Freezing-induced cellular and membrane dehydration in the presence of cryoprotective agents

Figures & data

Table I. Parameters and variables for Equations (1) and (2). Dimensions of 3T3 cells were taken from Akhoondi et al. (2011).

Symbol	Description	Value	SI unit
V	Volume	(variable)	m^3
T	Temperature	(variable)	K
TR	Reference temperature	273.15	K
Lp	Membrane hydraulic permeability	(variable)	m s^-1 Pa^-1
Lpg	Permeability of the membrane to water at TR	(parameter)	m s^-1 Pa^-1
ELp	Activation energy for the permeation process	(parameter)	J mol^-1
A	Membrane surface area of the cell	781 10^-12	m^2
R	Universal gas constant	8.314	J K^-1 mol^-1
B	Cooling rate	0.0167	K s^-1
vw	Molar volume of water	18 10^-6	m^3 mol^-1
Vo	Isotonic volume	2053 10^-18	m^3
Vb	Osmotically inactive volume	1068 10^-18	m^3
vs	Dissociation constant of salt	2	(-)
ns	Number of moles of solutes in the cell	2.710–13	mol
ΔHf	Heat of fusion of water	6011	J mol^-1
ncpa	Number of moles of CPA in the cell	(variable)	mol
vDMSO	Molar volume of DMSO	71 10^-6	m^3 mol^-1
vGLY	Molar volume of GLY	73 10^-6	m^3 mol^-1

Figure 1. Infrared absorption spectra of 3T3 cells, acquired at 20°C in the absence and presence of DMSO and glycerol. The OH-stretching, H₂O-libration and bending combination band, and the H₂O scissoring band arising from water, the CH₂-stretching bands mainly arising from membrane lipids, and the protein amide bands are indicated. Typical bands arising from glycerol and DMSO are visible in the 1200–900 cm^-1 fingerprint region of the spectrum.

Figure 2. Membrane phase behavior of 3T3 cells during cooling in the absence of cryoprotective agents (open squares) as well as in presence of 5% DMSO (black squares) or glycerol (gray squares). Cell pellets were cooled from 20 to −40°C at cooling rate of 1°C min^-1, and nucleated at either −4°C (A) or −10°C (B). The wavenumber position of the symmetric CH₂ stretching vibration band is plotted as a function of the sample temperature.

Figure 3. Cell volume response of 3T3 cells during cooling from 4°C down to −30°C at 1°C min^-1, with ice nucleation at −5°C, in the absence of cryoprotective agents (A) and in presence of 5% DMSO (B) or glycerol (C). The cell volume response was determined as changes in cell size using cryomicroscopy (open squares) as well as derived from changes in the membrane lipid band (FTIR-νCH₂; black squares) and water band (FTIR AνH₂O; gray squares) determined using FTIR. Cell volumes were normalized towards the initial value prior to ice nucleation, and FTIR data were scaled towards the end volume as assessed using cryomicroscopy.

Table II. Subzero hydraulic membrane permeability parameters of 3T3 cells. Membrane permeability parameters were derived from cryomicroscopic as well as lipid band position (νCH₂) and water band area (AνH₂O) measurements.

Method		Lpg (μm min^-1 atm^-1)	ELp (kcal mol^-1)
cryoMIC	Subzero	0.1256	56.0
FTIR-νCH2	Subzero (bound water)	0.0130	44.1
FTIR-νH2O	Subzero (free water)	0.0116	58.5
Coulter counter	Suprazero	0.0631	15.0

Figure 4. Arrhenius plot of the membrane hydraulic permeability of 3T3 cells as a function of the nucleation temperature, as derived from cryomicroscopic studies (open squares) and membrane lipid band (FTIR-νCH₂; black squares) and water band (FTIR- AνH₂O; gray squares) analysis from FTIR studies. The dashed line represents extrapolated Coulter counter data from previous experiments performed at suprazero temperatures (Akhoondi et al. 2011).

Table III. Subzero hydraulic membrane permeability parameters of 3T3 cells incubated in the presence of 5% DMSO or glycerol (GLY). Arrhenius plots derived from FTIR-νCH₂ data were constructed. E_Lp-1 and Lpg-1 values were determined using linear regression analysis at high subzero temperatures, whereas E_Lp-2 and Lpg-2 were determined at low subzero temperature.

Cryoprotectant	Method	Lpg-1 (μm min^-1 atm^-1)	ELp-1 (kcal mol^-1)	Lpg-2 (μm min^-1 atm^-1)	ELp-2 (kcal mol^-1)
5% DMSO	FTIR-νCH2	0.0151	44.6	0.0018	11.6
5% GLY	FTIR-νCH2	0.0167	41.7	0.0073	27.3

Figure 5. Arrhenius plots of the membrane hydraulic permeability of 3T3 cells as a function of the nucleation temperature in the presence of 5% DMSO (open squares) or glycerol (black squares), derived from membrane lipid band analysis from FTIR studies. Linear regression lines are indicated in the temperature ranges above and below −9 and −7°C for DMSO and glycerol, respectively.

Figure 6. Membrane hydraulic permeability as a function of the temperature, as calculated using Equation (1) and the parameter listed in Tables II and III, for cooling of 3T3 cells with 1°C min^-1 in the absence of cryoprotective agents (open squares) as well as in presence of 5% DMSO (black squares) or glycerol (gray squares). To illustrate differences more clearly, different scales were used for the y-axis in panels A and B. Lp was calculated for above and below −9 and −7°C for DMSO and glycerol, respectively, using the different E_Lp and Lpg values as determined for these temperature ranges.

Figure 7. Cell volume response of 3T3 cells during cooling with different cooling rates in the absence of cryoprotective agents (A) as well as in presence of 5% DMSO (B) or glycerol (C), as calculated using Equation (1) and the parameter listed in Tables II and III. Normalized cell volumes were calculated above and below −9 and −7°C for DMSO and glycerol, respectively, using the different E_Lp and Lpg values as determined for these temperature ranges. The dashed line illustrates the cell volume response under equilibrium conditions, as calculated using Equation (3).

Akhoondi M, Oldenhof H, Stoll C, Sieme H, Wolkers WF. 2011. Membrane hydraulic permeability changes during cooling of mammalian cells. Biochim Biophys Acta 1808:642–648.

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