CA2044494C - Methods and apparatus of a defined serumfree medical solution - Google Patents

Abstract

A defined serumfree medical solution for applications in Ophthalmology, that contains one or more cell nutrient supplements which maintains and enhances the preservation of eye tissues, including human corneal tissues at low temperatures (2°C to 15°C). This solution is composed of a defined aqueous nutrient and electrolyte solution, supplemented with a glycosaminoglycan(s), a deturgescent agent(s), an energy source(s), a butter system(s), an antioxidant(s), membrane stabilizing components, antibiolic(s), ATP precursors and nutrient cell supplements.

Description

wj '~~ ~2r ~ 2fl44494 Method and Apparatus for a Defined Serumfree Medical Sotutlon Background of the Invention 1. Fleid of the Invention:
The present invention relates to the preservation of eye tissue in a defined nutritive. aqueous medical solution, and more particularly, relates to the preservation and enhancement of human corneal llssue, specified as the 1 0 time between removal from the donor and transplantation.

2. Description of the Prior Art:
Keratoplasly, or transplantallon of the cornea, has baste elfective In 1 5 providing visual rehabilitation to many who sutler from corneal disorders.
This procedure has gained widespread acceptance but has been severely hampered by the universally Inconsistent availability of donor tissue. This problem made the development of a storage solution lmperalive. The development of MK~-preservation medium, and subsequent chondroitin 2 0 sulfate-containing media, has positively impacted the availability of quality donor tissue. Much research in this area has been undertaken with a view towards prolonging donor storage time and yet maintaining a viable endothelium, which is crucial to successful transpiantatlon. Storage of the cornea for up to 14 days ai 4°C has been reported, although the current 2 5 technology does not permit adequate tissue preservation beyond a few days.

w Storage longer than 96 hours is attended by epithelial decomposition and loss of corneal clarity, as demonstrated by increased swelling of the corneal slrorna. This stromal edema is attributed to the decreased maintenance of the barrier pump function of the corneal endothelium, a specific cell layer lining the corneal stroma.
The functional status of the endothelium and sustained corneal deturgescence after corneal preservation are of great clinical importance, and contribute primarily to the success of the surgical outcome. The ability of the cornea to maintain a relatively dehydrated state is essential to the 1 0 maintenance of corneal transparency. Corneal deiurgescence is an energy-dependent phenomenon performed primarily by the endothelial cells. In order for the cornea to remain viable, various enzymatic reactions must occur to carry out energy-dependent functions, maintained by high levels of ATI~.
The lower temperature of the 4°C corneal storage method reduces the 1 5 metabolic rate of the cornea, but the storage medium must still be able to support the basal requirements of the cornea. Thus, corneal storage media are a complex mixture of balanced sails, amino acids, energy sources, antioxidants, buffering agents, cell membrane stabilizers, giycosamino-gtycans, deturgescents and antibiotics. Temperature reduction changes the 2 0 membrane lipids, proteins and water structures, each of which could alter the active transport mechanism by hindering the ease of passive diffusion, carrier-substrate Interaction and energy-coupling relationships. Thus disturbances of membrane function, as well as morphological, and biochemical alterations, assume a greater consequence as the direct result of the lower 2 5 metabolic rate. Therefore, a critical evaluation of physiologic parameters '.:.J ~a~ '._.~~44494 such as ionic and amino acid composition, bicarbonate equilibrium, available energy sources, dissolved oxygen levels, osmolality and pH should be observed with respect to each preservation medium. parameters for extended 4°C storage should be defined as to the reversibility of cell damage incurred during storage.
Adult corneal endothelium have a limited regenerative capacity and mitotic figures have been rarely observed In vivo; human corneal endothelium In vJvo normally responds to trauma by sliding into the wounded area by cell migration. However, in vlvo endothelial cell mitosis has been 1 0 demonstrated tn rabbits, cats and primates. in tissue culture, mitosis has been observed in rabbits and human corneal endothellum~ Autoradiographic Ihymidlne uptake studies alter cryowounding or mechanical wounding oI
corneas in vitro has demonstrated existence of mitotic figures in the endothelial monolayer. Surgical trauma and disease can accelerate the loss of 1 5 endothelial cells and further compromise the cornea. Thus, the tong term preservation and enhancement of the corneal endothelium is a very important aspect of eye bank storage of eye tissue.
An overview of the issues surrounding the storage and handling of .
corneal tissue is found in Corneal Surgery, Chapters 1-4 , pages 1-128 2 0 edited by Federick S. Brightbill, M.D., published by C.V. Mosby Company, St.
Louis, M0,1986. A variety of storage media and techniques have been proposed, and current research continues to be directed towards maintaining and actually enhancing the quality of donor (issues, and increasing the duration of storage corneal (issues, as defined as the lime between excision 2 5 from a donor and transplantation.

~i /. ~ I 1 ~5~ 2044494 Accordingly, the present Invention is directed toward rnateriais and methods for enhancing ocular tissues, especially corneal iissues,~during storage prior to transplantaUon. One aspect of the invention provides for the enhancement of corneal tissue viability by maintaining normal physiologic metabolism and corneal deturgescence during low temperature storage.
Another aspect of the invention provides for increasing the length of time that eye tissues, especially corneal tissues, can maintain the attributes of fresh tissue.
summary of the Invention Intermediate-term cornea! storage at 4°C should provide tissue preservation which is capable of sustaining the functional status of the 1 5 endothelium.~Exper(mental work has demonstrated that both human and an(mai eye tissues, especially corneas, are protected from deterioration and actually are enhanced during low temperature eye bank storage in a delined serum-tree, nutrient supplemented preservation solution. The undesirable attributes of storage in serum-containing solutions are avoided, and the 2 0 potential of the corneal endothelial cells to maintain normal physiologic metabolism and corneal deturgescence during low temperature storage is increased.
The corneal endolheltum is responsible for preservation of the transparency of all corneal layers. The endothelium regulates the ion 2 5 composition of the various corneal layers, thereby maintaining osmotic ,, ~ (s) pressure, permitting permanent hydration of the cornea, and thus constant Ilrickness and Iransparoncy. Consoquontty, any disturbance of ondollrolial call function provokes corneal edema followed by partial or complete loss of transparency. The composition of synthetic media must address the increased stromal hydration that occurs with increased preservation lime and reduced temperatures.
The remarkable capacity of the corneal stroma io uptake water is due to the presence of glycosaminoglycans (GAGS), such as chondroitin sulfate, dermatan or keratan sulfate between the collagen fibers. Electron 1 0 microscopic studies comparing the collagen fibrils in swollen corneal stromas damonslrated that the diameter of collagen fibrils did not differ signilicantiy from chat of the normal fibrils. This linding suggests that it is, rather, the volume increase of the interttbrtllar substance which is responsible for the swelling of the slroma. Additional refraction studies 1 5 demonstrated that the hydration of the fibrils is unchanged despite the the tact the cornea can swell from a state of near dryness to three times Its normal Ihickness. When corneas are Ireated with hyaluronidase or cetylpyridinium chloride the stromal swe111ng is greatly reduced. These studies also suggest that the swelling takes place in the interflbrillar substances.
2 0 Glycosaminoglycans, such as chondroitin sulfate, are long, unbranched polysaccharide chains composed of repeating disaccharide units. .
Glycosaminoglycans are highly negatively-charged due to the presence of sulfate or carboxyl groups, yr both, on many of the sugar residues.
Glycvsaminoglycan chains tend to adopt highly extended, random coiled 2 5 conformations, and to occupy a huge volume for their mass. Being ..
c,~ ~~,.~ 244494 hydrophilic, they attract large amounts of water, thereby forming hydrated gels at even low concentrations. This tendency is markedly enhanced by their high density of negative charges, which attract osmoticaliy active cations.
This water-attracting property of giycosaminoglycans creates a swelling pressure, or turgor, in the extraceilular matrix chat resists compressive forces, in contrast to collagen fibrils, which resist stretching forces.
Because of their porous and hydrated organization, the glycosamlnoglycan chains allow the rapid diffusion of water soluble molecules.
Recent studies suggest that not only has the proteoglycan ground 1 0 substance as a whole been implicated as playing a significant role in corneal hydration, but that the specific distribution of the different proteoglycans, which have different hydrating power, may also play a role in the establishment of water gradients across the cornea. The distribution of keratan sulfate and chondroitin-a-sulfate across the cornea directly relates 1 5 to the asymmetric hydration of the cornea. There is a greater chondroitin-sulfate concentration near the epithelium than near the endothelium; keratan sulfate is more concentrated near the endothelium. Keratan sulfate and predominantly keratan sulfate-bearing proleoglycans have great water sorptive capacity, but meager wafer retentive capacity. It is therefore 20 plausible that the keratan sulfate-bearing proteoglycan gradient, highest at the endothelium, helps to set up the total water content gradient because of its great sorptfve capacity. In contrast, the chondroitin-4-sulfate and dermatan sul(ale-bearing proteoglycans, with their great water retentive capacity, can help establish the bound wafer gradient that is maximum near the epithelium.
2 5 This gradient would then serve to diminish the dehydration of the front of the . l., .l y ~.~ tee t~ '2x44494 cornea, which is exposed to the atmosphere. Therefore, the water gradient across the cornea Is highly correlative wish the distribution of proleoglycans and their water sorptive and relenttve capacities.
The present invention reduces intraoperative and postoperative rebound swelling associated with the use of chondroitin sulfate-containing preservation solutions. The increase of corneal swelling may be due to the influx of low molecular weight moieties of chondroitin sulfate into the stroma during prolonged low temperature storage. Additional fluid is imbibed through the cut edge of the scleral-corneal rim. The use of deturgescent 1 0 agents, such as dextran and increased concentrations of chondroitin sulfate, control cornea! hydration during low temperature storage. Dextran, a neutrally-charged molecule, osmoticaily restricts excess water from swelling the cornea during storage while chondroitin sulfate, a negatively charged molecule, actually binds to the cell membrane and provides a 1 5 membrane stabilizing effect. Chondroitln sulfate and dextran assist in the prevention of stromal hydration by increasing the colloidal osmotic pressure in the aqueous environment surrounding the stored cornea. Sustained corneal deturgescence during and after corneal preservation are of great clinical importance, reducing handling and suturing problems encountered by the 20 transplant surgeon, and consequently reducing the risk of grail failure.
The functional status of the endothelium and sustained corneal deturgescence after corneal preservation are of great clinical importance, and contribute primarily io the success of the surgical outcome. Other areas addressed in the present invention include the enhancement of corneal wound 2 5 healing, and the reduction or elimination of the normal progressive loss of c9~ ~ . ~ 204444 endothelial cells, through the use of nutritive cell supplements. Timely and adequate healing of corneal tissues is required to restore visual acuity.
There is a loss of corneal endothelial cells throughout life. In addition, endotheUal cells are frequently damaged or destroyed in operations involving the anterior chamber. Damage by trauma or loss through aging is compensated by growth in size of the endothelial cells, which migrate to cover denuded surfaces of Descemet's membrane. In clinical cases, endothelial dysfunction is associated with variations of cetl size rather than cell density.
The appearance of increased numbers of giant cells contributes greatly to 1 0 Increased corneal edema. The junctions of giant cells are abnormal. These abnormalities in cell Junctions increase the permeability of the intercellular spaces, thus increasing the fluid diffusion toward the cornea. The decreased density of organelles, such as mitochondria or rough endoplasmic reticulum, are diminished in giant cells. These organelles are essential for the adequate 1 5 functions of the biological pump. Insufficient pump function results in excess accumulation of fluid in the corneal stroma. Furthermore, these giant cells have extended external membranes, supporting functional changes associated with decreased biological pump sites, associated with increased corneal swelling. It should be noted Ihat disturbances in endothelial cell function 2 0 leading to corneal edema occur when endothelial cell density falls to 40%
of the normal value, when hexagonalily tells to 33%, when the coetllctent of variation of endothelial cell density increases three-to-lour load, and the size of giant cells has Increased by 7.5 times over normal endothelial cells.
It is evident from these studies that the anterior chamber 2 5 environment limits cell regeneration of the endothelium, and supports wound (, o) '~ X044494 healing via cell migration. Extreme cell loss is compensated by the formation of giant cells. Furthermore, it Is the complex Interaction of the human corneal endothelial cell and the extraceliular matrix that signal the cell to respond to cell loss In this manner.
The present invention further defines a nutritive solution that pravtdes the cornea with additional amino acids, vitamins, trace minerals, and energy promoting precursors to enhance cell metabolism, wound healing and viability. Cell proliferation is regulated by events leading to DNA
synthesis; whether or not a cell proceeds with DNA synthesis or is arrested 1 0 In the early stages of the cell cycle is dependent upon extracellular conditions. Cellular metabolism can be enhanced by the addition of essential nutritive components by Increasing hexose transport. glycogen transport, protein synthesis, amino acid and Ion transport.
The novel delined nutrient containing solutions are serumfree. While 1 5 serum-supplemented solution can stimulate limited mitosis in human corneal endolheHat cells in tissue culture, the presence of serum In products for use with tissues for human transplantation presents many disadvantages. Serum can be an agent for the transmission of diseases, such as viral diseases. Non-human-derived sera contains many substances capable of eliciting an immune 2 0 response, and a!I sera contain some substances such as endotoxins, and growth factors, that actually retard cell mitosis. These disadvantages are avoided by the present, serum-free solution.
Cornea preservation solutions are well known. In general, those employed herein contain an aqueous nutrient and electrolyte solution, a 2 5 glycosaminoglycan, a deturgesceni agent(s), an energy source(s), a bulfer ~ ~
«~ . (11) system(s), an antioxldanl(s), membrane stabilizing components, antibiotic(s), ATP precursors and nutrient cell supplements. Nutrient and electrolyte solutions are well defined in the art of tissue-culturing. Such solutions contain the essential nutrients and electrolytes at minimal concentrations necessary for cell maintenance and cell growth. The actual composition of the solutions may vary greatly. In general, they contain Inorganic salts, such as calcium, magnesium, Iron, sodium and potassium salts of carbonates, nitrates, phosphates, chloride and the like, essential and non-essential amino acids, vitamins and other essential nutrients.
1 0 Chemically defined basal nutrient media are commercially .available, for example from Gibco Laboratories (3175 Stanley Road, Grand Island, New York 14073) and Microblotogical Associates (P.O. Box 127, Briggs Ford Road, Watkersville, Maryland 21793) under the names Eagle's Minimal Essential Medium (MEM) and TC199. Corneal storage solutions have been 1 5 adapted from these nutrient media. The defined serumtree medical solution base of the present invention is composed of components found in both MEM
and TCi99 supplemented with ATP precursors, vitamins, amino acids and growth promoting supplements. The delined serumfree medical solution is compared with commercially available corneal storage medium CSM~
2 0 developed by R.L. Lindslrom, M.D. and Debra L. Skelnik, B.S., available Irom Chiron Ophthalmics, Inc. (Irvine, CA) and TC199 from Gibco Laboratories (Grand Island, NY) in Table i.

. r (12) ~v2044~94 Description of the Preferred Embodiments Preferred defined serumtree medical solutions for use in the composition and methods of this Invention contain an aqueous electrolyte solution (e.g. Minimal Essential Medium and/or TC199), a glycosaminoglycan (a.g. standard or purified high or low molecular weight chondrottin sulfate (A, B or C isomers), dermatan sulfate; dermatin sultaie, heparin sulfate, heparan sulfate, keratin sulfate, keratan sulfate and/or hyaluronic acid in a range of .01 mg/mt to 100 mg/ml; a deturgescent agent 1 0 (e.g, low or high molecular weight polysaccharide, such as dexlran, dextran sulfate, polyvinyl pyrrolidone, polyethylene glycol, polyvinyl acetate, hydroxypropyimethyl cellulose, carboxypropylmethyl cellulose) tn a range of .01 mg/ml to 100 mg/ml; an energy source and carbon source (e.g.
glucose, pyruvate, sucrose, fructose, dextrose) in a range of .05 mM to 10 1 5 mM; a buffer system (e.g. a bicarbonate buffer system and hydroxyethylpiperizene ethanesultonic acid, HEPES butter) fn a range of .i mM to 100 mM; to maintain a physiologic pH (desirably between 6.8 and 7.6), an antioxidant (e.g. ascorbic acid, 2-mercaptoethanol, glutathione, alpha locopherot), in a range of .001 mM to 10 mM; membrane stabilizing 2 0 agents (e.g. vitamins A and B, retinoic and/or cotactor~, elhanolamine, and phosphoethanolamine, selenium and iransferrin), in a range of .O1 mglml to 500 mg/mt; antibiotics and/or antimycotic agents (e.g. ampholericin-B, gentamycin sulfate, kanamycin sulfate, neomycin sulfate, nystatin, penicillin, tobramycln, streptomycin sulfate) in a range of .001 mM to 10 2 5 mM; and ATP precursors (e.g. adenosine, inosine, adenine) in a range of .001 .. f~ ~ ?
(13) ~.~. 2044494 mM to 10 mM; and nutrient cell supplements (e.g. cholesterol, L-hydroxyproline, d-biotin, calciterol, niacin, pare-aminobenzoic acid, pyridoxine HCI, Vitamin 612, Fe(NOg)3, non-essential amino acids) in a range of .001 mM to 10 mM.
The serumtree medical solution of this invention is composed of a defined aqueous nutrient and electrolyte solution, supplemented with a glycosaminoglycan(s), a deturgescent agent(s), an energy souroe(s), a buffer system(s), an antioxidani(s), membrane stabilizing components, antibiotic(s), ATP precursors and nutrient cell supplements in the amounts 1 0 sutlicient to enhance cell metabolism, cell viability, wound healing, and corneal deturgescence following low temperature eye bank storage. The excised corneas are aseptically transferred to containers of the corneal storage solution, which are then sealed. For storage and transport,these corneas are maintained at low temperature (e.g. 2°C to 15°C
optimally at 1 5 4°C) to minimize the risk of bacterial growth and to reduce corneal tissue metabolic damage. It has been found that even ai these low temperatures, the endothelial cells can be maintained for periods up to 14 days. At the time of iranspiant-ation, narmat corneal deturgescence (s maintained intraoperatively and postoperativety. Endothelial cell function and 2 0 metabolism is maintained, permitting permanent hydration of the cornea, and thus constant thickness and transparency postoperatively. In addition to providing a viable cornea for transplantation, wound healing is potentiated.
Various modifications can be made to the present invention without departing from the apparent scope thereol. For instance, the serumtree solution can be 2 5 used fn any medical application, and is not strictly limited to ophthalmology.

., c~4~ k'v2044494 The invention is turther illustrated by the foliowtng examples, which is not intended to be Itmiling.

'~~-~ 2044494 Brief Description of the Figures Table I: Formulation of TC-199, CSMTM and a representative formulation of the defined serumtree medical solution.
Figure 1: Corneal Thickness of Human Corneas After 4°C Storage Figure 2: Corneal Thickness After 12 Days Storage at 4°C and Pos!
Storage Warming to 24°C.
Figure 3: [3H]-Thymidine Incorporation of Human Corneal Endothelial Cells Incubated Wilh Defined Serumlree Medical Solution Components 1 0 Figure 4: Postoperative Corneal Thickness (mm) ~16~ ' ~ 2044494 Mode of Operation Example One A Defined Serumtree Medical Solution Intermediate-term corneal storage at 4°C should provide tissue preservation capable of sustaining the functional status of the endothelium and the maintenance of corneal deturgescence post-keratopiasty. CSMT"~ and K-SoiT"~ have become the standard media of intermediate-storage at 4°C.
As 1 0 demonstrated in Kaulman H.E., Varnell E.D., Kautman S. et al. K-SoIT"~
corneal preservation. Am J Ophthalmol 1985. 100:299-304; Bourne W.M., Endothelial cell survival on transplanted human corneas preserved at 4°C In 2.5% choniirottin sulfate for one to 13 days. Am J Ophthafmol 1986;
102:382-6; Lindstrom R.L., Skelnik D.L., Mindrup E.A. , et al: Corneal 1 5 preservation at 4°C with chondroitin sulfate containing medium.
Invest Oph(halmol Vls Sc! (Supply 1987; 28 (3): 167; Bhugra M.K., Sugar A., Meyer R. , et al: Results of a paired trial of MKT"" and K-SoITM storage.
Invest Ophthalmol Vis Scl (Supply 1988; 29 : 112 and Lass J.H., Reinhart W.J., Bruner W.E., et al. Comparison of corneal storage in K-Sole and Chondroitin 2 0 Suilale Corneal Storage Medium in human corneal transplantation.
Ophthalmology 1989; 96: 688-97.
Increased corneal thickening is associated with CSM~~"-stored corneas, with greater rebound swelling apparent at the time of surgery. However, normal corneal thickness is achieved during the first post-operative month.
2 5 The Increase of comeai swelling may be due to the influx of low molecular (1y weight moieties of chondroitin sullate Into the stroma during prolonged storage at 4°C. In an effort to reduce corneal swelling, studies were conducted to determine It the addition of dextran to a defined serumfree chondroitin sulfate-containing medium would minimize corneal hydration.
Dextran, an effective osmotic agent in MKT~~ medium, keeps the cornea thin and effectively maintains the barrier function of the corneal endothelium. Corneas stored in dextran-containing medium are inhibited from swelling because of the colloidal osmotic pressure of dextran. The dextran is present to osmotically restrict excess water from swelling the 1 0 cornea during slorago. Dextrart can ponolralo taro cornoal ondollroliurrt and enter the strama. This entrance and egress of dextran occurs rapidly at 4°C, with the degree of penetration of dexiran depending on the length of storage .
and the condition of the endothelium. Thus, dextran was an attractive agent to reduce the corneal swelling associated wish low temperature storage with 1 5 chondroitin sulfate containing medium.
The defined serumtree medical solution consisted of Eagle's Minimal EssenUai Medium (MEM) supplemented with Earle's salts, sodium bicarbonate, 25 mM HEPES, .1 mM non-essential amino acids, i mM sodium pyruvate, 2 mM L-glutamine, .5 mM 2-mercaptoethanol, 1.0 % dextran, 2 0 2.5% chondroitin sulfate and 100 pg/ml genlamycin sulfate . The base medium was further supplemented with the following components: Fe(N03)3 9H20, adenine sulfate, cholesterol, L-hydroxyproline, ascorbic acid, alpha iocopherol phosphate, D-biotin, caicilerol, niacin, paraminobenzotc acid, pyridoxine HCI, adenosine, inosine, and vitamin B12. These components were 2 5 added to more completely define the basal medium and potentials ceN growth .~ 1 \..i _. (18) ~ X444494 c and cell function (See Table I[.
In order to determine the safety and efficacy of this defined serumfree medical solution, a dose response curve of chondroitin sulfate concentration with human corneas stored for 12 days at 4°C was conducted. Chondroiltn sulfate concentrations consisted of 1.5%, 1.75%, 2.0% and 2.5%. Corneal thickness measurements were taken at 0, 1, 7 and 12 days storage at 4°C.
in addition, Isolation techniques developed in our laboratory have enabled the establishment of primary and subsequent subcultures of human corneal endothelium that retain the attributes of native endothelium. In vitro 1 0 conditions maintain these human corneal endothelial cells in a proliferative state, actively undergoing mitosis. A quantitative bioassay has been developed to determine the effects of various lest medium In the stimulation or inhibition of DNA synthesis as measured by [3HJ-thymidine incorporation.
Next a prospective pilot clinical trial was conducted, evaluating 1 5 corneal thickness and endothelial cell survival for corneas that had been stored in a defined serumfree medical solution (Formula A) and then transplanted Into patients.
20 Materials and Methods Chondrottin Sulfate Dose Response Curve With Human Corneas Human donor globes were Immersed in 1.0% povidone iodine in normal saline for"three minutes, followed by a one-minute immersion in 25 normal saline: The globes were then rinsed with 12 cc of normal saline with a (19) ' .' 2044494 syringe titled with a 18-gauge needle. Sixteen paired corneas from donors urrsuilabte for transplantation bocauso of ago or cause of doaih wore romovod at a certified eye bank an average of 12.0 hours after death, and placed in 20 ml of defined medical solution supplemented with 1.5%, 1.75%, 2.0%, and 2.5% chondroltin sulfate. Control media was commercial DexsoITM (Chiron Ophthalmlcs, inc., Irvine, CA). Supptemenied media was warmed to room temperature before the corneas were placed into the media, and corneal thickness measurements were taken. Corneal Ihickness trreasurements were made using a Leliz upright microscope tilted with a micrometer. The 1 0 microrneler dial indicator was attached to the microscope stand above the stage, with the set screw placement through the stage, directly under the foot of the dial indicator. The corneal Ihickness measurement involved focusing on the endothelium, setting the set screw to bring the dial to 'zero', raising the stage to bring the epithelium into focus, and recording the dial indicator 1 5 reading. The cornea was then cooled to 4°C, and stored for 12 days.
Corneas were removed from the storage medium and placed in 15 ml of MEM
supplemented with 2 mM L-glutamine and 100 pg/ml gentamycin. Corneas were then warmed to 34°C for 2 hours and central corneal thickness measurements were taken at 30, 60 and 120 post-warming. Corneal 20 endothelium was evaluated by staining with .1% trypan blue and alizarin red S after final corneal thickness measurements were taken.
(3H]-Thymidlne Incorporation of Muman Corneal Endotheilal Cells 2 5 Fourteen medium components were tested as follows: Fe(N03)3 . ..-.~
(20) '..' 20~~~~~
9H20, adenine sulfate, cholesterol, L-hydroxyprollne, ascorbic acid, alpha tocopherol phosphate, D-biotin, calciteroi, nfactn, para-aminobenzoic acid, pyridoxine HCI, adenosine, inosine, and vitamin 812. Components were added individually or In combination to a base medium consisting of Eagle's Minimal Essential Medium (MEM) supplemented with Earle's salts, 25 mM HEPES, sodium bicarbonate, .i mM non-ass~nllal amino acids, 1 mM°sodium pyruvate, 2 mM L-giutamlne, .5 mM 2-mercaptoethanot, 2.5% chondroitin sulfate and 100 p.g/ml gentamycin sulfate. Additional chondroitin sulfate concentrations of 1.75% and 2.0% were also tested. Control media consisted 1 0 of commercially available DexsoITM (Chiron Ophlhalrnics, Inc., Irvine, CA) and CSMTM supplemented with 10% fetal bovine serum. Ali test media samples were freshly made up and warmed to room temperature at the time of the experiment.
~uantltative Bioassay The quantitative bioassay is based on the incorporation of [3H]-thymidine into the DNA of human corneal endothelial cells incubated in 2 0 serumtree and serum containing medium. Costar 9f-well tissue culture plates were seeded with 3 X103 in a final volume of 200 p.l of designated medium. Fourth passage human corneal endolhelfal cells were maintained in a humfdltied Incubator at 35.5°C in a 95% air: 5% C02 atmosphere. Attar hours of incubation in CSMTM, supplemented with 10% fetal bovine serum, to 2 5 permit attachment, the medium was removed , and each well was rinsed once (.
.. c2') ._~ ~.04449~
with serumtree Minimal Essential Medium with Earle's salts and 25 mM
HEPES. The cells were then rinsed and incubated with the appropriate test solution. Human corneal endothelial cells ware then Incubated for an additional 72 hours in the presence of 1 mlcrocurie/well of [3H]-thymidine.
Uptake was ended by the aspiration of the radioactive medium and rinsing the cells twice with serumtree Minimal Essential Medium. The human corneal cells were detached with .5% trypsin and prepared for liquid scintillation counting. The [3H]-thymidine counts represent acid-Insoluble counts. One-way analysts of variance and the Newman-Keuls multiple range test were 1 0 used to evaluate statistical stgniticance (p<.05).
Clinical Trial Eye Bank Procedures Human donor globes were Immersed in 1.0% povidone iodine in normal saline for three minutes, toliowed by a one-minute Immersion in normal saline. The globes were then rinsed with 12 cc of normal saline with a syringe fitted with a 18-gauge needle. Corneas Irom suitable donors were 2 0 removed at the eye bank an average of 8.6 hours after death, and placed in a defined serumtree medical soluUon (Formula A). This solution was warmed to room temperature before the cornea was placed Into the solution. The cornea was then cooled to 4°C, and. stored for an average of 4.3 days (range 1-days).

. . ..j ~ , c2z) 2044494 Recipient Criteria The following recipterrl diagnoses were considered for entry Into lire study: aphakic bullous keratopathy, Fuchs' dystrophy, pseudophaklc bulious keralopalhy, corneal scar, keratoconus and failed graft. The preoperative examination consisted of measurement of best corrected visual acuity, intraocular pressure, slit lamp and funduscopic examination. Informed consent was obtained from all participants in clinical trials consistent with the United States Department of Heahh and Human Services guidelines. This randomized clinical trial was performed with Institutional Review Board 1 0 consent and monitoring.
Surgical Technique Corneas were warmed to room temperature at the time of transplantation. The donor buttons were cut from the endothelia! side with a 1 5 corneal trephine press. Sodium hyaluronate (Healon) or sodium hyaluronate with chondrottin sutlate (Viscoat) was used in all cases. Operative and postoperative care was similar for all cases. Suturing techniques consisted of a combination of 12 Interrupted 10-0 sutures with a running 11 ~0 nylon or mersilene suture. Gentamycin, Betamethasone and Ancef were 6nJected 2 0 subconjunctivaily at the end of each procedure.
Postoperative Treatment Postoperalively all patients received neomycin or gentamycin drops four times daily during the first month. Topical steroids were administered 2 5 as needed. Patients were evaluated during the tir~t two months ~2~> ~ 2044494 pastoperalively for complications, rejection, corneal vascularization, infection, wound leak, dehiscence of wound, persistent epithelial defects, and overall corneal condition. Ultrasonic pachymetry of the central cornea was performed preoperatively and posloperatively at one day, one week, one month and two months. The total number of patients Included in this study was 15. Between group differences in corneal thickness were analyzed to determine if there were significant differences using a paired t-test.
Results and Discussion Dextran Dose Response Curve Wtth Human Corneas The chondroitin sulfate dose response curve for human corneas incubated at 4°C for 12 days with respect to corneal thickness is shown in 1 5 Figure 1. Corneas Incubated with DexsotTM, containing 1.35% chondroitin sulfate, demonstrated effective thinning at 1, 7, and 12 days. Corneal thickness measurements at these time periods were .425 t .082 mm, .530 t .040 mm and .572 t .043 mm. Corneas Incubated with 1.5%-2.0%
chondroitin sulfate demonstrated increased corneal deturgescence at these , , 2 0 same time periods with the greatest corneal thinning occurring al 2.5%
chondrottin sulfate. Corneal thickness at 1, 7, and 12 days post-incubation was .405 1.021 mm, .480 t .042 mm, .480 ~ .028 mm, respectively.
Corneas stored in DexsolTM for 12 days exhibited a 19.6% increase in corneal swelling post warming l0 34°C. Corneas stored in 1.5%-1.75% chondrottin 2 5 sulfate demonstrated a stalisticalty similar increase in corneal swelling .
~'.:'~ (24) post-warming. Corneas stored In 2.0% and 2.5% chondroltln sulfate demonstrated a 15.6% and 13.5% Increase In corneal swelling post-warming (Figure 2).
Ali endothelial cell monolayers were Intact, with normal endothelial cell morphology for all concentrations of chondroitin sulfate tested. Corneas incubated wllh higher concentrations of chondrottin sulfate demonstrated fewer stromal folds, and fewer areas of alizarin red S staining of Descemet's membrane. All alizarin red S staining was minimal for ail corneas, and was confined to areas of stromal folding. In conclusion, all corneas stored in 1 0 1.35% -2.5% chondroltin sulfate had intact corneal endothelium alter 12 days preservation at 4°C. Corneas stored in the defined medical solulton (containing 2.5°!° chondroilln sulfate) maintained the greatest corneal deturgescence over the 12 day preservation period. Minimal corneal folding and swelling was also noted for Ihis test group alter rewarming to 34°C.
1 5 These results "support the use of ihls defined serumtree medical solution to preserve human corneas at 4°C for ~ransplantaUon.
[3Hj-Thymidine Incorporation of Human Corneal Endothelial Cells 2 0 This study was conducted to evaluate the components of a defined serumfree medical solution. The test medium was evaluated in a [3Hj-thymidine incorporation bioassay with human corneal endothelial cells. This bioassay provides a sensitive method to determine it the lest medium will inhibit or stimulate the incorporation of [3Hj-thymldine into the DNA of 2 5 these cells. The incarporation of [3Hj-thymidine by human corneal f~ L y (25j 2044494 endothelial cells incubated with test medium containing one or more of fourteen components was compared to serunotree DexsotTM medium and CSM~M
medium supplemented with 10% FBS (Figure 3j. One-way analysis of variarice and the Newman-Keuls multiple range test were used to evaluate statistical signit(cance (p<.05).
In this bioassay, the cells were kept in a proliterative slate, actively undergoing mitosis. Inhibition of [3Hj-thyrnidine Incorporation Into the DNA
of human corneal endothelial cells is an Indicator of decreased cell metabolism, decreased cell health and possible cellular toxicity. Human '! 0 corneal endothelial cells Incubated with CSM~M medium supplemented with 10% FBS exhibtied a statistically significant increase in [3H]-thymidine incorporation rate as compared to the freshly prepared control serumtree DexsoITM medium. HCE cells Incubated with 1.75% or 2.0% chondroitin sulfate exhibited statisitcaily similar [3H[-thymidine Incorporation rates as 1 5 HCE cells incubated with serumtree DexsoITM. The addition of 2.5%
chondroitin sulfate and 1% dexlran, in combination with the following individual components: Fe(N03)3~9H20, adenine sulfate, L-hydroxyproline, ascorbic acid, alpha tocopherol phosphate, D-biotin, pyridoxine HCI, inosine, and vitamin B12 exhibited statistically similar rates of [3H[-Ihymidine 2 0 incorporation as HCE cells incubated with serumtree DexsoITM. The addition of 2.5% chondroitin sulfate and 1% dextran, with adenosine or combination of adenosine, adenine, and inostne exhibited stalisticaliy greater [3HJ-thymidine Incorporation rates than HCE cells Incubated w(ih the Dexsol~
control medium. When all tourteen components were combined with 2,5%
2 5 chondroitin sulfate in a supplemented MEM base, a statistically greater (26) 2Q~4494 [3H]-ihymidine incorporation rate was demonstrated as compared to the Dexsol~M control. All tnadla tested rnaintainod normal endothelial cell morphology throughout the 72-hour tncubaiion period.
In conclusion, from the results of this [3H]-thymtdine incorporation study with human corneal endothelial cells, a defined serumiree solution (Formula A) containing: 2.5% chondroitin sulfate, 1% dextran, Fe(N03)3~
9H20, adenine sulfate, cholesterol, L-hydroxyproline, ascarblc acid, alpha tocopherol phosphate, D-biotin, caiciferol, niacin, para-aminobenzotc acid, pyridoxine HCI, adenosine, inostne, and vitamin B12 was capable of 1 0 stirnulattng [3H]-thyrnidine Incorporation rates stalisttcalty greater than serumtree Dexsol~ medium as defined by the parameters of this bioassay.
This dellned serumtree medical sotulion fs capable of enhancing the mitotic potential of human corneal endothelial cells, by providing a more comptelely defined solution than the control DexsoITM medium. This solution is therefore, 1 5 acceptable for use as a 4°C corneal preservation medium.
Clinical Study Fifteen corneas were transplanted utilizing the defined serumfree medical solution (Formula A). All patients were operated on by one surgeon 2 0 and were Included in the following study. The cornea donors had the following characteristics: donor age (mean age 53 t 19 years), death to enuclealion time (mean: 4.3 t 2.7 hours), and death to preservation time (mean: 4.3 t 3.2 hours). Storage time of corneas at 4°C was 4.3 days (range 1-7 days).
One-hundred percent of the Formula A transplanted corneas were clear after 2 5 2 months. No persistent epithelial detects were noted In this patient group.

~''~ (27) ''~1 ~0444g4 Intraoperatlve corneal thickness was .623 t .054 mm. Comparative corneal intraoperative thickness measurements of corneas stored in Dexsol~°
under similar parameters was .787 t .047 mm. Corneal thickness measurements at ane week for Formula A and Dexsol stored corneas was .650 t .084 mm and .743 t .093 mm, respectively. r=ormula A stored corneas were significantly thinner intraoperatively and at one week post-operatively (Figure 4).
Progressive corneal thinning occurred for ail patients during the 2 month follow-up period (corneal thickness: one month .6i2 t .167 mm; two months .544 t .062 mm). Post-operative Intraocular pressures were 1 0 within normal Ifmits for all patients. No primary donor failures occurred in this Formula A cornea group.
The defined serumlree medical (Formula A) solution was effective in maintaining normal corneal deturgescence Intraoperatively and post-operatively. Endothelial cell function and metabolism was maintained, 1 5 permitting normal hydration of the cornea, and Ihus sustaining constant corneal thickness and transparency posloperalivety.

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Claims (10)

1. The defined serumfree medical solution comprising:
a. an aqueous nutrient and electrolyte solution;
b, a glycosaminoglycan;
c. a deturgescent agent;
d. an energy source;
e. a butler system;
f. an antioxidant;
g. membrane stabilizing agents;
h. an antibiotic and/or an antimycotic agent;
i. ATP presursors; and j. nutrient cell supplements.

(29)

2. The defined serumfree medical solution comprising:
a. An aqueous nutrient and electrolyte solution selected from the group of:
1. Eagle's minimal essential medium (MEM) 2. TC199 medium

3. A combination of Eagle's minimal essential medium (MEM) and TC199 b. A glycosaminoglycan in the range of .01 mg/ml to 100 mg/ml selected from the group of:
1. chondroitin sulfate;
2. dermatan sulfate;
3. dermatin sulfate;

4. heparin sulfate;

5. heparan sulfate;

6. keratin sulfate;

7. keratan sulfate; and/or

8. hyaluronic acid.
c. A deturgescent agent in the range of .01 mg/ml to 100 mg/ml selected from the group of:
1. dextran;
2. dextran sulfate;
3. polyvinyl pyrrolidone;
4. polyethylene glycol;
5. polyvinyl acetate;

(30) 5. hydroxypropylmethyl cellulose; and 6. carboxypropylmethyl cellulose.
d. An energy source in a range of .05 mM to 10 mM
selected from the group of:
1. glucose;
2. pyruvate;
3. sucrose;
4. fructose; and 5. dextrose.
e. A buffer system in a range of .1 mM to 100 mM
selected from the group of:
1. Bicarbonate buffer; and 2. HEPES butter.
f. An antioxidant in a range of .001 mM to 10 mM
selected from the group of:
1. ascorbic acid;
2. 2-mercaptoethanol;
3. glutathione; and 4. alpha-tocopherol.
g. A membrane stabilizing component in a range of .01 mg/ml to 500 mg/ml selected from the group of:
1. vitamin A;
2. vitamin B;
3. retinoic acid;
4. ethanolamine;

(31) 5. phosphoethanolamine;
6, selenium; and 7. transferrin.
h. An antibiotic and/or antimycotic in the range of .1 µg/ml to i mg/ml selected from the group of:
1. amphotericin-B;
2. gentamycin sulfate;
3. kanamycin sulfate;
4. neomycin sulfate;
5. nyslatin;
6. penicillin;
7. tobramycin; and 8. streptomycin.
I. ATP presursors in a range of .001 mM to 10 mM
selected from the group of:
1. adenosine;
2. inosine; and 3. adenine.
j. Nutrient cell supplements in a range of .001 mM to 10 mM
selected from the group of:
1.cholestrol;

2.L-hydroxyproline;

3.d-biotin;

4.calciferol;

5.niacin;

(32) 6. para-aminobenzoic acid;
7. pyridoxine HCI;
8. vitamin B12;

9. Fe(N03)3; and

10, non-essential amino acids.

3. The defined serumfree medical solution containing components which maintain and enhance the preservation of eye tissues, including human corneal tissues at low temperatures 2°C to 15°C) with a physiological pH comprised of:
a. an aqueous nutrient and electrolyte solution;
b. a glycosaminoglycan;
c. a deturgescent agent;
d. an energy source;
e. a buffer system;
f. an antioxidant;
g. membrane stabilizing agents;
h. an antibiotic and/or an antimycotic agent;
i. ATP precursors; and j. nutrient cell supplements.
4. Use of a defined serumfree medical solution as defined in claim 3 for storing donor corneal tissue at 2°C to 15°C.

(34) 5. The defined serumfree medical solution containing components which maintain and enhance the preservation of eye tissues, Including human corneal tissues at low temperatures (2°C to 15°C) with. a physiological pH
comprised of:
a. An aqueous nutrient and electrolyte solution selected from the group of:
i. Eagle's minimal essential medium (MEM) 2. TC199 medium 3. A combination of Eagle's minimal essential medium (MEM) and TC199 b. A glycosaminoglycan in the range of .01 mg/ml to 100 mg/ml selected from the group of:
1. chondrollin sulfate;
2. dermatan sulfate;
3. dermalin sulfate;
4. heparin sulfate;
5. heparan sulfate;
6. keratin sulfate;
7. keratan sulfate; and/or 8. hyaluronic acid.
c. A deturgescent anent in the range of .01 mg/ml to 100 mg/ml selected from the group of:
1, dextran;
2. dextran sulfate;

(35) 3. polyvinyl pyrrolidone;
4. polyethylene glycol;
5. polyvinyl acetate;
5. hydroxypropylinethyl cellulose; and 6. carboxypropylmethyl cellulose.
d. An energy source in a range of .05 mM to 10 mM
selected from the group of:
1. glucose;
2. pyruvale;
3, sucrose;
4. fructose; and 5. dextrose.
e. A buffer system in a range of .1 mM to 100 mM
selected from the group of:
1. Bicarbonate buffer; and 2. HEPES buffer.
f. An antioxidant in a range of .001 mM l0 10 mM
selected from the group of:
1. ascorbic acid;
2. 2-mercaploethanol;
3. glulathione; and 4. alpha-tocopherol.
g. A membrane stabilizing component in a range of .01 mg/ml to 500 mg/ml selected from the group of:
1. vitamin A;

(36) 2. vitamin t3;
3. retinoic acid;
4. ethanolamine;
5. phosphoethanolamine;
6. selenium and 7. transterrin.
h. An antibiotic and/or antimycotic in the range of .1 µg/ml to 1 mg/ml selected from the group of:
1. amphotericin-B;
2. gentamycin sulfate;
3. kanamycin sulfate;
4. neomycin sulfate;
5. nystatin;
6. penicillin;
7. tobramycin; and 8. streptomycin.

i. ATP presursors in a range of .001 mM to 10 mM
selected from the group of:
1. adenosine;
2. inosine; and 3. adenine.

j. Nutrient cell supplements in a range of .001 mM to 10 mM
selected from the group of:
1. cholestrol;
2. L-hydroxyproline;

3. d-biotin;
4. calciferol;
5. niacin;
6. para-aminobenzoic acid;
7. pyridoxine HCl;
8. vitamin B12;
9. Fe(NO3)3; and 10. non-essential amino acids.

6. Use of a defined serumfree medical solution as defined in claim 5 for storing donor corneal tissue at 2°C to 15°C.

(38) 7. The defined serumfree medical solution containing components which maintain and enhance the preservation of eye tissues, including human corneal tissues at low temperatures (2°C to 15°C) with a physiological pH
comprised of:

a. An n aqueous nutrient and electrolyte solution:
1. Eagle's minimal essential medium (MEM) b. A glycosaminoglycan in the range of .01 mg/ml to 100 mg/ml 1. chondroitin sulfate;
c. A deturgescent agent in the range of .01 mg/ml to 100 mg/ml 1. dextran;
d. An energy source in a range of .05 mM to 10 mM
1. pyruvate;
2. dextrose.
e. A buffer system in a range of .1 mM to 100 mM
1. Bicarbonate buffer; and 2. HEPES buffer.

f. An antioxidant in a range of .001 mM to 10 mM
1. 2-mercaptoethanol; and 2. alpha-locopherol.

g. An antibiotic and/or antimycotic (n the range of .1 µg/ml to 1 mg/ml i. gentamycin sulfate;

h. ATP precursors in a range of .001 mM to 10 mM
1. adenosine;
2. inosine; and 3. adenine.
i. Nutrient cell supplements in a range of .001 mM
to 10 mM
1. cholesterol;
2. L-hydroxyproline;
3. d-biotin;
4. calciferol;
5. niacin;
6. para-aminobenzoic acid;
7. pyridoxine HCl;
8. vitamin B12;
9. Fe(NO3)3; and 10. non-essential amino acids.

8. Use of a defined serumfree medical solution as defined in claim 7 for storing donor corneal tissue at 2°C to 15°C.

(40) 9. The defined serumfree medical solution containing components which maintain and enhance the preservation of eye tissues, including human corneal tissues at low temperatures (2°C to 15°C) with a physiological pH
comprised of:
a. An aqueous nutrient and electrolyte solution:
1. Eagle's minimal essential medium (MEM) b. A glycosaminoglycan 1. 2.5% chondroltin sulfate;
c. A deturgescent agent 1. 1% dextran;
d. An energy source 1. 110 mg/L pyruvate;
2. 1000 mg/L glucose e. A butter system 1. 2200 mg/L Bicarbonate buffer; and 2. 25 mM HEPES buffer.

f. An antioxidant 1. .5 mM 2-mercaptoethanol; and 2. .01 mg/L alpha-tocopherol.
g. An antibiotic and/or antimycotic 1. 100 mg/L gentamycin sulfate;

h. ATP precursors 1. 5 mg/L adenosine;
2. 10 mg/L inosine; and 3. 10 mg/L adenine.

i. Nutrient cell supplements 1. 0.2 mg/L cholesterol;
2. 10 mg/L L-hydroxyproline;
3. 0.01 mg/L d-biotin;
4. 0.1 mg/L calciferol;
5. ~0.025 mg/L niacin;
6. ~0.05 mg/L para-aminobenzoic acid;
7. ~0.25 mg/L pyridoxine HCl;
8. ~1.36 mg/L vitamin B12;
9. ~0.5 mg/L Fe(NO3)3; and 10. ~.1 mM non-essential amino acids.

10. Use of a defined serumfree medical solution as defined in claim 9 for storing donor corneal tissue at 2°C to 15°C.