U.S. patent application number 11/276183 was filed with the patent office on 2006-06-08 for methods and solutions for storing donor organs.
This patent application is currently assigned to Human BioSystems. Invention is credited to Fernando Lopez-Neblina, Luis H. Toledo-Pereyra.
Application Number | 20060121438 11/276183 |
Document ID | / |
Family ID | 33313505 |
Filed Date | 2006-06-08 |
United States Patent
Application |
20060121438 |
Kind Code |
A1 |
Toledo-Pereyra; Luis H. ; et
al. |
June 8, 2006 |
METHODS AND SOLUTIONS FOR STORING DONOR ORGANS
Abstract
A method of preserving, storing and transplanting mammalian
donor organs. The method includes the cooling of refrigeration
preservation, loading pre-freezer preservation, cryopreservation
and washing solutions at least containing polyvinylpyrrolidone, a
calcium channel blocker, a nucleoside, potassium chloride,
polyethylene glycol, at least one amino acid, and a steroid to a
temperature of 2.degree. to 4.degree. C. and/or of 0.degree. to
2.degree. C., harvesting a donor organ, perfusing it with one or
more of the solution, immersing it in one or more of the solutions
and storing it at a temperature above 0.degree. C. or at a
temperatures below 0.degree. C. The cryopreservation solution also
contains cryopreservative agents. Preserved organs may be
transplanted directly from refrigeration storage or from freezer
storage by cooling the washing refrigeration preservation solutions
to 2.degree. to 4.degree. C., perfusing the organ with washing
solution and then preservation solution, and transplanting it.
Inventors: |
Toledo-Pereyra; Luis H.;
(Portage, MI) ; Lopez-Neblina; Fernando;
(Vicksburg, MI) |
Correspondence
Address: |
WILSON SONSINI GOODRICH & ROSATI
650 PAGE MILL ROAD
PALO ALTO
CA
94304-1050
US
|
Assignee: |
Human BioSystems
Palo Alto
CA
|
Family ID: |
33313505 |
Appl. No.: |
11/276183 |
Filed: |
February 16, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10830863 |
Apr 22, 2004 |
7029839 |
|
|
11276183 |
Feb 16, 2006 |
|
|
|
60471028 |
May 15, 2003 |
|
|
|
60465114 |
Apr 23, 2003 |
|
|
|
Current U.S.
Class: |
435/1.1 |
Current CPC
Class: |
A01N 1/02 20130101; A01N
1/0226 20130101 |
Class at
Publication: |
435/001.1 |
International
Class: |
A01N 1/02 20060101
A01N001/02 |
Claims
1. A method of preserving mammalian donor organs or tissues at
refrigeration temperatures comprising: cooling a refrigeration
preservation solution at least containing polyvinylpyrrolidone
(PVP-40), a calcium channel blocker, a nucleoside, potassium
chloride, polyethylene glycol, at least one amino acid, and a
steroid to a temperature between 2.degree. and 4.degree. C.;
harvesting a donor organ; perfusing the donor organ with the
refrigeration preservation solution; immersing the donor organ in
refrigeration preservation solution; and storing the donor organ in
the refrigeration preservation solution at a refrigeration
temperature above 0.degree. C.
2. The method of claim 1 wherein the calcium ion flux inhibitor is
verapamil.
3. The method of claim 1 wherein the nucleoside is adenosine.
4. The method of claim 1 wherein the amino acid or amino acids are
selected from a group consisting of N-acetylcysteine, glycine,
arginine, proline, glutamate, serine, alanine, histidine, leucine,
methionine, phenylalanine and tryptophan.
5. The method of claim 1 wherein the steroid is dexamethasone.
6. The method of claim 1 wherein the donor organ is a mammalian
heart, liver, kidney, pancreas or any other organ or tissue.
7. The method of claim 1 wherein the refrigeration preservation
solution has a pH between 7.0 and 7.5.
8. The method according to claim 1, including the further steps of
removing the preserved donor organ from a refrigeration temperature
above 0.degree. C.; and transplanting it.
9-23. (canceled)
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/471,028, filed May 15, 2003, and U.S.
Provisional Application No. 60/465,114 filed Apr. 23, 2003, both of
which are incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to organ storage systems.
More particularly, the invention relates to solutions and methods
for preserving donor organs and storing them for extended periods
of time before transplantation or other use in the future.
BACKGROUND OF THE INVENTION
[0003] One of the greatest problems in donor organ transplantation
is the storage and preservation of organs between the time of
harvest from a donor and the time of transplantation into a
recipient. The amount of time that can lapse between the two events
is quite limited because the cells and tissues of the donor organ
deteriorate over time, even if they are stored at refrigerated
temperatures. Once harvested, cells and tissues are deprived of the
oxygen that is required to maintain internal metabolism and cell
volume integrity. To counteract the ill effects of low oxygen,
standard techniques for modern organ preservation involve the
exposure of a harvested organ to preservation solutions at cold
temperatures not below 0.degree. C. Although colder temperatures
are a solution to oxygen deprivation in donor organ tissue, they
present their own problems. Cold or hypothermic conditions may lead
to cellular damage including a reduced ability to generate energy,
maintain cell volume integrity, and also swelling and/or cell
death.
[0004] A widely used preservation solution is commonly known as
University of Wisconsin (UW) solution or Viaspan, which is
manufactured by DuPont. However, the preservation of donor organs
using Viaspan is generally limited to a 36-hour period in kidneys
before the organs begin to deteriorate. For example, if kidneys are
perfused with UW solution and packed on ice, surgeons will attempt
to use them within 24 hours but not later than 36 hours after
harvesting. A principal problem however is that the viability of
the donor kidney decreases over time of storage so that by 36 hours
there is at least some damage to the tubular cells. This generally
results in decreased viability of the kidney cells so that urine
production and proper kidney function are delayed after transplant.
As a result, artificial kidney function or dialysis is generally
required for full recovery of a recipient after
transplantation.
[0005] Storage of organs at sub-zero temperatures is not possible
or extremely difficult because the tissue and water in the organ
usually freezes. These relatively lower temperature ranges cause
damage or destruction to the cells and tissues. Today there are
some solutions currently available for organ storage purposes such
as Viaspan, but their capacity to store organs effectively is
generally limited. There is a need for improved solutions and
methods for effective organ preservation for extended periods of
time.
SUMMARY OF THE INVENTION
[0006] The invention describes solutions and methods for preserving
donor organs for use in transplantation or other medical purposes
in the future. In accordance with one aspect of the invention, a
variety of storage methods at different temperatures are provided.
The invention provides for example a first series of methods for
cold storage or storage at refrigerator temperatures (about
0.degree. to about 6.degree. C.), and a second series of methods
for storage at sub-zero temperatures as low as about -20.degree.
C., which is generally the equivalent to a refrigerator freezer
temperatures, or even lower temperatures including cryopreservation
temperatures that drop to as low as about -80.degree. C. Other
aspects of the invention provide preservation solutions that can be
designed to provide low temperature organ storage benefits
including reduction of interstitial edema and endothelial swelling.
These solutions can also provide antioxidant and anti-proteolytic
protection, can preserve proper intracellular ion concentration,
and can offer an energy source to support cellular functions
including the Krebs cycle.
[0007] Other goals and advantages of the invention will be further
appreciated and understood when considered in conjunction with the
following description and accompanying drawings. While the
following description may contain specific details describing
particular embodiments of the invention, this should not be
construed as limitations to the scope of the invention but rather
as an exemplification of preferable embodiments. For each aspect of
the invention, many variations are possible as suggested herein
that are known to those of ordinary skill in the art. A variety of
changes and modifications can be made within the scope of the
invention without departing from the spirit thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0008] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0009] FIG. 1 is an overall flowchart illustrating the operation of
an embodiment of the invention that provides methods for organ
preservation and transplantation.
[0010] FIG. 2 is a flowchart illustrating the operation of two
embodiments of the invention wherein a donor organ can be stored at
refrigeration temperatures as in FIG. 2A or at freezing
temperatures as in FIG. 2B.
[0011] FIG. 3 is a flowchart illustrating the operation of
different embodiments of the invention wherein a preserved donor
organ can either be removed from cold storage or refrigeration
temperatures and transplanted as in FIG. 3A, or thawed from freezer
temperatures as in FIG. 3B and transplanted.
[0012] FIG. 4 is a table that lists the composition of a
refrigeration preservation solution, Solution #1, provided in
accordance with another aspect of the invention. In addition, a
range of concentrations is provided to illustrate some other
variations of the ingredients that may be used for Solution #1.
[0013] FIG. 5 is a table that lists the components of the loading
pre-freezer preservation solution, Solution #2, which may be used
before treatment with a cryopreservation solution. In addition, a
range of concentrations for these components is provided to
illustrate some other alternatives of Solution #2.
[0014] FIG. 6 is a table that lists another embodiment of the
invention that provides a cryopreservation solution, Solution #3.
In addition, a range of concentrations is provided to illustrate
some other variations of solution ingredients that may be used for
Solution #3.
[0015] FIG. 7 is a table that lists the composition of a washing
solution, Solution #4. In addition, a range of ingredient
concentrations is provided to illustrate some other variations of
Solution #4 that may be used in accordance with this aspect of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Current organ preservation, storage and transplantation
procedures are limited because organs are so vulnerable to damage
after removal from a donor. Once harvested, cells and tissues are
deprived of the oxygen that is required to maintain internal
metabolism and cell volume integrity. This low oxygen state is
called ischemia and leads to hypoxia, which prevents oxygen from
being delivered to the organ tissue. Without oxygen, cellular
tissue can suffer injury as cell metabolism fail and individual
cells can be subject to swelling or inflammation.
[0017] To counteract the ill effects of ischemia, standard
techniques for modern organ preservation involve the exposure of a
harvested organ to preservation solutions at cold temperatures not
below 0.degree. C. This treatment essentially creates hypothermic
conditions that reduce a cell's need for metabolic oxygen.
Components of the solution and the cold environment combine to
protect the cell from ischemic conditions and thereby prevent the
onset of injury. This procedure is known as cold flush
preservation, in which the preservation solutions are designed to
eliminate chemical potential gradients across the cell membranes of
the cells composing the organ. By doing so the solution tends to
mimic the intracellular environment and prevent the donor organ
cells from activating metabolic pathways. Although hypothermia is a
solution to oxygen deprivation in donor organ tissue, it presents
its own problems. The cells of an organ preserved under hypothermic
conditions lose their ability to source of ATP, and therefore
cannot produce the energy required to regulate the sodium-potassium
pump, which is one of the most important modulators of internal
cell volume. Also the hypoxic environment induces the release of
intracellular calcium and elevated concentrations of calcium can
lead to subsequent activation of multiple metabolic inflammatory
pathways. As a result, the cells may exhibit endothelial cell
swelling, a loss of blood vessel integrity, including the reduction
in the internal diameter of blood vessels called a vasospasm, and
even cell death in tubules.
[0018] One of the most widely used solutions in organ preservation
and storage is known as University of Wisconsin (UW) solution or
Viaspan, which is manufactured by DuPont. However, preservation of
donor organs using Viaspan is generally limited to a 36-hour period
in kidneys before the organs begin to deteriorate.
[0019] In general, the current limitations on donor organ
preservation time seriously hamper the capacity of organ
transplantation procedures. The 36-hour time frame allowed for
kidneys does not always provide sufficient time for accurate
cross-matching of donors and recipients, which is needed to
increase the chances of a successful transplant. In addition, the
time required for transnational and trans-international transport
of donor organs and of recipients may exceed the viability period
of organs preserved under current procedures.
Methods for Preservation Storage and Transplantation
[0020] The invention provides improved methods and solutions for
storing organs for future medical uses such as organ transplants
into a recipient. In one aspect of the invention, methods are
provided using various preservation solutions and cryopreservation
solutions to prepare and store a donor organ after it is harvested.
The preserved organ can then be placed in storage at an appropriate
temperature for prolonged periods of time that can be greater than
36 hours. With respect to another embodiment of the invention, when
the preserved organ is required for transplantation, the
combination of a preservation solution and a washing solution can
be utilized to thaw the organ from storage and transplant it into a
recipient.
[0021] FIG. 1 is an overall flowchart illustrating the operation of
one embodiment of the invention that provides organ preservation
and transplantation methods. At step 101, a mammalian organ is
removed or harvested from a donor. It may be a liver, kidney,
pancreas, heart or any other type of mammalian organ or tissue.
Step 102 represents the application of a preservation solution on
to a harvested donor organ. In another embodiment of the invention,
it may be preferable to have the solution perfused through the
donor organ. In step 103, the donor organ is prepared for storage
at an appropriate temperature. The donor organ can also be
maintained in the preservation solution and placed in storage. This
aspect of the invention provides for the application, perfusion,
infusion or immersion of the donor organ into a cryopreservation
solution before storing the donor organ at an appropriate
temperature.
[0022] At the time a donor organ becomes available, a potential
recipient may not yet have been identified. One embodiment of the
invention addresses such a problem as follows. The donor organ may
be prepared as above in steps 101-103, and then stored at an
appropriate temperature as in step 104. At a later date when the
appropriate recipient for the stored donor organ becomes available,
it can be thawed and transplanted as in step 105. One embodiment of
the invention utilizes a washing solution that is followed by a
preservation solution to thaw the stored organ in preparation for
the transplantation procedure.
[0023] FIG. 2 provides two flowcharts each illustrating an
embodiment whereby a donor organ can be stored at a different
temperature. The flowchart in FIG. 2A illustrates a series of steps
in accordance with this aspect of the invention for preserving a
donor organ at refrigeration temperatures, which are defined herein
as between approximately 0.degree. C. and 6.degree. C. Step 201
involves the cooling to 2.degree. and 4.degree. C. of Solution #1,
which is a refrigeration preservation solution provided in
accordance with another aspect of the invention. This mixture can
have a pH of 7.0 to 7.5, and can contain a variety of ingredients
such as a hydrophilic polymer, a saccharide, a vinyl polymer, a
calcium ion flux inhibitor, a dihydrofolate reductase inhibitor, a
bacteriostatic, antibacterial agent, a nucleoside, amino acids,
salts, an energy source for the citric acid cycle, a steroid
analogue, a membrane stabilizer, and a diuretic. Step 202
represents the harvesting of a donor organ. After cooling and
harvesting, the organ is perfused with Solution #1 as shown in step
203. Following perfusion, the organ is immersed in Solution #1 as
shown in step 204 and stored at 2.degree. and 4.degree. C. for 36
hours before transplantation, as shown in step 205.
[0024] The flowchart in FIG. 2B describes yet another embodiment of
the invention for storage of donor organs at freezer temperatures,
defined herein as between approximately -1.degree. and -80.degree.
C. Step 206 represents the cooling of both Solution #2, a loading
pre-freezer preservation solution, and Solution #3, a
cryopreservation solution. Solution #2 may contain a hydrophilic
polymer, a saccharide, a vinyl polymer, a calcium ion flux
inhibitor, a dihydrofolate reductase inhibitor, a bacteriostatic,
antibacterial agent, a nucleoside, amino acids, salts, an energy
source for the citric acid cycle, a steroid analogue, a membrane
stabilizer, and a diuretic. Solution #3 contains the same
ingredients as Solution #2 but also contains a number of
cryopreservatives, including glycerol, propanediol, an alcohol and
a cryoprotectant agent. A quantity of Solution #2 is cooled to a
temperature between 2.degree. and 4.degree. C. In addition, a
quantity of Solution #2 and #3 is further cooled to a temperature
between 0.degree. and 2.degree. C. As represented by step 207, a
needle such as a 27 g needle is then inserted into the isolated
arterial system of the organ before removal from the donor, and
Solution #2 cooled at 2.degree. to 4.degree. C., is infused via the
needle for approximately 1 minute. In step 208 the organ is removed
from the donor and immersed in Solution #2 which is cooled to
0.degree. and 2.degree. C., and maintained at that temperature for
30 minutes. In step 209, the organ is kept at 0.degree. to
2.degree. C. and a quantity of Solution #3 cooled to 0.degree. and
2.degree. C. is gradually infused via the needle. Then the donor
organ is immersed in Solution #3 cooled to 0.degree. and 2.degree.
C. for 30 minutes. Following this incubation, the donor organ is
stored in Solution #3 at a temperature below 0.degree. C. as in
step 210.
[0025] In another embodiment of the invention as shown in FIG. 2B,
an additional step can be provided that follows step 210. The donor
organ can be then transferred to cryofreezer temperatures, which
can be defined as about -80.degree. C. or lower as shown in step
211. However, it is preferable for the donor organ to be stored at
-20.degree. C. for at least 8 hours before it is transferred to
lower temperatures such as -80.degree. C.
[0026] FIGS. 3A-B provide flowcharts that illustrate the
methodology and operation of alternative embodiments of the
invention. A preserved organ can either be removed from
refrigeration temperatures and transplanted as shown in FIG. 3A. In
FIG. 3A, the donor organ is removed directly from storage at
refrigeration temperatures in step 301 and transplanted into a
suitable recipient in step 302. Alternatively, an organ can be
thawed from freezer temperatures, and subsequently transplanted as
indicated in FIG. 3B. FIG. 3B provides the steps for
transplantation of a donor organ stored at freezer temperatures.
Step 303 illustrates the first requirement of cooling Solution #1
and Solution #4 to a temperature between 2.degree. C. and 4.degree.
C. The organ is then removed from freezer temperature storage in
step 304 and perfused with cooled Solution #4 as shown in step 305.
Solution #4 is a washing solution containing a hydrophilic polymer,
a saccharide, a vinyl polymer, a calcium ion flux inhibitor, a
dihydrofolate reductase inhibitor, a bacteriostatic, antibacterial
agent, a nucleoside, amino acids, salts, an energy source for the
citric acid cycle, a steroid analogue, a membrane stabilizer, and a
diuretic. Following step 305, the organ is perfused with cooled
Solution #1 according to step 306. Step 307 represents the
transplantation of the organ into a suitable recipient.
[0027] An alternate embodiment of the invention provides suitable
solutions and methods for organ storage at cryofreezer
temperatures. The steps described in FIG. 3B can be first preceded
by an additional step. A preserved donor organ can be removed from
storage at cryofreezer temperatures and placed at freezer
temperatures for 8 hours or more. After this period, the donor
organ may be transplanted following steps 304-308 in FIG. 3B.
Solutions Used for Preservation, Storage and Transplantation
[0028] One of the most widely used solutions in organ preservation
and storage is known as University of Wisconsin (UW) solution or
Viaspan, which is manufactured by DuPont. However, preservation of
donor organs using Viaspan is generally limited to a 36-hour period
in kidneys before the organs begin to deteriorate. The solutions
described herein may allow for significantly longer storage
periods.
[0029] A variety of organ preservation and storage solutions are
provided herein in accordance with invention. These preservation
solutions may contain one or more of the following ingredients: a
large molecule hydrophilic polymer used for cellular protection of
the organ, agents for reducing interstitial edema or fluid buildup
inside cells, an energy source for cellular functions, agents for
maintaining cellular ion concentrations including a variety of
salts, and series of one or more amino acids that can help prevent
proteolysis and to scavenge free radicals as antioxidants. Other
solution additives may include cell membrane stabilizers and
anti-inflammatory agents.
[0030] The table in FIG. 4 lists a solution provided in accordance
with another aspect of the invention, Solution #1, a refrigeration
preservation solution. In addition, a range of concentrations is
provided to illustrate some other embodiments that may be used for
Solution #1. Solution #1 includes for example polyethylene glycol
(PEG), which is a large molecular hydrophilic polymer used to
protect the cells of the donor organ by preventing the passage of
extracellular solutes through an organ cells' membranes. The PEG
from Sigma-Aldrich, product P2263, may be preferably used but any
comparable or equivalent chemical can be used in place of PEG.
Polyvinylpyrrolidone or PVP-40 is a large molecular vinyl polymer.
PVP-40 can be used in a manner similar to PEG. PVP-40 protects
donor organ cells from an influx of excess solutes. Its large size
generally serves to prevent solute entry. A preferable form of
PVP-40 from Sigma-Aldrich, product P0930, or any other comparable
chemical may be used. Sucrose is a disaccharide and as a large
molecule also functions to prevent solute entry into the cells of
the donor organ. It also helps reduce the amount of interstitial
edema, or fluid buildup, inside the cells. Another ingredient of
Solution #1 is verapamil, which is a calcium ion influx inhibitor
for preventing the entry of extracellular calcium ions into the
donor organ cells. Veraparnil may protect donor organ cells by
preventing an elevation of intracellular calcium concentration,
which can limit the activation of inflammatory pathways after long
storage preservation periods. Moreover, it has been observed that
verapamil can also provide protection by down-regulating
infiltration of neutrophils or other immune response elements.
Lopez-Neblina F, et al. "Mechanism of protection of verapamil by
preventing neutrophil infiltration in the ischemic rat kidney" J.
Surg. Res. (March 1996) Volume 61(2), pages 469-72. The
trimethoprim ingredient consists of a solution containing 16 mg/ml
of treimthoprim and 80 mg/ml of sulfamethoxazole. Both are
antibacterial agents used to prevent infection of the donor organ.
Adenosine is a nucleoside that plays a role in metabolic energy
transfers. It serves as another energy source in Solution #1. Each
listed salt MgSO.sub.4, NaCl, KCl, MgCl can be present in Solution
#1 and used to preserve the proper intracellular concentration of
ions. Proper ionic gradients across the donor organ cell membranes
are maintained through the use of these salts. The amino acids,
glycine, arginine, serine, proline, glutamine and N-acetylcysteine
are used to prevent proteoloysis and to scavenge free radicals as
antioxidants. In addition, acetylcysteine itself enhances the
production of the enzyme glutathione, which is a powerful
antioxidant. Pyruvate is present in Solution #2 as the primary
energy source for the donor organ cells. It is the main input into
the citric acid cycle, which allows cells to utilize oxygen for
cellular respiration and the generation of energy. Lidocaine is a
local anesthetic used to stabilize cell membranes and to some
extent, to prevent ischemic and reperfusion damage, as well as
subsequent swelling of the donor organ cells. Dexamethasone is a
steroid that functions as an anti-inflammatory agent. It helps
reduce endothelial cell swelling. Ethacrynate is a diuretic that
serves to reduce interstitial edema or fluid buildup.
[0031] FIG. 5 provides a table illustrating another embodiment of
the invention, Solution #2. Solution #2 is a loading pre-freezer
preservation solution that includes an illustrated list of
ingredients that vary within a range of concentrations. This
solution contains a higher concentration of polyethylene glycol
(PEG) than Solution #1 because the storage conditions will be lower
than 0.degree. C. The additional PEG may provide additional
cryoprotection at these temperatures. In addition, the large
molecular size of PEG, sucrose and PVP-40 provide protection
against the influx of extracellular solutes into the donor organ
cells, and also produce a slight dehydration that allows better
cryoprotection. Verapamil serves as a calcium ion influx inhibitor,
just as it did in Solution #1. Verapamil is a phenylalkylamine
calcium channel blocker. There are a number of classes of calcium
channel blockers that might be used in place of verapamil. For
example, diltiazem (a benzothiazepine), nicardipine, nifedipine, or
nimodipine (all dihydropyridines), bepridil (a
diarylaminopropylamine ether) and mibefradil (a
benzimidazole-substituted tetraline) may all serve the same
function in Solutions #1-#4. Trimethoprim is a dihydrofolate
reductase inhibitor and is used as a bactericidal to stop folic
acid production in bacteria. Other bactericidals may be used in its
place, such as those in the following classes: penicillins,
cephalosporins, and aminoglycosides. Sulfamethoxazole serves as an
anti-microbial agent just as it did in Solution #1. This agent is
one of a group of drugs called sulfonamides, which prevent
bacterial growth in the body. Other members of this group may be
substituted for sulfamethoxazole in Solutions #1-4, such as
sulfadiazine. Adenosine and the salts MgSO.sub.4, NaCl, KCl, MgCl,
all serve the same function as they did in Solution #1 but the
higher concentration of NaCl causes a slight dehydration that is
protective in nature because it decreases the amount of water in
the cells and by doing so limits the formation of ice crystals. The
amino acids, glycine, arginine, serine, proline, glutamine and
N-acetylcysteine act as anti-proteolytic agents and/or
antioxidants. Pyruvate inputs into the citric acid cycle, lidocaine
stabilizes the donor organ cell membranes, dexamethasone provides
anti-inflammatory protection and ethacrynate helps to reduce
interstitial edema.
[0032] FIG. 6 is a table that lists another embodiment of the
invention, Solution #3, which is a cryopreservation solution. A
range of concentrations is provided to illustrate some other
variations of Solution #3 that can be used in accordance with the
invention. As in Solution #2, the PEG concentration can be higher
to cope with the lower temperatures at which the organ will be
stored. PEG, sucrose and PVP-40 play a similar role a
cryopreservants and their large molecular size prevents the entry
of extracellular solutes. Other disaccharides besides sucrose may
be substituted, such as lactose, maltose, isomaltose, or
cellobiose. In addition, PVP-40 may be substituted with alternate
macromolecules, such as the complex colloidal Dextran-40 or
gelatin. Trimethoprim and sulfamethoxazole are added as
anti-microbial agents, adenosine and pyruvate are added as energy
sources, and the salts are added to preserve safe ionic gradients
across the donor organ cell membranes. As in Solutions #1 and #2,
the amino acids, glycine, arginine, serine, proline, glutamine and
N-acetylcysteine are added to prohibit proteolysis of cellular
proteins. These amino acids, particularly serine and proline, may
be substituted with other amino acids of a similar function, such
as alanine, histidine, leucine, methionine, phenylalanine and
tryptophan. The members of the latter group all have
anti-proteolytic activity. As in Solutions #1 and #2, lidocaine
serves to stabilize cell membranes, dexamethasone prevents
inflammation of the donor organ and ethacrynate reduces
interstitial edema and the initial induction of diuresis or urine
excretion. Solution #3 is a cryopreservation solution and can
therefore contain ingredients not found in Solutions #1 and #2. For
example, a variety of anti-freeze components can be included such
as four different types in Solution #3. Two are glycerol and
ethanol, both alcohols with a low freezing point, which allows them
to prevent the organ from freezing at temperatures below freezing.
Propanediol, another anti-freeze agent, is a third ingredient and
dimethyl sulfoxide (DMSO) is the fourth. Besides being an organic
solvent that keeps all the ingredients in solution, DMSO is a
well-known cryoprotective agent that lowers the freezing point and
allows a slow cooling rate. It is effective at preventing donor
organ cells from freezing at subzero temperatures.
[0033] It is important to note that the NaCl concentration is
generally higher in Solution #2 than in Solutions #3 and #4 because
the organ is being dehydrated slightly. This removal of water from
the cell will reduce the likelihood that ice crystals will be
formed during the freezing process of the organ. The DMSO in
Solution #3 also plays a role by replacing the lost water from the
cell. As DMSO has a lower freezing point, the cell will be less
likely to form ice crystals.
[0034] FIG. 7 is a table that lists another embodiment of the
invention, Solution #4, which is a washing solution. In addition, a
range of concentrations is provided to illustrate some other
variations of Solution #4 provided herein. The washing solution
substantially contains the same macromolecules, PEG, PVP-40 and
sucrose to help prevent an influx of extracellular solutes.
Verapamil is present to block calcium ion entry, trimethoprim and
sulfamethoxazole are present as anti-microbial agents, pyruvate and
adenosine are present as energy sources, the same salts preserve
proper ionic gradients, and lidocaine stabilizes the cell
membranes. The adrenal cortical steroid, dexamethasone is included
to stop inflammation of the organ. However, other such steroids may
be substituted for dexamethasone, such as hydrocortisone,
aldactone, or aristocort. Ethacrynate is included in Solution #4 as
a diuretic to reduce interstitial edema.
[0035] Solution #4, the washing solution, generally contains the
same ingredients as found in Solutions #1 and #2, except the
concentration of NaCl is typically lower than in Solution #1. This
allows Solution #4 to wash out the cryosolution, Solution #3, and
rehydrate the cell. The DMSO from Solution #3 that had replace
water in the cell prior to freezing is washed out and water is
added back as the temperature of the stored organ is restored to
normal.
[0036] It is important to note that the concentrations and the
ranges of concentrations of each ingredient of Solutions #1-#4 are
relatively low compared to other organ preservation/storage
solutions currently available, such as Viaspan. Viaspan contains
ingredients in higher concentrations than the solutions described
herein. Higher ingredient concentration however generally increases
the toxicity of the solution to the donor organ.
Kit for Organ Preservation, Storage and Transplantation
[0037] In accordance with yet another aspect of the invention, each
of the Solutions #1, #2, #3 and #4 described herein can be stored
in separate containers within a single package or a kit. Such kits
may be marketed to entities engaged in the business or activities
of harvesting, storing, preserving and/or transplanting donor
organs. These kits may include instructions for methods of organ
preparation and storage as described elsewhere herein. In
accordance with an aspect of the invention, various types of
mammalian organs can be treated and prepared for storage over
extended periods of time. While experiments were conducted in the
following examples with rat kidneys, the invention here can be
applied to human subjects or other mammals and their respective
organs.
Organ Storage at Cold Storage or at Refrigerator Temperatures
EXAMPLE 1
[0038] Donor rat kidneys were harvested by usual methods and
perfused with Solution #1 (described below), comprised of
ingredients listed in the table below. This solution is a mixture
designed to reduce interstitial edema and endothelial swelling,
contains antioxidants and anti-proteolytic amino acids, and
preserves proper intracellular concentrations of ions including
magnesium, sodium, and potassium. This solution is comprised of
macromolecules, impermeable molecules, amino acids, energy sources
that support the Krebs cycle, and salts. The pH of this solution is
about 7.3+/-0.1.
[0039] The kidneys that were perfused with Solution #1 were stored
in a refrigerator at about 2.degree. to 4.degree. C. for 36 hours
and then transplanted into anephric rats using a published method.
The transplanted kidneys were observed to quickly turned pink with
fresh blood and immediately began producing urine.
[0040] In contrast, donor kidneys perfused with UW solution and
stored for 36 hours in the refrigerator did not turn as bright with
blood nor did they make appropriate amounts of urine after
transplantation.
[0041] Organs perfused or stored in a solution such as this
Solution #1 or equivalent solution can be stored for extended
periods and recover and function rapidly after transplantation.
Specifically, kidneys can be stored for 36 hours, 40 hours, 48
hours, 50 hours, or longer and then transplanted and are viable and
they function.
Organ Storage at Sub-Zero Temperatures (Below 0.degree. C.)
[0042] Two solutions are used in sequence to perfuse or wash organs
in preparation for sub-zero storage (Solution #2 and Solution #3
described below). After storage at sub-zero temperature, Solution
#3 is washed out with Solution #4 (described below) followed by
washing with Solution #1, and the organ is transplanted. These
solutions #2, #3, and #4 are similar to Solution #1 with some
modifications for use in cryopreservation (storage at sub-zero
temperatures). Solution #3 (cryosolution) contains cryoprotectants.
All solutions are cold at about 2 to 4.degree. C. when used for
perfusing or washing organs.
EXAMPLE 1
[0043] The donor rat kidneys were perfused for 30 minutes at
2.degree. to 4.degree. C. with Solution #2 which is the same as
Solution #1 except that sodium chloride is at 2.5% and amounts of
PEG and sucrose are increased. Then the donor kidneys were perfused
at 2.degree. to 4.degree. C. for 30 minutes with Solution #3
(cryopreservation solution). The kidneys were placed into a
refrigerator's freezer at about -20.degree. C. Kidneys were stored
for days, but could be stored for much longer periods or weeks or
months.
[0044] The kidneys did not freeze because of the use of the
perfusion solutions and the system utilized as indicated above.
[0045] After removing the kidneys from the freezer, the kidneys
were perfused with a washing solution (Solution #4), which is
identical to Solution #2 except that the amount of NaCl is 10 mM.
Finally, kidneys were perfused for 30 minutes with Solution #1 and
transplanted. The transplanted kidneys were observed to turn pink
in color as they were reperfused with blood and immediately
produced urine.
[0046] The solutions used in this example are comprised as listed
in the tables below.
[0047] By following the method stated above and using the invented
solutions described, or essentially equivalent solutions, organs
can be stored at sub-zero temperatures for extended periods of time
of days, weeks, or months, until used for transplantation by
following the stated method of this invention.
[0048] Solutions listed below are all at a pH of about 7.3+/-0.1 in
a phosphate buffer comprised of 950 ml water, 10 ml 1M monobasic
phosphate, and 40 ml 1M dibasic phosphate.
EXAMPLE 2
[0049] The following is a description of a procedure in accordance
with the invention for organ storage of four rat kidneys at
temperatures as low as about -80.degree. C. The same solutions
described herein may be applied for storage at temperatures
generally in this range.
[0050] Each of the kidneys can be dissected in a standard manner
isolating the renal flow with silk. The venous drainage is opened
which may be accomplished usually by sectioning the renal vein. A
27 g needle may be inserted in the isolated arterial system so
flushing of the kidney can start immediately. It is desirable to
avoid air circulation or the introduction of air bubbles into the
system during this process. An infusion with 1 ml of volume of the
loading hypertonic solution (Solutions #2) may be performed in
about 1 minute at 2 to 4.degree. C. Then the organ is removed and
immediately immersed in the same solution at 0 to 2.degree. C., and
is maintained at this temperature in the loading solution for 30
minutes. Then the infusion of 10 ml of the subzero solution
(anti-freeze Solution #3) is initiated at 0.300 ml per minute, and
the kidney is maintained at 0 to 2.degree. C. during this infusion.
The graft immersed in the antifreeze solution is subsequently
placed in the freezer at -20.degree. C. for 12 hours, and then
placed in the cryo-freezer at -80.degree. C. for
cryo-preservation.
[0051] To warm up the organ after its storage at -80.degree. C. for
cryo-preservation, it is initially placed in a -20.degree. C.
surrounding for 12 hours (regular freezer). Afterwards it is placed
at a 0 to 2.degree. C. environment, and then 10 ml of the washing
solution (Solution #4) is introduced at 0.300 ml per minute. The
kidney is maintained at 0 to 2.degree. C. during this infusion.
Subsequently the graft is placed in a refrigerator at 2 to
4.degree. C. for at least one hour. Each of the kidneys were then
transplanted in a customary fashion and then allowed circulation of
blood. The Reperfusion Damage Index was measured during the first
15 minutes. The graft was removed at 60 minutes of reperfusion and
placed in buffered formal in 10% for histology (H&E) and
pictures taken at 600.times.. The histology yielded positive
results with intact glomeruli and the cellular structure in the
kidneys were generally maintained following organ storage at
-80.degree. C. in accordance with the invention herein.
[0052] As evidenced by the examples mentioned above, the methods
and solutions herein provide organ storage at temperatures as low
as -20.degree. C. or even lower at about -80.degree. C. These
techniques may be applied to other mammalian organs including human
kidneys.
[0053] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *