U.S. patent application number 10/225274 was filed with the patent office on 2003-01-30 for method for preserving mammalian organs.
This patent application is currently assigned to Biobank Co., Ltd.. Invention is credited to Seki, Kunihiro.
Application Number | 20030022148 10/225274 |
Document ID | / |
Family ID | 25175064 |
Filed Date | 2003-01-30 |
United States Patent
Application |
20030022148 |
Kind Code |
A1 |
Seki, Kunihiro |
January 30, 2003 |
Method for preserving mammalian organs
Abstract
The method of the invention for preserving mammalian organs
comprises two steps, one being the step of dehydration in which an
organ having a physiologically normal water content is deprived of
water in an amount of at least about 25% by weight of the total
weight of the organ before dehydration such that water is left
intact in an amount of at least from about 10 to about 20% by
weight of the total content of water before dehydration, the step
of dehydration being followed by the step of immersing the
dehydrated organ in an inert medium and maintaining it at a chill
temperature.
Inventors: |
Seki, Kunihiro;
(Kanagawa-Ken, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Biobank Co., Ltd.
|
Family ID: |
25175064 |
Appl. No.: |
10/225274 |
Filed: |
August 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10225274 |
Aug 22, 2002 |
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09799112 |
Mar 6, 2001 |
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6475716 |
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Current U.S.
Class: |
435/1.3 ;
128/898 |
Current CPC
Class: |
A01N 1/02 20130101; A01N
1/021 20130101 |
Class at
Publication: |
435/1.3 ;
128/898 |
International
Class: |
A01N 001/00; A01N
001/02; A61B 019/00 |
Claims
What is claimed is:
1. A method for preserving mammalian organs which comprises two
steps, one of dehydrating an organ to remove water but leave intact
an amount of water that permits later resuscitation and the other
of immersing the organ in an inert medium and maintaining it at a
chill temperature or below.
2. A method for preserving mammalian organs which comprises two
steps, one being the step of dehydration in which an organ having a
physiologically normal water content is deprived of water in an
amount of at least about 25% by weight of the total weight of the
organ before dehydration such that water is left intact in an
amount of at least from about 10 to about 20% by weight of the
total content of water before dehydration, the step of dehydration
being followed by the step of immersing the dehydrated organ in an
inert medium and maintaining it at a chill temperature.
3. A method for preserving mammalian organs which comprises three
steps, the first for removing blood from the heart by flushing with
physiological saline until the blood in the heart is replaced by
the physiological saline, the second for depriving the flushed
heart of water in an amount of from about 25 to about 60% by weight
of the total weight of the heart before dehydration, and the third
for immersing the dehydrated heart in an inert medium and
maintaining it in the state of apparent death at a chill
temperature between about 2 and about 4.degree. C.
4. The method of preservation according to claim 1, wherein said
step of dehydration includes bringing a dehydrator into contact
with the organ.
5. The method of preservation according to claim 1, wherein said
step of dehydration includes withdrawing water from within the
organ through channels in the vascular system in said organ.
6. The method of preservation according to claim 1, wherein said
step of dehydration includes feeding a gas into the vascular system
in the organ.
7. The method of preservation according to claim 1, wherein said
step of dehydration includes feeding air into the vascular system
in the organ.
8. The method of preservation according to claim 1, wherein said
step of dehydration includes feeding an O.sub.2-based gaseous
mixture of O.sub.2 and CO.sub.2 into the vascular system in the
organ.
9. The method of preservation according to claim 6, wherein a gas
is flushed into the aorta of the organ and the resulting flow of
said gas is used to withdraw water from within blood vessels.
10. The method of preservation according to any one of claims 1-3,
wherein trehalose is dissolved in said physiological saline.
11. The method of preservation according to claim 1, wherein a
liquid perfluorocarbon is used as said inert medium.
12. The method of preservation according to claim 1, wherein said
organ is selected from the group consisting of the heart, liver,
kidneys, pancreas and lungs.
13. A preserved mammalian organ which is deprived of water in an
amount of at least about 25% by weight of its physiologically
normal, total weight while leaving water intact in an amount of at
least from about 10 to about 20% by weight of the total water
content in the organ which is then immersed in an inert medium and
maintained at a chill temperature or below.
14. The preserved organ according to claim 13, which is selected
from the group consisting of the heart, liver, kidneys, pancreas
and lungs.
15. A method for organ transplantation, in which an organ preserved
by the method according to claim 1 or the preserved organ according
to claim 13 is resuscitated by irrigating the vascular system in
either of said organs with body fluid or a substitute body fluid,
each being near at body temperature, and the resuscitated organ or
a tissue thereof is transplanted into the human body.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] Japanese Patent Public Disclosure No. 72601/2000: This
application was filed with the Japanese Patent Office on Aug. 31,
1998 as Japanese Patent Application No. 245052/1998. The title of
the invention was "a method for preserving extracted mammalian
organs" and the inventor was Kunihiro Seki, D. Sc., the same as the
inventor of the present invention. The application was laid open to
public inspection in Japan on Mar. 7, 2000 and is incorporated
herein by reference to the specification and the drawings.
BACKGROUND OF THE INVENTION
[0002] 1.Technical Field of the Invention
[0003] The present invention relates to a method for prolonged
storage of extracted mammalian organs and such organs that have
been preserved for use in transplants.
[0004] 2.Prior Art
[0005] Clinical transplants of human organs such as lungs, heart,
liver, kidneys and pancreas are routinely performed today. However,
as the number of patients waiting for organ transplants increases
yearly, the shortage of donors has become a serious problem and the
waiting time to surgery is also increasing. Even if a donor is
found, his or her organs cannot be effectively used in transplants
since nothing like blood banks exist for organs that are fully
equipped with the ability to preserve organs for prolonged periods
and allow for efficient supply of organs.
[0006] Organs to be transplanted are most commonly preserved by
cold storage but the preservation limit is about 4-24 hours (Cooper
J D, Patterson G A, Trulock E P et al.; J. Thorac. Cardiovasc.
Surg. 107, 460-471, 1994). In experiments using University of
Wisconsin Solution (UWS) as a medium for cold storage of hearts
from rats, rabbits and baboons before resuscitation, the time limit
was 6-18 hours (Makowka I, Zerbe T R, Champman F et al.; Transplant
Proc. 21, 1350, 1989 and Yen T, Hanan S A, Johnson D E et al.; Ann.
Thorac. Surg. 49, 932, 1990). Transplanting of rat hearts (n=5)
immersed in a combination medium of UWS and perfluorocarbon was
found to be successful at both 24 hours (100%) and 48 hours (4 out
of the 5 animals) (Kuroda Y, Kawamura T, Tanioka T et al;
Transplantation, 59, 699-701, 1995). The reason for these short
time limits is that when removed hearts are exposed to the low
temperature of 4.degree. C. or ischemic injury, their cell
membranes are damaged to make tissue cell resuscitation impossible
(Pegg D E; Organ Preservation Surg. Clin. North Am. 66, 617, 1986:
Oz M C, Pinsky D J, Koga S et al.; Circulation 88, 291-297, 1993:
and Heffner J E, Pepine J E; Rev. Pespir. Dis. 140, 531-554,
1989).
[0007] The technology for storing mammalian living tissues over
prolonged periods before resuscitation have seen marked advances
only in the area of single cells such as blood, sperm and ova.
Efforts to develop practically feasible methods for the cold
storage of living tissues which are aggregates of cells and organs
which are composed of several tissues are also in progress but they
have to meet the inexorable requirement that transplant be
performed within 24 hours of storage (Kalayoglu M, Sollinger H W,
Strarra R J et al.; Lancet 2, 617, 1988).
[0008] As regards the technology of organ preservation and
resuscitation, trehalose (C.sub.12H.sub.22O.sub.11) is an
interesting substance to mention. This is a nonreducing disacharide
found widely in nature and it has been reported to have the ability
to stabilize or protect the structure of cell membranes under
various types of stress (Crowe J H, Crowe L M, Chapman D; Science
233, 701-703, 1984 and Wiemken A; Antinei Van Leeunwenhoek 58,
209-217, 1990). It was also reported that trehalose had the ability
to protect cell membranes of the heart when it was exposed to the
low temperature of 4.degree. C. or ischemic injury (Stringham J C,
Southhard J H, Hegge J et al.; Transplantation 58, 287-294, 1992
and Hirata T, Fukuse T, Liu C J et al.; Surgery 115, 102-107,
1994).
[0009] According to reports of experiments with tardigrades under
high hydrostatic pressure, trehalose increased 10-fold in an
anhydrous state (Crowe J H, Crowe L M, Chapman D; Science 233,
701-703, 1984 and Crowe J H, Crowe L M, Chapman D, Aurell Wistorm;
Biochemical Journal 242, 1-10, 1987). Tardigrades are multicellular
organisms composed of ca. 40,000 cells including nerve cells.
[0010] The present inventor previously found that tardigrades in a
desiccation state had the viability to withstand high hydrostatic
pressures up to 600 MPa (Kunihiro Seki et al.; Nature Vol. 395, No.
6705, pp. 853-854, 29 Oct. 1998 and Japanese Patent Public
Disclosure No. 289917/1999 which is incorporated herein by
reference to the specification and the drawings). Tardigrades
become "desiccate" when they are in the "tun" state. The
physiological mechanism behind their tun state has not been fully
unravelled but it is at least clear that desiccated tardigrades
have lost an extremely large amount of water in their body to
become dehydrated.
SUMMARY OF THE INVENTION
[0011] As described above, the shortage of donors and the
increasing time for which patients have to wait before surgery are
two serious problems with organ transplants and a strong need
exists to develop feasible techniques for preserving organs and
later resuscitating them.
[0012] In the conventional storage of organs by refrigeration, the
temperature of the organ is lowered so that its metabolism is
suppressed to a level that maintains its viability. While the
metabolism of the organ is suppressed by reducing temperature,
water as a polar medium is a rich supply of ions which cause
self-disintegration of cells, their death and necrosis over time.
Hence, the longer the period of storage by refrigeration, the
higher the frequency of the occurrence of serious thrombus
formation and dysfunction. Organs cannot be stored cold for an
indefinite period.
[0013] An object, therefore, of the invention is to provide a novel
technique by which organs can be stored in vitro for a
significantly increased number of days while preventing their cells
and tissues from undergoing self-disintegration over time.
[0014] Another object of the invention is to provide a basic
technique of such substantial utility that it can extend the
duration of preservation of mammalian organs for use in
transplanting into humans.
[0015] The present inventor found that the ability of desiccated
tardigrades to withstand extreme environments in an inert medium
could be applied to the purpose of preserving mammalian organs for
an extended period. The organs preserved by the present invention
can be later resuscitated for collecting viable nerves or stem
cells. The resuscitated organs or the collected tissues can be used
in transplants. For histopathological studies, it is quite
significant that resuscitable biomaterials rather than necrotic
specimens can be stored for a long period.
[0016] Animal tissue cells generally are not viable in the absence
of water. One may readily imagine that organs of higher animals
which are composed of heterogeneous tissues can never be
resuscitated from a desiccation state. Techniques for preserving
plants and various bacteria in a desiccation or dry state have
already been developed but not a single experiment has been
reported in which organs of higher animals were successfully
resuscitated after storage in a desiccation or dry state.
[0017] To his surprise, the present inventor found that when
extracted mammalian organs were deprived of much water under
specified conditions and later stored at low temperature within an
inert medium, they had apparent death of the same nature as
experienced by tardigrades which remained viable in the tun state
for a prolonged period.
[0018] Particularly surprising was that multi-cell and multi-tissue
mammalian organs resuscitated from an extremely dehydrated state
and that the resuscitated heart was found to beat. The cells of the
resuscitated organ are believed to be in apparent death
characterized by either an extreme drop in oxygen consumption (no
greater than {fraction (1/1000)} of the normal level) or
substantial arrest of oxygen consumption.
[0019] The method for preserving mammalian organs according to the
first aspect of the invention comprises two steps, one of
dehydrating an organ to remove water but leave intact an amount of
water that permits later resuscitation and the other of immersing
the organ in an inert medium and maintaining it at a chill
temperature or below.
[0020] In a preferred case of the dehydration step, an organ having
a physiologically normal water content is deprived of water in an
amount of at least about 25% by weight of the total weight of the
organ before dehydration such that water is left intact in an
amount of at least from about 10 to about 20% by weight of the
total content of water before dehydration. This step of dehydration
is preferably followed by the step of immersing the organ in an
inert medium and maintaining it at a chill temperature.
[0021] The preserved mammalian organ according to the second aspect
of the invention is such that it is deprived of water in an amount
of at least about 25% by weight of its physiologically normal,
total weight while leaving water intact in an amount of at least
from about 10 to about 20% by weight of the total water content in
the organ which is then immersed in an inert medium and maintained
at a chill temperature or below. Examples of such stored mammalian
organs include heart, liver, kidneys, pancreas and lungs.
"Depriving of water in an amount of at least about 25%" means
removing the body fluid in the vascular system, as well as the free
water present in and between individual cells. "Leaving water
intact in an amount of at least from about 10 to about 20%" shall
be taken to mean that after removal of water, the organ still
contains a sufficient amount of water to permit later
resuscitation, inclusive of the bound water in the living
tissue.
[0022] When free water is removed from the tissues and cells of the
organ, biostructures such as biomembranes become less susceptible
to the attack of substances, particularly metal ions, that can be
activated in the aqueous phase. The biostructures are presumably
protected by the surrounding water in a crystalline state called
"bound water". As a result of the removal of the polar medium that
degrades the living tissue, the tissues and cells of the organ
become immune to degradation with time and the organ can be
preserved in a significantly improved state. In this case,
trehalose in the preserving solution can contribute to stabilizing
the biostructure.
[0023] The freshness of stored organs is believed to depend
primarily on the amount of free water and to prolong the
preservation period, it is theoretically preferred to remove free
water as much as possible. To maintain resuscitability, it is
preferred to ensure that water is left intact in a range of amounts
that enable the maintenance of bound water. Bound water may be
defined as the water in which the state of hydration or
crystallization can be observed, and free water as the water other
than bound water.
[0024] Animal organs are generally understood to have a water
content in the range from about 60 to about 80 mass %. A suitable
state of dehydration will depend on the inherent water content of a
specific kind of organs but all that is required by the present
invention is that the organ to be preserved should be deprived of
water in an amount of at least about 25% by mass of the total
weight of the organ before dehydration so that the organ contains
water in an amount of at least about 10 to about 20% by weight of
the total water content before dehydration.
[0025] If the method of the invention is to be applied to
preserving heart, it comprises three steps, the first for removing
blood from the heart by flushing with physiological saline until
the blood in the heart is replaced by the physiological saline, the
second for depriving the flushed heart of water in an amount of
from about 25 to about 60% by weight of the total weight of the
heart before dehydration, and the third for immersing the
dehydrated heart in an inert medium and maintaining it in the state
of apparent death at a chill temperature between about 2 and about
4.degree. C. By removing about 25 to about 60% of water from the
heart, water can be left intact in an amount of at least from about
10 to about 20% by weight of the total water content before
dehydration.
[0026] Dehydration of organs can conveniently be accomplished by
bringing the organ to be preserved into contact with a dehydrator
and absorbing water from within the organ. Specifically, the washed
and flushed organ is surrounded by the required amount of
dehydrator and immersed in an inert medium together with it. The
immersed organ is gradually dehydrated in the inert medium until it
suffers apparent death. Having been dehydrated to an extremely low
water content, the organ stored cold in the inert medium can
maintain chemical stability in all tissues including nerve tissue.
In the experiments conducted by the present inventor, rat hearts
could actually be preserved for as many as 10-20 days without
suffering excessive damage to the nerve system.
[0027] In order to increase the resuscitation ratio and achieve
further improvements in the state of preserved and resuscitated
organs, a method of dehydration by withdrawing water from within
the organ through channels in the vascular system may be employed
in practicing the preservation method of the invention.
[0028] Specifically, this can be achieved by irrigation perfusion
or flushing with a specified gas medium that can flow through
arteries or veins connecting to capillaries in the organ until it
displaces the water in the organ. The gas medium gets into
capillaries from one end of the vascular system and creates a flow
pressure that allows the gas medium to infuse all parts of the
organ tissues at substantially the same rate; thereafter, the gas
medium circulates through the capillaries (for example, from
arterial to venous vessels) until it reaches the other end of the
vascular system. As a result of this gas perfusion of the vascular
system, the body fluid in the organ is pushed forward so that water
is withdrawn from every one of the cells via capillaries. Gas
perfusion can be effected with an irrigation apparatus for flushing
physiological saline if the gas is pumped in instead of the
physiological saline.
[0029] The gas to be supplied into the vascular system may be air
or a gaseous mixture of O.sub.2 and CO.sub.2. Alternatively, inert
gases may be used, as exemplified by N.sub.2, He, Ar, Ne, Kr and
Xe.
[0030] In anatomy, vascular systems are classified into two groups,
blood vessels and lymphatics. For the purposes of the invention,
nutrition blood vessels through which water and nutrients are
supplied to individual cells in the organ of interest can
preferably be used as the "vascular system". In the case of the
heart, the irrigation apparatus may be connected to inherent
vascular vessels leading to the atria and ventricles so that flow
pressure is applied indirectly to the coronary arteries and veins
leading to the nutrition blood vessels in the heart. Besides the
nutrition vessel system, organs such as the liver have a functional
blood vessel system associated with portal vein circulation; in
such organs, the functional blood vessel system may be substituted
for the nutrition vessel system.
[0031] Liquids to be supplied to the vascular system include
solvents that make use of osmotic pressure difference to displace
water from cells, as exemplified by hypertonic liquids more
concentrated than the body fluids in organs. These may be
substituted by the inert medium to be described later, or
alcohols.
[0032] Dehydration via the vascular system utilizes the water
supply passages inherent in organs and can hence create a uniform
dehydrated state at slow speed in the desired tissues or cells.
Even in the case of mammalian, multi-cell and multi-tissue organs,
transfer to a highly dehydrated state can be achieved smoothly
without undue stress on the living tissue. The living tissue is
usually placed under stress by 25 mass % or more dehydration;
however, dehydration via the vascular system can bring organs to
apparent death in an extremely stable state without causing
ischemic injury or damaging the living tissue. Upon refilling with
water, functional resuscitation occurs not only in the cells and
tissues but also in the organs themselves.
[0033] Apparent death usually means biological apparent death. The
term "apparent death" as intended by the invention should be taken
to mean such a state that the external signs of "life" are lost as
a result of enhanced dehydration but can be restored upon refilling
with water. The term "resuscitation" as used herein means such a
phenomenon that upon refilling with water, a dehydrated tissue or
organ resumes recognizable electrophysiological reactions or
biological life activities, respectively.
[0034] In the method of the invention, the step of dehydration
using gases is preferably preceded by blood removal using
physiological saline. Blood removal is effective in avoiding the
problem of blood coagulation upon contact with the flushing
gas.
[0035] The "organ having a physiologically normal water content" is
typically an organ as extracted from the living body. If the method
of the invention includes the step of blood removal, this term can
be taken to mean an organ whose blood has been replaced by
physiological saline as a result of irrigation performed to effect
blood removal. The degree of dehydration can be specified with
reference to the weight of the "organ having a physiologically
normal water content".
[0036] The physiological saline to be used in blood removal is a
substitute body fluid having similar physiological activity to
blood and typical examples are known Ringer's solutions such as KH
(Kreps-Henseleit) solution. Polysaccharides that will help
stabilize the dehydrated biostructure may be dissolved in
physiological salines of this class. A preferred polysaccharide
having this ability is trehalose. Other biostructure stabilizing
substances that can be dissolved in physiological saline include
malic acid, mannitol, glycerol, and amino acids such as glycine
betaine, proline and ectoine.
[0037] The inert medium to be used in the invention is a medium
that is insoluble in water and oils and fluorocarbons that are
liquid at the temperature for preservation are preferred, with a
liquid perfluorocarbon being particularly preferred. If similar
conditions are satisfied, other forms of inert medium may be used
such as gas, sol and gel. Other inert media that are believed to be
useful include mercury and silicone oil.
[0038] The heart, liver, kidneys, pancreas and lungs preserved by
application of the present invention can be resuscitated by
flushing their vascular system with body fluid or substituted body
fluids that have been warmed to near body temperature, as
exemplified by the physiological saline described above, artificial
blood and/or natural blood. The resuscitated organs or tissues
collected from them are believed to be transplantable to the human
body. Nerve tissue and other living tissues and cells can be
collected from the resuscitated organs and used for testing
purposes as in a pharmacological test.
[0039] Another possible application of the invention is to organs
of mammals except humans that can be used as heterologous
transplants to the human body and by this application the invention
provides an effective technique for preserving animal organs that
are expected to find increasing demand in clinical settings to deal
with the shortage of human donors. In particular, the invention is
also useful for preserving mass-producible organs such as the
heart, liver and pancreas from cultured pigs; thus, the invention
will provide an effective preservation technique that can withstand
transport for a long time, particularly air transport for a period
longer than ten-odd hours.
[0040] The invention will also find utility in application to the
storage of tissues and organs that have been reconstituted by
cultivating stem cells having totipotency such as embryonic stem
cells taken from blatocysts. The invention may find further
applicability to the storage of nerve tissues in the brain and
other organs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 is a surface cardiac electrocardiogram (SECG) for an
extracted rat R016 heart as resuscitated after 10-day
preservation;
[0042] FIG. 2 is an SECG for an extracted rat R020 heart as
resuscitated after 20-day preservation;
[0043] FIG. 3 is an SECG for an extracted rat R029 heart as
resuscitated after 10-day preservation;
[0044] FIG. 4 is an SECG for an extracted rat R033 heart as
resuscitated after 10-day preservation;
[0045] FIG. 5 is an SECG for an extracted rat R034 heart as
resuscitated after 10-day preservation;
[0046] FIG. 6 is an SECG for an extracted rat R041 heart as
resuscitated after 11-day preservation; and
[0047] FIG. 7 is an SECG recording for an extracted rat R051 heart
as a comparison.
THE PREFERRED EMBODIMENT OF THE INVENTION
[0048] Step of Blood Removal
[0049] Upon contact with gases or exposure to low temperature,
blood usually clots to form thrombi and this is an obstacle to the
subsequent steps of dehydration and resuscitation. To eliminate
this possibility, the organ to be preserved, for example, an organ
extracted in surgery is washed to remove blood. The known
Langendorff apparatus may be used to effect perfusion for blood
removal. The technique of irrigation for blood removal is known to
the skilled artisan as the Langendorff method (see Doring H. J. and
Dehnert H.; Biomesstechnik-Verlag Match GmbH, Germany 1988).
According to the Langendorff method, an irrigation catheter (or
cannula) is attached to the aorta in the blood vessel system of the
organ and a mixture of trehalose and KH solution is sent into the
blood vessel system to replace the blood in the organ with the
physiological saline. The physiological saline is preliminarily
aerated to ensure that the organ is supplied at all times with a
fresh substitute body fluid. During the blood removal by
irrigation, the organ is cooled to 1-8.degree. C. to stop its
activity.
[0050] Step of Dehydration
[0051] The step of blood removal is followed by dehydration.
[0052] i) Dehydration with Dehydrator
[0053] If dehydration is primarily performed using a dehydrator, a
convenient way is by adjusting the dehydrator to a desired volume
(which can remove water in an amount of at least 50% by weight of
the total weight of the organ before dehydration) and immersing it
in an inert medium (see below) together with the organ.
Specifically, the organ as surrounded with silica gel, molecular
sieve, zeolite or the like is placed in a metal cage, a synthetic
fiber net or other casings that are made of materials neutral to
the inert preserving medium, and then immersed in the inert
medium.
[0054] The amount of the dehydrator to be used can be calculated
from the percentage of water removal that is required by the organ
of interest. After immersion in the inert medium, the dehydrator
may be removed or replaced on a suitable timing; if necessary, an
additional amount of the dehydrator may be added. If contacted by
the dehydrator within the inert medium, the organ gets the internal
water to be slowly absorbed by the dehydrator via the outer surface
until the intended state of dehydrastion is reached.
[0055] ii) Dehydration Via the Vascular System
[0056] Dehydration with gas can be accomplished by means of the
Langendorff apparatus used in the step of blood removal. For
example, the aorta in the organ that has been subjected to the
necessary blood removal is supplied with a suitable gas rather than
physiological saline at a specified flow pressure from this
irrigation apparatus. It will be readily understood by the skilled
artisan that the artificial means of irrigation include not only a
device for one-way supply of the gas through the aorta but also a
closed circulation system connected between the aorta and venae
cavae. Thus, the perfusion with gas is not limited to the forcing
of the gas and it may be aspirated through the vascular system.
[0057] If the flow pressure created by the irrigation apparatus is
applied to the vascular system of the organ, the physiological
saline flows out of the organ through the venae cavae to cause
progressive dehydration. Preferably, the gas entering blood vessels
flow through individual capillaries toward the venae cavae and the
water both within and between individual cells is forced under its
own vapor pressure to enter the gas flowing through the blood
vessels. Eventually, most of the free water within tissues and
cells flows through the blood vessel system to be discharged to the
outside of the organ.
[0058] Unlike the treatment by contact with the dehydrator,
dehydration via the blood vessel system utilizes the extremely
large surface area of the capillaries distributed uniformly in the
organ, and the intended tissue and individual cells can be
dehydrated in a fairly short period of time although slowly. This
advantage is obvious from the smaller extent of unevenness in
dehydration and from the by-no-means unbearable change in the color
of the dehydrated organ. As will be shown in the examples to be
given later in this specification, dehydration by irrigation with
gas achieved markedly high resuscitation ratio, indicating
significant improvements in the state of resuscitation. As further
advantages, dehydration with gas is an active treatment that can be
controlled externally, which puts less stress on organs that must
be handled rapidly but carefully, and which is very efficient.
[0059] Inexpensive compressed gases packed in commercially
available containers may be forced into the vascular system. In the
case of an O.sub.2--CO.sub.2 gaseous mixture, the CO.sub.2 content
is preferably less than 5%. Dry gases were used in the Examples to
be described later in this specification but humid gases may of
course be used. More preferably, the dehydrating gas may be chosen
from inert gases such as N.sub.2, He, Ar, Ne, Kr and Xe. Among the
inert gases, Xe is expensive but considering its reported
anesthetic action on living tissues, the advantage of using Xe as
the dehydrating gas would be great. The use of inert gases has the
added advantage of avoiding damage that may be caused to living
tissues by active oxygen.
[0060] The dehydration by perfusion with gas may be combined with
the ancillary method of dehydration by bringing the organ into
contact with a dehydrator. In the experiments the present inventor
conducted using tardigrades, 100% resusictation was achieved when
dehydration was performed in a highly humid environment, preferably
at a humidity of 80%. Considering this fact, it would also be
preferred to dehydrate organs at high humidity.
[0061] To give guide figures as the conditions for perfusion with
gas, the aorta in the heart of a rat is perfused with air at a flow
rate of ca. 0.05-0.2 kgf/cm.sup.2 (ca. 4.9-19.6 kPa), preferably
ca. 0.1 kgf/cm.sup.2 (ca. 9.8 kPa), for a period of at least 1-1.5
hours. By means of perfusion with this amount of air, at least 25
mass % water removal can be achieved as contrasted with the total
weight of the organ that is about to be dehydrated.
[0062] The organ about to be dehydrated contains the substitute
body fluid and has a physiologically normal water content. For
extended storage of the organ, the largest possible amount of free
water must be removed. In the present invention, the amount of
water to be removed is defined in terms of percent water removal
(weight ratio) with reference to the total weight of the organ and
expressed by the following equation:
Percent water removal (weight ratio)=100-[(total weight of the
organ after dehydration/total weight of the organ before
dehydration).times.100]
[0063] The state of dehydration may be defined by other methods
such as NMR which specifies the absolute amount or the state of the
molecules of water present in the organ of interest. NMR is a
technique most widely used by scientists to study the state of
water In living tissues.
[0064] The first NMR-based study of the water in living tissues was
reported by Belton, P. S., Jackson, R. R. and Packer, K. J. in
Pulsed NMR Studies of Water in Strained Muscle, I. Transverse
Nuclear Spin Relaxation Times and Freezing Effects; Biochem.
Biophys. Acta. 286:16-25 (1972). A structural analysis of water by
NMR was reported by Hazlewood, C. F., Chang, D. C., Woessner, D. E.
and Nichols, B. C. in Nuclear Magnetic Resonance Transverse
Relaxation Times of Water Protons in Skeletal Muscle; Biophys. J.
14:583-605 (1974).
[0065] Belton et al. concluded that the molecules of water in
living tissues consisted of three states, ca. 8% of which was bound
water, or water bound to biomolecules such as the intima of cell
membranes, as well as proteins and nucleic acids, ca. 82% being
free water, and the remaining 10% was occupied by free water
bordering on the outside of cell membranes. The bound water differs
from other molecules of water in that it has certain preferential
orientations in several molecular layers based on the molecules of
water directly bound to biopolymers.
[0066] Typically, it is understood that ca. 10-ca. 20% of the total
water content in the living tissue is occupied by bound water and
ca. 80-ca. 90% by free water. The total content of water in the
heart of a rat is typically ca. 80 mass % of the total weight of
the organ, so the contents of bound water and free water are
respectively 8-16 mass % and 64-72 mass %.
[0067] In the step of dehydrating the heart of a rat, water is
removed in an amount of ca. 25-60 mass % of the total weight of the
organ and if this is the case, it is reasonable to think that all
the water removed is free water. Hence, as the result of
dehydration, the mass of the free water remaining in the rat's
heart will drop to a level on the order of several to forty percent
but the absolute amount of bound water remains the same. Although
the actual water content slightly varies depending on the animal
species and the type of the organ to be preserved, removing free
water in an amount of at least ca. 25% by weight of the total
weight of the organ before dehydration is sufficient to leave at
least ca. 10-ca. 20% by weight of water (including bound water)
intact on the basis of the total water content before
dehydration.
[0068] While the percentage of free water removal that is tolerated
by organs may vary with the type of the organ to be preserved, the
intended duration of storage and other conditions for preservation,
ca. 25-35% is currently considered safe for the heart of rats and
if prolonged storage is intended, as much as ca. 45-50% of free
water is preferably removed. Removing less than 25% of water makes
little contribution to extending the duration of storage.
Theoretically, removing water to a level near 60% where the
presence of free water is very much limited would make great
contribution to extended storage.
[0069] As of today, no full explanation has been proposed for the
mechanism connecting the above-described state of dehydration to
the increase in the period of organ preservation. The present
inventor has at least shown that the increase in the period of
organ preservation does not depend on the composition of the
preserving fluid but depends largely on the absolute amount of
water molecules in the tissues or cells of the organ. Since the
organ that ceased to show signs of substantial life activity as the
result of dehydration of its tissues and cells later resuscitated,
the organ may well he considered to have reached "apparent death",
which may be called "immortal state" or more academically
"cryptobiotic state" (Vreeland H. R. et al; Nature, Vol. 407, pp.
897-900, Oct. 19, 2000: Cano J. R. et al.; Science, Vol. 268, pp.
1060-1064, 19 May 1995).
[0070] Step of Preservation
[0071] The dehydrated organ is preserved in a perfluorocarbon which
is an inert medium insoluble in water and oils. The organ is
immersed in the inert medium generally in a closed state at
atmospheric pressure, optionally under superatmospheric pressure.
As already mentioned, the organ may be immersed in the inert medium
together with the dehydrator. The inert medium is preferably
aerated with pure oxygen.
[0072] The term "at a chill temperature or below" as used herein
means the range of from ca. +1 to ca. +8.degree. C., preferably in
the neighborhood of ca. +2 to ca. +4.degree. C. The organ is
maintained in this preserved state for a predetermined number of
days. If the free water in the organ is adequately removed, it can
theoretically be stored frozen as in liquid nitrogen.
[0073] Step of Resuscitation
[0074] To resuscitate the organ from the preserved state, the
already mentioned Langendorff method can be employed. First, the
preserved organ is taken out of the perfluorocarbon and the
dehydrator, if any, is also removed. The organ is then immersed in
a KH solution, preferably aerated with pure oxygen, within a Petri
dish at +4.degree. C. A perfusion catheter is fixed to the aorta in
the organ and the KH solution, preferably aerated continuously with
a gaseous mixture of O.sub.2 and CO.sub.2 and warmed to 37.degree.
C., is forced by an irrigation pump into the catheter at a constant
flow rate. The organ is resuscitated by this procedure of
irrigation.
[0075] Verification of Resuscitation
[0076] One of the important aspects of preserving mammalian organs
is the need for post-storage verification of viable tissues in the
organ. This can be accomplished by several methods including tissue
autopsy, actual transplant and electrophysiology. Whichever method
is adopted, it must be determined whether each type of tissue cell
is alive. In the present invention, electrophysiological
verification of tissue cell resuscitation was conducted by taking
SECGs because they could provide real-time recording of the
activity of nerve cell tissue and cell death could be found to have
occurred at the point in time when neuron activity disappeared. As
an ancillary means, the visual change in the color of the organ
tissues and the presence of beats (in the case of the heart) were
monitored to complete the verification of organ resuscitation.
[0077] The following examples are provided for the purpose of
further illustrating the present invention but are in no way to be
taken as limiting.
EXAMPLE 1
[0078] Seven-week old male-Wistar rats (300 g) were artificially
reproduced in compliance with the American NIH standards for
laboratory animals. The rats were anesthetized with Nembutal (Na
solution) and their SECGs were recorded. Thereafter, the heart was
extracted from each animal, immersed in a KH solution aerated with
pure oxygen and mixed with 117 mmol of trehalose and had the
adhering blood rinsed off. Moisture was only wiped off from the
outer surface of the heart before its total weight was
measured.
[0079] Catheters were inserted into the aorta and vena cavae in the
extracted heart and the same KH solution mixed with trehalose was
irrigated through the heart to remove blood. Silica gel (9-10 g
sufficient to remove nearly 60% of the water in the heart) was put
into a ball-shaped metal cage and the pretreated heart was placed
on the silica gel, immersed in a liquid perfluorocarbon (C8F17;
FLUORINERT FC77 of Sumitomo 3M) at 4.degree. C. which had been
aerated with pure oxygen for one minute; the heart was then placed
in a hermetically sealed 500-mL jar and stored in a
refrigerator.
[0080] After 10 days, the heart was recovered from the preservation
fluid; after removing the silica gel with tweezers, the recovered
heart was placed in a KH solution in a Petri dish at 4.degree. C. A
catheter for perfusion was attached to the aorta with cotton thread
and the heart was set in a fixed-flow Langendorff perfusion
apparatus. A KH solution continuously aerated with a gaseous
mixture of O.sub.2 and CO.sub.2 at a volume ratio of 95:5 was
supplied from the storage tank, warmed to 37.degree. C. within the
glass coil tube in the homothermal tank and forced by an irrigation
pump (Masterflex Model No. 7520-10 of Cole-Palmer Instrument Co.)
into the aortic catheter at a constant flow rate of 6 mL/g (heart's
weight) per minute.
[0081] As is well known, isolated hearts resuscitate spontaneously
and nerve response appears if the tissue cells are alive.
Irrigation of the extracted hearts was started at predetermined
temperatures and SECG electrodes were attached at the left
ventricle and the opening of the aorta, and with a bipolar lead,
SECGs were recorded continuously using an organic amplifier
(Bioview-E of NEC-Sanei). This sequence of treatments and
operations was specifically applied to the following
experiments.
[0082] i) Experiment 1
[0083] At 15:10 on Jul. 7, 1998, an R016 rat under Nembutal
anesthesia was incised in the chest to recover the heart which was
subjected to irrigation for removing blood. Upon completion of this
preliminary treatment, the extracted heart weighed 1.240 g.
[0084] Ten days later (Jul. 16, 1998), the heart was recovered from
the preserving perfluorocarbon fluid at 4.degree. C. and had the
silica gel removed at 17:20; just before irrigation started, the
heart weighed 0.774 g. During the storage, about 38% of the water
in the cells of the heart tissue had been absorbed by the silica
gel.
[0085] Thereafter, a catheter was inserted into the aorta of the
heart and fastened with cotton thread. The heart was then set in
the fixed-flow Langendorff irrigation apparatus and irrigation
started at 17:25 at 37.degree. C. The heart resuscitated to give
the SECG shown in FIG. 1 (recorded at 18:31 with 40 beats per
minute). The heart rate later dropped to 27 beats per minute at
18:47.
[0086] ii) Experiment 2
[0087] At 17:30 on Jul. 10, 1998, an R020 rat was incised in the
chest to recover the heart which was subjected to irrigation for
removing blood. Upon completion of this preliminary treatment, the
extracted heart weighed 1.521 g.
[0088] Twenty days later (Jul. 30, 1998), the heart was recovered
from the preservation fluid and had the silica gel removed at
15:12; just before irrigation started, the heart weighed 1.063 g.
During the storage, about 30% of the water in the cells of the
heart tissue had been absorbed by the silica gel.
[0089] Thereafter, a catheter was inserted into the aorta of the
heart and fastened with cotton thread. The heart was then set in
the fixed-flow Langendorff irrigation apparatus and irrigation
started at 15:13 at 32.degree. C. The heart resuscitated to give
the SECG shown in FIG. 2 (recorded at 15:19 with 42 beats per
minute).
[0090] iii) Experiment 3
[0091] At 14:40 on Jul. 26, 1998, an R029 rat was incised in the
chest to recover the heart which was subjected to irrigation for
removing blood. Upon completion of this preliminary treatment, the
extracted heart weighed 1.291 g.
[0092] Ten days later (Aug. 5, 1998), the heart was recovered from
the preservation fluid and had the silica gel removed at 16:55;
just before irrigation started, the heart weighed 0.832 g. During
the storage, about 36% of the water in the cells of the heart
tissue had been absorbed by the silica gel.
[0093] Thereafter, a catheter was inserted into the aorta of the
heart and fastened with cotton thread. The heart was then set in
the fixed-flow Langendorff irrigation apparatus and irrigation
started at 17:19 at 34.degree. C. The heart resuscitated to give
the SECG shown in FIG. 3 (recorded at 17:39 with 42 beats per
minute). The activity of the heart was also visible.
[0094] iv) Experiment 4
[0095] At 14:40 on Jul. 29, 1998, an R033 rat was incised in the
chest to recover the heart which was subjected to irrigation for
removing blood. Upon completion of this preliminary treatment, the
extracted heart weighed 1.293 g.
[0096] Ten days later (Aug. 7, 1998), the heart was recovered from
the preservation fluid and had the silica gel removed at 15:58;
just before irrigation started, the heart weighed 0.808 g. During
the storage, about 38% of the water in the cells of the heart
tissue had been absorbed by the silica gel.
[0097] Thereafter, a catheter was inserted into the aorta of the
heart and fastened with cotton thread. The heart was then set in
the fixed-flow Langendorff irrigation apparatus and irrigation
started at 16:15 at 36.degree. C. The heart resuscitated to give
the SECG shown in FIG. 4 (recorded at 16:19 with 108 beats per
minute). As in Experiment 3, the activity of the heart was also
visible.
[0098] v) Experiment 5
[0099] At 16:01 on Jul. 29, 1998, an R034 rat was incised in the
chest to recover the heart which was subjected to irrigation for
removing blood. Upon completion of this preliminary treatment, the
extracted heart weighed 1.375 g.
[0100] Ten days later (Aug. 7, 1998), the heart was recovered from
the preservation fluid and had the silica gel removed at 17:48;
just before irrigation started, the heart weighed 0.808 g. During
the storage, about 38% of the water in the cells of the heart
tissue had been absorbed by the silica gel.
[0101] Thereafter, a catheter was inserted into the aorta of the
heart and fastened with cotton thread. The heart was then set in
the fixed-flow Langendorff irrigation apparatus and irrigation
started at 17:57 at about 30.degree. C. The heart resuscitated to
give the SECG shown in FIG. 5 (recorded at 18:11 with 66 beats per
minute).
[0102] vi) Experiment 6
[0103] At 14:30 on Aug. 1, 1998, an R041 rat was incised in the
chest to recover the heart which was subjected to irrigation for
removing blood. Upon completion of this preliminary treatment, the
extracted heart weighed 1.295 g.
[0104] Eleven days later (Aug. 12, 1998), the heart was recovered
from the preservation fluid and had the silica gel removed at
17:40; just before irrigation started, the heart weighed 0.797 g.
During the storage, about 39% of the water in the cells of the
heart tissue had been absorbed by the silica gel.
[0105] Thereafter, a catheter was inserted into the aorta of the
heart and fastened with cotton thread. The heart was then set in
the fixed-flow Langendorff irrigation apparatus and irrigation
started at 17:56 at 36.degree. C. The heart resuscitated to give
the SECG shown in FIG. 6 (recorded at 18:13 with 162 beats per
minute).
[0106] vii) Reference Experiment
[0107] At 13:16 on Jul. 20, 1998, an R051 rat was incised in the
chest to recover the heart which was subjected to irrigation for
removing blood. Upon completion of this preliminary treatment, the
extracted heart weighed 1.162 g.
[0108] Thereafter, a catheter was inserted into the aorta of the
heart and fastened with cotton thread. The heart was then set in
the fixed-flow Langendorff irrigation apparatus and irrigation
started at 13:25 to give the SECGs shown in FIG. 7A (recorded at
13:45 with 171 beats per minute; irrigation temperature, 34.degree.
C.), FIG. 7B (recorded at 17:25 with 161 beats per minute;
irrigation temperature, 32.degree. C.), FIG. 7C (recorded at 19:25
with 134 beats per minute; irrigation temperature, 32.1.degree.
C.), and FIG. 7D (recorded at 22:02 with 17 beats per minute;
irrigation temperature, 31.1.degree. C.). At the points in time
when the SECG showed an amplitude (output) of 4 mV per centimeter,
the vigorous activity of the heart was visible. At 22:22, no
potential appeared, indicating the cessation of neuron
activity.
[0109] The SECGs shown in FIGS. 1-6 which were recorded after
storage for 10-20 days had amplitudes (outputs) of 2-4 mV which
were of the same order as the amplitude of the SECG shown in FIG. 7
for the control experiment. Thus, even after 10- to 20-day storage,
the heart produced comparable outputs to the value at which the
vigorous activity of the heart was visible in the control
experiment and this indicates the absence of excessive damage to
the nerve system in the preserved heart.
EXAMPLE 2
[0110] Rats were administered Nembutal (0.25 mL) and heparin sodium
salt (5 mg) by intraperitoneal injection. After measurement of
their body weight, the heart was extracted from each animal. The
extracted heart was placed in a homothermal tank and a catheter was
inserted into the aorta; thereafter, using a KH solution mixed with
trehalose (117 mmol) and aerated with a gaseous mixture of 95%
O.sub.2 and 5% CO.sub.2, irrigation for blood removal was performed
by the Langendorff method. The temperature of the homothermal tank
was lowered to stop the beating of the heart, thereby completing
the process of blood removal by irrigation. After measuring the
weight of the heart, the same irrigation apparatus was used to feed
the gaseous mixture of 95% O.sub.2 and 5% CO.sub.2 into the heart,
thereby starting dehydration. While monitoring the percent water
removal as calculated from the measurements of heart's weight,
dehydration was continued for about 1 hour and a half, whereupon
the gas irrigation was stopped. After measuring its weight, the
heart was immersed in a liquid perfluorocarbon aerated with the
gaseous mixture of O.sub.2 and CO.sub.2. After the passage of a
predetermined time, the preserved heart was taken out of the liquid
perfluorocarbon. A catheter was inserted into the aorta of the
heart and an attempt was made to resuscitate the heart by
Langendorff irrigation using a warm KH solution aerated with the
gaseous mixture of O.sub.2 and CO.sub.2.
[0111] i) Experiment 7
[0112] R349(5-week old male Wistar rat): Storage for 4 hours
1 Sept. 11, 2000 at 24.degree. C. 15:39 body weight measured: 130 g
15:51 extraction of the heart started 15:53 extraction of the heart
and insertion of catheter ended 15:53 blood removal by irrigation
started: homothermal tank held at 5.0.degree. C. and irrigation
effected at flow rate of 1.0-3.8 mL/min 16:14 the heart stopped
beating and blood removal by irrigation ended: homothermal tank
held at 5.0.degree. C. and the heart was at 18.2.degree. C. 16:14
heart's weight measured: 0.663 g 16:18 dehydration started by
irrigation with O.sub.2/CO.sub.2: gas pressure at 0.05 kgf/cm.sup.2
and homothermal tank held at 2.8.degree. C. 16:48 heart's weight
measured: 0.516 g at 22.2% water removal 16:50 dehydration
continued: gas pressure at 0.05 kgf/cm.sup.2 and homothermal tank
held at 1.1.degree. C. 17:20 heart's weight measured: 0.458 g at
30.9% water removal 17:21 dehydration continued: gas pressure at
0.05 kgf/cm.sup.2 and homothermal tank held at 1.6.degree. C. 17:51
heart's weight measured: 0.394 g at 40.6% water removal 17:55 4-hr
storage in PFC: 0.6.degree. C. 21:54 storage ended and the heart
recovered at 26.degree. C. 21:55 heart's weight measured: 0.436 g
21:55 irrigation for resuscitation started: homothermal tank held
at 34.4.degree. C. and irrigation effected at flow rate of 1.0-3.8
mL/min 23:40 irrigation for resuscitation ended: homothermal tank
held at 33.7.degree. C.
[0113] Assessment
[0114] Faint contraction of the cardiac muscle occurred at 4
minutes after the start of KH irrigation but there was no atrial
movement. The cardiac muscle continued to move until the irrigation
was stopped. The heart swelled slightly.
[0115] ii) Experiment 8
[0116] R350 (5-week old male Wistar rat): Storage for 8 hours
2 Sept. 12, 2000 at 25.degree. C. 19:10 body weight measured: 150 g
19:20 extraction of the heart started 19:22 extraction of the heart
and insertion of catheter ended 19:22 blood removal by irrigation
started: homothermal tank held at 5.0.degree. C. and irrigation
effected at flow rate of 1.0-3.8 mL/min 19:37 the heart stopped
beating and blood removal by irrigation ended: homothermal tank
held at 4.6.degree. C. and the heart was at 15.7.degree. C. 19:39
heart's weight measured: 0.718 g 19:43 dehydration started by
irrigation with O.sub.2/CO.sub.2: gas pressure at 0.1 kgf/cm.sup.2
and homothermal tank held at 4.4.degree. C. 20:13 heart's weight
measured: 0.647 g at 9.9% water removal 20:15 dehydration
continued: gas pressure at 0.1 kgf/cm.sup.2 and homothermal tank
held at 2.4.degree. C. 20:35 heart's weight measured: 0.550 g at
23.4% water removal 20:48 dehydration continued: gas pressure at
0.05 kgf/cm.sup.2 and homothermal tank held at 4.8.degree. C. 20:18
heart's weight measured: 0.483 g at 32.7% water removal 17:55 8-hr
storage in PFC: 0.6.degree. C. Sept. 13, 2000 05:21 storage ended
and the heart recovered at 27.degree. C. 05:23 heart's weight
measured: 0.523 g 05:24 irrigation for resuscitation started:
homothermal tank held at 33.6.degree. C. and irrigation effected at
flow rate of 1.0-3.8 mL/min 07:25 irrigation for resuscitation
ended: homothermal tank held at 36.6.degree. C.
[0117] Assessment
[0118] Starting at 05:29, the cardiac muscle contracted vigorously
but that event stopped in about 10 seconds. Around 06:00, the right
side of the heart was found to move faintly in the area near the
pulmonary artery. The heart swelled considerably and from
appearances it was hardly found beating; however, when the
irrigation was stopped and the heart was drained of the KH
solution, distinct beating was observed.
[0119] iii) Experiment 9
[0120] R350 (5-week old male Wistar rat): Storage for 16 hours
3 Sept. 13, 2000 at 245.degree. C. 16:22 body weight measured: 120
g 16:28 extraction of the heart started 16:31 extraction of the
heart and insertion of catheter ended 16:31 blood removal by
irrigation started: homothermal tank held at 26.9.degree. C. and
irrigation effected at flow rate of 1.0-3.8 mL/min 16:44 the heart
stopped beating and blood removal by irrigation ended: homothermal
tank held at 4.6.degree. C. and the heart was at 16.2.degree. C.
16:45 heart's weight measured: 0.605 g 16:50 dehydration started by
irrigation with O.sub.2/CO.sub.2: gas pressure at 0.1 kgf/cm.sup.2
and homothermal tank held at 3.3.degree. C. 17:20 heart's weight
measured: 0.534 g at 11.7% water removal 17:23 dehydration
continued: gas pressure at 0.1 kgf/cm.sup.2 and homothermal tank
held at 1.3.degree. C. 17:53 heart's weight measured: 0.517 g at
14.5% water removal 17:55 dehydration continued: gas pressure at
0.2 kgf/cm.sup.2 and homothermal tank held at 0.5.degree. C. 18:25
heart's weight measured: 0.522 g at 13.7% water removal 18:30 16-hr
storage in PFC: 2.8.degree. C. Sept. 14, 2000 10:28 storage ended
and the heart recovered at 25.degree. C. 10:30 irrigation for
resuscitation started: homothermal tank held at 32.9.degree. C. and
irrigation effected at flow rate of 1.0-3.8 mL/min 17:25 irrigation
for resuscitation ended: homothermal tank held at 34.5.degree.
C.
[0121] Assessment
[0122] The heart swelled so extensively that the state of its
beating was not clear at all and this made the recording of an SECG
necessary.
EXAMPLE 3
[0123] An experiment for the resuscitation of the rat's heart was
performed as in Example 2, except that dehydration was effected by
flushing with air and that the duration of storage was extended to
24 hours.
[0124] i) Experiment 10
[0125] Rat R443
4 Feb. 6, 2001 16:10 the heart extracted 16:20 blood removal by
irrigation started: homothermal tank first held at 26.8.degree. C.
and ended at 7.9.degree. C. 16:45 blood removal by irrigation ended
and heart's weight measured: 0.582 g 16:45 the heart was immersed
in PFC as it was flushed with air (0.1 kgf/cm.sup.2) through the
aorta Feb. 7, 2001 16:48 the heart recovered from PFC 16:50 heart's
weight measured: 0.430 g at 26.1% water removal 16:50 irrigation
with KH solution started: homothermal tank first held at
27.0.degree. C. and ended at 35.5.degree. C. 18:30 the heart was
found to resuscitate with consistent beasting
[0126] ii) Experiment 11
[0127] Rat R444
5 Feb. 7, 2001 18:55 Nembutal (0.2 mL) administered 18:57 heparin
sodium (5 mg) administered 18:57 body weight measured 19:00 the
heart extracted 19:03 flushing with trehalose in KH solution
started: homothermal tank first held at 27.6.degree. C. and ended
at 5.5.degree. C. 19:23 irrigation ended and heart's weight
measured: 0.767 g 19:27 flushed with air in jar (0.1 kgf/cm.sup.2,
0.8.degree. C.) 19:57 irrigation ended and heart's weight measured:
0.483 g at 34.8% water removal 20:00 the heart immersed in PFC Feb.
8, 2001 20:25 the heart recovered from PFC (0.5.degree. C.) 20:27
heart's weight measured 20:27 flushing with KH solution started:
homothermal tank first held at 26.9.degree. C. and ended at
35.4.degree. C. 21:27 the heart was found to resuscitate and beat
several times before coming to complete stop
EXAMPLE 4
[0128] Five-week old male Wistar rats were reproduced in compliance
with the American NIH standards for laboratory animals. Five rats
were used in each of the following experiments. Perfluorocarbon
(PFC) was used as an inert medium.
[0129] i) Experiment 12 (4-hr Storage)
[0130] The rats were administered intraperitoneally first with
Nembutal (0.2 mL), then with a solution of heparin sodium (5 mg) in
physiological saline (0.3 mL). Each rat was incised in the chest
and the heart was removed. A catheter was inserted into the aorta
of the heart and ligated with cotton thread. The heart was then set
on a fixed-flow perfusion apparatus and flushed with a KH solution
having trehalose (117 mol) dissolved therein; the irrigation
temperature was 27.degree. C. and the flow rate of the KH solution
was 3.2 mL/min. The KH solution was constantly aerated with a
gaseous mixture of 95% O.sub.2 and 5% CO.sub.2. The temperature of
the flushing fluid was gradually lowered until the heart stopped
beating. After measuring the its weight, the heart was dried by
flushing with air gas through the catheter at a flow rate of 0.1
kgf/cm.sup.2. After measuring its weight, the dry heart was
preserved within PFC held at 4.degree. C. as it was constantly
aerated with an O.sub.2/CO.sub.2 mixture. After 4 hours, the heart
was taken out of the preservation fluid, set on the fixed-flow
irrigation apparatus and flushed with a KH solution as it was
constantly aerated with an O.sub.2/CO.sub.2 mixture; the
temperature of the KH solution was 27.degree. C. and it was flushed
at a flow rate of 3.2 mL/min. As is well known, when the preserved
heart is subjected to another irrigation, it will resuscitate
spontaneously and neuron activity will appear if the tissue cells
are still alive. Electrodes were attached to the resuscitated heart
and an SECG was recorded with a pen oscillograph.
[0131] ii) Experiment 13 (16-hr Storage)
[0132] The procedure of Experiment 12 was repeated except that the
duration of storage was extended to 16 hours.
[0133] The time of gas irrigation and the percent water removal
achieved for each rat are shown below.
6 Experiment 12 1-1 1 hr and 34 min, 44.1% 1-2 2 hr and 37 min,
32.8% 1-3 2 hr and 37 min, 44.4% 1-4 2 hr and 37 min, 47.5% 1-5 3
hr and 7 min, 41.1% Experiment 13 2-1 1 hr and 35 min, 44.4% 2-2 2
hr and 37 min, 41.2% 2-3 1 hr and 3 min, 45.8% 2-4 3 hr and 12 min,
41.3% 2-5 2 hr and 9 min, 41.4%
[0134] Result
[0135] The resuscitation ratio was 100% in both
[0136] Experiments 12 and 13.
[0137] In each of Experiments 12 and 13, there were significant
variations in the relationship between the time taken to remove
water and the actual percent water removal. A probable reason is
that one extracted heart had a different state from another sample.
Air as supplied through the catheter inserted into the aorta would
pass through the openings of the right and left coronary arteries
to fill the capillaries in the cardiac muscle, thereby drying its
cells. However, if the slightest thrombus formation occurs in
capillaries, the air gas will not reach the cells farther ahead;
this would be the reason for the lack of proportionality between
the time taken to remove water and the actual percent water
removal. Whatever the reason, it was quite surprising that the
hearts of rats deprived of water at 40% or more resuscitated at
100% probability after preservation for 4 or 16 hours.
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