CryoLetters 33 (2), 95-106 (2012)
© CryoLetters, [email protected]
COMPARISON OF CELL MEMBRANE WATER PERMEABILITY IN
MONOLAYERS AND SUSPENSIONS
Adam Z. Higgins1 and Jens O.M. Karlsson2*
1
School of Chemical, Biological and Environmental Engineering, Oregon State University,
Corvallis, Oregon 97331, USA
2
Department of Mechanical Engineering, and Cellular & Molecular Bioengineering Research
Group, Villanova University, 800 Lancaster Avenue, Villanova, Pennsylvania 19085, USA
*Corresponding author
email: [email protected]
Abstract
We previously measured the membrane water permeability of monolayers and
suspensions of MIN6 mouse insulinoma cells at room temperature, and found that water
transport was faster in monolayers. Here, we compare water transport kinetics in monolayers
and suspensions over a range of temperatures for two different cell types, MIN6 cells and
bovine pulmonary artery endothelial cells (BPAEC). At room temperature the results for
BPAEC and MIN6 cells were similar, with approximately 2-fold faster water transport in
monolayers than suspensions. The activation energy for water transport (Ea) was estimated
from Arrhenius plots of the water permeability data. The values of Ea for monolayers and
suspensions of MIN6 cells were not significantly different. However, the activation energy
was significantly lower for BPAEC monolayers (Ea = 49±2 kJ/mol) than suspensions (Ea = 70
± 4 kJ/mol). Predictions of water transport during cryopreservation revealed substantial
differences in supercooling between monolayers and suspensions.
Keywords: beta cell, endothelial cell, hydraulic conductivity, isolation, trypsinization
INTRODUCTION
Formation of extracellular ice during cryopreservation creates an osmotic driving force
for water to flow from the cytoplasm to the extracellular space. If cooling is too rapid, not
enough water will be removed and the cytoplasm will freeze (18). On the other hand,
excessively slow cooling leads to extensive cell dehydration and prolonged exposure to high
solute concentrations, which can cause damage due to solution effects (18). The optimal
cooling rate depends on the rate at which intracellular water can be removed during cooling.
Thus, the cell membrane water permeability is a critical biophysical parameter for designing
cryopreservation procedures.
Because most mammalian cell types are found as components of tissue, with physical
connections to the surrounding matrix and to neighboring cells, it is difficult to measure the
membrane permeability of cells in their native state. Therefore, it is a common experimental
approach to disaggregate tissue to form cell suspensions, which are more amenable to
measurement of membrane permeability parameters (1, 3, 14, 20). The implicit assumption in
95
such studies is that the biophysical properties of the cell membrane are not affected by the
disaggregation process. Several previous investigations have found differences in the
magnitude of membrane permeability parameters for cells in suspension and in intact tissue,
suggesting that in situ techniques are needed to obtain accurate permeability estimates for
cells within tissue (2, 4, 19, 23, 28, 29). However, this effect has only been studied in a small
number of cell types, and the reported results have sometimes been inconsistent.
The objective of the present investigation was to determine the effect (if any) of cell
monolayer disaggregation on the membrane water permeability by comparing water transport
kinetics in monolayers and suspensions of two different cell types, bovine pulmonary artery
endothelial cells (BPAEC) and MIN6 mouse insulinoma cells. In previous studies, we
measured the water permeability of MIN6 monolayers (11) and suspensions (10) at room
temperature, revealing a significant effect of monolayer disaggregation at this temperature.
Here, we present permeability data for both BPAEC and MIN6 cells over a range of
temperatures, allowing comparison of water permeability values at each temperature, as well
as the activation energy for water transport. We show that monolayers and suspensions of
these two cell types have different membrane water permeability properties, leading to
substantial differences in the predicted response to cryopreservation.
THEORY
When cells are exposed to an anisotonic solution containing membrane impermeable
solutes, they reach a new equilibrium volume by osmotic transport of water across the cell
membrane. The cell volume (V) can be divided into two compartments: the water volume,
Vw, and the osmotically inactive volume, Vb. In practice it is common to define an
osmotically inactive volume fraction, Vb , equal to the ratio Vb/V0, where V0 is the cell volume
under isotonic conditions. Assuming negligible solute transport and an ideal and dilute
intracellular solution, the equilibrium cell volume (Ve) after exposure to a given extracellular
osmotic pressure (e) can be determined from the Boyle-van’t Hoff equation:
Ve

 1  Vb  0  Vb
V0
e
(1)
where 0 is the isotonic osmotic pressure. The Boyle-van’t Hoff equation can also be
expressed in terms of the cell water volume as follows:
Vw,e
Vw,0

0
e
(2)
where Vw,e is the water volume at equilibrium and Vw,0 is the isotonic water volume.
The rate at which the cell volume changes after exposure to an anisotonic solution is
proportional to the osmotic pressure driving force, as described by the equation (12):
dV
 Lp A   i   e 
(3)
dt
where t is time, Lp is the membrane water permeability, A is the cell surface area available for
water transport (assumed constant), and i is the intracellular osmotic pressure. The Boylevan’t Hoff relationship relates the instantaneous value of the intracellular osmotic pressure
(i) to the corresponding cell volume:
i 
 0V0 1  Vb 
(4)
V  VbV0
96
Substituting this expression into Eq. 3 results in:
  0V0 1  Vb 

dV
 Lp A 
 e 
 V  VbV0

dt


(5)
~
Introducing a nondimensional cell water volume ( Vw  Vw Vw, 0 ), Eq. 5 can be rewritten:


dVw
 Lp  0   e 

dt
 Vw

(6)
where the coefficient Lp’ (≡ LpA/Vw,0) is an effective permeability parameter.
The temperature dependence of the water permeability is described by an Arrhenius
relationship (13):
E  1
1 
L p  L p ,ref exp  a 
 
(7)
   Tref T  
where Lp,ref is the water permeability at a reference temperature Tref, Ea is the activation
energy for water transport,  is the ideal gas constant, and T is the absolute temperature.
MATERIALS AND METHODS
Cell Culture
Mouse insulinoma (MIN6) cells were generously provided by Dr. Paolo Meda
(University of Geneva, Switzerland) and were cultured as described previously (10, 11). To
prepare cell monolayers for permeability experiments, cells were seeded onto 40-mm
diameter glass coverslips and cultured for 3–4 days, as described elsewhere (11). Cell
suspensions were prepared as previously described (10). Briefly, cells cultured in 35-mm
Petri dishes for 3–4 days were exposed to 0.2% (w/v) trypsin solution at 37°C for 15 min,
followed by trituration in culture medium 20 times with a 20-gauge needle. Cell suspensions
were used in water permeability experiments within 5 minutes of preparation.
Bovine pulmonary artery endothelial cells were obtained from Cambrex (San Diego, CA)
and cultured as described previously (24). To prepare cell monolayers for membrane
permeability experiments, BPAEC were seeded onto 40-mm diameter glass coverslips in
60-mm Petri dishes, and cultured for 2 days, at which point they had achieved a confluency of
approximately 50%. Cell suspensions were prepared as follows. Cell monolayers that had
been cultured in 35-mm Petri dishes for 2 days were first rinsed with Hepes buffered saline
solution (Cambrex). The surface of the Petri dish was then coated with 0.5 ml of 0.2% (w/v)
trypsin solution (Invitrogen) and excess trypsin solution was aspirated. After incubation for
10 min at 37°C, trypsinization was stopped by addition of 1 ml of culture medium, and the
resulting suspension was triturated gently 20 times with a 20-gauge needle. The quality of the
resulting cell suspension was assayed by counting cells on a hemocytometer; viable cells were
identified by their ability to exclude trypan blue dye.
Osmotic Test Solutions
Test solutions were prepared as described in our previous studies (10,11). Purified water
or sucrose (Fisher Scientific, Fair Lawn, NJ) was added to isotonic (300 mOsm/kg) phosphate
buffered saline (PBS, Mediatech, Inc., Manassas, VA) to create hypo- or hypertonic solution,
respectively. The PBS used in experiments with cell monolayers contained Mg2+ and Ca2+,
but the PBS used with cell suspensions did not. The osmolality (m) of all solutions was
measured using a freezing point depression osmometer (Advanced Micro Osmometer Model
97
3300, Advanced Instruments, Norwood, MA); to convert osmolality to osmotic pressure, we
assumed that solutions were ideal and dilute, for which case  =  Tρm (where the density of
water was assumed to have a value ρ = 1 kg/L). Solutions for membrane permeability
experiments with suspended cells were passed through a 0.2 m filter before use in order to
remove particles that would interfere with Coulter counter measurements.
Measurement of Membrane Water Permeability in Cell Monolayers
The membrane water permeability of cells cultured in monolayers was determined for
BPAEC and MIN6 cells using a fluorescence quenching method (11). Cell monolayers on
glass coverslips were incubated in PBS containing 1.25 g/ml calcein acetoxymethyl ester
(calcein-AM, Molecular Probes, Eugene, OR) for 15–20 min at 37°C, assembled into a flow
chamber (Focht Chamber System 2, Bioptechs), and mounted onto an upright microscope
(Eclipse ME600, Nikon, Tokyo, Japan) using a custom stage adapter. Monolayers were
perfused with isotonic solution for 15 min, anisotonic solution for 3 min, and then returned to
isotonic conditions for 5 min. Fluorescence intensity was recorded at 1 second intervals
throughout this process using a cooled charge-coupled device camera (SensiCam, Cooke
Corporation, Romulus, MI). The flow chamber temperature was controlled using a
refrigerated water bath and monitored throughout the experiments using a thermocouple
inserted into the perfusate outlet tubing; temperature control was accurate to within ±1°C.
The cell membrane water permeability was estimated from the cell fluorescence data by
correlating changes in fluorescence intensity with changes in cell volume, as described
previously (11). First, the cell fluorescence was corrected for the effects of non-volumedependent fading (e.g., photobleaching or leakage of intracellular calcein) by fitting an
exponential decay model to fluorescence intensity measurements made while cells were in
equilibrium with isotonic perfusate. The data were normalized to this best fit exponential,
yielding a nondimensional cell fluorescence intensity, F , which was assumed to vary linearly
with the cell water volume (11):
~
~
F  a Vw  1  1
(8)


where a is a phenomenological constant. Combining Eqs. 6 and 8 one obtains the following
differential equation describing transient changes in the normalized cell fluorescence (11):
 a 2 0

dF
 Lp 
 a e 

dt
 F  a 1

(9)
The values of a and Lp’ were estimated from the measured cell fluorescence data as follows:
Eq. 9 was integrated numerically (assuming a step-change in extracellular osmotic pressure at
t = 0) and best-fit parameter values were determined by minimizing the sum of the squared
residuals between measured and predicted values of F .
Measurement of Membrane Water Permeability in Cell Suspensions
The membrane water permeability in suspensions of BPAEC and MIN6 cells was
determined using a Coulter counter (8–10). A volume of 100 L of cell suspension in
isotonic cell culture medium was injected into 10 mL of well-mixed osmotic test solution, and
volume measurements were obtained using a Z2 series Coulter counter with a 100-m
aperture tube. For each osmotic solution, four replicate measurements were performed, each
using cell suspensions created by trypsinization of distinct monolayer cultures. The peak
voltages from the Coulter counter sensor were digitized using a Cell Size Analyzer device
(Great Canadian Computer Company, Spruce Grove, Alberta, Canada), and converted to
volumes by calibration with 10-m diameter latex beads (Beckman Coulter, Fullerton, CA).
98
The sample solution was continuously mixed using a magnetic stirrer (Instec Laboratories,
Plymouth Meeting, PA) and temperature was controlled to within ± 0.5°C by circulating
liquid from a refrigerated water bath through the jacket of the sample beaker.
Coulter counter data were analyzed using methods similar to those described in our
previous studies (8–10). Because Coulter counter measurements are susceptible to error
caused by the coincidence phenomenon, we estimated the error due to coincidence that would
be expected for the BPAEC and MIN6 cell suspensions used in the present study. For both
cell types, the cell concentration and the degree of cell aggregation were low enough to keep
the predicted coincidence error below 6% (8). Therefore, the effects of coincidence were
neglected in analysis of the Coulter counter data. To determine the water permeability from
the transient cell volume measurements, the value of the osmotically inactive volume fraction
must be known. For MIN6 cell suspensions, we used our previously published value (9). To
determine the value of Vb for BPAEC suspensions, steady-state cell volumes were measured
after suspensions had been exposed to anisotonic solution for at least 3 min. The distribution
of measured volumes was then fit with the sum of an exponential distribution and a lognormal
distribution, as described previously (9), and the mean volumes of the best-fit lognormal
distributions were used to generate a Boyle-van’t Hoff plot. The membrane water
permeability was determined by fitting Eq. 5 to the transient cell volume data using the
volume limit adjustment method (10). The cell membrane area (A) was assumed to be equal
to the area of a sphere with volume V0: i.e., A = (36)1/3V02/3.
Data Analysis
Whereas the experiments with cell suspensions yielded estimates for the water
permeability Lp, the experiments with cell monolayers resulted in estimates for the effective
permeability Lp’. To allow comparison between suspensions and monolayers, the Lp values
for suspended cells were multiplied by a representative surface to volume ratio, A/Vw,0, which
was calculated from the average isotonic cell volume μ0 as follows:
A
Vw, 0

36 1 3  1 3
1  Vb
(10)
0
Values of the effective permeability Lp’ are reported as averages and the standard error of the
mean; logarithmically transformed data were analyzed by two-way ANOVA, followed by
Tukey’s tests for pairwise comparisons. Activation energy (Ea) values were determined from
Arrhenius plots and were compared using two-tailed t-tests. Differences were considered to
be significant at a 95% confidence level (i.e., p < 0.05).
Simulation of Water Transport During Freezing
Assuming that the extracellular solution is in equilibrium with ice, and that the
intracellular solution is ideal, the water transport equation can be written (13, 16):
 T 
 H f
dVw
TVw
 Lp 
ln 

    v
dt
v

TV
w
w
w
0
w




T
1 
 Tm



(11)
where Hf = 6016.5 J/mol, Tm = 273.15 K and vw = 1.8 ×10-5 m3/mol are the latent heat of
fusion, melting temperature and molar volume of pure water, respectively.
The
nondimensional cell water volume was predicted as a function of temperature by numerically
integrating Eq. 11, using a constant cooling rate B = –dT/dt. The intracellular supercooling
was then calculated using the following equation:
99
1
1


TVw

T   
ln 
  T

 Tm H f  TVw  w  0  
(12)
RESULTS
Membrane Water Permeability in BPAEC Monolayers at Room Temperature
The fluorescence quenching technique relies on an assumed linear relationship between
the measured cell fluorescence and the cell water volume (Eq. 8). To validate the linear
relationship for BPAEC monolayers, the equilibrium cell fluorescence was measured after
exposure to various anisotonic solutions. The Boyle-van’t Hoff relationship (Eq. 2) was then
used to compute the equilibrium cell water volume at each osmotic pressure. As shown in
Fig. 1A, the cell fluorescence decreased with decreasing water content, as expected for
fluorescence quenching behavior. This effect of osmotic strength on the cell fluorescence was
statistically significant (p < 0.0001) and the data were reasonably well-described by a linear
model (R2 = 0.94) with a best-fit slope a = 0.20. These data confirm the linear relationship in
Eq. 8 and validate the use of Eq. 9 for estimation of the cell membrane water permeability
from the transient fluorescence data.
Figure 1B shows representative transient cell fluorescence data for BPAEC monolayers
exposed to various osmolalities at 21°C. To quantify the kinetics of water transport, Eq. 9
was fit to the data, yielding best-fit values of the permeability parameter Lp’ and the
fluorescence quenching constant a. As shown in Fig. 1B, the data are well described by the
best-fit model predictions. The osmotic strength of the solution had a significant effect on the
best-fit value of a (p = 0.003), yielding a values of 0.213 ± 0.004, 0.192 ± 0.008 and 0.164 ±
0.009 for solution concentrations of 1000 mOsm/kg, 600 mOsm/kg and 430 mOsm/kg,
respectively. However, osmotic strength did not have a statistically significant effect on the
best-fit water permeability (p = 0.54). Pooling of data across all osmotic pressures yielded an
average value Lp’ = (5.1 ± 0.2) × 10-8 Pa-1 s-1.
A
B
1.2
Cell Fluorescence
Cell Fluorescence
1.3
1.1
1.0
0.9
0.8
0.7
0.0
0.5
1.0
1.5
2.0
Vw,e / Vw,0 =  / e
1.0
0.9
0.8
0
10
20
30
40
50
60
Time (s)
Figure 1. Measurement of water transport in BPAEC monolayers at 21°C. (A) Steady-state
cell fluorescence as a function of solution concentration. Each open symbol represents the
mean of 4 replicate experiments. The line is a regression of Eq. 8 to the data; the best-fit line
was constrained to intersect the point (1,1), which is shown as a closed symbol. (B)
Transient response of the normalized cell fluorescence during exposure to hypertonic
solutions with concentrations 430 mOsm/kg (triangles), 600 mOsm/kg (squares) and 1000
mOsm/kg (circles). Solid curves represent predictions from Eq. 9 using the best-fit
parameter values.
100
100
Count
80
60
40
20
0
0
1
2
3
Volume (pL)
B
C
2.0
3
1.6
Volume (pL)
A
Normalized Cell Volume
Membrane Water Permeability in BPAEC Suspensions at Room Temperature
The water permeability of suspended BPAEC was determined from volume
measurements obtained using a Coulter counter. Because this technique can be confounded
by the presence of multicellular aggregates, we optimized the monolayer disaggregation
procedure by varying the trypsin concentration, exposure time and trituration process. The
optimal procedure for BPAEC (see Methods for details) yielded a cell suspension with less
than 4% of the cells in multicellular aggregates and an average viability of 97 ± 2%.
Figure 2A shows a representative volume distribution for BPAEC suspensions in isotonic
solution, along with the best-fit curve comprising the sum of an exponential distribution and a
lognormal distribution (9). The best-fit lognormal distribution under isotonic conditions had
an arithmetic mean 0 = 1380±40 fL and a standard deviation 0 = 530±4 fL. Figure 2B
shows a Boyle-van’t Hoff plot for suspended BPAEC. To generate the plot, the means of the
best-fit lognormal distributions were normalized to the isotonic mean 0. Linear regression of
the normalized cell volume data yielded an osmotically inactive volume fraction Vb = 0.29 ±
0.03 (R2 = 0.98).
Figure 2C shows representative transient volume data for BPAEC suspensions exposed
to a 1000 mOsm/kg solution at 21°C. Each data point represents a volume measurement for a
distinct particle, and the gap in the data at ~35 seconds is a result of the refractory period
between Coulter counter runs. The data show a clear trend of decreasing volume with time,
as expected after exposure to hypertonic solution. The membrane water permeability in
BPAEC suspensions at 21°C was determined using the volume-limit adjustment method (10)
for exposure to hypotonic solution (200 mOsm/kg), yielding Lp = (7.2 ± 1.4) × 10-14 m Pa-1
s-1, as well as two different hypertonic concentrations (600 mOsm/kg and 1000 mOsm/kg),
yielding Lp = (3.1 ± 0.6) × 10-14 m Pa-1 s-1 and Lp = (2.8 ± 0.3) × 10-14 m Pa-1 s-1, respectively.
Solution concentration had a significant effect on the best-fit value of Lp (p = 0.013), with
significant differences in pairwise comparisons between Lp values for the hypotonic
concentration and each of the two hypertonic concentrations. However, the Lp values for the
two hypertonic solutions were not significantly different from each other (p = 0.97). To allow
comparison to the permeability data for monolayers (which are based on measurements of
1.2
0.8
0.4
2
1
0
0.0
0.0 0.5 1.0 1.5 2.0
/ e
0
20
40
60
Time (s)
Figure 2. Measurement of water permeability in BPAEC suspensions. (A) Representative
volume distribution under isotonic conditions. The line shows the best-fit curve comprising
the sum of an exponential distribution and a lognormal distribution (9). (B) Boyle-van’t Hoff
plot. Each data point represents the average of 4 replicates. The line shows the regression
of Eq. 1 to the data; the best-fit line was constrained to intersect the point (1,1). (C)
Representative transient of measured volumes during exposure to a 1000 mOsm/kg solution
at 21°C. The line shows the best-fit curve from the volume-limit adjustment method (10),
which was used to estimate Lp.
101
water efflux after exposure to hypertonic solution), we pooled the data across the hypertonic
osmotic pressures, yielding an average water permeability at 21°C of Lp = (2.9 ± 0.3) × 10-14
m Pa-1 s-1.
A
B
-15
-15
-17
-18
-19
-20
-21
0.0032
0.0034
-1 -1
A
-16
ln [LpA / Vw,0 (Pa s )]
-1 -1
ln [LpA / Vw,0 (Pa s )]
Effect of Temperature on Membrane Permeability in Monolayers and Suspensions
The water permeability in monolayers and suspensions of BPAEC and MIN6 cells at
various temperatures are compared in Fig. 3. ANOVA revealed significant main effects of
monolayer disaggregation and temperature on the effective permeability parameter Lp’, as
well as a significant interaction, for both BPAEC and MIN6 cells. The effective permeability
was significantly higher in monolayers than suspensions at all temperatures investigated for
both cell types (p < 0.05). The activation energy for water transport (Ea) was calculated from
the linear regression of the Arrhenius-transformed data using Eq. 7; the results are presented
in Table 1. The activation energy for BPAEC monolayers was significantly lower than the
activation energy for suspensions (p < 0.05). However, the difference between the activation
energies for MIN6 monolayers and suspensions was not significant (p = 0.12). The data for
MIN6 monolayers were adequately described by the regression line, but the accuracy of the
linear regression for MIN6 suspensions
Table 1. Activation energy (Ea) and coefficient of
was relatively poor. In particular, the
determination (R2) from Arrhenius fits.
permeability at 37°C appears to deviate
Ea/kJ mol-1 (R2)
from the remaining data, suggesting a
Cell Type
Monolayer
Suspension
possible discontinuity in the activation
energy between 21°C and 37°C (21).
BPAEC
49 ± 2a (0.96)
70 ± 4b (0.94)
Thus, a revised activation energy for
35 ± 6c (0.62)
MIN6
46 ± 3a,c (0.82)
the low-temperature regime was
59 ± 6a,b (0.84)†
obtained by restricting the Arrhenius
a,b,c
Means differ significantly if they do not share
fit to the temperature range 4°C–21°C,
a common superscript letter.
†
yielding Ea = 59 ± 6 kJ/mol.
Alternative Arrhenius fit for T ≤ 21°C.
0.0036
1/T (K-1)
B
-16
-17
-18
-19
-20
-21
0.0032
0.0034
0.0036
1/T (K-1)
Figure 3. Arrhenius plots for monolayers (open symbols) and suspensions (closed symbols)
of BPAEC (A) and MIN6 cells (B). Circles show data from the present study and each data
point represents the average of n = 4–12 replicates. Square symbols represent previously
published data (10, 11). The lines show linear regressions to the data using Eq. 7; the dotted
line is the linear regression with the data point at 37°C excluded. Permeability estimates at
4°C, 12°C and 37°C were obtained using osmolalities of 600 mOsm/kg (MIN6 monolayers,
MIN6 suspensions, BPAEC suspensions) or 1000 mOsm/kg (BPAEC monolayers).
102
Simulation of Water Transport During Freezing
Figure 4 compares predictions of intracellular supercooling for monolayers and
suspensions of BPAEC cooled at different rates to –40°C. Membrane water transport is faster
in monolayers than suspensions, resulting less supercooling for a given cooling rate. This is
particularly evident at a cooling rate of 10°C/min, for which monolayers exhibited a
maximum supercooling that is less than 5°C, whereas suspensions reached a supercooling
greater than 30°C. Supercooling predictions for MIN6 cells are not shown because of
ambiguity in the measured value of Ea, as explained above. Nonetheless, MIN6 cells also
exhibit lower supercooling in monolayers than suspensions, if the activation energy value Ea
= 59 kJ/mol is used for subzero extrapolation of the permeability of suspended cells.
o
Supercooling ( C)
30
o
B = 80 C/min
25
10
20
20
15
80
10
5
5 10
0
0
20
-10
5
-20
-30
-40
o
Temperature ( C)
Figure 4. Theoretical predictions of supercooling in suspensions (dashed lines) and
monolayers (solid lines) of BPAEC during freezing at various rates of cooling (B), as marked.
DISCUSSION
The goal of this study was to elucidate the differences in membrane permeability
properties between monolayers and the cell suspensions created by monolayer disaggregation.
We found that membrane water transport was faster in monolayers than suspensions over the
temperature range 4°C to 37°C for both BPAEC and MIN6 cells. Moreover, the water
permeability was more strongly affected by temperature in BPAEC suspensions than
monolayers, as evidenced by the higher activation energy for suspended cells. The activation
energy for MIN6 suspensions over the temperature range 4°C to 21°C was also higher than
the activation energy for monolayers (although the difference was not statistically significant).
These results have important implications for the design of cryopreservation procedures.
It is common to use permeability properties obtained from measurements on suspensions
of isolated cells to design cryopreservation procedures for tissue (1, 3, 14, 20). However, this
practice may yield erroneous results if the cell isolation process alters the membrane
permeability. Our results demonstrate that monolayer disaggregation alters the effective
water permeability parameter Lp’, leading to substantial differences in the predicted
supercooling during freezing.
These results underscore the importance of in situ
measurement of permeability in tissue.
To evaluate the effect of monolayer disaggregation on the kinetics of membrane water
transport, we made comparisons between measured values of the effective permeability
parameter Lp’, which comprises the product of the water permeability Lp and the surface-tovolume ratio A/Vw0. Thus, one possible explanation for the variation in Lp’ observed in this
study is differences in A/Vw0 arising from geometric differences between cells in monolayer
103
and suspension. Scanning electron microscopy studies show that suspended cells have highly
convoluted membranes, whereas adherent cells have relatively smooth surfaces (6).
Consequently, it has been estimated that suspended and adherent cells have similar membrane
surface areas (6). Moreover, cells in monolayer are connected to the matrix and to adjacent
cells, and as a result, a large portion of their membrane area is not available for water
transport. Accordingly, a recent confocal microscopy study reported slightly less membrane
area available for water transport in adherent cells than in suspended cells (29). Thus, it is
unlikely that our observation of approximately two-fold higher Lp’ in monolayers than
suspensions can be explained based on differences in cell surface area. Moreover, differences
in surface-to-volume ratio cannot explain the observed differences in activation energy, as the
surface-to-volume ratio would be unlikely to vary significantly with changes in temperature.
To obtain membrane permeability estimates, we assumed that the cell surface area A was
constant for both monolayers and suspensions. This common assumption has previously been
justified based on the argument that cell shrinkage and swelling are accommodated by folding
or unfolding of the membrane (16, 17). An alternative approach is to assume the surface area
is dependent on cell volume in a manner that depends on the geometry of the cell. For
example, spherical cells would have a surface area proportional to V2/3 (15). Using such a
variable-area model to re-fit a representative subset of our data (BPAEC suspensions at 21°C
and 37°C), the resulting permeability values deviated by no more than 15% from values
estimated using a constant-area model. Thus, the observed differences between the values of
Lp’ for monolayers and suspensions are too large to be caused by an artifact due to the
assumption of constant surface area in cell suspensions.
Consequently, the differences observed in water transport kinetics are most likely
attributable to changes in the cell membrane permeability to water. It is possible that watertransporting membrane channels were inactivated during the enzymatic disaggregation
process. This would explain the slower water transport in suspensions. Inactivation of
membrane channels would also explain the higher activation energy in BPAEC suspensions,
as water transport through the membrane bilayer is associated with a higher activation energy
than transport through membrane pores (5, 26). It is also possible that increased membrane
tension in the spread state leads to a higher water permeability. Membrane tension is affected
by cytoskeletal organization (22), which is known to change upon creation of a cell
suspension, and it is also affected by tethering to the matrix and adjacent cells.
A potential limitation of the current study is that we used different techniques to measure
the membrane water permeability in cell suspensions and monolayers. Because of the
physical differences between suspended cells and monolayers, it is difficult to measure the
membrane permeability using the same technique. For example, the fluorescence quenching
technique cannot be used with suspensions because suspended cells that have settled at the
base of the perfusion channel cannot withstand the high flow rates necessary for rapid
solution exchange (unpublished observations). Furthermore, the Coulter counter method
cannot be used with adherent cells, because cells must be suspended in order to be drawn
through the instrument’s aperture. While the use of different techniques does introduce some
uncertainty in comparisons of permeability properties, both techniques used in this study have
been carefully validated, and potential sources of error were minimized (7–11). In particular,
a common source of error in membrane permeability measurements is so-called unstirred
layer effects, caused by limitations in the rate at which the extracellular solution concentration
can be changed (26). For both the Coulter counter method and the fluorescence quenching
technique, we performed experiments to validate that unstirred layer effects were negligible
(7,11); the time required to change the extracellular solution composition was less than 1 s,
whereas the fastest cellular osmotic response observed in this study was approximately 10 s.
Thus, it is unlikely that the observed differences in membrane permeability can be attributed
104
to unstirred layer effects. Examination of the data provides further evidence that the
differences in water permeability were not caused by unstirred layer effects. Mixing
limitations would be expected to decrease the apparent value of the water permeability
parameter Lp’ and also decrease the activation energy for water transport (as the activation
energy for the self-diffusion of water is approximately 20 kJ/mol) (27). The value of Lp’ was
lower for cell suspensions than monolayers, but the activation energy was higher, suggesting
that the permeability differences cannot be explained by differences in unstirred layer effects
for the two different measurement techniques.
When estimating BPAEC permeability at room temperature, we found that a hypotonic
solution yielded a significantly larger value of Lp’ than did hypertonic solutions. Such
differences between shrinking and swelling experiments have previously been reported by us
(11) and others (e.g., 25). Possible causes of this effect include time-varying cell surface area,
unstirred layer artifacts (11), hydration state of the plasma membrane (25), flow rectifying
membrane channels (25), or stretch-activated channels. However, in the case of our present
results with BPAEC suspensions, we have presented evidence above to rule out confounding
effects due to variable cell surface area or unstirred layers, suggesting that the increased Lp’ in
hypotonic solutions is due to real differences in the physiological or biophysical state of the
membrane. Further studies are necessary to elucidate the mechanisms of this phenomenon.
Table 2 compares the results of the present study to permeability parameters reported in
the literature. At room temperature, we observed approximately two-fold slower water
transport in suspensions than monolayers for both BPAEC and MIN6 cells. Previously
reported effects of monolayer disaggregation on water transport kinetics are highly variable.
For instance, Balasubramanian et al. (2) compared fibroblast cell suspensions to cells that had
been cultured for 24 h in a collagen gel, using an indirect technique for estimating the cell
membrane water permeability based on cryomicroscopic observations of intracellular ice
formation. The resulting estimate of the effective permeability parameter Lp’ for suspended
cells was more than 1000-fold lower than that for cells cultured in a collagen gel. Using
similar cryomicroscopy techniques, Yarmush et al. (28) compared hepatocytes cultured in a
collagen matrix to the corresponding cell suspensions, and observed a 60-fold higher
permeability in the cell suspensions. The effect of tissue disaggregation on the activation
energy for water transport is also unclear, as both increases (28) and decreases (2) have been
reported in the literature. In the present study, we observed a significant increase in the
activation energy for BPAEC after monolayer disaggregation, whereas the effect of
monolayer disaggregation on the activation energy of MIN6 cells was not statistically
significant. While our results help to clarify the effect of tissue disaggregation on the water
permeability, further studies will be necessary to establish general trends.
Table 2. Relative magnitude of permeability parameters for suspensions and intact tissue.
References
Cell Type
Relative Lp’ a,b
Relative Ea a
Current study
BPAEC
0.35
1.4
Current study; (10); (11) MIN6 cells
0.60
0.76 (1.3d)
c
(2)
Human fibroblasts
0.0006
0.57
(4)
J774 macrophages
1.4
(19); (23)
Rat hepatocytes
2.1
1.1
(28)
Rat hepatocytes
60c
3.1
(29)
PC-3 adenocarcinoma cells
1.0
a
b
Ratio of suspension parameter to tissue parameter; At temperatures in the range 21–23ºC;
c
Values of A/Vw,0 were not given, so equal values were assumed for tissue and suspension;
d
Alternative calculation of relative Ea using suspension data between 4ºC and 21ºC only.
105
SUMMARY
We have investigated the differences between the membrane permeability properties of
cell monolayers and suspensions. We observed slower membrane water transport kinetics in
suspensions than monolayers over the temperature range 4°C to 37°C for BPAEC and MIN6
cells. Although the activation energy for water transport was not significantly different in
monolayers and suspensions of MIN6 cells, BPAEC suspensions exhibited a higher activation
energy than monolayers. Simulations indicated that BPAEC suspensions and monolayers
have different levels of supercooling during cryopreservation. Our results highlight the
importance of in situ measurement of permeability parameters for cells within tissue.
Acknowledgements: This work was supported in part by the National Science Foundation
(NSF) under awards CBET-0954587 and CBET-0541530 (to JOMK), as well as the Georgia
Tech/Emory Center for the Engineering of Living Tissues, an NSF Engineering Research
Center (EEC-9731643). Fellowship support (for AZH) was provided by the NSF, the Howard
Hughes Medical Institute, the Medtronic Foundation and the George Family Foundation.
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Accepted for publication 21/11/2011
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