Research Paper
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Acta Biochim Biophys Sin
2005,37: 814–818
doi:10.1111/j.1745-7270.2005.00121.x
Response of Cytoskeleton of Murine Osteoblast Cultures to Two-step
Freezing
Bao-Lin LIU1* and John McGRATH2
1
Institute
of Cryobiology, University of Shanghai for Science and Technology, Shanghai
200093, China;
2
Department
of Aerospace and Mechanical Engineering, University of Arizona, Tucson 85721,
USA
Received: August 4,
2005
Accepted: October
20, 2005
This work was
supported by the grants from the Strategic Partnership Grant from Michigan
State University Foundation, NSF (DMR 0074439), NSFC (50436030), Shanghai
Leading Academic Discipline Project (P0502, T0503), and SRF for ROCS, SEM
*Corresponding
author: Tel, 86-21-65688765; Fax, 86-21-65682376; E-mail, [email protected]
Abstract Understanding the
ultrastructural response of cells to the freezing process is important for designing cryopreservation
strategies for cells and tissues. The cellular structures of attached cells are
targets of cryopreservation-induced damage. Specific fluorescence staining was
used to assess the status of the actin filaments (F-actin) of murine
osteoblasts attached to hydroxyapatite discs and plastic coverslips for a
two-step freezing process. The F-actin of dead cells was depolymerized and
distorted in the freezing process, whereas that of live cells had little
change. The results suggest that the cytoskeleton may support the robustness
of cells during cryopreservation. The present study helps to investigate the
damage mechanism of attached cells during the freezing process.
Key words cytoskeleton; osteoblast;
actin filament; two-step freezing
The evolving field of tissue engineering has created the need for
cryopreservation processes to store tissue-engineered products. Osteoblast (OB)-seeded
hydroxyapatite (HA) implants are a new type of engineered cortical bone
substitutes that have a mineral content and crystal structure similar to that
of bone [1]. To successfully cryopreserve this new bone construct, it is
necessary to understand the cellular response of attached cells to the
cryopreservation process. In tissue engineering, it is crucial for cells to remain attached on
the surface of biomaterials because most tissue-derived cells are
anchorage-dependent and require attachment to a solid surface for viability
and growth [2]. Also, cell-substrate attachment precedes cell spreading,
migration, differentiation, mineralization and expression of phenotype [3], all
of which are critical for the final formation of engineered tissues.
Cytoskeleton, one of the ultrastructures of cells, is a network of protein
fibers extending throughout cells, used for support, transport and motion [4].
Cell locomotion and muscle fiber contraction could not take place without it.
Cytoskeleton is one of the fundamental parameters to evaluate the attachment
of cells and it is also the target of damage in the cryopreservation process.
Changes in the ultrastructure of oocytes from rabbit [5] and monkey [6] in suspension after
chilling or freezing have been observed. Chilling can cause degradation of
microtubules in cultured human fibroblasts [7]. Hall et al. [8]
reported the loss of actin filament (F-actin) stress fibers in arterial
endothelial cells attached to glass coverslips after cooling to 4 ?C. The previous
studies mainly focused on cells in suspension with a relatively high
temperature range of 0–4 ?C. But in cryopreservation, the minimum temperature may reach as
low as –196 ?C. At this extremely low temperature, the biochemical and biophysical
properties of cells should be quite different from those above 0 ?C.Although much work has been done in cryopreservation on how to
maintain the viability of cells, little attention has been focused on the
molecular or ultrastructure level [9], especially at liquid nitrogen
temperature (–196 ?C). No current information exists on the response of cytoskeleton
to freezing for attached osteoblast cells. It is important to understand the
ultrastructure response of cells to the freezing process in order to design
cryopreservation strategies for cells and tissues. In this study, specific fluorescence staining and green fluorescence
protein (GFP) transfection techniques were used to assess the status of the
F-actin attached to HA discs or plastic coverslips in murine osteoblast cells
using a two-step freezing method.
Materials and Methods
Culture of OB cells
Mouse calvaria-derived OB cell line MC3T3-El cells [10] were
cultured in a-minimum essential medium (a-MEM; Gibco BRL, Grand Island, USA) supplemented
by 5% fetal bovine serum (Gibco BRL) at 37 ?C with 5% CO2.
Cells were harvested by rinsing with 1?phosphate-buffered saline (PBS), then exposing to 0.25% trypsin
solution (Gibco BRL) for 3 min at room temperature. Cells were then suspended in
a-MEM
at a concentration of 4?104
cells/ml.
Seeding of OB cells on the surface of HA discs
HA discs with a diameter of 11 mm and a thickness of 1 mm were
fabricated in Material Laboratory at Michigan State University (East Lansing,
USA). HA discs were sterilized and
placed on the bottom of 24-well plates (Costar, Cambridge, USA) before seeding
of cells. OB cell suspensions containing 4?104 cells/ml were seeded on HA discs and the plates were incubated at
37 ?C with 5% CO2. The medium was changed every other day. All the OB:HA discs were
incubated for 2 d to avoid the effect of cultivation time on F-actin. Thermanox coverslips (Nunc, Rochester, USA) of 13 mm in diameter
were used as the control. Thermanox coverslips were placed on the bottom of 100
mm Petri dishes for cell seed on their surfaces.
Two-step freezing process
Two-step freezing process
Ten percent of dimethyl sulfoxide (DMSO; JT Baker,
Phillipsburg, USA) in a-MEM was used for two-step freezing. The addition and removal of DMSO
was performed as described previously [1] except that the temperature was 4
?C. When freezing the cell monolayers, the holding time in 10% DMSO was 10 min
to ensure the thorough penetration of cryoprotective agents (CPA) into the
attached cells. After the addition of 10% DMSO, the cell monolayers were cooled
to –80
?C at the cooling rate of 1, 3, 5, 10 or 20 ?C/min then quenched into liquid
nitrogen. The modified cooling method was used as described elsewhere [1]. The
samples were cooled in 25 mm diameter vials and warmed in a 37 ?C water bath,
generating a warming rate of approximately 60 ?C/min.
Viability assay
A live/dead viability/cytotoxicity kit (L-3224; Molecular Probes,
Eugene, USA) was used to stain cells as a viability assay. Twenty microliters
of 2 mM EthD-1 stock solution and 5 ml of 4 mM calcein AM stock solution were added
to 10 ml of sterile tissue culture grade D PBS, vortexing to ensure thorough
mixing. The resulting working solution containing 2 mM calcein AM and 4 mM EthD-1 was added
directly to the cells. The stained cells were transferred to a microscope with
an ultraviolet light source. A digital camera (Spot RT; Diagnostic
Instruments, Sterling Heights, USA) was attached to the microscope and used to
produce digital images of live cells (green) and dead cells (red) by focusing
on different locations on the discs with a 25? phase contrast objective.
Specific fluorescence staining
All the cells ready for specific fluorescence staining were rinsed in
PBS at 37 ?C, immediately fixed, permeabilized simultaneously in
formaldehyde-Triton solution (4% formaldehyde, 0.2% Triton-X 100, 60 mM PIPES,
25 mM HEPES, 10 mM EGTA, 3 mM MgCl2, pH 6.1) for
10 min, and washed with PBS again. To visualize F-actin, the fixed cells were
incubated with 100 nM fluorescent phalloidin (Molecular Probes) in PBS for 30
min at room temperature in dark. After two additional washes in PBS, the
F-actin stained cells were mounted on glass slides. Nuclei were stained with ethidium bromide for double staining.
GFP-actin plasmids transfection of cell
GFP-actin plasmids were obtained from the physiology laboratory at
Michigan State University. Attached OB cells were transfected with GFP-actin plasmid
using Lipofectamine 2000 (Gibco BRL) according to the manufacturers
instructions. Approximately 75% transfection efficiency of cells was obtained.
Cells with the most intense fluorescence signal were selected to perform the cryomicroscopy test.
Cryomicroscopy
A computer-controlled cryomicroscopy system [11] was used to study
the effect of freezing and thawing on the F-actin of OB cells. The GFP-actin
plasmid transfected live OB cells were mounted on the cryostage of the cryomicroscopy
system. The FITC filter was selected to detect the green fluorescent F-actin of
the transfected cells. The cooling rate of 20 ?C/min was programmed for the
freezing of the cells and the warming rate of 60 ?C/min was used to thaw the
cells. The whole process was recorded by a video cassette recorder through a
Spot RT color camera. The video was then used for image analysis.
Image processing
The specific fluorescence-stained samples were photographed using a Nikon
E800 (Tokyo, Japan) microscope with a Spot RT color camera mounted on the top.
A 40? objective was used and the number of
cells was 19.0+/–1.7 cells per image. Twenty images were used to calculate the
spreading area index (SAI) for each cooling rate. Photographs were taken
only at the fields where cells were uniformly distributed. The F-actin
spreading area was automatically obtained using Image Pro software (Media
Cybernetics; Silver Spring, USA). To quantitatively describe the effect of CPA
exposure or freezing on F-actin, SAI was defined as in Equation 1:
Eq. 1
where SAfreeze is the spreading area of the
freezing group, and SAcontrol is the spreading area of the
control group.
Statistics
All data were expressed as mean+/–SD and subjected to
statistical analysis using Student’s t-test. Student’s t-test for
independent samples with unequal or equal variances was used to test the
equality of the mean values at a 95% confidence interval (P<0.05).
Results
F-actin damaged by two-step freezing
The F-actin was depolymerized and distorted after the two-step
freezing process (Fig. 1). The F-actin in the control group was well
stretched and spread [Fig. 1(A)]. However, in the freezing group, the F-actin
was cut, curled and depolymerized [Fig. 1(B)]. The F-actin/nuclei double
staining [Fig. 1(C)] clearly shows that the severe distortion of
F-actin was only related to dead cells, the F-actin of live cells had little
change compared with that in the control group.Fig. 2 represents the SAI of
F-actin at various cooling rates for HAs and plastic coverslips. Cells frozen
at 3 ?C/min retained the best attachment. Increasing the cooling rate from 3
?C/min to 5 ?C/min produced no significant change (P>0.05) in the SAI.
But in the group with a cooling rate of 10 or 20 ?C/min, the SAI
decreased significantly compared to that of the 3 ?C/min group for both HA
discs and plastic coverslips (P<0.05). Interestingly, the SAI
of F-actin at 1 ?C/min was smaller than that at 3 ?C/min (P<0.05), which does not follow the decreasing pattern from lower cooling rates to higher cooling rates. Students t-test between HAs and plastic coverslips
showed that significant differences existed only at 20 ?C/min.
F-actin and viability affected by minimum temperature
To investigate the damage mechanism of attached OB during freezing
process, cells were frozen to different minimum temperatures at 10 ?C/min then thawed
to 20 ?C (Fig. 3). The viability and SAI of F-actin showed
similar decreasing patterns at various minimum temperatures.
Inspection of GFP-actin plasmids transfected F-actin during freezing
process
To directly inspect the F-actin changes during the two-step freezing
process, Live cells with GFP plasmids transfected F-actin were frozen and
thawed using a computer-controlled cryomicroscope. The F-actin was very clear
in the fresh cells [Fig. 4(A)]. A small distortion of F-actin was
observed when the cells were frozen [Fig. 4(B)]. After thawing, most of
the F-actin was depolymerized or damaged [Fig. 4(C)], which showed a
similar pattern to that in Fig. 1(B).
Discussion
Our previous results showed that the survival of cells attached to a
substrate decrease greatly compared to those in suspension [12]. The present
study helps to investigate the damage mechanism of attached cells during the freezing
process. F-actin is one of the three cytoskeletons in animal cells. It is
generally believed to provide the molecular basis for many of the mechanical
properties of cytoplasm, a complicated viscoelastic material [13]. F-actin is
highly concentrated in the cortex, just beneath the plasma membrane. This
actin-rich layer controls the shape and surface movements of most animal cells
[4]. Because of their close proximity, any mechanical damage to the membrane
might also disrupt the F-actin. The viability assay kit used in this study is
based on membrane damage. If the F-actin does not depolymerize after exposure
to CPA and subsequent freezing, the structural changes of F-actin might, to
some extent, reveal the viability of cells. Depolymerization of microtubules and F-actin occurred after 2 h at 4
?C for arterial endothelial cells [8]. Because there was no ice formation at 4
?C, these cytoskeletal changes might be induced by the temperature change inside
the cells. In our study, OB cells were frozen to temperatures as low as –196 ?C, the
damage mechanism was different to that in arterial endothelial cells. Fig.
1(B) clearly shows that the F-actin was cut or distorted after freezing, revealing
some mechanical forces exerting on the cells, such as extracellular ice,
mechanical stress and differential thermal contraction between cells and
substrates. The F-actin was distorted in dead cells, but retained good structure
in live cells [Fig. 1(C)]. The fact that the F-actin spreading area and
viability decreased in the same pattern as the temperature changes (Fig. 3)
indicates that F-actin is a target of damage to attached cells. This provides
more evidence of mechanical damage. We assume that some mechanical forces
damaged the cell membrane leading to cells death. It is well known that the shape and size of ice crystals change with
the cooling rate. The ice crystal front moves quickly with increasing cooling
rate, which may lead to more cell damage. Cells attached to a surface are fixed
and will be sheared as the ice crystals pass through, as is the case for mouse
oocyte [14]. With increasing cooling rates, the ice crystal damage,
differential thermal contraction injury and mechanical stress disruption become
more serious (Fig. 2). At 1 ?C/min, the mechanical damage should be the
smallest of all the five cooling rates (Fig. 2). But its spreading area
(survival) was lower than that at 3 ?C/min. This was probably caused by the
toxicity of the DMSO, in which the cells stayed a relatively long time at the
cooling rate of 1 ?C/min. There was significant difference in SAI
between HA discs and plastic coverslips at 20 ?C/min (Fig. 2), which
shows that the type of material affects the cellular response of cells to
freezing at relatively high cooling rates.Because the two-step freezing method includes CPA addition and
removal, freezing and thawing, it is uncertain whether the ultrastructure
damage occurred in the freezing or thawing process. Fig. 4 provides the
direct observation of ultrastructural change of live cells during the
cryopreservation process. A small distortion of F-actin can be seen when the
cell was frozen [Fig. 4(B)], but this change was not very clear because
of the low magnification of objective used in this study and the existence of
ice. The image in Fig. 4(C) clearly shows that the F-acin was severely
damaged after thawing, revealing the same results as in the specific
fluorescence staining method. High magnification objective (100?) and powerful tools (such as scanning
electronmicroscopy) should be used in the future to inspect GFP-actin changes
during the freezing process.In conclusion, the distortion of F-actin helps explain the damage
mechanism for attached OB cells during the two-step freezing process.
Mechanical forces, such as extracellular ice and differential thermal
contraction, were the main damage factors in our
study. The protocols for the cryopreservation of the tissue-engineered bone constructs
should be designed to decrease this mechanical damage. It is recommended to
optimize the cooling and warming rates, the substrates to which the cells are
attached, the types of CPA solutions, and the minimum temperatures. These
factors will form the basis of our future research.
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