# Patent

## Title
Tissue preservation method with polyoxyethylene/polyoxypropylene copolymer

## Abstract

Tissue preservation media comprising a polyoxyethylene/polyoxypropylene copolymer are used to preserve tissues and organs for storage and transplantation. In particular embodiments, the polyoxyethylene/polyoxypropylene copolymer is Pluronic F68 or FLOCOR (CRL-5861; purified poloxamer 188), and the medium is Steinhardt medium, polyoxyethylene/polyoxypropylene copolymer-supplemented Optisol GS or polyoxyethylene/polyoxypropylene copolymer-supplemented ViaSpan.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation of Ser. No. 11/015,180, filed Dec. 17, 2004 now U.S. Pat. No. 7,371,513, which is continuation-in-part of Ser. No. 10/738,331, filed Dec. 17, 2003, now U.S. Pat. No. 7,087,369.


This work was supported by National Institutes of Health Grants AR44066 and EY 13436. The U.S. government has rights in any patent issuing on this application.


FIELD OF THE INVENTION
The field of the invention is tissue preservation media.
BACKGROUND OF THE INVENTION
We have previously disclosed that the mechanism of cell membrane repair requires an active process of calcium regulated exocytosis (Steinhardt R A, Bi G, Alderton J M. Cell Membrane Resealing by a Vesicular Mechanism Similar to Neurotransmission. Science 1994; 263: 390-393). This lead to our idea that membrane breaks could be repaired by artificial means under conditions where normal metabolism is curtailed, such as the storage of donated tissues for transplantation.
We have devised new tissue preservation media, including corneal preservation media, and tested them against the American standard, Optisol GS (Bausch & Lomb). Optisol was developed for low temperature storage of corneas and other eye tissues by Richard L. Lindstrom and Debra Skelnik (U.S. Pat. Nos. 5,104,787; 5,407,669). This tissue preservation solution was originally marketed by Chiron Ophthalmics, Irvine, Calif. Bausch & Lomb acquired Chiron Corp.'s vision-care product line in 1997. Optisol GS is commercially available from: Bausch & Lomb Surgical, Inc. (San Dimas, Calif.). Our media also outperform ViaSpan (Barr Laboratories, Pomona, N.Y.) in side-by-side tissue preservation studies.
Relevant Literature
Togo T, Alderton, J M, Bi G, Steinhardt R A. The mechanism of facilitated cell membrane resealing. Journal of Cell Science 1999; 112: 719-731; Togo T, Krasieva T B, Steinhardt R A. A Decrease in Membrane Tension Precedes Successful Cell-Membrane Repair. Molecular Biology of the Cell 2000; 11: 4339-4346; Bi G, Morris R L, Liao G, Alderton J M, Scholey J M, Steinhardt R A. Kinesin- and Myosin-driven Steps of Vesicle Recruitment for Ca2+-regulated Exocytosis. The Journal of Cell Biology 1997; 138(5): 999-1008). Greenbaum A, Hasany S M, Rootman D. Optisol vs Dexsol as storage media for preservation of human corneal epithelium, Eye. 2004 May; 18(5):519-24′ Peter H. Laverty, et al., A Preliminary Study of Intravenous Surfactants in Paraplegic Dogs: Polymer Therapy in Canine Clinical SCI, Journal of Neurotrauma December 2004, Vol. 21, No. 12: 1767-1777.
SUMMARY OF THE INVENTION
The invention provides methods and compositions for preserving tissues and organs, particularly corneal tissue, for storage and transplantation. The compositions include media comprising a polyoxyethylene/polyoxypropylene copolymer in an amount sufficient to increase tissue storage-ability, particularly concentrations of about 0.5 to 5 mg/ml. In particular embodiments, the polyoxyethylene/polyoxypropylene copolymer is Pluronic F68 or FLOCOR(CRL-5861; purified poloxamer 188), and the medium is Steinhardt medium, Optisol GS supplemented with the polyoxyethylene/polyoxypropylene copolymer, or ViaSpan supplemented with the polyoxyethylene/polyoxypropylene copolymer.
In other embodiments, the invention provides kits for preserving tissue including:
kits comprising a premeasured amount of a disclosed preservation medium;
kits comprising a premeasured amount of a disclosed preservation medium, and recorded instructions copackaged or associated with the premeasured amount describing use of the medium to preserve a tissue; and
kits for making a disclosed medium comprising premeasured amounts of the ingredients, or a plurality of the ingredients; and recorded instructions copackaged or associated with the premeasured amounts describing how to combine the ingredients to make the medium.
The invention also provides methods of making a disclosed medium, comprising the step of combining the recited ingredients to make the medium; and methods of using a disclosed medium comprising incubating a tissue in the medium, preferably cornea tissue, preferably at 4 degrees C., and preferably for at least 7 days, typically not more than 21 days. The incubating step is typically followed by an assessment of post-incubation survival utility to determine whether the tissue has retained its suitability for its intended use, typically transplantation.


DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
The invention provides methods and compositions for preserving tissues and organs, particularly transplantable tissues and organs including kidney, liver, pancreas, heart, lung, bone marrow, skin grafts, and particularly corneal tissue, for warm and cold storage and transplantation. The compositions include media comprising or supplemented with a polyoxyethylene/polyoxypropylene copolymer in an amount sufficient to increase tissue storage-ability, particularly concentrations of about 0.5 to 5 mg/ml. Tissue storage-ability may be measured by post-storage cell and tissue viability, transplantability, etc. In particular embodiments, the polyoxyethylene/polyoxypropylene copolymer is Pluronic F68 or FLOCOR(CRL-5861; purified poloxamer 188), and the medium is Steinhardt medium, Optisol GS supplemented with the polyoxyethylene/polyoxypropylene copolymer, or ViaSpan supplemented with the polyoxyethylene/polyoxypropylene copolymer.
Optisol GS and ViaSpan are commercially available products. Steinhardt medium is a tissue preservation medium based on fundamental physiological principles and on research findings from our laboratory regarding maintenance of cell membrane integrity. After testing many different iterations, we have established a particular formulation that provides exceptional tissue preservation of a panel of human and large animal organs and tissues, including cornea, kidney and heart tissue. The medium is typically provided in sterile solution; the ingredients and their final sterile solution concentrations are as follows:
potassium sulfate K2SO4 (5 mM)
L-aspartic acid (110 mM)
magnesium sulfate (1.2 mM)
calcium hydroxide (2.0 mM)
potassium phosphate monobasic KH2PO4 (20 mM)
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (5 mM)
ethylenediaminetetraacetic acid (EDTA) (1 mM)
taurine (20 mM)
zinc sulfate (1 microM)
N-(tert-butyl)hydroxylamine HCL (200 microM)
dextran M.W. 60,00-90,000 (5.5% w/v)
N-acetyl-cysteine (0.5 mM)
gentamycin sulfate (0.02% w/v)
creatine phosphate (5 mM)
Lipid Concentrate (GIBCO/INVITROGEN #11905-031) (1% v/v) at pH adjusted to 7.45 with 1 M KOH, and having an osmolarity of 310 to 320 mOsmoles.
The ionic composition of Steinhardt medium is designed to approximate intracellular ionic composition for potassium, sodium, and magnesium ions.
The HEPES (SIGMA #H-9136)(N-2-Hydroxyethylpiperazine-N′-2-ethanesulfonic acid) provides a physiologically compatible buffer widely used in cell culture media. The potassium salts of the sulfate ion (K2SO4) and the monobasic phosphate ion (H2PO4) are used in place of the sodium salts of these ions that are typically used in cell culture media.
L-aspartic acid is provided based on observations in our laboratory that cells microinjected with solutions containing high concentrations of the potassium salt of L-aspartic acid continue to grow and divide. L-aspartic acid is a key amino acid in cellular metabolism that links the urea cycle to the citric acid cycle. Steinhardt medium uses 110 millimolar L-aspartic acid to yield 110 millimolar aspartate. 13 millimolar aspartate has been used in cardioplegia (Rosenkranz et al., Journal of Thoracic Cardiovascular Surgery 1986; 91: 428-435).
Potassium hydroxide is used to adjust the pH, causes the acids such as L-aspartic acid and ethylenediaminetetraacetic acid (EDTA) to dissolve, and also provides high potassium ion levels characteristic of the intracellular environment.
The chelator ethylenediaminetetraacetic acid (EDTA) is used to buffer the level of calcium, magnesium, and zinc ions. Extracellular free calcium ion at about 1 millimolar and free magnesium ion at about 1.2 millimolar result in optimal cell membrane repair (Steinhardt R A, Bi G, Alderton J M. Cell Membrane Resealing by a Vesicular Mechanism Similar to Neurotransmission. Science 1994; 263: 390-393). This paper demonstrates that cell membrane repair is an active biological process that requires calcium-dependent exocytosis of vesicles at the cell membrane. Excessive free magnesium ion antagonizes the vesicle exocytosis and membrane resealing. EDTA also chelates trace amounts of iron and copper that may be present in the medium. Iron and copper are known to be catalytic for free radical reactions that are damaging to cells (Evans et al. Catalytic Metal Ions and the Loss of Reduced Glutathione from University of Wisconsin Preservation Solution. Transplantation 1996; 62(8): 1046-1049)
The zinc ion is an essential cofactor for DNA repair and other enzymatic cellular processes (Chimienti F, Aouffen M, Favier A, Seve M. Zinc homeostasis-regulating proteins: new drug targets for triggering cell fate. Current Drug Targets 2003; 4(4):323-38).
The amino acid taurine is cytoprotective (Eppler B, Dawson R Jr. Cytoprotective role of taurine in a renal epithelial cell culture model. Biochemical Pharmacology 2002; 63: 1051-1060). It is an abundant intracellular amino acid and provides multiple homeostatic functions (Lourenco et al., Taurine: a conditionally essential amino acid in humans? An overview in health and disease. Nutr Hosp. 2002; 17(6):262-70).
The 5.5% dextran is consistent with the corneal preservation medium described by McCarey B E and Kaufman H E. Improved Corneal Storage. Investigative Ophthamology 1974; 13(3): 165-173; and U.S. Pat. No. 5,370,989. Optisol GS contains 1% dextran. The dextran used has an average molecular weight between 64,000 and 76,000.
The N-acetyl-cysteine is a cell permeable precursor of glutathione. (Ceconi C, Curello S, Cargnoni A, Ferrari R, Albertini A, Visioli O. The role of glutathione status in the protection against ischemic and reperfusion damage: effects of N-acetyl cysteine. Journal of Molecular and Cellular Cardiology 1988; 20(1): 5-13). The 0.5 mM concentration is consistent with that used in U.S. Pat. No. 5,370,989.
N-tert-Butyl hydroxylamine is an antioxidant that prevents free radical-induced toxicity to mitochondria (Liu J, Atamna H, Kuratsune H, Ames B N. Delaying brain mitochondrial decay and aging with mitochondrial antioxidants and metabolites. Ann N Y Acad. Sci. 2002; 959:133-66). Also, Atamna et al. N-t-Butyl hydroxylamine is an antioxidant that reverses age-related changes in mitochondria in vivo and in vitro. FASEB J. 2001; 15(12):2196-204; also, U.S. Pat. No. 6,455,589. N-t-butyl hydroxylamine, a hydrolysis product of alpha-phenyl-N-t-butyl nitrone, is more potent in delaying senescence in human lung fibroblasts. J Biol. Chem. 2000; 10; 275(10):6741-8.
The Lipid Concentrate (GIBCO/INVITROGEN catalog number 11905-031) is formulated for use in suspension cultured cells grown for recombinant protein production, and allows for serum-free growth that facilitates purification of the secreted recombinant protein. An ingredient in the Concentrate is Pluronic F68 (BASF, Ludwigshafen, Germany; also called Poloxamer 188 and Lutrol F68, RheothRx, (Glaxo) and Flocor (CytRx)), a non-ionic surfactant included by GIBCO to help dissolve the lipids.
Pluronic F68 also provides a beneficial effect on cell survival in suspension culture (Kilburn D G, Webb F C. The Cultivation of Animal Cells at Controlled Dissolved Oxygen Partial Pressure. Biotechnology and Bioengineering 1968; 10: 801-814), and has been used to protect animal cells from damage caused by shear and the effects of sparging (the aeration bubbles used in bioreactors; Hua et al., Critical Reviews in Biotechnology 1993; 13(4): 305-28). Its use has also been proposed as an intravenous agent for treatment of sickle cell disease and acute vaso-occlusive disorders, and to preserve organs for transplantation (e.g. U.S. Pat. No. 5,990,241).
Pluronic F68 is a mixture of polyoxyethylene and polyoxypropylene; commercial preparations are mixtures of the polymers with a range of molecular weight from 7680 to 9510. See BASF Technical Bulletin “Pluronic Block Copolymer NF Grades (Poloxamer NF Grades).” U.S. Pat. No. 6,359,014 describes one purification method for commercial preparations of Pluronic F68. FLOCOR(CRL-5861; purified poloxamer 188; CytRx Corporation, Los Angeles, Calif.) is another purified form of Pluronic F68; see, e.g. WO9216484; U.S. Pat. No. 5,990,241; Moghimi S M, et al., 2004, Biochim Biophys Acta, 1689(2):103-13.
The subject media may also be used to improve organ transplantation success by perfusing the organ before surgical removal from the donor. This timing is important because we have previously shown that cell membranes must be repaired within 30-90 seconds for a cell to survive a disruption of this protective interface (Steinhardt R A, Bi G, Alderton J M. Cell Membrane Resealing by a Vesicular Mechanism Similar to Neurotransmission. Science 1994; 263: 390-393).
The invention provides methods of making and using the subject media, and various kits for making and using the subject media. Methods of making a disclosed medium include methods comprising combining the recited ingredients to make the medium; and methods of using a disclosed medium include methods comprising incubating a tissue in the medium, preferably cornea tissue, preferably at 4 degrees C., and preferably for up to at least about 7 days, typically not more than about 21 days.
Kits for using the subject media include kits comprising a premeasured amount of the disclosed medium; kits comprising a premeasured amount of a disclosed medium and instructions copackaged or associated with the premeasured amount describing use of the medium to preserve a tissue in the medium, preferably cornea tissue, preferably at 4 degrees C., and preferably for up to at least about 7 days, typically not more than about 21 days. Preferred instructions disclose that a subject medium can provide, or has been shown to provide cold cornea tissue storage for at least about 7 days, preferably for up to about 21 days. The premeasured amounts are preferably contained in a container labeled with the instructions.
Kits for making a disclosed medium include kits comprising premeasured amounts of the recited ingredients, one or more of the ingredients, or a plurality of the ingredients, and recorded instructions copackaged or associated with the premeasured amounts describing the medium and/or how to combine the ingredients to make the medium. The premeasured amounts are preferably contained in a container labeled with the instructions.
EXAMPLES
Evaluations of the endothelial cell layer in bovine corneas in different media. post-incubation survival utility or usefulness for transplantation is defined as less than 3% dying and less than 1.5% missing. Missing cells leave a gap in the layer; calcein-AM staining for esterase activity is used to determine cell health. Transplant surgeons typically use missing cell counts and morphology alone as the index of health and post-incubation survival utility.







TABLE I







Corneas stored in Optisol at 4° C.


All experiments were performed double-blind.


Conclusion: Optisol does not provide useful


tissue beyond 6 days cold storage.











Days at

percent
percent
total cells


4° C.
Percent live
dying
missing
examined














6
98.0
2.0
0.0
892


6
98.6
1.0
0.3
937


9
88.6
5.4
6.0
1338


9
82.3
11.6
6.1
1386


12
67.1
16.5
16.3
1393


12
70.7
16.4
12.9
566
















TABLE II







Comparison of Steinhardt medium with a defined composition


Optisol medium. Corneas stored at 4° C. in Optisol(F) defined


media (1) vs. Steinhardt medium. Conclusion: Steinhardt medium


stored corneas retain utility at least 21 days.

















Total


Days




cells


at


Percent
Percent
exam-


4° C.
Medium
Percent Live
Dying
Missing
ined


















4
Steinhardt
99.4
(1083)
0.6
(7)
0

1090


4
Steinhardt
100
(776)
0

0

776


4
Optisol(F)
99.0
(576)
1.0
(6)
0

582


4
Optisol(F)
95.0
(623)
3.4
(22)
1.7
(11)
656


10
Steinhardt
97.8
(840)
2.0
(17)
0.2
(2)
859


10
Steinhardt
100
(635)
0

0

635


10
Optisol(F)
92.6
(598)
5.9
(38)
1.5
(10)
646


10
Optisol(F)
91.0
(1154)
4.9
(62)
4.1
(52)
1268


14
Steinhardt
98.7
(869)
0.9
(8)
0.4
(3)
880


14
Steinhardt
99.3
(1216)
0.7
(9)
0

1225


14
Optisol(F)
85.3
(1039)
8.4
(102)
6.3
(77)
1218


14
Optisol(F)
93.3
(1228)
4.0
(52)
2.7
(35)
1315


21
Steinhardt
96.4
(888)
2.6
(24)
1.0
(9)
921


21
Steinhardt
97.8
(1271)
1.6
(21)
0.6
(8)
1300


21
Optisol(F)
63.6
(743)
13.5
(158)
22.9
(268)
1169


21
Optisol(F)
66.7
(933)
17.2
(241)
16.1
(225)
1399





(1) Optisol(F) is formulated as commercial Optisol, but is freshly made and provides better results than commercial Optisol after several days of cornea storage.


















Formulation I: Steinhardt Cornea Preservation Medium












Manufacturer
[ ]
MW
for 200 ml





RT Ingredients


Potassium Sulfate K2SO4
Mallinckrodt
5 mM
174.26
0.174 g


L-Aspartic Acid
Sigma A-7219
110 mM
133.10
2.93 g


Magnesium Sulfate
Sigma-1880
1.2 mM(1)
246.5
59 mg


Calcium Hydroxide
Allied Chemical
2.0 mM(2)
74.1
29.6 mg


Potassium Phosphate,
Mallinckrodt
20 mM
136.1
0.544 g


Monobasic KH2PO4


HEPES
Sigma H-9136
5 mM
238.3
0.238 g


EDTA
Sigma EDS
1 mM
292.2
58.44 mg


Taurine
Sigma T-8691
20 mM
125.1
0.50 g


Zinc Sulfate
Allied Chemical
1 μM(3)
287.54
200 μl of 2.88 mg/10 ml







(1) free Mg2+: 1.2 mM at 4 C; 1.17 mM at 36 C.


(2) free Ca2+: 1 mM at 36 C.


(3) free zinc: 1.4 × 10−12 M at 36 C.; 1.7 × 10−12 M at 4 C.; stock is 1 mM (1000x)


Adjust to about pH 7.4 using 1 M KOH.


Add 1 M KOH slowly to dissolve the salts completely.











N-(tert-Butyl)
Aldrich 19,475-1
200 μM
125.6
5 mg


hydroxylamine HCL


Refrigerator ingredients:


Dextran
Sigma D-4751(4)
5%
68,800
10 g


N-acetyl-cysteine
Sigma A-9165
0.5 mM
163.2
16.3 mg


Gentamycin Sulfate
Biowhittaker


20 mg


Freezer ingredient:


Creatine Phosphate
Calbiochem 2380
5 mM
255.1
0.255 g







(4) Takes about 1 hr. to dissolve at RT with stirring


Adjust volume to 190 ml. Adjust pH to 7.45. Bring volume to 200 ml. Check


osmolarity. = 314 mOsmoles. Filter sterilize 0.2 μ


Add 1 ml lipid concentrate (Gibco 11905-031(5))/100 ml medium after the medium is


aliquoted into the corneal storage vials.








(5) Lipid Concentrate Composition:
100x


Arachidonic Acid
2 mg/liter


Cholesterol
0.22 g/liter


DL-alpha-Tocopherol Acetate
70 mg/liter


Linoleic, Linolenic, Myristic, Oleic,
All 10 mg/liter


Palmitoleic, Stearic, and Palmitic Acids


Pluronic F-68
100 g/liter


Tween 80
2.2 g/liter



















Formulation II: Optisol(F) Medium









Ingredient; storage.
Source
Quantity





Medium 199, with Earle's salts,
GIBCO 12340-030
490 ml


with L-glutamine, with 2,200 mg/L


sodium bicarbonate, with 25 mM


HEPES; 4 C.


Chondroitin sulfate A, sodium salt
Calbiochem
12.5 g/500 ml


from bovine trachea; RT.
#230687


Dextran, clinical grade.
Sigma D-4751
  5 g/500 ml


Av. MW 64.76 K; 4 C. and


desiccated.







Stir and intermittently shake the above ingredients at RT and protect from


light for several hours or until solids are dissolved. Then add:









Choline, Chloride salt; RT.
Sigma C-1879
 0.5 mg/500 ml


Folic acid; RT.
Sigma F-8758
 0.5 mg/500 ml


i-Inositol myo-inositol; RT.
Sigma I-7508
  1 mg/500 ml


Inosine; RT.
Sigma I-4125
  5 mg/500 ml


L-Asparagine; RT, desiccated.
Aldrich
 6.6 mg/500 ml



#A9,300-3


Riboflavin; RT.
Sigma R-4500
0.05 mg/500 ml


Nicotinamide; RT.
Sigma −0636
 0.5 mg/500 ml


L-glutamine; RT.
Sigma G-3126
 100 mg




additional/




 500 ml


Vitamin B12; 4 C. and desiccated.
Sigma V-6629
0.68 mg/500 ml


D-Pantothenic acid; 4 C. and
Sigma P-5155
 0.5 mg/500 ml


desiccated.


Adenosine, free base 4 C. and
Sigma A-9251
0.75 mg/500 ml


desiccated.


Alpha-tocopherol phosphate,
Sigma T-2020
  25 mg/500 ml


disodium salt; 4 C. and desiccated.


Pyridoxal HCl; −20 C. and
Sigma P-9130
 0.5 mg/500 ml


desiccated.


Gentamycin sulfate; 4 C. liquid
BioWhittaker
  50 mg/500 ml


stock @50 mg/ml
#17-518Z


Sodium pyruvate liquid; 4 C.
GIBCO #11360-
  5 ml stock/


and protected from light; yields
070
500 ml


1 mM final concentration.


2-mercaptoethanol liquid stock;
GIBCO BRL
0.45 ml/500 ml


4 C. Thiamine, HCl Sigma T-1270;
#21985-023
1 microliter/ml


rapidly destroyed above pH 5.5;


1000× stock in MES = 2[N-


Morpholino] ethane Sulfonic Acid


buffer Sigma −8250, pH 5.5.


Yields 0.5 mg Thiamine, HCl/500


ml Add just before cornea is placed


in the medium.









In another series of demonstrations, the preservation solutions, Steinhardt Medium and Optisol-like Medium were made using the ingredients in Formulation I and III, respectively. Initial experiments used dextran from Leuconostoc mesenteroides of average molecular weight of 64,000-76,000 (Sigma D 4751). Data from these experiments are shown in Table 4. The source of the dextran used for Steinhardt Medium in Table 3 was Amersham Biosciences (Catalog #US14495, Lot 108318, average molecular weight 60,000-90,000). Optisol-GSTM was purchased from Bausch & Lomb Surgical, Inc., San Dimas, Calif.
Bovine eyes were obtained from Rancho Veal, Petaluma, Calif. Two to 3 hours elapsed between death of the cattle and excision of the cornea with a 1 to 2 millimeter (mm) scleral rim from the enucleated eyes. During the approximately 80 min. transit time, 12 bovine eyes were packaged without medium in a plastic bag that rested on bubble paper above crushed ice in a styrofoam box. (Submersing the corneas in cold Ringer's during transport did not improve the endothelial preservation). The bovine corneas were dissected in a sterile laminar flow biological cabinet and then transferred to the corneal preservation media at 22° C. Each cornea was transferred with the epithelial side down to an ultraviolet light-sterilized Nalgene polycarbonate screw-capped jar # 2116-0015 (Nalge Nunc International, Rochester, N.Y.) filled with 22 ml of medium. Corneas in the preservation media were then stored in a refrigerator for 3 to 21 days before being assayed for endothelial cell viability.
The viability of the corneal endothelium was quantified with the vital stain Calcein AM (Molecular Probes, Eugene, Oreg.). The hydrophobic and non-fluorescent Calcein AM readily diffuses across the plasma membrane and into the cytoplasm. In the cytoplasm, the AM moiety is hydrolyzed by cellular esterases to release the highly fluorescent Calcein. Live cells exhibit a bright green fluorescence with an especially bright nucleus. The bright nuclei are easily counted. Dead cells are dark, and dying cells are pale green. Calcein AM was purchased in 50 microgram aliquots. An aliquot was freshly mixed with cell culture-tested dimethylsulfoxide (DMSO, Sigma D2650, Sigma-Aldrich, St. Louis, Mo.) to yield a 4 millimolar (mM) Calcein AM stock. To aid dispersion of the hydrophobic Calcein AM in the Ringer's solution, 1 microliter (μl) of the Calcein stock was thoroughly mixed with 1 μl of Pluronic F-127 solution (25%, weight to weight, in DMSO). Pluronic F-127 was from Molecular Probes. This mixture was thoroughly mixed with 2 ml Ringer's in a 35 mm diameter Corning # 430165 petri dish (Corning, N.Y.). The cornea was gently transferred, epithelial side down, in the tipped petri dish to ensure that the Calcein AM in Ringer's covered the endothelial layer. The Ringer's was 138 mM Sodium Chloride; 2.7 mM Potassium Chloride; 12.4 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES); 5.6 mM D-glucose (dextrose); 1.06 mM Magnesium Chloride; 1.8 mM Calcium Chloride, pH 7.25 with Sodium Hydroxide (all salts from Sigma). The half cornea in Calcein AM Ringer's was placed in a 36° C. incubator for 1 hour. The half cornea was then carefully transferred, endothelial side down, to a 24×60 mm number one coverglass. A Zeiss IM-35 inverted fluorescence microscope (Thornwood, N.Y.) equipped with excitation and emission filters for Green Fluorescent Protein (filter set XF100-2 from Omega Optical, Brattleboro, Vt.) was used to illuminate the half cornea. The lens used was a Fluor 10 0.5 160/0.17 10× (Nikon, Melville, N.Y.). The central endothelial layer was photographed using a Nikon Coolpix 5000 digital camera with 3× optical zoom. 8 by 11 inch images were printed using a S900 color printer (Canon, Lake Success, N.Y.).
For analysis, each photograph was overlaid with a sheet of transparency film. A felt-tip lab pen was used to mark cells as they were counted and scored as bright (live), dim (dying), or dark (dead or missing). Dark areas were outlined. Later these dark, outlined areas were placed over normal, bright areas so that the number of missing cells could be estimated by counting the bright nuclei encompassed by the outline. The percentages of cells in each category were used to compare endothelial viability among preservation media.
Bovine corneas stored in a new experimental cold-preservation solution, Steinhardt Medium, show enhanced long-term viability of the endothelial cell layer compared to corneas stored in Optisol-GSTM Medium (Table 3). After 4 days at 4° C., the corneal endothelial qualitative viability is similar for corneas stored in Steinhardt Medium or in Optisol-GSTM. After 14 days at 4° C., we found that that dead and dying cells are present in corneas preserved in Optisol-GSTM, but virtually absent from corneas stored in Steinhardt Medium. By 21 days, corneas stored in Optisol-GSTM show conspicuous cell loss and a high percent of pale green dying cells. In contrast, corneas stored in Steinhardt Medium for 21 days at 4° C. show very few dead or dying cells. Black areas that are smaller than the diameter of an endothelial cell were probably created when adjacent cells repaired their cell membrane following mechanical damage.
A progressive and significant advantage for Steinhardt Medium over Optisol-GSTM Medium is seen (Table 3) when large numbers of cells in many corneas are scored as live, dying or missing (dead). A large advantage for Steinhardt Medium over Optisol-GSTM Medium is seen for corneas stored longer than 4 days. The percent viability is 99.7 in Steinhardt Medium versus 98.5% in Optisol-GSTM Medium after 4 days at 4° C. By 10 days in the different storage media, corneas in Steinhardt Medium have an endothelial viability of 98.9% while corneas stored in Optisol-GSTM Medium have only 93.5% viability. At 14 days, corneas stored in Steinhardt Medium still retain an average high endothelial cell viability of 98.5% while Optisol-GSTM-stored corneas have an average endothelial cell viability of 86.9%. Finally, corneas stored in Steinhardt Medium for 21 days average 96.3% endothelial cell viability compared to about 67.9% endothelial viability for corneas stored in Optisol-GSTM Medium.
The advantage of Steinhardt Medium over Optisol-GSTM may be largely due to the addition of Pluronic F68 (Poloxamer 188). This non-ionic surfactant facilitates the resealing of disrupted membranes in the absence of normal mechanisms of cell membrane repair (Togo et al. 1999). In order to compare the effect of Pluronic F68 on endothelial membrane resealing at 4° C., we used a completely defined medium that is similar to Optisol-GSTM. This Optisol-like Medium was composed using the published patent literature (U.S. Pat. No. 5,104,787). Optisol-GSTM may contain proprietary ingredients that are undisclosed in the patent literature so we chose to work with a defined solution for this direct comparison of the effects of pluronic. A higher level of endothelial cell viability was maintained in the corneas stored in the defined medium with Pluronic F68 (Table 4). After 3 days at 4° C. in the Optisol-like Medium, the corneas stored in both the medium plus Pluronic F68 and the medium without Pluronic F68 were equally viable (98.8% vs 98.7%). A large difference in endothelial cell viability was evident after 6 days storage at 4° C.: the endothelial cell viability in the Optisol-like Medium plus Pluronic F68 was 99.0% compared to 93.2% for corneas stored in the Optisol-like Medium without Pluronic F68. The inclusion of Pluronic F68 in the preservation medium continued to maintain endothelial cell viability for corneas stored for 18 days at 4° C. (97.7% viable). In contrast, corneas stored for 18 days at 4° C. in an Optisol-like Medium without Pluronic F68 showed only 79.9% viability. Optisol-like Medium appears to be as good as or better than Optisol-GSTM and the addition of pluronic has a large positive effect on endothelial viability.
The solvent used to dissolve Pluronic F68 was dimethylsulfoxide (DMSO) at 0.5%. DMSO has been shown to have a beneficial effect on cell survival in some systems, but had no effect on corneal endothelial cell survival in our experiments using bovine corneas. Corneas stored for 10 days in an Optisol-like Medium without Pluronic F68, but with 0.5% DMSO, showed a similar loss in endothelial cell viability (90.3% viability with DMSO vs 90.1% viability without DMSO).










Formulation III: Optisol-like Medium








Ingredient (source)
quantity/liter












Medium 199 (GIBCO 12340-030), with Earle's salts, with
980
ml


L-glutamine, with 2,200 mg/L sodium bicarbonate, with


25 mM HEPES.


Chondroitin sulfate A, sodium salt (Calbiochem #230687)
25
g


from bovine trachea.


Dextran, clinical grade. Av. MW 64.76 K (Sigma D-4751).
10
g


Stir the above ingredients at RT and protected from light


until solids are dissolved. Then add:


Choline, Chloride salt (Sigma C-1879).
1
mg


Folic acid (Sigma F-8758).
1
mg


i-Inositol (myo-inositol) (Sigma I-7508).
2
mg


Inosine (Sigma I-4125).
10
mg


L-Asparagine (Aldrich #A9,300-3).
13.2
mg


Riboflavin (Sigma R-4500).
0.1
mg


Nicotinamide (Sigma N-0636).
1
mg


L-glutamine (Sigma G-3126).
200
mg


Vitamin B12 (Sigma V-6629).
1.36
mg


D-Pantothenic acid (Sigma P-5155).
1
mg


Adenosine, free base (Sigma A-9251).
1.5
mg


Alpha-tocopherol phosphate, disodium salt (Sigma T-2020).
50
mg


Pyridoxal HCl (Sigma P-9130).
1
mg


Gentamycin sulfate (BioWhittaker # 17-518Z)
100
mg


stock @ 50 mg/ml.


Sodium pyruvate liquid (GIBCO #11360-070), 1 mM final
10
ml


concentration.


2-mercaptoethanol liquid (GIBCO BRL #21985-023).
0.9
ml


Thiamine, HCl (Sigma T-1270); 1000x stock in
1
mg


2[N-Morpholino] thane Sulfonic Acid buffer


(Sigma M-8250), pH 5.5*.


Add the above thiamine stock to the medium in the corneal


storage vial at 1 microliter/ml


Just before the cornea is placed in the medium.





*Thiamine is rapidly destroyed in solutions above pH 5.5.













TABLE 3







Corneal Endothelial Viability











Days at 4 C.
Medium
Percent Live
Percent Dying
Percent Missing














4
Steinhardt
99.7 +/− 0.14*
0.26 +/− 0.13* 
0.02 +/− 0.02


4
Optisol
98.5 +/− 0.42*
1.12 +/− 0.25* 
0.42 +/− 0.28


10
Steinhardt
98.9 +/− 0.40*
0.90 +/− 0.36**
 0.20 +/− 0.16*


10
Optisol
93.5 +/− 1.14*
4.12 +/− 0.50**
 2.32 +/− 0.49*


14
Steinhardt
98.5 +/− 0.29*
1.12 +/− 0.23**
 0.42 +/− 0.16**


14
Optisol
86.9 +/− 2.15*
7.56 +/− 0.93**
 5.52 +/− 0.80**


21
Steinhardt
96.3 +/− 0.61*
2.68 +/− 0.45* 
 1.00 +/− 0.16*


21
Optisol
67.9 +/− 1.38*
11.12 +/− 2.03* 
20.92 +/− 1.49*





Viability was assessed by the accumulation of fluorescent Calcein in the corneal endothelial cells. Live cells were bright green with bright nuclei. Dying cells were pale. Missing cells were dark. Four or five bovine corneas were assayed for each medium at each preservation time. A total of 41,657 central corneal endothelial cells were scored. The statistical analysis software was InStat 2.0 from GraphPad.com.


*indicates significant difference between Optisol and Steinhardt media (P < 0.05).


**indicates very significant difference between Optisol and Steinhardt media (P < 0.01).













TABLE 4







The effect of 1 mg/ml Pluronic F68 on corneal endothelial viability.












Days
Pluronic
Percent
Percent
Percent
Total Cells


at 4° C.
F68
Live
Dying
Missing
Examined















3
plus
98.8
1.2
0
2496


3
minus
98.7
1.3
0
2479


6
plus
99.0
1.0
0
2424


6
minus
93.2
4.2
2.6
2852


9
plus
98.6
1.2
0.2
2885


9
minus
79.4
17.8
2.8
3467


12
plus
98.9
1.1
0.1
3385


12
minus
80.8
9.0
10.2
3605


18
plus
97.7
0.6
1.7
3107


18
minus
79.7
7.1
13.2
3333









Bovine corneas were stored at 4 C in Optisol-like defined media. These experiments were done double blind. Calcein accumulation was used to classify cells as live, dying, or missing. Cell counts are pooled from 3 corneas for each treatment, except for 18 days, which are from 2 corneas.











Formulation IV. ViaSpan (Barr Laboratories, Pomona, NY)









Component
Concentration
Function





Raffinose
30 mM
Impermeant: suppression of



(17.83 g/L)
hypothermic cell swelling


Lactobionic
100 mM
Impermeant: suppression of


acid
(35.83 g/L)
hypothermic cell swelling


Pentafraction
50 g/L
Colloid: reduction of interstitial


(hydroxyethyl

edema and endothelial cell


starch)

swelling


Glutathione
3 mM (0.992 g/L)
Antioxidant


Allopurinol
1 mM (0.136 g/L)
Inhibition of xanthine oxidase activity




and purine metabolism/reduction of




oxygen free radicals


Adenosine
5 mM (1.34 g/L)
Restoration of high energy phosphate


Potassium
25 mM (3.4 g/L)
pH buffer: maintenance of


phosphate

intracellular sodium and potassium




concentrations: restoration of high




energy phosphate


Magnesium
5 mM (1.23 g/L)
Preservation of intracellular


sulfate

magnesium concentration


Potassium
100 mM
Maintenance of intracellular sodium


hydroxide
(5.61 g/L)
and potassium concentrations


Sodium
27 mM
Maintenance of intracellular sodium


hydroxide

and potassium concentrations





Solution is pH adjusted to 7.4 with either sodium hydroxide or hydrochloric acid. Final: Sodium = 29 mM; Potassium = 125 mM; mOsm/L = 320 + 10 Immediately prior to use, to formulate the final solution, aseptically add: Penicillin G 200,000 units, regular insulin 40 units, and dexamethasone 16 mg.






The foregoing descriptions of particular embodiments and examples are offered by way of illustration and not by way of limitation. All publications and patent applications cited in this specification and all references cited therein are herein incorporated by reference as if each individual publication or patent application or reference were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

## Claims

1. A method of preserving tissue, comprising the step of:
incubating a tissue in a tissue preservation medium at 4 degrees C. for between 7 and 21 days, the medium containing a polyoxyethylene/polyoxypropylene copolymer in final concentration of 0.5 to 5 mg/ml.

2. The method of claim 1 wherein the polyoxyethylene/polyoxypropylene copolymer is FLOCORTM (also known as CRL-5861TM or purified poloxamer 188).

3. The method of claim 1 wherein the olyoxyethylene/polyoxypropylene copolymer is Pluronic F68TM.

4. The method of claim 1 wherein the medium is Steinhardt MediumTM.

5. The method of claim 1 wherein the medium is Optisol GSTM supplemented with the polyoxyethylene/polyoxypropylene copolymer.

6. The method of claim 1 wherein the medium is ViaSpanTM supplemented with the polyoxyethylene/polyoxypropylene copolymer.

7. The method of claim 1 further comprising the step of verifying post-incubation survival and transplant utility of the tissue.

8. The method of claim 1 wherein the polyoxyethylene/polyoxypropylene copolymer is Pluronic F68TM, and the final concentration is 1 mg/ml.