Cryobiology 49 (2004) 241–249
www.elsevier.com/locate/ycryo
Survival of mammalian cells under high vacuum condition
for ion bombardmentq
Huiyun Fenga, Lijun Wua,*, An Xua, Burong Hua, Tom K. Heib, Zengliang Yua,*
a
Key Laboratory of Ion Beam Bioengineering of Chinese Academy of Sciences, Institute of Plasma Physics, P.O. Box 1126,
Hefei 230031, Anhui Province, PeopleÕs Republic of China
b
Center for Radiological Research, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA
Received 14 October 2003; accepted 23 August 2004
Available online 2 November 2004
Abstract
An ion beam has been used to irradiate various organisms and its effects have been studied. Because of the poor tolerance that mammalian cells have for vacuum, such studies have not been carried out on living mammalian cells until now.
However, this work is important both for elucidating the mechanism of mutation in response to low-energy ions and in
exploring possible new applications of ion beam technology. The current paper describes an investigation of the survival
of mammalian cells (the AL cell line) in a high-vacuum chamber in preparation for ion bombardment studies. The ion
beam facility is described and the actual vacuum profile that the cells endured in the target chamber is reported. Cells were
damaged immediately following vacuum exposure; the injury was characterized by alteration of the membrane permeability, loss of firm adhesion to the dish, and increased fragility. Three cryoprotective agents were tested (glycerol, propylene
glycol, and trehalose) and of these, glycerol showed the highest potency for protecting cells against vacuum stress. This
was revealed by an increase in the cell survival level from <1 to >10% with a glycerol concentration of 15 and 20%.
Two glycerol-based protocols were investigated (freezing-vacuum vs. non-freezing-vacuum), but there was no significant
difference (P > 0.1) in their ability to improve cell survival, the values being 10.31 ± 4.5 and 12.7 ± 3.37%, respectively
with 20% glycerol concentration. These cells had a normal growth capability, and also retained integrity of the cell surface
antigen CD59. These initial experiments indicate that mammalian cells can withstand vacuum to the degree that is needed
to study the effect of the ion beam. In addition to the improvements made in this study, other factors are discussed that
may increase the survival of mammalian cells exposed to a vacuum in future studies.
2004 Elsevier Inc. All rights reserved.
Keywords: Ion bombardment; Vacuum survival; Mammalian ALcells
q
This work was supported by the National Natural Science Foundations of China (Project Nos. 30170234 and 10225526).
Corresponding authors. Fax: +86 551 5591310 (L. Wu and Z. Yu).
E-mail addresses: [email protected] (H. Feng), [email protected] (L. Wu), [email protected] (Z. Yu).
*
0011-2240/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.cryobiol.2004.08.003
242
H. Feng et al. / Cryobiology 49 (2004) 241–249
In the past three decades, studies of the biological effects of energetic heavy ions have rarely included reports of the effects of low-energy ions
(E < 100 keV) on organisms. Because of the assumed short range of low-energy ions it was generally assumed that they would have little or no
effect on organisms. However, the discovery in
1989 and 1991 of genetic effects that were induced
in rice by ion bombardment opened a new area of
study of ion beam effects in the life sciences [20,21].
Recent advances have included the consideration
of a possible role for low-energy ions in the origin
and evolution of life [5,10,13], in mechanisms of
damage and mutation due to low-energy ions
[8,14,15], and the application of ion beam technology to genetic modification [2,3,16,22].
For most kinds of biological sample that can
tolerate vacuum and desiccation, such as dry crop
seeds, bacteria, deoxyribosenucleic acid (DNA),
and some organic molecules (amino acids, pyrimidines, and purines, for example), it is convenient to
bombard them with ion beams generated in a high
vacuum condition (at least 10 2 10 3 Pa pressure). However, mammalian cells have not been
used as target materials due to their poor vacuum-tolerance. In this paper we describe the use
of our low-energy ion beam bioengineering facility
(LE-IBBF). We have tested the survival of mammalian cells under various high vacuum conditions
in preparation for ion bombardment studies. We
achieved greater than 10% survival by using glycerol as a stabilizer against vacuum-induced stress
and freezing injury. This success makes the study
of the effects of ion-beams on mammalian cells
plausible, but room exists for further
improvements.
Materials and methods
Reagents
All biochemical reagents were purchased from
the Shanghai Sangon Company. Cell culture reagents and materials were from Gibco BRL. Propidium iodide (PI) is a product of Molecular
Probes, and CD59 antibody is from BD
Bioscience.
Cell line and culture condition
The human-hamster hybrid AL cell line contains a standard set of CHO (Chinese hamster
ovary cell) chromosomes and a single copy of human chromosome 11; it was created by Waldren
and colleagues [12], and was used in this study.
An outstanding feature of the AL cell is that
the gene that encodes the surface antigen CD59
is located at 11p13.5, and provides a highly sensitive marker for mutation analysis. For this reason
AL has been one of the widely used model cell
lines for evaluation of genetic mechanisms of toxicity and carcinogenesis and the investigation of
environmental factors such as radiation [17], heavy metals [7], electro-magnetic fields (EMF) [1],
and chemical carcinogens [19]. For the same reason, we chose AL cells to assess the mutagenic effects of low-energy heavy ions on mammalian
cells.
The cells were cultured in standard HamÕs F-12
medium (Gibco BRL, 21700-018) supplemented
with 8% heat-inactivated fetal bovine serum
(FBS, Hyclone, SH30071.03), 25 lg/mL gentamycin, and 0.2 mM glycine (Sigma–Aldrich, USA)
at 37 C in humidified 95% air + 5% CO2 in an
incubator (SANYO MCO-18AIC) and passaged
as described. Cells for vacuum exposure and ion
bombardment were plated in 35 or 60 mm tissue
culture dishes (Falcon, BD Labware) and cultured
for 2 days to 90% confluence.
Low-energy ion beam bioengineering facility for
biological materials bombard
The LE-IBBF comprises a gas ion source, a vacuum system, a vertical beam line, two target chambers, and control systems. It was developed
specifically for ion bombardment of biological
materials. The pivotal components of the facility
are the two sample chambers, as shown in Fig. 1.
A 7000 L/s diffusion pump and mechanical pumps
were used to create the vacuum: the target chamber base pressure of 10 3–10 4 Pa could be
achieved routinely. Ion beams of gases such as
H, He, Ar, and N, were produced by a heated
cathode, duo-plasma ion source. Beam energy
could be varied between 10 and 40 keV.
H. Feng et al. / Cryobiology 49 (2004) 241–249
Fig. 1. Schematic diagram of LE-IBBF. 1, Ion beam; 2, main
chamber; 3, small chamber; 4, rotating wheel for loading crop
seeds; 5, main vacuum system; 6, pre-pump for small chamber;
and 7, isolating valve.
The main chamber was designed for ion bombardment in dry crop seeds. A wheel, 900 mm in
diameter, was installed on the base of the chamber
and could be rotated. The wheel contained six
sample plates and each of the plates could be
loaded with 1500 dry seeds. The bombardment
parameters for each sample plate could be programmed in advance and the ion bombardment
carried out sequentially. The small chamber, located under the main chamber, was specifically designed for bombardment of water-rich living cells
and samples of small volume, such as microbes,
seeds of Arabidopsis, and mammalian cells. The
dimensions of this chamber were 100 mm in diameter and 150 mm in height. The volume of the
small chamber is about 1.2 L; the volume ratio
of the small chamber and the main chamber is
about 1 : 600. The two chambers, separated by
an isolating valve, formed a differential pumping
system capable of creating a vacuum rapidly in
the small chamber.
Protocol for applying a given vacuum and pressure
profile upon cells in the vacuum chamber
Before each experiment, the main chamber was
pumped to < 5 · 10 3 Pa. A prepared cell dish was
243
Fig. 2. Pressure profiles of main and small vacuum chambers.
The pressure profiles represent the actual pressure during the
application of vacuum after cell loading. The upper and lower
panels are the pressure profiles measured in the small and main
chambers, respectively. Data are averages of 20 independent
experiments. Bar represents SEM.
loaded into the small chamber, and the air in this
chamber was pumped out for 12 s before opening
the isolating valve. Due to volume expansion, the
pressure in the main chamber would jump to 40–
50 Pa once the valve was open. Then, the pressure
of both chambers gradually reached 10 2 Pa in
about 100 s due to the high pumping rate of the
diffusion pump. Ion bombardment was carried
out immediately: the total time for which the cells
remained under vacuum conditions was around
200 s. The actual pressure profile to which the cell
dish was subjected was recorded (Fig. 2).
Temperature measurement of cells under vacuum
The temperature of the cell population under
vacuum was sensed by a thermocouple. The detailed procedure is as follows: 3 · 106 AL cells were
collected in an Eppendorf tube by centrifugation
at 1000 rpm and the supernatant was removed.
The tube was then put in the small sample chamber and the sensing point of a 0.4 mm Type T copper/copper–nickel thermocouple was embedded in
the center of the cell pellet. Vacuum was then applied as described above. The decreasing temperatures of the cell pellet were recorded as well as the
pressure descent.
244
H. Feng et al. / Cryobiology 49 (2004) 241–249
Anti-vacuum treatment protocols
PI membrane integrity assay by flow cytometry
Two glycerol-based protocols, freezing and
non-freezing, were tested for their ability to enhance the vacuum tolerance of the cells. For all
tests, 90% confluent cells in 35 mm dishes were
incubated with 0, 5, 10, 15 or 20% (v/v) glycerol/F12 medium at 4 C for 30 min. Afterwards,
in the non-freezing protocol (F V+), the medium was removed, and the cell dishes were placed
on ice before the process of vacuum application
and ion bombardment (the time needed for this
was about 3 min). F stood for not frozen and
V+ represented vacuum exposure. In the freezing
protocol, after removing the medium containing
glycerol, the cell dishes were frozen overnight in
freezers set to 25, 30, or 70 C. The dishes
were either directly placed in the freezers (for rapid freezing) or were first placed in ÔstyrofoamÕ
boxes (wall thickness = 20 mm) and the boxes
then placed inside the freezers (slow freezing).
On the second day, the frozen cell dishes were taken out one at a time from the freezers, immediately thawed by floating on a 37 C water bath,
and pre-warmed phosphate-buffered saline
(PBS) was added to each dish. These groups were
designated as the (F+V ) group. The freezing
conditions were important factors in the freezing-vacuum procedure, and cell viability in response to various freezing conditions was also
assessed.
The results from the (F+V ) test indicated
that the 25 C fast freezing procedure produced
the highest survival levels (Fig. 5), so for the
(F+V+) tests, only the 25 C fast freezing condition was used. The details of the process are as
follows: the cell dishes were prepared in the same
way as the (F+V ) samples, and were directly
frozen at 25 C overnight. On the second day,
they were taken out one at a time, kept on an
ice bag that had been frozen to 25 C, and
then loaded into the small chamber as quickly
as possible and the vacuum applied as described
above.
All vacuum-exposed samples were thawed by
water bath and warm PBS was added immediately
after the 200 s vacuum exposure.
Immediately after thawing, cells were assayed
for membrane integrity using a fluorescent staining method with PI. PI is a membrane-impermeable dye and is excluded by viable cells. It is
commonly used for identifying dead cells in a
population (Product information, Molecular
Probes). After thawing, frozen and/or vacuum-exposed cells were washed once with PBS. As a portion of cells became detached from the dish due
to the freezing or vacuuming process, the PBS
used for washing was also collected. Then the
cells still attached to dishes were detached, harvested, and combined with the PBS cell suspension. PI staining was performed at a
concentration of 2 lg/mL at room temperature
for 15 min. The fluorescent intensity was detected
in the FL2 channel of a FACSCalibur flow
cytometer (Becton Dickinson, USA) and analyzed
by Cellquest software.
Evaluation of CD59 cell surface antigen expression
by flow cytometry
The most important character of the AL cell
line is its expression of the CD59 cell surface antigen. To evaluate the effects of glycerol plus vacuum exposure on CD59 expression, both the
untreated cells and the cells that experienced
20% glycerol treatment (4 C for 30 min) and a
200 s vacuum exposure were cultured for 7 days,
and then evaluated by flow cytometry for the
expression of the CD59 surface antigen. In detail,
cells were completely detached from the dishes
with 1 mM sodium EDTA/PBS-(without Ca2+
and Mg2+), and the reaction was stopped with
PBS+ (PBS containing Ca2+ and Mg2+). Then
the suspended cells were washed with PBS+, then
stained with R-phycoerythrin conjugated mouse
anti-human CD59 monoclonal antibody (BD
Pharmingen, 555764) in PBS+BS buffer (1%
BSA, 10 mM sodium azide in PBS+, pH 7.0) on
ice for 30 min. Stained cells were washed twice
to remove uncombined antibody, resuspended in
PBS+BS buffer and fluorescence detected
immediately.
H. Feng et al. / Cryobiology 49 (2004) 241–249
245
Statistics
Statistical analysis of the data was carried out
using a one-way analysis of variance (ANOVA)
and StudentÕs t test. Differences between means
were regarded as significant if P < 0.05.
Results
Pressure profile in vacuum chambers
The real time vacuum condition that the cells
experienced is shown in Fig. 2, with the upper
and lower panels showing the pressure in the
small chamber, and the main chamber, respectively. As described above, each cell dish was
loaded into the small chamber and air was evacuated for 12 s. Curve 1 showed that the pressure
to which the cells were subjected dropped rapidly
from 1 atm to near 1000 Pa, a decrement of ·100.
Curve 2 indicated that after the initial evacuation
of air from the main chamber, the facility was
ready for experiment, with a base pressure of
7.0 · 10 3 Pa. After the opening of the isolating
valve between the two chambers at the time point
zero, the pressure jumped to 45 Pa (curve 3) due
to volume expansion. Then the pressure descended gradually, reaching 10 2 Pa in about
100 s. Ion bombarding could be applied to the
cells from this point on. Cells were allowed to
stay in this vacuum condition, with a steady pressure at 2.5 · 10 3 Pa, for 100 s (this time was sufficient for ion bombardment) until they were
taken out.
Temperature in cell pellet during vacuum exposure
The curve for cell temperature in the vacuum
chamber, as recorded by the thermocouple, is displayed in Fig. 3. First, the temperature showed a
gradual decrease from 10 C to near 0 C as the
vacuum was generated from 1 atm to 1000 Pa;
then there was a sharp drop from 0 to 25 C as
the pressure was decreased from 1000 to 100 Pa.
Following this the temperature leveled off and
remained constant at 28 C.
Fig. 3. Temperature profile in cells during the application of
vacuum. The temperatures measured inside the cell pellet were
recorded during the process of generating the vacuum. Data are
the average of three independent experiments. Bar
represents ± SEM.
Microscopic observations of vacuum-exposed cells
Visible light and PI fluorescent microscopy were
used to estimate the cell status after exposure to
the vacuum. Fig. 4A, C, and E shows representative examples of non-viable cells after vacuum
exposure without any protective pretreatment.
Although these cells were still attached to the dish,
their morphology was evidently different from that
of control cells: the cells were flattened and dry
(Fig. 4A). PI staining indicated that very few cells
had intact plasma membranes (Fig. 4C). Moreover, the ability of cells to adhere had been destroyed, as shown in Fig. 4E: few cells were still
attached to the dish after rinsing with PBS. The
cells that were detached by rinsing were mostly
fragmented. Fig. 4B, D, and F shows the morphology and PI staining characters of vacuum-exposed
cells that were pretreated with 20% glycerol.
Although some cells were distorted and had an
irregular shape (Fig. 4B), many cells appeared
healthy, were firmly attached, round or spindleshaped, with morphology similar to that seen in
normal control dishes (Fig. 4G). PI staining confirmed that the cells with normal morphology were
alive (Fig. 4D) and remained firmly attached to the
dish (Fig. 4F). After washing off the non-viable
cells, those that remained adherent were cultured
to determine the recovery capacity and surface
246
H. Feng et al. / Cryobiology 49 (2004) 241–249
Fig. 4. Light and PI fluorescent microscopic images of cells.
Control and vacuum-exposed cells were stained in situ with
2 lg/mL PI, then separate light transmission and fluorescent
images of the same field were acquired and merged. (G) Control
cells, showing no PI fluorescence. (A, C, and E) Vacuumexposed cells, without any protective pretreatment. Image (A)
shows cell morphology before PI staining; image (C), all cells
were stained with PI; image (E) illustrates that almost no cells
were left in the dish after pipet-rinsing with PBS. (B, D, and F)
Vacuum-exposed cells, with pretreatment with 20% (v/v)
glycerol/media at 4 C for 30 min. Image (B), shows the cell
morphology which is distinct from that in Image (A); Image
(D), some cells were PI-stained, while many healthy, firmly
attached, round or spindle-shaped cells excluded PI. Image (F),
after twice rinsing with PBS, the detached, dead or fragmented
cells were cleared away, only surviving cells being left. (H)
Recovery of the viable vacuum-exposed cells after 3 h incubation. Dead cells and debris had been washed out.
antigen expression. Fig. 4H shows cells with clear
edges and a three-dimensional morphology similar
to that of untreated cells (Fig. 4G) after 3 h incubation. There were no significant differences in
growth rate between the untreated cells and the
cells that had survived vacuum exposure (our
unpublished data) during the following two weeks.
Glycerol enhances the ability of cells to withstand
vacuum stress
All protocols were tested using monolayers
grown on 35 mm culture dishes. The cells to be
Fig. 5. Cell survival comparison of various freezing procedures.
Cell survival rates for five freezing procedures without vacuum
exposure were measured. The purpose of this experiment was to
find the most survival-favorable freezing procedures and to test
the effect of vacuum on cells using these freezing conditions
for later experiments. The best procedure was fast freezing
at 25 C with 20% glycerol pretreatment. Please refer to
‘‘Materials and methods’’ section for details. The data are
the average of three independent experiments. Bar
represents ± SEM.
ion bombarded had to be in a monolayer without
a covering of medium or any other material since
that would have prevented cells from being bambarded by the ion beam. Previous experiments
clearly indicated that there were dramatic changes
in temperature inside the cell pellet during the process of generating a vacuum (Fig. 3). Therefore,
temperature was an important parameter to measure as well as the actual vacuum. We tested various freezing procedures to find the most favorable
one, and then used that procedure in combination
with vacuum exposure to test the protective effect
of glycerol against vacuum-induced damage to
cells. Fig. 5 shows the survival of cells frozen
according to five protocols using five glycerol concentrations of between 0 and 20%. It is evident
that fast-freezing at 25 C was more favorable
to cell survival than the other freezing protocols
tested. The fast-freezing procedure yielded significantly higher survival levels (more than 70% PI
negative cells compared to untreated cells) than
all the other freezing procedures in which cells
were exposed to 10, 15, or 20% glycerol for
30 min at 4 C. Therefore, 25 C fast freezing
H. Feng et al. / Cryobiology 49 (2004) 241–249
Fig. 6. Survivals of vacuum-exposed cells with or without the
protective pretreatment. The efficiency of the two kinds of
protocols (F V+) vs. (F+V+) was compared. F+ represented
fast freezing at 25 C and F stood for no freezing step before
vacuum exposure. The vacuum procedure was the same for all
the tests. Please refer to Fig. 2 and related description in the
text. The significant levels shown above each pair of bars were
relative to the cells without glycerol pretreatment.
was chosen as the preferred procedure for the rest
of the freezing-vacuum experiments.
Cell viability after vacuum exposure with or
without protective treatment is shown in Fig. 6.
The results indicate that the higher concentrations
of glycerol (15 and 20%) had significant cell protecting effects against vacuum exposure. To our
surprise, at two glycerol concentrations there was
no significant difference in viability between cells
treated with the freezing step and cells that were
not frozen; 10.31 ± 4.5% vs. 12.7 ± 3.37%;
(P > 0.1) with 20% glycol. It appears that freezing
is not necessary for experiments using ion beam
bombarding in mammalian cells; pretreatment
with 20% glycerol dramatically increased the cell
survival level from < 1 to > 10%, and this survival
level is sufficient for the study of the genetic effects
of low-energy ion-beam on mammalian cells.
CD59 cell surface antigen expression
A group of representative flow cytometry histograms, evaluating the expression of CD59 surface
antigen were shown in Fig. 7. Both control and
vacuum-exposed cells expressed the CD59 antigen
normally, with almost the same CD59 negative
247
Fig. 7. CD59 surface antigen expression of control and vacuum
survival cells. The surviving cells following freezing-vacuum or
non-freezing-vacuum treatment had a normal positive expression of CD59 surface antigen as determined by flow cytometry.
(B) Cells were probed with R-phycoerythrin conjugated mouse
anti-human CD59 monoclonal antibody. (A) Cells were probed
with R-phycoerythrin conjugated isotype mouse IgG unable to
bind to human CD59.
population percentages (P > 0.05) on average of
3.72 and 3.83, respectively.
Discussion
Energetic particles in the environment, whether
produced naturally or originating in human activities, become low-energy particles due to slowing
down in matter. It is important to understand
the biological effects of low-energy heavy particles
on organisms, especially on living mammalian cells
because of the probable health implications and
potential applications. However, no data are available on low-energy ion beam bombardment of living mammalian cells: neither have attempts been
made to design high-vacuum, low-temperature
methods for ion bombardment. To our knowledge, the only report of ion sputtering of mammalian cells was carried out using fixed dry cells [11]
in order to expose internal cellular structures for
surface-sensitive microscopies. The present work
is the first effort to improve the vacuum resistance
of mammalian cells, aiming to obtain sufficient
248
H. Feng et al. / Cryobiology 49 (2004) 241–249
viable cells for further analysis after ion bombardment under vacuum conditions: this is a prerequisite for low-energy ion bombardment at the
present stage.
Microscopy indicated that vacuum damaged
cells in a direct manner, characterized by the alteration of membrane permeability and reduced
adhesion to culture dishes. Glycerol is an effective
cryoprotective additive for some cells (but toxic
for other cells); it replaces some of the water and
reduces adverse effects of freezing and dehydration. The pretreatment with glycerol substantially
counteracts the harmful effects of vacuum on AL
cells. Cells equilibrated with 15 and 20% glycerol
were more resistant to the application of high vacuum, as revealed by the increase in survival from
<1% to typical values >10% (P < 0.05). Those viable cells not only had normal growth capability,
but also retained their characteristic CD59 surface
antigen panel. We conclude that AL cells can be
used to study the genetic impact of low-energy
ion beams. Other kinds of cryoprotectants, including propylene glycol, which is regarded as a good
vitrification solute [6,18], and trehalose, which
has been reported to be a potent enhancer of desiccation-tolerance [4,9], were also tested in the pilot study, either individually or in combination
with glycerol. Not all the resulting data are shown,
but the results do not indicate any advantage over
glycerol alone.
Although various factors may be involved,
freezing and dehydration are the main factors
responsible for the lethal effects of vacuum for
mammalian cells. For any moisture-containing
sample, exposure to a high vacuum will result in
the evaporation of water from the surface and consequently the sample temperature decreases sharply in a short period. The trend in temperature
shown in Fig. 3 provides an approximate image
of this process. The distinct contrast of viability
between the vacuum-exposed and control cells that
were frozen by the same procedure, together with
the equivalence of freezing-vacuum and non-freezing-vacuum protocols, proved that low temperature was only partly responsible for the observed
vacuum-induced stresses. It is not completely clear
whether negative pressure itself also damages cells
by mechanisms other than freezing and dehydra-
tion. If the latter two factors are the sole reasons,
then there are opportunities to apply cryobiological methods to develop the most effective reagents
and protocols. Another possible solution to the
challenge of vacuum may derive from the anhydrobiotic engineering of mammalian cells, enabling
desiccated cells, like other dry materials, to be directly exposed to a vacuum.
In conclusion, our initial experiments clearly
demonstrate that mammalian cells have the potential to withstand vacuum stress, allowing studies of
ion bombardment in living cells to be carried. While
these preliminary findings are encouraging, we still
need to improve the efficiency of the vacuum protocol by considering factors such as the degree of cell
confluence, water content remaining in the dish
prior to vacuum-exposure, the optimal rate of pressure decrease, and the pre-treatment protocols.
Acknowledgments
We thank our colleagues Shuqing Zhang, Lixiang Yu, Xinhai Liu, and Hang Yuan for their kind
assistance in the vacuum experiments. We are
grateful to Prof. Haiying Hang and the Cryobiology Editorial Office for valuable discussions and
improvements to this paper.
References
[1] R.M. Ansari, T.K. Hei, Effects of 60 Hz extremely low
frequency magnetic fields (EMF) on radiation and chemical-induced mutagenesis in mammalian cells, Carcinogenesis 21 (6) (2000) 1221–1226.
[2] S. Anuntalabhochai, R. Chandej, B. Phanchaisri, L.D. Yu,
T. Vilaithong, I.G. Brown, Ion-beam-induced deoxyribose
nucleic acid transfer, Appl. Phys. Lett. 78 (2001) 2393–2395.
[3] C.M. Ge, S.B. Gu, X.H. Zhou, J.M. Yao, R.R. Pan, Z.L.
Yu, Breeding of L(+)-lactic acid producing strain by lowenergy ion bombardment, J. Microbiol. Biotechnol. 14 (2)
(2004).
[4] N. Guo, I. Puhlev, D.R. Brown, J. Mansbridge, F. Levine,
Trehalose expression confers desiccation tolerance on
human cells, Nat. Biotechnol. 18 (2000) 168–171.
[5] W.D. Huang, Z.L. Yu, Y.H. Zhang, Reactions of solid
glycine induced by keV ion irradiation, Chem. Phys. 237
(1998) 223–231.
[6] M. Kasai, Simple and efficient methods for vitrification of
mammalian embryos, Anim. Reprod. Sci. 42 (1996) 67–75.
H. Feng et al. / Cryobiology 49 (2004) 241–249
[7] S.X. Liu, M. Athar, I. Lippai, C.A. Waldren, T.K. Hei,
Induction of oxyradicals by arsenic: implication for mechanism of genotoxicity, Proc. Natl. Acad. Sci. USA 98
(2001) 1643–1648.
[8] B. Phanchiaisri, L.D. Yu, S. Anuntalabhochai, R. Chandej, P. Apavatjrut, T. Vilaithong, I.G. Brown, Characteristics of heavy ion beam-bombarded bacteria E.coli and
induced direct DNA transfer, Surf. Coat. Tech. 158–159
(2002) 624–629.
[9] I. Puhlev, N. Guo, D.R. Brown, F. Levine, Desiccation
tolerance in human cells, Cryobiology 42 (2001) 207–217.
[10] H.B. Shi, C.L. Shao, Z.L. Yu, Preliminary study on the
way of formation of amino acids on primitive earth under
non-reducing conditions, Rad. Phys. Chem. 62 (2001) 393–
397.
[11] G.D. Stasio, B.H. Frazer, M. Girasole, L.M. Wiese, E.K.
Krasnowska, G. Greco, A. Serafino, T. Parasassi, Imaging
the cell surface: argon sputtering to expose inner cell
structures, Microsc. Res. Tech. 63 (2004) 115–121.
[12] C.A. Waldren, L. Correll, M.A. Sognier, T.T. Puck,
Measurement of low levels of X-ray mutagenesis in relation
to human disease, Proc. Natl. Acad. Sci. USA 83 (1986)
4839–4843.
[13] X.Q. Wang, C.L. Shao, J.M. Yao, Z.L. Yu, Mass and
energy deposition effects of implanted ions on solid sodium
formate, Radiat. Phys. Chem. 59 (2000) 67–70.
[14] Y.G. Wang, G.H. Du, J.M. Xue, F. Liu, S.X. Wang, S.
Yan, W.J. Zhao, The primary target model of energetic
[15]
[16]
[17]
[18]
[19]
[20]
[21]
[22]
249
ions penetration in thin botanic samples, Phys. Lett. A 300
(2002) 611–618.
Q. Wang, G. Zhang, Y.H. Du, Y. Zhao, G.Y. Qiu, Lowenergy (30 keV) carbon ion induced mutation spectrum in
the Lacz a gene of M13mp18 double-stranded DNA, Mut.
Res. 528 (2003) 55–60.
L.F. Wu, H. Li, H.Y. Feng, L.J. Wu, Z.L. Yu, Introduction of rice chitinase gene into wheat via low energy Art
beam implantation, Chin. Sci. Bull. 46 (2001) 318–322.
L.J. Wu, G. Randers-Pehrson, A. Xu, C.A. Waldren, C.R.
Geard, Z.L. Yu, T.K. Hei, Targeted cytoplasmic irradiation with alpha particles induces mutations in mammalian
cells, Proc. Natl. Acad. Sci. USA 96 (1999) 4959–4964.
M.C. Wusteman, D.E. Pegg, M.P. Robinson, L.H. Wang,
P. Fitcha, Vitrification media: toxicity, permeability, and
dielectric properties, Cryobiology 44 (2002) 24–37.
A. Xu, L.J. Wu, R.M. Santella, T.K. Hei, Role of
oxyradicals in mutagenicity and DNA damage induced
by crocidolite asbestos in mammalian cells, Cancer Res. 59
(1999) 5922–5926.
Z.L. Yu, X.D. Wang, J.G. Deng, J.J. He, J. Zeng, Primary
studies of mutational mechanism for rice induced by ion
bombardment, Anhui Agricult. Sci. 16 (1) (1989).
Z.L. Yu, J.G. Deng, J.J. He, Y.P. Huo, Mutation breeding
by ion bombardment, Nucl. Instrum. Methods Phys. Res.
B 59/60 (1991) 705–708.
Z.L. Yu, Ion beam application in genetic modification,
IEEE Trans. Plasma. Sci. 28 (2000) 128–132.