins
R. G.
,
1992
, “
Hepatic tissue engineering. Development of critical technologies. [Review]
,”
Annals N.Y. Acad. Sci.
, Vol.
665
, pp.
238
252
.
38.
Zaleski
J.
,
Richburg
J.
, and
Kauffman
F. C.
,
1993
, “
Preservation of the rate and profile of xenobiotic metabolism in rat hepatocytes stored in liquid nitrogen
,”
Biochem. Pharm.
, Vol.
46
, pp.
111
116
.
This content is only available via PDF.
You do not currently have access to this content.