FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to the controlled freezing of biological

samples consisting of cells and tissues, such as semen, oocytes, and

embryos; and, more particularly, to a directional freezmg device that sets

up a laterally varying temperature gradient, and freezing and thawing

protocols allowed by the device.

When a biological sample containing living cells in a freezing

solution is frozen, the first portion of the sample to freeze is the

intercellular fluid. The formation of ice in the intercellular fluid increases

the salt concentration there. If the sample is frozen too slowly, the high

concentration of salt in the intercellular fluid may kill the cells, by osmotic

shock or by chemical toxicity. Conversely, freezing the sample too

rapidly may lead to the formation of intracellular ice crystals, which also

kill the cell, by internal mechanical damage. In addition, the rate of

cooling affects the morphology of the intercellular ice crystals.

Morphologies such as closely packed needles also kill cells, by external

mechanical damage. Thus, maximizing the survival rate of cells subjected

to freezing and thawing requires careful control of the freezing process.

An alternative method of freezing biological samples, which totally

avoids the problems associated with ice crystal formation, is to cool them

so fast that the intercellular and intracellular fluids vitrify instead of

crystallizing as ice. This method has dangers of its own, however. In

particular, the rate of cooling is so fast that, because of thermal shock,

glass fractures may form within the sample at temperatures below its glass

transition temperature. To prevent ice crystal formation upon thawing,

vitrified samples must be warmed as fast as they were cooled, so thermal

shock may cause fracture formation either during the cooling process or

during the warming process.

The conventional method for freezing biological samples is to place

them in a chamber and lower the temperature of the chamber in a

controlled manner. Samples frozen in this manner freeze from the outside

in. The thermal gradient within the sample is determined implicitly by the

temperature of the chamber and the thermal conductivities of the materials

within the sample, and is not explicitly controllable. This makes it

difficult to achieve the optimal cooling rate, which minimizes both the

toxicity associated with cooling too slowly and the mechanical damage

associated with cooling too fast.

Rubinsky, in U.S. Patent No. 4,531 ,373, introduced controlled

directional freezing, in which a sample is placed on a microscope slide,

and the microscope slide is moved longitudinally through a region of

substantially constant temperature gradient dT/dx (T denoting temperature

and x denoting distance). If the microscope slide is moved through the

temperature gradient at a constant speed V = dx/dt, where t denotes time,

then each point in the sample cools at a rate of dT/dt = V*(dT/dx).

Using Rubinsky's method, the rate of cooling of each point in the sample

is subject to explicit control. In addition, if the cooling is done on a

microscope stage, the sample can be monitored in detail for undesired

phenomena such as the formation of intracellular ice.

Rubinsky's method, having only one uniform thermal gradient, is

inherently limited to cooling at a single rate. Thus, it is unsuitable for

cooling protocols that require different rates in different temperature

ranges. For example, Arav ("Vitrification of oocytes and embryos", in

Embryonic Development and Manipulation (Lavria and Gandalfi, editors),

Portland Press, 1992, pp. 255-264) recommends that vitrification be done

with rapid cooling above the glass transition temperature and slower

cooling below the glass transition temperature. In addition, Rubinsky's

use of a microscope stage for monitoring makes his device unsuitable for

commercial or industrial scale production, or for the use of commercial

cell packaging ("straws").

There is thus a widely recognized need for, and it would be highly

advantageous to have, a device for directional cooling of a biological

sample by moving the sample through regions of laterally varying

temperature gradient, and associated freezing and thawing protocols that

exploit the ability to cool and thaw at different rates in different

temperature ranges.

SUMMARY OF THE INVENTION

According to the present invention there is provided a device for

freezing a biological sample, comprising: (a) a track; (b) refrigeration

means for imposing a laterally variable temperature gradient along the

track; and (c) a mechanism for moving the biological sample along the

track.

According to the present invention there is provided a method for

freezing a biological sample having a freezing temperature, comprising the

steps of: (a) placing the biological sample inside a straw having a leading

end; (b) moving the straw from a warm region having a first temperamre

higher than the freezing temperamre to a cold region having a second

temperamre lower than the freezing temperamre, the leading end of the

straw entering the cold region before any other part of the straw; and (c)

freezing the leading end of the straw before the leading end of the straw

enters the cold region.

According to the present invention there is provided a method for

freezing a semen sample having a lipid phase transition temperamre, the

sample being initially at a temperature above the lipid phase transition

temperature, the method comprising the steps of: (a) cooling the sample

to an intermediate temperature slightly below the lipid phase transition

temperamre at a rate sufficiently slow to prevent chilling injury; and (b)

cooling the sample below the intermediate temperature at a rate of between

about 30°C per minute and about 1500°C per minute.

According to the present invention there is provided a method for

freezing a biological sample having a glass transition temperature, the

sample being initially at a temperamre above the glass transition

temperamre, the method comprising the steps of: (a) cooling the sample

to about the glass transition temperamre at a rate sufficiently fast to

prevent ice formation; and (b) cooling the sample below about the glass

transition temperamre at a rate sufficiently slow to prevent glass

fracturing.

According to the present invention there is provided a method of

warming a biological sample having a glass transition temperamre, the

sample being initially at a temperature below the glass transition

temperamre, the method comprising the steps of: (a) warming the sample

to about the glass transition temperature at a rate sufficiently slow to

prevent glass fracturing; and (b) warming the sample above about the glass

transition temperamre at a rate sufficiently fast to prevent devitrification.

The preferred embodiment of the device of the present invention is

a series of copper blocks arranged in a line, with a straight track running

through the blocks. Each block is equipped with a refrigerator to cool the

block, and optionally one or more heaters to warm the block. In the

simplest configuration, the refrigerator is on one side of the block and one

heater is on the other side of the block, thereby imposing a temperamre

gradient on the portion of the track contained in the block. In another

configuration, the refrigerator cools the block as a whole, and two or more

heaters impose a temperamre gradient along the portion of the track

contained in the block. The blocks are separated by gaps, and the

temperature of the block on one side of the gap typically is different from

the temperature on the other side of the gap, thereby imposing a

temperature gradient across the gap. Biological samples to be frozen or

thawed are placed inside straws, and the straws are moved along the track

at speeds such that the samples are frozen or thawed at rates specified by

protocols specific to the samples. Monitoring devices, such as CCD video

cameras coupled to microscope objectives, and such as infrared

thermographs, are deployed at the gaps to monitor the progress of the

freezing or thawing.

In a variant of this preferred embodiment, the blocks are mounted

in the neck of a dewar of liquid nitrogen, with the entrance (high

temperature end) of the track at or above the top of the neck, and the exit

(low temperamre end) of the track within the neck or at the base of the

neck. When the straws reach the exit, they fall into the liquid nitrogen for

long term storage.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with

reference to the accompanying drawings, wherein:

FIG. 1A is a schematic side view of a preferred embodiment of the

device of the present invention, based on thermally conductive blocks;

FIG. IB is a schematic cross sectional view of the preferred

embodiment of FIG. 1A.

FIG. 2 is a schematic diagram of a variant of the device of FIG.

1A.

FIG. 3 shows the manner in which a sample of embryos or oocytes

is loaded in a straw.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of a cooling device which can be used for

the controlled freezing and thawing of biological samples, and of protocols

for its use with various kinds of samples. Specifically, the device of the

present invention can be used to move biological samples through regions

of laterally varying temperamre gradients, thereby effecting cooling and

thawing at controlled rates.

The principles and operation of a cooling device according to the

present invention may be better understood with reference to the drawings

and the accompanying description.

Referring now to the drawings, Figure 1A is a schematic side view

of one preferred embodiment of the device of the present invention. Three

blocks 12, 14, and 16, of a thermally conductive material, preferably

copper, are arranged in a line. Block 12 is about 16 centimeters long.

Block 14 is about 2.5 centimeters long. Block 16 is about 10 centimeters

long. Blocks 12 and 14 are separated by a gap 18. Blocks 14 and 16 are

separated by a gap 20. Gaps 18 and 20 may be between .01 centimeters

and 1.5 centimeters wide. A tunnel 36, preferably of rectangular cross

section, runs through blocks 12, 14, and 16. Tunnel 36 defines a track

along which a sled 40 is moved. Sled 40 preferably is made of a

thermally conductive material, preferably copper, and bears one or more

straws 38 that contain biological samples to be frozen or thawed. Straws

38 typically are hollow tubes of circular or rectangular cross section, about

14 to 30 centimeters long. Sled 40 is moved through tunnel 36 by a piston

42 to which is attached a helically threaded rod 44. Rod 44 is moved to

the right by a screw drive (not shown).

Blocks 12 and 14 include refrigerators 50 and 52. Blocks 12 and

16 include heaters 56, 57 and 58. Refrigerators 50 and 52 operate

conventionally, by compressing and expanding cryogenic fluids. Heaters

56, 57 and 58 typically are electrical resistance heaters. Block 16 includes

a channel 54 through which liquid nitrogen is circulated. Refrigerator 50

and heater 56 serve to impose a temperamre gradient on the portion of

tunnel 36 that runs from warm side 22 of block 12 to cold side 24 of block

12. Refrigerator 52 imposes a substantially constant temperamre on block

14. The effect of the liquid nitrogen in channel 54 and heaters 57 and 58

is to impose a temperature gradient on the portion of tunnel 36 that runs

from warm side 30 of block 16 to cold side 32 of block 16. The

temperatures within blocks 12, 14, and 16 are monitored by thermocouples

(not shown) and controlled by feedback loops (not shown) that include

refrigerators 50 and 52 and heaters 56, 57 and 58.

In general, gaps between blocks of the present invention, such as

gaps 18 and 20 separating blocks 12, 14, and 16, preferably are no wider

than 1.5 centimeters. In that way, the tunnel, such as tunnel 36, through

the blocks encloses substantially all of the track along which the biological

samples move, isolating the samples from the outside environment and

helping to impose the thermal gradients of the blocks on the biological

samples.

At gap 18, a video camera 60 and an infrared thermograph 64 are

deployed for monitoring the condition of the biological sample in straw 38

as straw 38 traverses gap 18. Suitable infrared thermographs 64 include

those made by Elbit Ltd. of Haifa, Israel, and the Microscanner D501

made by Exergen Co. of Newton MA. Similarly, another video camera

62 is deployed at gap 20 for monitoring the condition of the biological

sample in straw 38 as straw 38 traverses gap 20. Video cameras 60 and

62, and infrared thermograph 64, transmit signals to a monitor (not

shown), on which an operator can observe the visual appearance and the

temperamre contours of the contents of straw 38. In production mode,

most straws 38 are opaque. For the purpose of visual monitoring for

quality control, some of straws 38 are special transparent (typically glass)

straws of rectangular cross section. Infrared thermograph 64 monitors the

temperamre profile of all straws 38.

Block 14 is provided with a channel 72 for applying liquid nitrogen

to straw 38 to seed freezing, as described below.

Figure IB is a schematic cross sectional view of the preferred

embodiment of Figure 1A, taken along cut A-A. Figure IB shows that the

geometry of channel 54 in block 16 is that of a coil open at both ends,

with liquid nitrogen entering channel 54 at the end nearer to cold side 32

of block 16 and exiting channel 54 at the end nearer to warm side 30 of

block 16. Figure IB also shows that channel 72 in block 14 is provided

with an electrically activated valve 74 to admit liquid nitrogen. The

variant of channel 72 shown in Figure IB is capable of seeding six straws

38 at once.

Figure 2 is a schematic view of a variation of the device of Figures

1A and IB. In this variation, blocks 12, 14, and 16 are mounted in a neck

104 of a dewar 100 of liquid nitrogen 102. Refrigerators 50 and 52, and

heaters 56, 57 and 58 are thermoelectric devices, rather than conventional

cryogenic refrigerators or resistance heaters. Tunnel 36 is circular in

cross section; and, instead of providing sled 40 to move straw 38 laterally

along the track defined by tunnel 36, as in Figure 1 A, pairs of rollers 120

are provided that grip straw 38 frictionally and move straw 38 downwards

through tunnel 36. When straw 38 emerges from exit 37 of tunnel 36,

straw 38 falls into liquid nitrogen 102 for preservation.

The device of the present invention enables the implementation of

controlled seeding in the freezing of biological samples. When any liquid

is cooled below its freezing point, it remains a liquid, in an unstable

supercooled state, for at least a short time. Freezing starts at nucleation

sites that are distributed substantially randomly throughout the volume of

the liquid, and spreads through the rest of the liquid. In the conventional,

equiaxial (nondirectional) method of freezing biological samples, ice grows

with uncontrolled velocity and morphology, and may disrupt and kill the

cells of the samples.

Directional freezing allows controlled nucleation, or seeding, of

freezing, at least in principle. As a straw containing a biological sample

is moved forward along a thermal gradient, from a temperamre above the

freezing point of the sample to a temperature below the freezing point of

the sample, at some point in time, the leading edge of the straw reaches

a point in space at which the temperature is below the freezing point of the

sample. The leading end of the straw is now frozen, for example by

touching it with a cold object such as a small amount of liquid nitrogen.

Uncontrolled freezing proceeds backwards along the straw to the point in

space at which the temperamre is equal to the freezing point of the sample.

As the straw continues to move forward, the frozen part of the sample

nucleates freezing of the liquid part of the sample as the liquid part of the

sample passes the point in space at which the temperamre is equal to the

freezing point of the sample. Thus, uncontrolled freezing, with

consequent random destruction of cells, is confmed to a small region at the

leading end of the straw, and controlled freezing occurs at a freezing front

that moves backwards along the straw but remains Substantially stationary

with respect to the thermal gradient, substantially at the point along the

thermal gradient at which the temperature is equal to the freezing point of

the sample.

Ideally, the velocity of the freezing front should be such that the ice

morphology does not disrupt the cells of the biological sample. This is

difficult to achieve using the directional freezing devices of the prior art,

which have laterally constant gradients, because the rate of cooling

consistent with favorable ice morphology may not be consistent with other

desired cooling rates of a sample's freezing protocol. The laterally

varying gradient of the device of the present invention allows cooling at

different rates in different temperamre regimes, thereby allowing fully

controlled nucleation at the freezing front. For example, a short part of

the thermal gradient, immediately to the cool side of the point at which the

temperature is equal to the freezing point of the sample, can be set equal

to zero, providing a short region of constant temperamre slightly below the

freezing point of the sample. If the sample is a suspension of separate

cells, then this constant temperature is slightly below the freezing

temperature of the freezing solution in which the cells are suspended. If

the sample is a tissue sample, then this constant temperamre is slightly

below the freezing temperature of the tissue sample. Note that in the

context of the present invention, "slightly below" means lower in

temperature by between about 1°C and about 10°C. This now will be

illustrated in the context of the use of the device of Figure 1 for freezing

oocytes and embryos.

The prior art protocol for freezing oocytes and embryos is to cool

from 0°C to -7°C at a rate of between about 0.5°C per minute and about

10°C per minute, but most preferably at a rate of about 1°C per minute;

and from -7°C to -35°C at a rate of between about 0.1 °C per minute and

about 1.5 °C per minute, but most preferably at a rate of about 0.3°C per

minute. The present invention allows this protocol to be effected

directionally. The present invention also allows this protocol to be applied

to the directional freezing of ovarian cortical tissue. The ovarian cortical

tissue is removed surgically from the patient (typically a woman about to

undergo chemotherapy or radiation therapy) and sliced into slices having

a dimension of about 1 cm x 1 cm x 0.5 mm. These slices are frozen

inside specially dimensioned flat straws 38 having rectangular cross

sections about 1 cm wide and about 2 mm high.

Straw 38 is loaded as shown in Figure 3. A sample 150, containing

one or more oocytes or embryos, is placed in the middle of straw 38,

surrounded by about 10 to 100 microliters 160 of freezing solution. One

end 39 of straw 38, which is the leading end as straw 38 travels through

tunnel 36, is plugged with a cotton plug 180 saturated with freezing

solution. The other end of straw 38 is filled with sucrose solution 190 and

sealed with seal 200. Plug 180, sample-bearing freezing solution 160,

sucrose solution 190, and seal 200 are separated by air bubbles 170 as

shown.

Refrigerator 50 and heater 56 are set to give block 12 a temperature

of 22°C at warm side 22 and a temperamre of 0°C at cold side 24.

Refrigerator 52 is set to give block 14 a uniform temperamre of -7°C .

Heaters 57 and 58 are set to give warm side 30 of block 16 a temperamre

of -10°C and cold side 32 of block 16 a temperamre of

-35°C. For this protocol, the width of gap 18 is set to 0.84 centimeters,

and the width of gap 20 is set to 1.2 centimeters.

Straw 38 is placed on sled 40, the side of sled 40 bearing leading

end 39 of straw 38 is placed inside tunnel 36 at warm end 22 of block 12,

and sled 40 is moved through tunnel 36, using piston 42 and rod 44, at a

speed of 20 microns per second. When leading end 39 of straw 38 enters

end 26 of block 14, valve 74 is opened for between about 5 seconds and

about 10 seconds, allowing a small amount of liquid nitrogen to touch

leading end 39 of straw 38, thereby seeding the freezing of the contents

of straw 38. Because blocks 12 and 14 and sled 40 are made of a

thermally conductive material such as copper, the imposition of a

temperamre of 0°C at cold end 24 of block 12 and of -7°C throughout

block 14 sets up a substantially linear temperature gradient, of -8.3°C per

centimeter, in the portions of sled 40 and straw 38 that occupy gap 18.

As straw 38 moves across gap 18, freezing proceeds in the opposite

direction within straw 38, with a freezing front established at the point in

gap 18 where the temperature is the freezmg point of the solutions

contained in straw 38, about -3°C. (In air bubbles 170, the freezing front

propagates via fluid that wets the inner wall of straw 38.) As sample 150

crosses gap 18, it is cooled from 0°C to -7°C at the desired rate of 1°C per

minute. Video camera 60 and infrared thermograph 64 are used to

monitor the morphology and location of the freezing front in gap 18, so

that the speed at which straw 38 is moved across gap 18 can be fine-tuned.

The temperamre of sample 150 stays constant at -7°C as sample 150 moves

through block 14. As sample 150 enters block 14, the speed of sled 40 is

increased to about 40 microns per second, so that sample 150 spends about

10 minutes inside block 14 at a constant temperamre of -7°C. When

sample 150 emerges from block 14, it is in a region, including both gap

20 and block 16, in which the temperamre gradient is about -2.5°C per

centimeter. At this point the speed of sled 40 is reduced to the original 20

microns per second, so that sample 150 reaches cold end 32 of block 16,

at which the temperamre is -35°C, in about 93 minutes, i.e., at the desired

rate of -0.3°C per minute. Video camera 62 monitors the morphology of

the contents of straw 38 as straw 38 emerges from block 14 into gap 20,

to make sure that the contents of straw 38 are entirely frozen and that

sample 150 has not been damaged mechanically by the freezing process.

In the case of other kinds of biological samples, such as bull semen,

straw 38 is substantially entirely filled with the sample to be frozen. In

that case, seeding by quickly freezing one end of straw 38 inevitably kills

the part of the sample being frozen in the seeding process. Nevertheless,

the rest of the sample may be frozen in a controlled manner, and

significantly more of the sample survives freezing and thawing than in the

conventional, nondirectional freezing method.

The above protocol is conventional; the advantage of implementing

it using the device of the present invention is that it can be implemented

directionally. The present invention also includes other freezing and

thawing protocols that can be effected only through the use of the device

of the present invention. These include:

Semen (including bull, ram, goat, stallion, and human semen):

Cool from 30°C to an intermediate temperature slightly below the lipid

phase transition temperamre of the semen at a rate slow enough to prevent

chilling injury, preferably about PC per minute. Cool from the

intermediate temperamre to -50°C at a rate of between about 30°C per

minute and about 1500°C per minute. In the case of bull semen, the

preferred intermediate temperamre is about 5°C. This is faster than the

conventional protocol for bull semen, which prescribes a cooling rate of

only 30°C per minute between 0°C and -50°C. The preferred range of

velocities for directional cooling under this protocol is between about 50

microns per second and about 3000 microns per second.

Vitrification: Cool from 30°C to slightly below the glass transition

temperamre (typically about -110°C) at a rate fast enough to prevent ice

formation, at least about 100°C per minute, but preferably at a rate of

about 8400°C per minute. Cool from slightly below the glass transition

temperamre to the temperature of liquid nitrogen at a rate of at most about

10°C per minute, to avoid fracturing the sample by thermal shock.

Warming a vitrified sample stored in liquid nitrogen: Warm from

the temperamre of liquid nitrogen to slightly below the glass transition

temperamre at a rate of at most about 10°C per minute, to avoid fracturing

the sample by thermal shock. Warm from slightly below the glass

transition temperature to 30°C at a rate fast enough to prevent

devitrification, at least about 100°C per minute, but preferably at a rate of

about 8400°C per minute. This is safer than the conventional method of

heating the sample in a water bath at a temperature between 55°C and

75°C, because the danger of overheating inherent in the conventional

method is avoided.

While the invention has been described with respect to a limited

number of embodiments, it will be appreciated that many variations,

modifications and other applications of the invention may be made.