Cell, tissue or organ storage solution

The present invention relates to a method of maintaining viability of a cell, tissue or organ. The method involves maintaining the cell, tissue or organ in contact with a storage solution comprising transferrin and selenium at a subambient temperature in a non-frozen state. The invention further relates to a storage solution suitable for use in the above-described method. In one embodiment, the solution comprises insulin, transferrin, hydrocortisone, selenium and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

BACKGROUND OF THE INVENTION 
Technical Field 
The present invention relates in general, to the preservation of organs, 
tissues and cells during storage and transport and, in particular, to a 
method of maintaining organs, tissues and cells in a viable state prior to 
transplantation, and to a composition suitable for use in such a method. 
Background Information 
Ischemia, a localized tissue hypoxia resulting from partial or complete 
loss of blood circulation, ensues rapidly upon death of an organism. In 
designing a protocol for storing a tissue prior to transplantation, the 
susceptibility of the particular tissue to ischemia must be considered. 
One factor that influences the rate at which ischemia produces cellular 
injury, and subsequently cell death, is temperature. Kidneys, for example 
must be procured immediately after cessation of donor heartbeat, and can 
be stored for only 1-3 days at 0.degree.-4.degree. C., using current 
technology. The exact time is dependant upon whether or not continuous 
perfusion is employed. This is in contrast with bone marrow, which can 
tolerate at least 12 hours of warm ischemia post mortem and 3 days of cold 
ischemia at 0.degree.-4.degree. C. 
In order to extend the period for which particular cells, tissues and 
organs can be maintained in a state which will permit subsequent 
successful transplantation into a recipient host, new methods must be 
developed. One such method is provided by the present invention. 
SUMMARY OF THE INVENTION 
It is a general object of the invention to provide a method of maintaining 
cells, tissues and organs in a viable state. 
It is a specific object of the invention to provide a method of storing 
cells, tissues and organs at refrigerated temperatures prior to 
transplantation. 
It is another object of the present invention to provide a tissue storage 
solution that permits storage of cells, tissues and organs at refrigerated 
temperatures for periods of time longer than is possible using present 
clinically accepted solutions. 
Further objects and advantages of the present invention will be clear from 
a reading of the description that follows. 
The present invention relates to a method of delaying the detrimental 
effects of ischemia on organ, tissue and cell viability, and to a storage 
solution suitable for use in such a method. 
In one embodiment, the present invention relates to a method of storage 
comprising the steps of: 
i) contacting a cell, tissue or organ to be stored with a solution 
comprising transferrin and selenium; and 
ii) maintaining the cell, tissue or organ in contact with the solution at a 
sub-ambient temperature in a non-frozen state. 
In another embodiment, the present invention relates to a storage solution 
comprising insulin, transferrin, hydrocortisone, selenium and a Goodes 
buffer, for example, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 
(HEPES).

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a method of storing cells, tissues and 
organs, and to a storage solution suitable for use in such a method. The 
present storage method is such that viability of the material is 
maintained. Maintenance of viability permits subsequent successful 
transplantation of the stored material into a recipient host. 
In the present method, material to be stored is placed in contact with a 
storage solution comprising transferrin and selenium, advantageously at 
concentrations in the ranges of 2.5 .mu.g/ml to 10 .mu.g/ml and 2.5 ng/ml 
to 7.5 ng/ml, respectively. In a preferred embodiment, insulin and 
hydrocortisone are also present in the storage solution, advantageously at 
concentrations in the ranges of 2.5 .mu.g/ml to 7.5 .mu.g/ml and 25 ng/ml 
to 40 ng/ml, respectively. In a most preferred embodiment, a Goodes 
buffer, for example HEPES, is also present in the solution at a 
concentration in the range of 10 mM to 30 mM (corresponding to 2.92-8.85 
g/l in the case of HEPES). The inclusion of a Goodes buffer in the storage 
solution is particularly advantageous where storage for more than two days 
is required. For shorter storage periods, other pharmaceutically 
acceptable buffers, for example, a bicarbonate buffer, can be used. 
The above-described components of the tissue storage solution of the 
present invention can be present, for example, in a medium capable of 
supporting cellular metabolism in vitro at 37.degree. C. or the components 
can be present in a buffered physiological salt solution that is incapable 
of supporting cellular metabolism at 37.degree. C. Such salt solutions can 
include a carbohydrate source (for example, glucose). 
In the present method, cell viability is maintained by storing the cells, 
tissues or organs in the above-described solution at sub-ambient 
temperatures, in a non-frozen state. Advantageously, temperatures in the 
range of -4.degree. to 4.degree. C. are used. 
Materials suitable for storage according to the present method include, but 
are not limited to, heart, kidney, lung, liver, cornea, pancreas, skin, 
blood vessels, tendons, ligaments, bone, bone marrow, endocrine and 
exocrine glands, gametes, ova, nerves, gastrointestinal tract, ureter, 
bladder, or structures or cellular components derived from any of the 
above. Where intact organs are to be stored, such organs are flushed with 
the above-described solution prior to storage. 
The following non-limiting Examples further describe the present invention. 
EXAMPLE 1 
Comparison of Six Storage Solutions 
Confluent cultures of canine anterior cruciate ligament(ACL)-derived 
fibroblasts were placed, for five days at 0.degree.-4.degree. C., in: 
physiological saline (0.9% (w/v) NaCl) (designated NaCl); 2) a culture 
medium containing fetal calf serum (Dulbecco's Modified Eagle Medium plus 
10% v/v fetal calf serum) (designated PS); 3) a buffered physiological 
salt solution (0.10 g/l CaCl.sub.2 (anhydrous), 0.20 g/l KCl, 0.20 g/l 
KH.sub.2 PO.sub.4, 0.10 g/l MgCl.sub.2.6H.sub.2 O, 8.00 g/l NaCl, and 2.16 
g/l Na.sub.2 HPO.sub.4.H.sub.2 O) (designated DPBS); 4) Hanks Balanced 
Salt Solution as in Table I without hydrocortisone, insulin, transferrin 
and selenium and containing 15 mM HEPES instead of 25 mM (designated H); 
5) the solution of Table I (designated HITS); or 6) the solution of Table 
I plus chondroitin sulfate (25 g/l) and sucrose (47.922 g/l) (designated 
HITS.). 
At the end of this period, the solutions were removed from the cultures, 
the cells were washed, and placed in contact with fresh serum-free culture 
medium. After 2 hrs. of incubation in the serum-free medium, the cells 
were labeled with tritiated glycine at 37.degree. C. in a 5% CO.sub.2 and 
air incubator using a technique adopted from that detailed below in 
Example 2. The relative protein incorporation between the experimental 
groups is an indication of the level of cellular viability. 
TABLE I 
______________________________________ 
g/l 
______________________________________ 
CaCl.sub.2 (anhydrous) 0.14 
KCl 0.40 
KH.sub.2 PO.sub.4 0.06 
MgCl.sub.2.6H.sub.2 O 0.10 
MgSO.sub.4.7H.sub.2 O 0.10 
NaCl 8.00 
NaHCO.sub.3 0.35 
Na.sub.2 HPO.sub.4.7H.sub.2 O 
0.09 
D-Glucose 1.00 
Phenol Red 0.01 
Hepes 4.425 
Transferrin 0.005 
Insulin 0.005 
Selenium 0.000005 
Hydrocortisone 0.000036 
______________________________________ 
The results shown in FIG. 1 clearly indicate the superiority of the 
solution of the present invention (that given in Table I) over the others 
tested. (C=control--no storage at 0.degree.-4.degree. C.; storage 
medium=Dulbecco's Modified Eagle Medium plus 10% v/v fetal calf serum. 
EXAMPLE 2 
Comparison of Hydrocortisone, Insulin, Transferrin and Selenium-Containing 
Storage Solution and Euro-Collins Storage Solution 
Bisected human heart valve leaflets were placed in either the solution 
described in Table I or Euro-Collins solution at 4.degree. C. for 1-5 
days. (Euro-Collins=KH.sub.2 PO.sub.4 (2.05 g/l), KHPO.sub.4 (7.40 g/l), 
KCl (1.12 g/l), NaCO.sub.3 (0.84 g/l), and glucose (38.5 g/l).) After cold 
storage, the leaflets were washed and placed in tritiated glycine and 
incorporation of the isotope into protein was determine after 48 hrs of 
incubation using the following protocol: 
Tritiated Labelling of Tissue 
Day 1--Make up a 16 .mu.Ci/ml H-glycine solution in serum free Dulbecco's 
Modified Eagle Medium (DMEM). Cut tissue up into small pieces and place in 
a 5 ml snap-cap tube. Add 0.5 ml of the H-glycine to each tube. Incubate 
for 48 hours at 37.degree. C. 
Day 2 - Decant medium from the tubes and wash tissue quickly twice with 
phosphate buffered saline (PBS). Add PBS and let sit for 30 minutes. 
Remove PBS and add more PBS. Incubate overnight at 4.degree. C. 
Day 3--Decant PBS off of tissue and place tissue into 15.times.100 mm glass 
tubes. Wash for 15 minutes in alcohol, then wash for 15 minutes with 
ether. Remove ether and allow the tissue to dry for at least one hour. 
Weigh and record weight of tissue from each tube. Place all tissue into 
clean 12.times.75 mm glass tubes. 
Add 200 .mu.l H.sub.2 O to each tube and let rehydrate for 30 minutes to 1 
hour. 
Add 500 .mu.l 1M NaOH to each tube and place tubes in a heating block at 
60.degree. C. Allow 60 minutes incubation for valve tissue. 
Pipette samples into microtubes and sonicate twice for 20 seconds each time 
Centrifuge in microfuge for 2 minutes. 
Apply 100 .mu.l of each sample to glass fiber filter discs. Allow to dry 
for at least one hour. Move filter discs to glass scintillation vials and 
add 2 ml ice cold 10% trichloroacetic acid (TCA) to each vial. Refrigerate 
for 30 minutes minimum. Remove TCA and wash four times with 3 ml ice cold 
alcohol. Then wash twice with 3 ml ice cold ether. Allow filter discs to 
dry for at least one hour. Add 130 .mu.l H.sub.2 O to each filter disc. 
Add 1 ml Protosol to each vial. Vortex vigorously. Add 10 ml scintillation 
fluid and 100 .mu.l glacial acetic acid. Transfer vials into racks, place 
racks in counter and allow to dark adapt for 30 minutes before counting. 
Count each vial for 5 minutes. 
The results are summarized in FIG. 2. A total of 27 comparisons were done 
after 1-5 days of incubation. In 23 out of the 27 comparisons, the 
solution described in Table I was clearly superior. In only one instance 
did the Euro-Collins solution support protein synthesis levels greater 
than the solution described in Table I. In three cases, the numbers were 
similar. 
These results demonstrate a highly significant maintenance of cellular 
viability by the storage solution of the present invention, relative to 
the current clinically accepted alternative. 
EXAMPLE 3 
Effects of Removal of Hydrocortisone, Insulin, Transferrin and Selenium on 
Cell Storage at 4.degree. C. 
Human kidney-derived proximal tubule cells were plated on bovine type I 
collagen/fetal calf serum-coated Costar 24 well plates and placed in 
either complete solution (C) (see Table II) or in the solution shown in 
Table I from which either one or all of the following had been removed: 
hydrocortisone (H), insulin (I), transferrin (T), or selenium (S). The 
plates were then placed in a refrigerator at 4.degree. C. for 72 hours. 
Viability was assayed by the neutral red spectrophotometric assay 
described below in Example 4. 
TABLE II 
______________________________________ 
Complex culture medium used for testing HITS components. 
COMPONENTS mg/L 
______________________________________ 
INORGANIC SALTS: Vitamins 
CaCl.sub.2 (anhyd.) 
100 Biotin 0.00365 
CaCl.sub.2.2H.sub.2 O 
22 D-Capantothenate 
2.24 
CuSO.sub.4.5H.sub.2 O 
0.001245 Choline chloride 
8.98 
FeSO.sub.4 :7H.sub.2 O 
0.417 Folic acid 2.65 
KCl 311.8 i-Inositol 12.60 
MgCl.sub.2.6H.sub.2 O 
61 Niacinamide 2.0185 
MgSO.sub.4.7H.sub.2 O 
100 Pyridoxine HCl 
0.031 
NaCl 6999.5 Riboflavin 0.219 
NaHCO3 2438 Thiamine HCl 2.17 
Na.sub.2 HPO.sub.4.7H.sub.2 O 
134 Vitamin B.sub.12 
0.68 
ZnSO.sub.4.7H.sub.2 O 
0.4315 Pyridoxal HCl 
2.0 
Fe(NO.sub.3).sub.3.9H.sub.2 O 
0.05 
NaH.sub.2 PO.sub.4.H.sub.2 O 
62.5 
OTHER COMPONENTS 
D-Glucose 1401 
Hypoxanthine 2.05 
Linoleic acid 0.042 
Lipoic acid 0.105 
Phenol red 8.1 
Putrescine 2HC1 
0.0805 
Sodium pyruvate 
110 
Thymidine 0.365 
AMINO ACIDS: 
L-Alanine 4.45 
L-Arginine HCl 147.5 
L-Asparagine.H.sub.2 O 
7.505 
L-Aspartic acid 
6.65 
L-Cysteine 24 
L-Cysteine HCl.H.sub.2 O 
17.56 
L-Glutamic acid 
7.35 
L-Glutamine 365 
Glycine 18.75 
L-Histidine HC1.H.sub.2 O 
31.48 
L-Isoleucine 54.47 
L-Leucine 59.05 
L-Lysine HCl 91.25 
L-Methionine 17.24 
L-Phenylalanine 
35.48 
L-Proline 17.25 
L-Serine 26.25 
L-Threonine 53.45 
L-Tryptophan 9.02 
L-Tyrosine 38.70 
L-Tyrosine(disodium salt) 
-- 
L-Valine 52.85 
______________________________________ 
Data shown in FIG. 3 are expressed as the mean survival .+-.1 S.E. of 4-6 
experiments in percent of 37.degree. C. controls. 
EXAMPLE 4 
Influence of Divalent Cations (Ca.sup.+2 and Mg.sup.+3) on Cell Viability 
in the Presence of HEPES and Bicarbonate Buffers. 
In order to access the effects of HEPES and bicarbonate buffers on the 
viability of cells stored in the absence of divalent cations, human 
proximal tubule cells were plated on bovine type 1 collagen/fetal calf 
serum coated Costar 24 well plates and placed in the solutions indicated 
below in Table III (which solutions are based on that shown in Table I) 
for 24, 48, or 72 hours under cold (4.degree. C.) ischemic conditions. N=4 
for each solution in each of the four experiments performed. All data is 
expressed in Table III as a percent survival compared to a 37.degree. C. 
control as assessed by the neutral red spectrophotometric assay described 
as follows: 
Neutral Red Assay 
Highest quality Neutral Red dye was obtained from Aldrich Chemical Co. 
(Milwaukee, Wis.). A 0.5% solution was prepared in tris-buffered saline 
(TBS). A ten fold concentrated TBS stock was prepared as follows: to 1.0 
liter of distilled deionized water was added 24.2 g Trizma 7.7 (Sigma 
Chemical Co., St. Louis, Mo.), 68.0 g NaCl, 2.0 g KCl, 2.0 g 
MgCl.sub.2.6H.sub.2 O, and 1.0 g CaCl.sub.2 (anhydrous). The saline was 
then filter sterilized using a 0.22 .mu. nitrocellulose filter. The saline 
stock was diluted to 1 X using distilled deionized water. To prepare the 
Neutral Red solution, 0.5g Neutral Red was added to 100 ml 1X TBS 
solution, care being taken to minimize the light exposure of this 
photosensitive dye. The dye solution was filtered using Whatman No. 42 
paper just prior to use. 
Kidney tubule cells were gently washed 4X with TBS which had been warmed to 
37.degree. C. Following suction removal of the last saline wash, 0.5 ml of 
0.5% Neutral Red in TBS warmed to 37.degree. C. was added to each well. 
The plates were then floated in a covered 37.degree. C. water bath for 30 
min to allow maximum dye uptake. The unabsorbed Neutral Red solution was 
then aspirated off and the cells were washed 4X with cold TBS at 4.degree. 
C. The dye was extracted with cold 50% ethanol at 4.degree. C. for 15 min. 
A 0.15 ml aliquot was then drawn from each well and placed in the wells of 
Costar 96-well flat-bottom plates. Controls consisted of 50% ethanol 
blanks and 1X TBS blanks. Samples were read using a 450 nm filter on a 
Titertek Multiskan ELISA plate reader. Three rows of serial dye dilutions 
in 50% ethanol were used to generate a concentration curve. 
TABLE III 
______________________________________ 
BUFFER CATIONS DAY 1 DAY 2 DAY 3 
______________________________________ 
HEPES Ca, Mg 74% 61% 41% 
HEPES Ca 98% 93% 80% 
HEPES Mg 76% 60% 44% 
HEPES NONE 100% 97% 88% 
HCO.sub.3.sup.- 
Ca, Mg 79% 65% 45% 
HCO.sub.3.sup.- 
Ca 97% 89% 52% 
HCO.sub.3.sup.- 
Mg 80% 64% 48% 
HCO.sub.3.sup.- 
NONE 99% 92% 59% 
______________________________________ 
Ca.sup.2+ = CaCl.sub.2 (anhydrous) (0.14 g/l) 
Mg.sup.2+ = MgCl.sub.2.6H.sub.2 O [0.10 g/l] and MgSO.sub.4.7H.sub.2 O 
[0.10 g/l]- 
HEPES = 5.66 g/l (20 mM) 
HCO.sub.3.sup.- = NaHCO.sub.3 [0.35 g/l]- 
These results clearly demonstrate the superiority of HEPES buffer in the 
absence of magnesium or both calcium and magnesium after 3 days of 
storage. 
The foregoing invention has been described in some detail for purposes of 
clarity and understanding. It will be clear to one skilled in the art from 
a reading of the present disclosure that various changes can be made in 
form and detail without departing from the true scope of the invention.