CRYOBIOLOGY - IV
Chairman: J. Karlsson
THREE-DIMENSIONAL BEHAVIOR OF ICE CRYSTALS AND
BIOLOGICAL CELLS DURING FREEZING OF CELL SUSPENSIONS
Hiroshi Ishiguro and Kazuhito Koike
Institute of Engineering Mechanics and Venture Business Laboratory
University of Tsukuba, Tsukuba, Ibaraki 305-8573, JAPAN
(Ph) +81-298-53-5267, (Fax) +81-298-53-5207, (E-mail) email@example.com
To understand the mechanisms of both the freezing injuries of cells and the
protection of cells due to cryoprotectants in cryopreservation of cells, the microscopic
behavior of the ice crystal and the cells during the freezing of cell suspension has been
actively studied using optical and electron microscopes. However, the freezing of cell
suspensions generally proceeds transiently and spatially in three-dimensions(3D).
Therefore, 3D observation in real time is required to understand the details of
microstructure in the freezing process of cell suspensions. For such observation, optical
and electron microscopes have limitations because these microscopes produce only 2D
images of the sample. Furthermore, the required fixation of the sample does not allow
observation of the same sample. This lack of information means that presumptions must
be made concerning the phenomena that occur during the freezing.
A confocal laser scanning microscope (CLSM) with a high-performance computer
was recently developed. This is a noninvasive method that produces optical tomograms
of biological materials without fixation and slicing of a sample. In this study, the
behavior of ice crystals and human red blood cells during extracellular-freezing was
visualized in 3D in real time using a CLSM and a fluorescent dye. The influence of the
addition of cryoprotectant and the cooling rate were investigated on the morphology of
ice crystals and the interaction between ice crystals and cells near the freezing interface.
MATERIALS AND METHODS
Suspensions of human red blood cells stained with a fluorescent dye were frozen. For
the solutions, physiological saline (PS) alone and physiological saline with 2.4M glycerol
(PS+Gly) were used. For fluorescence observation, a fluorescent dye, acridine orange
(AO, CH20N3Cl, molecular wight=301.82), was added to the solutions.
A sample was set between a glass microslide and a glass coverslip, and then was
frozen on the surface of the directional solidification stage. This method allows us to
independently control the temperature gradient G and advancing velocity V of the
gradient at the freezing interface, resulting in the cooling rate H=GV. At a constant
G=13.5oC/mm, H was varied in the range up to 20.0oC/min.
A Leica erect-type CLSM (TCS4D) was used. This has an Ar-Kr (argon-krypton)
laser (488nm, 568nm and 643nm in wavelength) and two photomultipliers. The scanning
of laser beam on a horizontal section combined with the vertical movement of the stage
with a sample on it produced continuous, noninvasive, 3D optical tomograms.
The AO has a maximum-excitation wavelength of 492nm(blue), and two maximum-
emission wavelengths of 530nm(green) and 640nm(red). It is a monomer in solution and
becomes a dimer when taken into a cell membrane. The monomer and dimer generate
green and red fluorescences, respectively. The ice does not generate fluorescence because
during the freezing the dye is not trapped in ice. Therefore, the ice crystal, cells, and
unfrozen solution can be distinguished with different colors.
RESULTS AND DISCUSSION
The morphology of the ice crystals depends on the cooling rate and the concentration
of chemical additives. For the range of the parameters studied, optical microscopy shows
that the ice crystals in PS typically have two morphologies in 2D: a flat interface and a
cellular interface, and that the ice crystals in PS+Gly typically have a dendritic
appearance. For these typical morphologies, the 3D microstructure was measured by the
Freezing of Red Blood Cells in Physiological Saline (PS)
The ice crystals have a flat interface at lower cooling rates and a cellular interface at
higher cooling rates than the critical cooling rate for transition from a flat one to a cellular
one. The interface which appeared flat in 2D at H=3.0oC/min, had a wedge-shaped interface
in 3D, whose upper part protruded into the higher-temperature region. The unfrozen
solution remained in a thin layer on and under the ice. The solid-liquid interface
advanced into the unfrozen solution with red blood cells dispersed in the bottom region of
the sample while maintaining nearly the morphology. The red blood cells were pushed by
the interface, causing them to accumulate in the unfrozen solution in front of the
interface. These accumulated red blood cells were trapped in the unfrozen solution
between the ice crystals and the upper or lower glass substrate, and then became flatter in
the colder region.
An increase in cooling rate at a constant temperature gradient promotes constitutional
supercooling in the unfrozen solution adjacent to the freezing interface, resulting in
morphological instability of the flat interface in 2D. The instability changes the interface
from flat into cellular. As the cellular ice crystals grew in the unfrozen solution with the
red blood cells in the bottom region, most of the cells were pushed aside by the cellular
ice crystals and thus accumulated and compacted in the unfrozen solution between the
finger-like ice crystals. Each finger-like ice crystal in the horizontal section appeared as a
cluster of ice in the vertical section of uniform temperature. The clusters of ice were
arranged at roughly periodic intervals and were inclined to the vertical direction. The
change of the shape of the cross section in the direction of temperature-decrease clearly
indicates that the growth of ice crystal in the vertical section was anisotropic.
Freezing of Red Blood Cells in Physiological Saline with Glycerol (PS+Gly)
Secondary and tertiary arms of the dendritic structure are clearly seen in the
horizontal section. The red blood cells remained dispersed during the freezing. This
independence of the dispersion of red blood cells results from the "flexible" interaction
between ice crystals and cells. In this "flexible" interaction, as the ice crystal grows, the
morphology of the ice-solution interface changes in response to the shape of cells, and
the cells are almost not moved by the interface. This contrasts with the interaction
between ice crystals and cells in PS.
The microstructure in the vertical section of uniform temperature in PS+Gly has the
following similarities with the cellular ice crystals in PS: 1)arrangement of clusters of ice
crystals at nearly periodic intervals and with an inclination, and 2) anisotropic growth of
ice. However, a few cracks and red blood cells were also in the clusters of ice crystals in
the vertical section. The cracks were filled with unfrozen solution and corresponded to
the narrow space between the primary arm and the branch of the dendrites. At a higher
cooling rate, the branching of ice happens more frequently, and correspondingly the
number of cracks in the vertical section of ice increased. The outline of the ice was not
only wavy but also conformed locally to the shape of the red blood cells whereas the
outline of ice in PS was a monotonic curve.
Ice Fraction in the Freezing Region near the Tip of Ice Crystal
Ice fraction, defined as the ratio of the area occupied by the ice in each vertical
section, was estimated from a series of vertical tomograms for the cellular ice crystals in
PS and the dendritic ice crystals in PS+Gly. The ice fraction increased with distance and
temperature-decrease from the tip of ice crystal, reaching near saturation in each cell
suspension. The ice fraction in PS has a larger value than that in PS+Gly due to the lower
initial-concentration of solute in the PS than in the PS+Gly. For reference, the ice fraction
was also calculated from the liquidus of solution at thermal equilibrium. For PS, the
experimental value tends to be slightly smaller than the calculated value, while for
PS+Gly, the values are similar. The cause may be mainly that the mass transfer in the
direction of temperature-decrease in PS proceeded due to the diffusion during the
freezing more rapidly than in PS+Gly and consequently the solute-concentration at the tip
of ice crystal became higher than the initial value.
The 3D behavior of ice crystals and red blood cells during the extracellular-freezing
was made clear for all flat, cellular, and dendritic solid-liquid interfaces, and the ice
fraction in the freezing region was obtained. The results also indicates that using a CLSM
with a fluorescent dye is very effective for the purpose of this study.
PREVENTION OF HEMOLYSIS RAPIDLY FROZEN
ERYTHROCYTES BY USING A LASER PULSE
Alex J. Fowlera,b and Mehmet Tonerb
a Mechanical Engineering Department, University of Massachusetts Dartmouth, North
Dartmouth, Massachusetts 02747, USA
b Center for Engineering in Medicine and Surgical Services, Massachusetts General
Hospital, Harvard Medical School and Shriners Burns Hospital, Boston, Massachusetts
In this paper we report on our attempt to recover unprotected erythrocytes frozen
at 10,000 oC/min. To this end we vitrified the intracellular solution of frozen erythrocytes
using a laser pulse before thawing the sample. Electron microscopy studies have already
shown that unprotected erythrocytes frozen at the rate of 104 oC/min have substantial IIF;1
and numerous studies, including ours, indicate that rapid warming of erythrocytes frozen
at this rate results in 100% hemolysis. The hypothesis driving this work, however, is that
the formation of ice crystals during the rapid freezing of cells is completely innocuous, but
the kinetics of crystal growth and reformation during warming increase dramatically as the
ice crystal fraction within the cell increases. The apparent existence of a level of lethal IIF
is really a kinetic barrier beyond which so called "rapid warming" is still too slow to
prevent lethal damage during warming. By vitrifying the intracellular solution prior to
thawing the cells, we can partially decouple the damaging effects of crystal formation from
the damage incurred during warming.
The technique used to vitrify the intracellular solution is to use a pulse from a Q-
switched laser at a wavelength that selectively targets the intracellular solution. This
allows for the intracellular solution to be "instantaneously" melted while the surrounding
solution remains at cryogenic temperatures. Conductive cooling of the intracellular
solution results in cooling rates of more than 1 million degrees per second which is fast
enough to vitrify pure water.2 The efficacy of this technique in creating amorphous ice and
a numerical analysis of the resulting cooling rates has been reported elsewhere.3
Erythrocytes were chosen for this study because of their naturally occurring chromophore
(hemoglobin) which allows them to be selectively targeted by 532 nm. radiation when they
are frozen in a PBS solution. The pulse width was 7 ns.
Human blood was diluted with PBS and loaded in cryostraws. The cells were
frozen by immersion in a methanol slurry and subsequent transfer to liquid nitrogen. After
the administration of the laser pulse the straws were thawed by immersion in warm water
in a manner identical to control experiments. Different energy fluxes were administered to
the straws to study the effect of energy density on hemolysis and to find the optimal energy
flux. The diameter of the incident beam was held constant at 3mm.
All of the cells that were frozen using the described protocol and were then thawed
without laser treatment lysed. In samples that were laser irradiated up to 80% of the cells
remained intact after thawing. Studies were performed to determine the percentage of
lysed cells as a function of laser energy flux. Below a certain threshold energy (2.5 J/cm2)
all of the cells lysed. For energies between 3 and 6 J/cm2 the percentage of cells that did
not lyse increased roughly monotonically. As the incident energy was increased beyond 6
J/cm2 the percentage of non lysed cells decreased rapidly, but appeared to stabilize at
The cells that did not lyse after laser vitrification and thawing exhibited intact
membranes that were stable over time. The cells were osmotically active; and in particular
erythrocyte ghosts could be formed by exposing them to a hypotonic solution just as is true
for control cells. The cellular retention of hemoglobin, and proper cell morphology were
verified by using Wright's stain. The eosin in Wright's stain binds to hemoglobin. Dark
staining of the cells indicated clearly that they retained their hemoglobin. This was also
confirmed by the observation of red spots in centrifuge tubes after the cells were spun
down. The morphology of the cells was excellent. The cells maintained their discoid
shape, and showed no signs of crenelation.
A number of controls were run to prove that the beneficial effect of the laser
irradiation was the vitrification of the intracellular solution. Identical procedures were
used for the freezing and laser irradiation of fibroblasts, which do not selectively absorb
the radiation; and the laser pulses had no effect on their survival. In addition erythrocytes
were exposed to laser pulses with much longer pulse widths, thereby resulting in much
slower cooling rates; and they also experienced no beneficial effect from the laser pulses.
Only 7 ns. pulses of radiation that selectively targeted intracellular solution provided
protection form cell lysis during warming.
These findings do not prove that a new and improved cryopreservation technique
for unprotected erythrocytes can be developed based on laser recovery. They do indicate,
however, that the primary damage to cells that experience large amount of IIF may be
solely a result of damage incurred during the warming process. They also indicate that
this damage can be avoided if the cells are warmed fast enough.
- Nei, T. (1976), "Freezing injury to erythrocytes I. Freezing patterns and post-thaw hemolysis,"
Cryobiology, v. 13, pp. 278-286.
- Hallbrucker, A., Mayer, E. and Johari, G.P. (1989), "Glass-liquid transition and the enthalpy
of devitrification of annealed vapor-deposited amorphous solid water. A comparison with
hyperquenched glassy water," Journal of Physical Chemistry, v. 93, pp. 4986-4990.
- Fowler, A.J. and Toner M. (1997), "Cryopreservation of Cells using Ultra-Rapid Freezing,"
Advances in Heat and Mass Transfer in Biotechnology, BED-Vol. 37, pp. 179-183.
A CONTINUOUS HYBRID MICROWAVE PROCESS FOR PRODUCING RAPID AND
UNIFORM REWARMING OF CRYOPRESERVED TISSUES
Q. Zhang, T.H. Kackson, A. Ungan
ABSTRACT IS NOT RECEIVED.
RAPID COOLING OF SMALL SAMPLES QUENCHED INTO LIQUID NITROGEN:
THE EFFECTS OF SUBCOOLING AND QUENCHING SPEED
Qian Cao, Tse-Chao Hua
Institute of Refrigeration & Cryogenic Engineering,
University of Shanghai for Science and Technology, Shanghai 200093,P.R.China
14 small metal spheres of diameter 0.3~3.0mm are plunged into subcooled LN2 at speeds
of 0~6m/s,and the moving distances inside the LN2 are 5cm. As the results of the
increasing of the subcooled degree, or the decreasing of the diameter of sphere, the curves
of boiling heat flux or cooling rate would be shifted upward and rightward dramatically.
As the subcooling is 13K, the heat flux is up to 1.4 106W/m2, the cooling rate is also up
to 8200 K/s at the CHF point of boiling curves for sphere of diameter 0.287mm. It is
resulted that if the time of moving inside the liquid nitrogen is not longer than the time
required for forming stable vapor in the liquid, the quenching boiling heat transfer is not
influenced by the quenching speed. And when the subcooling is large enough, or the
sphere diameter is small enough, the curve shapes of the boiling heat flux would be
changed from "M" style to "L" style. As the subcooling beyond 13k, "L" shape boiling
curves are recorded for sphere of diameter 0.287mm.