Method and apparatus for ultra-fast preparation of the cornea for vitrification

Abstract

A method for handling, treating, and storing a cornea is disclosed that enables vitrification and rewarming in air and / or liquid nitrogen vapor without causing significant cell loss, cell death, or dysfunction after transplantation, as compared to untreated control corneas. The method utilizes osmotic imbalance and extreme corneal shrinkage due to corneal dehydration, and enables avoidance of cryoprotectant toxicity, even when exposed to the peak (vitrifiable) concentration of cryoprotectant above 0 °C for an extended period of time or when washing the cryoprotectant without performing osmotic buffering. The corneal device enables ideal handling and storage and air cooling inside or outside the container used for storage.

Description

Technical Field

[0001] The present invention relates to the preservation of human, animal, and bioartificial corneas by vitrification, and to methods and devices therefor.

[0002] Cross - Reference to Related Applications The present invention claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 390,074, filed on July 18, 2023, the entire disclosure of which is incorporated herein by reference.

Background Art

[0003] The Need for Long - Term Preservation of the Cornea Vitrification can have a positive impact on the majority of corneas transplanted annually in the United States and has the potential to export tens of thousands of natural corneas and an unlimited number of bioartificial corneas from the United States to countries in need of high - quality corneas.

[0004] Worldwide, there are 10 million cases of corneal blindness that could be alleviated by corneal transplantation (Kingsley 2002, Anonymous 2003, McGinn 2003), but only 100,000 transplants are performed each year (Kingsley 2002, Anonymous 2003). There are many countries where cultural and religious restrictions prevent the supply of corneas, but there is sufficient medical sophistication and wealth to permit transplantation if imported corneas can be obtained in a transplantable state. Notable examples include Taiwan (Hu, Tsai et al. 1999), Japan (Goto, Fukuhara et al. 1999), and India (Anonymous 2003). In India alone, as of 2002, there were 1.3 million visually impaired people whose vision could be restored by corneal transplantation (Anonymous 2003), and at least an additional 40,000 cases are increasing each year (Anonymous 2003, Anonymous 2003), but the supply of eyes available locally was only 15,000 - 18,000 per year (Anonymous 2003). In the United States, in 2001, there were 1,367,460 deaths of people 75 years of age and older (Anonymous 2003), and approximately 34% of them may be potential corneal donors for full-thickness corneal transplantation (Probst, Halfaker et al. 1997). Also, there were 1,048,965 deaths of people under 75 years of age (Anonymous 2003), and presumably 71% of them may be potential corneal donors (Armitage and Easty 1997). This indicates that the number of potentially available natural corneas exceeds domestic demand and that export could be supported if they could be preserved to an exportable extent.

[0005] In the United States, all corneas are stored at 4°C (Bourne 2001). Corneas stored for only about four days have a reduced endothelial cell density (ECD, the number of corneal endothelial cells per unit area of the corneal endothelial surface) to the extent that they are not suitable for transplantation, but in vitrified corneas, the ECD does not decrease. Similar deterioration occurs even in corneas that are refrigerated but not cryopreserved, clearly limiting the export of corneas to remote locations on Earth (Goto, Fukuhara et al. 1999, Hu, Tsai et al. 1999, Hollins 2000, McGinn 2003). For example, despite great efforts to ensure the rapid international delivery of exported corneas, the corneas exported from the United States to Taipei, Taiwan, looked "significantly different" from the photos taken in the United States when they arrived in Taipei. An estimated 590 endothelial cells per mm2 were lost during transportation (Hu, Tsai et al. 1999), and many were so severely edematous that they could not be transplanted, or they malfunctioned immediately after transplantation, or the ECD was lost by 72% four years after transplantation, and they were on the verge of malfunction. Clearly, a better method for stabilizing the cornea is needed.

[0006] In Europe, corneas are maintained in an "organ culture" system at 31-37°C for up to about 5 weeks (Armitage and Easty 1997, Bourne 2001, Thuret, Chiquet et al. 2003), but only 57% are suitable for transplantation after 4 weeks, and only 53% maintain an "excellent" rating within 2 weeks [9]. However, according to Redmond et al. (Redmond, Armitage et al. 1992), "Immediately after transplantation, organ-cultured corneas were thicker and more milky white than corneas stored at 4°C. 'And,' the transparency of the grafts was relatively slow, taking 3-4 weeks to obtain a good transparency." Since the cornea swells slowly during culture storage, it is necessary to "remove the swelling" in a concentrated polymer solution such as dextran T500 for about 2 days until it is suitable for transplantation (Thuret, Chiquet et al. 2003). This situation complicates the long-distance transport of cultured corneas. Because flaccid corneas are expected to be more sensitive to transport damage than corneas stored in Optisol-GS at 4°C. However, it is impossible to send the cornea while it is in the deflating agent unless it is transplanted within 2-3 days of arriving at its destination. Because leaving it in dextran (or other deflating agents) for a long time is harmful due to the irreversible uptake of dextran into the cornea. Another problem regarding the transport of cultured corneas is the difficulty of maintaining the corneal temperature during transport. Clearly, the current situation where damage cannot be prevented during corneal storage above 4°C leads to waste, extra cost, a decrease in the quality of transplantation, some late graft failures, and a reduction in the opportunity for corneal sharing between continents.

[0007] Despite the "immunological privilege" of the cornea (Niederkorn 2003), 28% of all graft failures up to 10 years in the low-risk population were due to rejection (relative risk, 17.3, p<0.001) (Ing, Ing et al. 1998). Clearly, vitrification makes it possible to gradually accumulate banks of corneas of different HLA and ABO types, enabling future matching to reduce rejection.

[0008] Commercial incentives for the development of long-term corneal preservation methods The potential commercial incentives for the development of successful clinical corneal banking are quite large. Around 2000, the price of corneas available for research was approximately $2000 for fresh transplant-grade corneas, $600 for corneas less than 4 days old, $350 for corneas less than 6 days old, and $200 for corneas 6 days or older. Corneas exported for transplantation due to their short shelf life were usually discounted to up to $500 (Campbell 2000). Thus, immediate vitrification of corneas upon arrival at the eye bank could potentially have brought a value of $1400 - $1800 per cornea for many or most corneas in 2000 dollars. Assuming a license fee of $800 per cornea, applied to 33,000 corneas used domestically in the United States and at least 43,000 corneas that could be fully utilized from elderly corneal donors for export, and adding the $400 / cornea for an additional 100,000 exported vitrified bioartificial corneas to meet 1% of the estimated annual overseas backlog, revenues in excess of $100 million per year would be obtained.

[0009] Considering the need for better corneal preservation and the economic incentives to develop it, the fact that there has been no commercially successful method for long-term corneal banking despite more than 70 years of effort (see below) is clearly due to a lack of sufficient technology, not a lack of sufficient incentives. This provides some evidence, albeit surprisingly, that the method of the present invention, which provides a solution to the long-standing problem of adequate cryopreservation of corneas, is not obvious compared to the prior art.

[0010] Prior art efforts for cryopreserving corneas The history of cryopreservation of the human cornea dates back at least to 1944, when Weiss and Taylor attempted freeze-drying and transplantation of rat corneas and claimed sustained transparency after transplantation (Weiss and Taylor 1944). Henaff also claimed success after freeze-drying canine corneas and transplanting them into human recipients (Henaff 1960), although freeze-drying is not considered a viable approach today.

[0011] The first attempt to preserve the cornea by slow freezing using a cryoprotectant was reported by Rob and Eastcott in 1954 (Rob and Eastcott 1954). They used 15% glycerol to freeze the cornea to the temperature of dry ice (-79°C), and it was observed that transparency persisted for up to 6 weeks after thawing and transplantation. Over time, numerous additional studies on cryopreservation of the cornea by freezing were published. Among these, the most popular method was developed by Capella, Kaufman, and Robbins in 1965 (Capella, Kaufman et al. 1965) (commonly called the Kaufman-Capella or K-C method), which involved freezing at 2-3°C / min to -80°C using a combination of dimethyl sulfoxide and sucrose, followed by transfer to liquid nitrogen. Corneas cryopreserved by the K-C method and other clinical freezing methods can usually maintain a clear state postoperatively after cryopreservation, but up to 15% of such corneas fail immediately after transplantation (Brunette, Le Francois et al. 2001), and the 10-year graft survival rate using the K-C method is as low as 47% (Brunette, Le Francois et al. 2001), compared to 78% for non-cryopreserved grafts in one study (Ing, Ing et al. 1998). Freezing inevitably results in differences in the degree of loss of corneal endothelial cells before and after transplantation, even if the time from death to freezing is limited to within 8 hours (Van Horn and Schultz 1974, Ehlers, Sperling et al. 1982, Ehlers and Sperling 1983, Taylor 1986, Brunette, Le Francois et al. 2001).Endogenous graft failure (failure to maintain transparency despite no recent rejection episodes) is associated with endothelial cell loss, so any cell loss is important (Ing, Ing et al. 1998, Bourne 2001, Armitage, Dick et al. 2003, Thuret, Chiquet et al. 2003). The authors of a 2001 retrospective study on the results of transplantation of corneas cryopreserved by the K-C method stated that "cryopreservation of corneas is not recommended for planned surgery" because of the high initial failure rate of cryopreserved corneas (Brunette, Le Francois et al. 2001). Canals et al. concluded: "Systematic cryopreservation of corneas in eye banking remains impossible until this high variability can be dramatically reduced" (Canals, Costa et al. 1996).

[0012] All attempts at the state-of-the-art for developing corneal vitrification methods have ended in failure (Armitage 1989, Brunette, Nelson et al. 1989, Rich and Armitage 1991, Rich and Armitage 1991, Bourne and Nelson 1994, Bourne, Shearer et al. 1994, Armitage, Hall et al. 2002). The fundamental problem associated with all these efforts is illustrated by the following summary of their specific findings by Bourne and Nelson: "To allow complete vitrification and rewarming of the cornea without ice formation, it was necessary to immerse the human cornea in four stepwise solutions at 0 °C for 25 minutes each. However, long-term exposure to this modified VS41A caused unacceptable damage to the corneal endothelium. A safe method of equilibrating the cornea with cryoprotectants is required to successfully vitrify the human cornea with this solution" (Bourne and Nelson 1994).

[0013] In 2002, Armitage reported that when rabbit corneas were vitrified and rewarmed in a solution containing 50% v / v (6.8 M) 1,2-propanediol, 0.25 M sucrose, 6% w / v polyethylene glycol, and 2.5% w / v chondroitin sulfate, they swelled during the first 3 hours of ex vivo perfusion but then, with active water restriction, achieved an essentially stable thickness value of approximately 0.6 mm compared to a control value of approximately 0.37 mm (Armitage, Hall et al. 2002). After thinning in 5% dextran for 2 hours, alizarin red and trypan blue staining was somewhat abnormal but there was no decrease in endothelial cell density. Furthermore, a single human cornea vitrified and rewarmed in the same way and thinned with dextran showed normal cell density and normal alizarin staining, but isolated cells sometimes showed trypan blue staining (Armitage, Hall et al. 2002). These results are promising and suggestive, but the evidence regarding human corneas is very limited (n = 1), there is no long-term study of corneal function after transplantation, and since this method employs a 12-step, 2.5-hour cryoprotectant loading and unloading procedure that requires seven different temperature-controlled baths, these results have not led to clinical utility. Considering how difficult it would be to perform this process in an eye bank, the authors themselves later judged their method to be unrealistic (Routledge and Armitage 2003).

[0014] Two patents claim methods for the cryopreservation of whole corneas by vitrification, in particular.

[0015] Linner and Goosey claimed a device and method for "freeze fixation in less than one second" (lines 26 - 27, column 2) by ultra - rapid cooling without using cryoprotectants (Linner and Goosey 1986). The so - called (but not actually almost certain) "vitrified" cornea is then dehydrated by sublimation of water near - 130 °C under high vacuum, obtaining the advantages of freeze - drying without forming ice crystals. There are no documents regarding long - term function after transplantation, nor documents regarding in vitro function tests and viability tests. As far as the inventor knows, since this method was introduced in this 1986 patent, this method has not been adopted by ophthalmic surgeons. This method has no similarity, either conceptually or practically, to the currently claimed invention.

[0016] In 1996, Wilkins and Watson of Organogenesis obtained a patent for a method of cryopreserving a living skin equivalent (LSE) with agitation, and speculated that this method was also applicable to the cornea (Wilkins and Watson 1996). This method involves a non - isothermal process (lines 4 - 13, 20, and 25, column 5) of immersing the specimen in a "cryoprotectant solution for a time sufficient to equilibrate the cells with the cryoprotectant" (lines 66 - 67, column 4 to line 1, column 5), where the "cryoprotectant solution is agitated for a time sufficient to allow effective penetration of the cryoprotectant" into the tissue (lines 31 - 33, column 5). Next, warming is performed "within about 1 - 3 seconds" (line 6, column 6), and the "cryoprotectant solution is removed within about 15 minutes after thawing" (lines 14 - 15, column 6). However, the invention regarding the cornea is essentially completely theoretical, and no actual experiments were conducted on any cornea or artificial cornea, and all experimental work was limited to LSE. As will be seen in the following description of the invention, this method did not anticipate the present invention, and as is clear from the above discussion, the invention of Wilkins and Watson has not been clinically successful for the cornea.

[0017] The third patent titled "Corneal vitrification, methods and devices for producing corneal vitrification, and methods of using the same" (Serdarevic, Berry et al. 2015) is not related to cryopreservation of the cornea by vitrification. Instead, this patent aims to form a vitreous state in the cornea in situ using optical means for the purpose of improving vision. In this process, the cornea is always kept warm and cryoprotectants are never used to cool it below -100°C to induce a vitreous state.

[0018] To date, cryopreservation of bioartificial corneas has not been reported to our knowledge. However, with our method, bioartificial corneas are also preserved in the same way as natural corneas.

[0019] There is prior art for preserving corneal endothelial cells not attached to the actual cornea and for vitrification of general tissues, but it is not useful. This is because a) it is impossible to predict the results of a whole cornea preservation protocol using this information (since attached cells show different reactions from unattached cells, the cornea is composed of three organized structures and is not composed of just unattached cells or isolated cell layers, and it is well known in the art that methods effective for isolated cells or one tissue type are generally not effective for cell aggregates or completely different tissue types), and b) this information, although generally available for over a decade, has not so far been able to produce a useful method for whole cornea vitrification.

[0020] In conclusion, this technique can be summarized by citing the current available up-to-date review on corneal cryopreservation (2021). In this review, it is stated that "cryopreservation is currently only recommended in case of emergency for structural corneal transplantation" (Rodriguez-Fernandez, Alvarez-Portela et al. 2021). Therefore, the need for an appropriate method of corneal cryopreservation is not met by the prior art, and considering the strong motivation to overcome the current deficiencies, the successful technique is not obvious compared to the prior art.

Summary of the Invention

[0021] In one aspect, the present invention is a method for preserving animals, humans, and bioartificial corneas in a substantially undamaged state by vitrification, not by achieving cryoprotection and osmotic balance of the cornea as attempted and required by all conventional methods, but by promoting vitrification at a very low cooling rate, preventing devitrification at a very low warming rate, and inducing extremely non-equilibrium corneal dehydration intentionally by the reverse approach of preventing the toxicity of cryoprotectants. The method of the present invention is preferably implemented using specially designed equipment that enables proper handling, storage, and rewarming of the cornea while preventing mechanical damage to the cornea. The present invention dramatically raises the glass transition temperature of the cornea by extreme dehydration and, surprisingly, provides conditions for cryoprotectant exposure that block the toxicity of cryoprotectants during long-term exposure above 0°C. The present invention includes the rapid introduction and washing of a highly vitrifiable concentration of cryoprotectant with a high theoretical tonicity under practical isothermal conditions that can be easily achieved in a normal eye bank.

[0022] The present invention enables truly selective timing of the use of both locally procured corneas and corneas shared globally, eliminates the deterioration of corneas over time, eliminates seasonal variations in the availability of corneas for transplantation, ensures the availability of corneas in emergencies, reduces logistics costs, and in addition to eliminating corneal aging, it may also reduce rejection reactions as a cause of graft failure by enabling tissue matching between donors and recipients during the corneal storage period.

[0023] In one embodiment, the present disclosure provides a method for cryopreserving a human, animal, or bioartificial cornea, the method comprising placing the cornea in a cryoprotectant solution and increasing the cryoprotectant concentration in the solution around the cornea from 0% of the target cryoprotectant concentration for vitrification to 100% of the target cryoprotectant concentration for vitrification in 0 to 25 minutes, most preferably in less than 20 minutes, and continuously or in 1 to 3 steps, more preferably in 5 to 15 minutes, even more preferably in 8 to 12 minutes, and preferably continuously or in 1 to 3 steps, and then maintaining the cornea in a solution containing 100% of the target cryoprotectant concentration for vitrification for 9 to 30 minutes before vitrification, most preferably for 10 to 25 minutes before vitrification, and then vitrifying the cornea, storing it for a desired time, and then rewarming it, and then decreasing the concentration of the cryoprotectant solution around the cornea from 100% of the target concentration for vitrification to 0% of the target concentration for vitrification in 0 to 25 minutes, preferably in less than 20 minutes, and continuously or in 1 to 3 steps, most preferably in 0 to 15 minutes, and continuously or in 1 to 3 steps, including the method is preferably carried out under nominal isothermal conditions of 0 to 10 °C, and the cornea can be cooled and warmed at a rate of 5 °C / min or less at 0 °C to -100 °C without ice formation, and a different carrier solution can be used when decreasing the cryoprotectant concentration than when increasing the cryoprotectant concentration, and The cornea is manipulated during the method by manipulation means comprising a ring and suspension means for the ring, or the cornea is maintained immobile within the ring of the manipulation means, during which time the concentration of the cryoprotectant in contact with the cornea changes continuously without manipulation of the cornea.

[0024] Another embodiment of the invention comprises manipulation means for the cornea, comprising a ring for supporting the cornea and means for positioning the ring on the floor of any container in which the cornea can be placed so as to prevent contact between the cornea and the floor of the container. The means for positioning the ring on the floor of any container in which the cornea can be placed may consist of a stand extending downward from the ring to the floor of the container, or The means for positioning the ring on the floor of any container in which the cornea can be placed may consist of a handle attached to the ring that can be manually grasped and held, or by a positioning device. The means for positioning the ring on the floor of any container in which the cornea can be placed may comprise both a stand and a handle for facilitating movement of the ring and the stand, and / or The means for positioning the ring may comprise a vertical member extending upward from the ring and attached to a movable lid of a container in which the cornea can be placed, the vertical member positioning the cornea above the floor of the container without contact between the cornea and the floor of the container when the movable lid of the container is used to close the container. The means for positioning the ring may, in addition to the vertical member extending upward from the ring and attached to a movable lid of a container in which the cornea can be placed, comprise a grippable handle disposed above the lid to further facilitate movement of the lid, the vertical member, the ring, and the cornea within the ring.

[0025] The patent or application file contains at least one drawing, preferably executed in color. Copies of this patent or patent application publication that include color drawings are provided by the United States Patent and Trademark Office upon request and payment of the required fee.

Brief Description of the Drawings

[0026]

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DETAILED DESCRIPTION OF THE INVENTION

[0027] In a preferred embodiment, the present invention includes a method for cryopreserving a human, animal, or bioartificial cornea by immersing the cornea in a concentrated cryoprotectant solution for a time sufficient to induce significant outward osmosis of water from the cornea, but insufficient to allow equilibration of the cornea with the cryoprotectant solution, thereby inducing an extreme dehydration state of the cornea. "Extreme" in this context means increasing the glass transition temperature (TG) of the cornea by at least 7 °C relative to the TG of the vitrification solution used to vitrify the cornea, decreasing the change in heat capacity at the glass transition of the cornea by at least 40% relative to the change in heat capacity at the glass transition of the vitrification solution used to vitrify the cornea, and showing dehydration sufficient to reduce the critical cooling and warming rates of the cornea to 5 °C / min or less between 0 °C and -100 °C. Specific means for achieving these endpoints can be reliably applied without the need to measure the endpoints achieved and are described in the following examples.

[0028] In one embodiment of the present invention, M22 and solutions having a composition and concentration comparable to M22 (Fahy, Wowk et al. 2004, Fahy 2005) are used and are preferred for use in the present invention. However, no specific cryoprotectant solution is required to practice the method of the present invention as long as a sufficiently non-toxic cryoprotectant solution is introduced in accordance with the method of the present invention described herein. In essence, this process involves a loading process in which the concentration of the cryoprotectant is increased from 0% VS to 100% VS, preferably in less than 20 minutes and either continuously or in less than 4 steps, preferably over 8 - 12 minutes and either continuously or in 2 - 3 steps. This process further includes an unloading process in which the VS is removed, preferably in less than 20 minutes and either continuously or in 2 - 3 steps, preferably over 8 - 15 minutes and either continuously or in 2 - 3 steps. This process is preferably also carried out under nominal isothermal conditions of 0 - 10 °C. This method further includes exposing the cornea to 100% VS for 10 - 30 minutes, most preferably 20 - 25 minutes, prior to vitrification, after the process of increasing the concentration to 100% VS. Based on the examples and other guidance provided herein, one of ordinary skill in the art will be able to practice the invention as claimed in many different specific embodiments.

[0029] In a preferred embodiment of the present invention, LM5 optimized for vitrification or a similar carrier is used as the carrier solution for adding cryoprotectants (Fahy, Wowk et al. 2004, Fahy 2005), and CPTES plus 2.5% chondroitin sulfate (Taylor and Hunt 1989) or another carrier optimized for stabilizing the viability of the cornea is used as the carrier for washing the CPA. To promote vitrification or for use in organs, the solution optimized for use in organs is not necessarily ideal for the cornea. Therefore, shortening the exposure time to LM5 or other carriers not optimized for use in the cornea is optional but considered beneficial in the present invention.

[0030] The method of the present invention is optimally carried out using a device comprising at least one ring for supporting the cornea and one means for positioning the ring on the floor of any container in which the cornea can be placed so as to prevent contact between the cornea and the floor of the container.

[0031] The means for positioning the ring may consist of a stand extending downward from the ring to the floor of the container.

[0032] Alternatively, the means for positioning the ring may consist of a handle attached to the ring that can be manually grasped and held, or by a positioning device.

[0033] Alternatively, the means for positioning the ring may consist of a combination of a stand and a handle to facilitate the movement of the ring and the stand.

[0034] Alternatively, the means for positioning the ring may comprise a vertical member extending upward from the ring and attached to the movable lid of the container in which the cornea can be placed. When the movable lid of the container is used to close the container, the vertical member positions the cornea above the floor of the container without contact between the cornea and the floor of the container.

[0035] Alternatively, the means for positioning the ring may comprise a vertical member extending upwardly from the ring and attached to a movable lid of a container in which the cornea can be placed. The vertical member positions the cornea above the floor of the container without contact between the cornea and the floor of the container when the movable lid of the container is used to close the container. The movable lid further includes a grippable handle located above the lid to facilitate movement of the lid, the vertical member, the ring, and the cornea within the ring.

[0036] The method of the present invention is also optimally carried out either using a series of containers containing cryoprotectants of different concentrations in which the cornea on the positioning ring is continuously moved, or using a continuous cryoprotectant concentration gradient provided by gradient-forming means, where the cornea is positioned in a bath in which the concentration continuously changes by the gradient-forming means.

[0037] The method of the present invention may also optionally be carried out by transferring the cornea on the ring to a pre-cooled storage container located in a low-temperature environment, which may be an environment having a temperature lower than the TG of the corneal vitrification solution, thereby vitrifying the cornea in a gaseous environment where the cooling rate may be as low as less than 5 °C / min between 0 °C and -100 °C, and sealing the pre-cooled storage container.

[0038] Alternatively, the present invention may be carried out by transferring the cornea on the ring to a low-temperature environment, which may be an environment having a temperature lower than the TG of the corneal vitrification solution, thereby vitrifying the cornea in a gaseous environment where the cooling rate may be as low as less than 5 °C / min between 0 °C and -100 °C, and then transferring the cornea on the ring to a pre-cooled storage container and sealing the pre-cooled storage container.

[0039] The following description of the details of the present invention is intended to be exemplary rather than exhaustive or limiting. Those skilled in the art will recognize that variations of the methods, solutions, and devices specifically described herein that do not depart from the spirit, scope, and meaning of the described invention are merely different embodiments of the same invention and will clearly fall within the scope of the claimed invention. For example, introducing a temperature change that is not critical for the sole purpose of avoiding the present invention, for the reason that the present invention is preferably an isothermal process, will still result in a process that falls within the spirit and scope of the present invention. The components of the present invention are illustrated by the following examples. The present invention itself includes the entirety of these examples and suitable variations thereof, such as those described above.

[0040] Definitions First, it is useful to precisely understand the present invention by defining certain terms used herein.

[0041] A "cryoprotectant," also referred to as a "cryoprotective agent" or "CPA," is a molecule that reduces or prevents freeze injury by reducing or preventing the formation of ice below 0°C. The properties and characteristics of cryoprotectants are well known in the art (Meryman 1971, Fuller 2004, Fahy 2005, Abazari, Meimetis et al. 2015, Elliott, Wang et al. 2017). These are composed of permeating cryoprotectants (pCPA) and non-permeating cryoprotectants (npCPA). The use of pCPA and npCPA in the present invention will be described in more detail below. The term "cryoprotectant" or "CPA" as used herein can refer to one or more cryoprotectants and either pCPA alone or a mixture of pCPA and npCPA. For example, adding or washing a solution known as M22 (Fahy, Wowk et al. 2004) or a related solution (Fahy 2005), which is a preferred solution for use in the present invention, can be referred to as adding or washing a "cryoprotectant" or "CPA," even if these solutions are composed of multiple pCPA and multiple npCPA.

[0042] The "carrier" solution is a component of the cryoprotectant solution other than the cryoprotectant (Fahy 2005, Fahy and Wowk 2021). It is composed of solutes necessary to maintain cell viability and health, regardless of the presence or absence of cryoprotectants. A non-exhaustive list of carrier solutions used in past studies includes LM5 (Fahy 2005), Eurocollins solution (Khirabadi and Fahy 1994), CPTES and CPTES plus chondroitin sulfate (Taylor and Hunt 1989), culture media, organ preservation solutions, and simple salt solutions.

[0043] "TG" is the glass transition temperature, which indicates the temperature range where a transition occurs from a liquid state to a glass state upon cooling, or from a glass state to a liquid state upon warming (Fahy and Wowk 2021).

[0044] "Vitrification" is the conversion of a liquid to a solid without crystallization. The solid formed by vitrification is known as a glass and is structurally similar to a liquid but has little translational molecular motion. "Vitrification" is also a method of cryopreservation that does not involve ice formation.

[0045] "Devitrification" is the formation of ice when a previously vitrified system is reheated. This is not the reverse of vitrification. The reverse of vitrification is liquefaction.

[0046] A "vitrification solution" or "VS" is a cryoprotectant solution at a concentration sufficient to prevent ice formation during cooling to TG at the cooling rate employed to preserve the biological system being preserved (Fahy, MacFarlane et al. 1984, Fahy and Wowk 2021).

[0047] "Equilibration" or "equilibrium" is achieved when diffusion stops due to the leveling of the concentration gradient between the cornea and its environment.

[0048] "Osmotic pressure equilibrium" is considered to be achieved when, after the previous osmotic pressure volume change of the cornea induced by changing the pCPA concentration of the medium around the cornea, the cornea has recovered to its original volume by equilibration of the cryoprotectant concentration between the cornea and the cryoprotectant medium to which the cornea is exposed. When one or more npCPA are present in the medium around the cornea, it is considered that osmotic pressure equilibrium has been reached when the cornea has reached a stable reduced equilibrium volume induced by exposure to the npCPA, and the stable reduced volume is within 20% of Vsr = (Vi - b) × πi / πf + b. Here, Vsr is the stable reduced volume of the cornea, Vi is the corneal volume in a normal isotonic medium and in the absence of a cryoprotectant, b is the non-osmotic volume of the cornea (constant), πi is the osmotic pressure of an isotonic carrier solution without npCPA, and πf is the sum of the osmotic pressure of the CPA carrier solution and the osmotic pressure of the npCPA.

[0049] "External osmosis" is the movement of water out of the cornea by immersing the cornea in a solution having an osmotic pressure higher than the osmotic pressure of the cornea before immersion in the solution.

[0050] "Rewarming" means returning the temperature of the vitrification system to a temperature near 0°C or higher.

[0051] Unless otherwise specified or clear from the context, the "a", "an", and "the" used in this specification are understood to be singular or plural.

[0052] Furthermore, the "and / or" used in this specification should be construed as a specific disclosure of each of the two specified features or components regardless of the presence or absence of the other. Thus, the term "and / or" used in phrases such as "A and / or B" in this specification is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A (alone), B (alone), and C (alone).

[0053] Unless otherwise specified or not apparent from the context, the term "about" as used herein is understood to be within the normal tolerance range in the art, for example, within two standard deviations of the average value. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise apparent from the context, all numerical values provided herein are modified by the term about.

[0054] Any composition, apparatus, system, or method provided herein can be combined with any one or more of the other compositions, apparatuses, systems, or methods provided herein.

[0055] In the present disclosure, terms such as "comprising," "including," "containing," and "having" can have the meanings given to them in the United States Patent Law, and can also mean "comprising," "including," etc. The terms "consisting essentially of" or "consisting essentially" similarly have the meanings given in the United States Patent Law, and this term is an open-ended one that allows the presence of more than what is described, provided that the basic or novel characteristics of what is described are not changed by the presence of more than what is described, except for prior art embodiments.

[0056] The ranges provided herein are understood to be a shorthand for all values within the range. For example, a range from 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0057] Example 1: Human Cornea Handling Device The inventors first observed that the cornea tends to be damaged if not handled properly. Ultraperfusion systems such as those previously used in the Bone Laboratory at the Mayo Clinic (Brunette, Nelson et al. 1989, Bourne, Shearer et al. 1994) hold the cornea safely, but due to their large mass, they cannot be used for corneal vitrification or rewarming. In the prior art, the cornea was probably simply placed on the floor of a small container with the endothelial side up, using forceps perhaps, and transferred from one cryoprotectant solution to another solution or other environment. However, this technique was found to cause epithelial damage due to the contact between the epithelium and the floor of the container.

[0058] The inventors were first able to prevent this damage by supporting and transferring the cornea with the endothelial side up on a square plastic O-ring that suspends the bottom of the cornea above the floor of the container when the O-ring is placed on the floor of the container, through a series of 10 ml solutions at 4°C. This is useful for the introduction and removal of cryoprotectants, but does not allow for easy vitrification and rewarming and does not allow for free diffusion of the cryoprotectant around the epithelial surface of the cornea.

[0059] Accordingly, the O-ring was changed to the round (torus-shaped) ring A of FIG. 1. The preferred inner diameter of the O-ring is 12 mm, although this can vary in the range of 11-13 mm, and the preferred thickness of the O-ring is 1 mm, although this can vary in the range of 0.5-4 mm. The O-ring is also attached to the bottom of the vertical member (wand) B of FIG. 1, facilitating transfer from solution to solution, from VS to liquid nitrogen vapor, and from liquid nitrogen vapor to VS, and enabling rapid warming. This allowed for short-term experiments, but did not include a function to protect the cornea from potential damage due to mechanical contact with storage containers during long-term storage or transportation. This latter issue was resolved by incorporating the cornea loop and wand into the lid C of the long-term storage container F of FIG. 1. This system allows for a) the cornea to be transferred from container to container without the need to manually hold or otherwise secure the wand, b) each solution to be protected from contamination by using the cornea transfer device A+B+C as the lid of each container during the addition and removal of cryoprotectant, c) contact between the cornea and the floor of each cryoprotectant container to be prevented and the cornea to be freely diffusible around the epithelial side during contact with the cryoprotectant, d) the cornea to be vitrified passively within the atmosphere of container F by placing the cornea handling device on top of the pre-cooled storage container F and fixing it to protect the cornea from contact with condensed water vapor around container F, e) the cornea to be transferred to another low-temperature environment (liquid nitrogen vapor, cold gas, or a combination of liquid nitrogen vapor and cold gas) for vitrification and then transferred to its storage container F by screwing or snap-fitting the cornea handling device A+B+C into the storage container, and finally f) the cornea to be pre-warmed or fully re-warmed (slow warming stage: pre-warming; rapid warming stage: re-warming) by pre-warming or re-warming its storage container as shown in FIG. 6B and / or the cornea to be re-warmed by transferring the cornea to a container of VS at an appropriate temperature on its handling device A+B+C. Optionally, for ease of handling, the extension D of the wand B can pass through the lid C and can be decorated with an additional member E to be easily gripped by hand or an appropriate gripping device as needed.

[0060] Any other variations of this device and the use of continuous means for adding and removing cryoprotectants are described above. The gradient forming means is well known in the art and may be a much simplified version of the technology developed for cryoprotective perfusion of organs (Fahy 1994).

[0061] Example 2: Human corneas survive extreme dehydration followed by vitrification: in vitro results (loading / unloading method 1) Corneas. In the experiments of Example 2, a total of 75 human corneas stored at 4 °C for 2 - 9 days in Optisol-GS were obtained from the Texas Lions Eye Bank in Texas or the Doheny Eye Tissue Transplant Bank in Los Angeles, California. These corneas were considered unsuitable for clinical transplantation due to arcus or systemic disease of the donor, but were suitable for our purposes as the endothelium remained intact.

[0062] Protocol. The steps for adding and removing cryoprotectants were as described in Method 1 of Figure 3. In all experiments involving cryoprotection, M22 was used as the vitrification solution, LM5 was used as the carrier solution for loading M22, and CPTES and 2.5% chondroitin sulfate were used as the carrier solutions for the M22 wash step.

[0063] For reference, in the absence of other cryoprotectants, the total effective molar concentration of M22 when the osmotic pressure contribution of its polymer component is made equal to the molar concentration (milliosmoles per millimole) is 9.45 M (Fahy, Wowk et al. 2004). The melting point of M22 is approximately -54.9 °C (Fahy, Wowk et al. 2004). Considering the approximation of a standard dilute solution where the freezing point drops by 1.86 °C for every 1 osmolar increase in solute concentration, the osmotic pressure of M22 can be roughly estimated as 54.9 °C / (1.86 °C / osmole) = 29.5 osmoles (Osm). Considering that the freezing point of plasma is approximately -0.55 °C, the normal osmotic pressure of most human cells is about 0.55 / 1.86 = 0.296 Osm or 296 milliosmoles (mOsm). Therefore, it can be said that M22 is approximately 100 times more concentrated than plasma based on osmotic pressure.

[0064] As background, studies on many cells have shown that most cells cannot withstand exposure to an increase (or a solution of 4 times the isotonic osmotic pressure) that exceeds 4-fold (4X) the extracellular osmotic pressure in one step (Meryman, Williams et al. 1977). However, surprisingly, the human cornea can be directly immersed in semi-strength M22 (about 4.73 M M22) that is isotonic with an osmotic pressure of at least (4.73 + 0.296) / 0.296 = 17-fold for 10 minutes, transferred to full-strength M22 for 25 minutes, vitrified and rewarmed as described below, immersed in 30% full-strength M22 for an additional 10 minutes, and finally transferred to M22-free medium without an osmotic buffer (Method 1 in Figure 3), as determined based on multiple in vitro endpoints. All procedures for loading and unloading M22 were performed at 4 °C (isothermal process). Control corneas were kept at 4 °C in CPTES + 2.5% CSA during loading and unloading of the treated corneas.

[0065] In vitro results and discussion. Figures 2A and 2B show the results of vital staining and ECD of this protocol, respectively. Syto 13 stains the nuclei of living cells green (Yang, Acker et al. 1998), and ethidium homodimer-1 stains the nuclei of non-living cells red. In addition to providing a vital staining score, the selective binding of Syto-13 to the nuclei provides a convenient way to identify and thus count individual corneal endothelial cells for calculating ECD. The cornea was stained with Syto 13 and ethidium homodimer-1 in D-PBS at room temperature for 40 minutes, and then a 7.5-mm button was excised from the central region of the cornea for observation and ECD measurement. The endothelium was observed with a fluorescence microscope (Olympus, IMT-2 inverted research microscope). As shown in Figure 2A, virtually all corneal endothelial cells (CECs, corresponding to the faint spots in the figure) were stained green, and there were few obvious red blood cells after vitrification and M22 washing.

[0066] Furthermore, few cells were lost from the corneal endothelial monolayer (Figure 2B). We tested the accuracy of our own ability to determine ECD by comparing the ECD counts of control corneas (white dots) and vitrified corneas (black dots) with the ECD counts reported from the original eye bank for the same corneas before we received them [using the method of Karnama et al (Karnama and Khodadoust 1986)]. As you can see, there is a data cluster near the line of ECD 21CM =ECD EB (with 0% cell loss in our facility), where ECD 21CM is the corneal endothelial cell density measured in our facility, and ECD EBis the corneal endothelial cell density measured by the eye bank. This holds true regardless of whether endothelial cells were counted in vitrified corneas (n = 8) or control (non-vitrified) corneas (n = 7), and deviation from the line is not a negligible change induced by vitrification, osmotic stress, corneal "aging" or transport, or mishandling in our laboratory, but rather measurement error. There are two exceptions. In one case (black dot), the vitrified cornea showed an ECD just over 2000 against a predicted ECD of approximately 2900. On the other hand, in another case (white dot), the control cornea showed an ECD of approximately 1900, while the ECD of the same cornea in the eye bank was approximately 2750 cells / mm 2 which indicates that loss of ECD is at least as likely in control corneas as in vitrified corneas, and again it is demonstrated that there is no difference in ECD between the entire vitrified and control corneas. Statistically, the p-value for the difference between the average of the eye bank count and our count was p = 0.49 for control corneas and p = 0.62 for vitrified corneas. Furthermore, notably, even the ECD just over 2000 in the vitrification-outlier corneas is above the cut-off line for transplant compatibility (the 2000 horizontal line).

[0067] After immersion in a solution at least 17 times more concentrated than the LM5 carrier solution in which the cornea was initially placed, a large-scale osmotically induced corneal dehydration is expected. Further, the cornea was immersed in this solution for only 10 minutes, but Bourne reported that 20 minutes or more were required at each of four loading steps for the human cornea to take up sufficient cryoprotectant to vitrify (Bourne and Nelson 1994). Thus, after exposure to 50% full strength M22, when the cornea was transferred to M22 and exposed to twice the concentration, the cornea was still very dehydrated and, in the prior art, this procedure was expected to be lethal to the cornea. For example, Taylor found that corneas immersed in 3M dimethyl sulfoxide (Me2SO, approximately 15.8x isotonic) were severely damaged, whereas corneas exposed to the same concentration recovered by first transferring them to IM and then to 2M Me2SO before exposure to 3M (Taylor and Hunt 1989). This is presumably due to avoidance of osmotic (shrinkage) stress. Nevertheless, in our experiments, surprisingly, the corneas recovered not only after direct exposure to 4.73M M22 but also after an additional 25 minutes of exposure to 9.45M M22 at 4°C.

[0068] Although not wishing to be bound by any theory, it is speculated that the reason for this unpredictable survival being observed is that a large amount of water loss occurred during the first stage of this two-step procedure, and then, when the permeability of the corneal cryoprotectant substantially disappeared and a certain minimum amount of permeation occurred, a substantial renewal of the M22 cryoprotectant to the cornea was possibly hindered. Alternatively, and more likely, following the first large-scale dehydration stage, another large-scale dehydration stage to peak concentration (which is expected to be fatal and has never been considered in the prior art) was carried out, and thus the suppression of CPA uptake might have been completed. The maximally shrunk cells containing relatively little intracellular water might be too dense and viscous inside (the intracellular protein concentration is too high) for additional cryoprotectant to enter, or the cell membrane permeability has changed, or both. This also explains the abnormal resistance of these corneas to dilution. A substantial equilibrium of M22 to the cornea during M22 loading should result in large-scale osmotic swelling of the whole cornea and corneal cells, especially when rapidly diluted without an osmotic buffer (Taylor and Hunt 1989, Meryman 2007), but this was not observed. However, importantly, the inventors found that ice was formed during cooling when the cornea was held in M22 for only 20 minutes before cooling, but ice formation was hindered when held in M22 for 25 minutes, which suggests that in the described protocol, the outflow of water from the cornea continues for 20 to 25 minutes of contact with M22. Therefore, for this protocol, the most preferred holding time in M22 is 21 to 25 minutes, especially 23 to 25 minutes.

[0069] However, in another protocol that includes exposure to 50% M22 for 3 - 5 minutes, exposure to M22 for 9 - 10 minutes, and exposure to 30% full M22 for 5 minutes before dilution to 0% M22, ice was not formed and a good recovery rate was obtained (data not shown). Therefore, overall, the minimum holding time at the peak concentration of the protocol is at most 9 - 10 minutes.

[0070] Another factor that may promote survival is the short total CPA exposure time enabled by a very short loading and very short unloading process. However, typically, the toxicity of CPA progresses most rapidly at the highest concentration used, and in this case, the full exposure to M22 lasts 2.5 times longer than the exposure to the loading or unloading solution, which is another unique feature of this method. Prolonged exposure to M22, or the highest concentration used in the process if it is not M22, is thought to be beneficial in ensuring sufficient extraction of water from the cornea by osmosis and preventing ice formation during cooling and warming, and the evidence of dilution tolerance above and DSC evidence below indicate that it is this dehydration, rather than the uptake of M22 into the cornea, that is primarily involved in stabilizing the cornea against ice formation.

[0071] However, it was surprising that the corneal endothelium was able to withstand such large-scale shrinkage and such large-scale dilution without osmotic buffering between the latter. This tolerance could not be predicted from studies on isolated corneal endothelial cells that predicted cell death after exposure to step changes to 4.4-fold isotonic medium (<<17-fold isotonic) or after diluting cells equilibrated in 0.25M Me2SO (corresponding to only 2.6% of the concentration of M22, or 8.8% of the concentration of full-strength M22) to 0M Me2SO in one step (Taylor and Hunt 1989). Separate from cell survival, it was also remarkable that the endothelium remained attached to the cornea. M22 was so named because it is intended to be in contact with live cells only at a temperature close to -22°C to eliminate its toxicity at high temperatures, but as described, our corneas withstood prolonged exposure at 4°C when treated with M22.

[0072] The theory of cryoprotectant uptake inhibition is independent of the nature of the cryoprotectant, as long as a sufficiently high concentration is introduced sufficiently rapidly. However, prevention of CPA uptake into cells is also promoted by the presence of npCPA sufficient to raise the tonicity of the CPA carrier solution considerably. The carrier used in past corneal studies, CPTES, contains 2.5% chondroitin sulfate (CS), a negatively charged glycosaminoglycan considered a non-permeating CPA. However, considering the molecular weight of this molecule (average of about 40,000 daltons, range of about 25,000 - 80,000 daltons), the osmotic pressure contribution of 2.5% CS is predicted to be about 25 / 40,000 - 0.6 mOsm, which is not enough to measurably change the tonicity of CPTES. On the other hand, the exemplary M22 solution contains an npCPA polymer mixture that raises the tonicity of the carrier solution to about 1.5 times isotonic, and the present invention can be usefully practiced by optionally but beneficially including VS npCPA that raises the tonicity of the carrier solution to 1.2 - 1.7 times isotonic or more [as calculated in the absence of cryoprotectant (Fahy, Wowk et al. 2004)] to inhibit CPA uptake.

[0073] The cooling and warming methods used in these studies are described in Example 3.

[0074] Example 3: Human corneas survive extreme dehydration and subsequent vitrification: In vivo results with xenografts from human to rabbit (load / unload method 2) To further define the scope of the present invention and to rigorously verify the survival and functional recovery of the vitrified cornea, a second extreme dehydration protocol was investigated and evaluated by transplanting 15 vitrified corneas into rabbits and comparing their performance with that of 4 control human corneas. In these experiments, the loading and unloading process of M22 was changed to the following steps: 25% M22 for 5 minutes, 50% M22 for 5 minutes, 100% M22 for 25 minutes; 50% M22 for 5 minutes, 25% M22 for 5 minutes, 0% M22 for 10 minutes (Method 2 in Figure 3). Here too, the carrier for M22 addition was LM5 (excluding calcium and magnesium as usual), and the carrier for M22 removal was CPTES and 2.5% chondroitin sulfate, and all steps were carried out again at 4°C. What is common to the two protocols shown in Figure 3 is that the time required to go from no CPA to 100% of the VS full concentration is 10 minutes in both cases, which is unprecedented in the art. Similarly, in both methods, the time taken to go from 100% VS to 0% VS is 10 minutes, which is also unheard of in the art, especially when no osmotic buffer is used, which is not adopted in either method. The transplanted vitrified corneas were stored at -145°C for 2 to 174 days (mean and standard deviation: 35 ± 47 days) before rewarming and transplantation. The effect of the storage time was not obvious.

[0075] All animal use was conducted with the approval of the Institutional Animal Care and Use Committee of 21st Century Medicine, Inc. and in accordance with current USDA and NIH guidelines. Male New Zealand white rabbits (Harlan Sprague Dawley, Indianapolis, Indiana) weighing 2.8 - 3.5 kg were premedicated and anesthetized according to standard approved procedures, and human xenografts were transplanted into only the right eye, leaving the left eye with normal vision. The surgical method was similar to that of Beyer et al (Beyer, Lin et al. 1990) and Khodadoust et al (Khodadoust 1968). The globe was exposed and fixed, and a central corneal disk 7.5 mm in diameter was removed from the recipient cornea and replaced with a corneal button 7.5 mm in diameter from vitrified / warmed human corneas. The anterior chamber was allowed to reform spontaneously, and atropine eye drops were applied at the end of the procedure. The necessary postoperative care was to instill atropine occasionally when the pupil was constricted. Antibiotics such as gentamicin, neomycin / polymyxin B / dexamethasone ointment, and steroids such as prednisolone acetate eye drops were used as needed. All grafts were observed daily and photographed for graft transparency, wound dehiscence, infection, etc. To avoid rejection, the transplanted corneas were removed at 10 ± 3 days (mean ± standard deviation) postoperatively. The corneas were fixed with 2.5% glutaraldehyde and sectioned for examination by light microscopy (histopathology), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), and all corneal evaluations were performed blindly by a consulting ophthalmic pathologist affiliated with another institution.

[0076] Visually, 10 out of 19 grafts remained clear from the first day after surgery, while 5 showed slight clouding during the first 2 days and became clear on days 4 - 7, affecting the epithelium rather than the endothelium. Overall, no difference was seen between the control corneas and the vitrified corneas. One clear control and one clear vitrified graft are shown in Figures 4A and 4B, respectively. The circular dotted line indicates the junction between the graft and the recipient cornea. Histologically, the vitrified corneas showed a clearly intact layer after vitrification, although there was some loss of epithelium. By SEM, it was found that the endothelium of the vitrified / re - warmed grafts (Figure 4C) was preserved similarly to that of the control grafts (not shown). The endothelial cells appeared flat and intact, and the boundaries were easily distinguishable. Transmission electron microscopy of vitrified and xenografted human corneas showed relatively normal endothelium with some changes consistent with proper adhesion of the endothelium to Descemet's membrane and continued good function even in the absence of a xenograft reaction (results not shown).

[0077] The transparency of the graft after transplantation demonstrates the survival of the donor endothelium. Since replacement of the donor graft endothelium by the growing host endothelium in rabbits does not commence until after the 4th postoperative day (Nakahori, Katakami et al. 1996), endothelial insufficiency prior to 4 days necessarily causes graft turbidity, which was not observed. In fact, the functional performance of these vitrified human grafts exceeds that of human corneas transplanted after organ culture and remains milky white for up to 4 weeks postoperatively (Redmond, Armitage et al. 1992). Secondly, evaluation by our consulting pathologist indicated that the peripheral turbidity observed on the 15th postoperative day represented infiltration by host immune cells. However, this also implies survival of the donor endothelium sufficient to cause an immune response on the 15th day. Notably, although inflammatory infiltration was dramatically reduced in the vitrified rewarmed grafts, there was no obvious reduction in ECD compared to controls. Furthermore, in rabbit-to-rabbit corneal transplantation, it has been observed that when donor endothelial cells are replaced by host endothelial cells, corneal turbidity occurs for 10 - 14 days (Chi, Teng et al. 1965), which was not observed. Therefore, human endothelial cells on vitrified rewarmed corneas may be able to maintain stromal hydration within a range compatible with normal corneal transparency for several days after transplantation, and appear to be less immunogenic than control corneal endothelial cells.

[0078] Differential Scanning Calorimetry Study of Corneas Vitrified by Method 2. To confirm the ability of the corneas processed as described above to escape ice formation during both cooling and warming, a differential scanning calorimetry (DSC) study was conducted on three available corneal samples processed with a second M22 load and unload protocol. Two corneal samples [4.8 mg for Cornea HC05-17(2) and 12.7 mg for Cornea HC05-4(1)] were cooled from +10 °C to -150 °C at 5, 10, 20, 40, and 80 °C / min and then reheated to the starting temperature at the same rate. No evidence of freezing or melting was seen in the thermograms, indicating that the corneal tissue remained completely ice-free during both cooling and reheating at all rates examined, and that the corneas were definitely stable against ice formation even at cooling and warming rates below 5 °C / min. The only events observed were glass transitions at -110 °C in one cornea and -97 °C in the second cornea. The third cornea (16.2 mg) was cooled to -150 °C at 100 °C / min and then reheated at 5, 10, 20, 40, or 80 °C / min. No melting peak of ice was seen in this cornea either, indicating, as in the other corneas, that this cornea was definitely stable against ice formation even at warming rates below 5 °C / min. A glass transition was seen at -110 °C in this cornea. Representative thermograms of the first and third corneal samples are shown in Figure 5. These results were also consistent with the macroscopic observations. After cooling to -134 °C, no visible ice formation or cracks were seen in the corneas or in the M22 contained in the "bowl" of the corneas. Furthermore, no cracks or obvious devitrification were visually observed during warming.

[0079] Importantly, the glass transition temperatures observed in the three samples (-110 °C, -110 °C, and -97 °C) were all dramatically higher than that of the pure M22 cryoprotectant solution (-124 °C) used to prevent corneal freezing. It is abnormal for the glass transition temperature of the tissue to be significantly different from that of the cryoprotectant solution penetrating the tissue, and this difference can only be explained by water extraction from the cornea that is not proportional to the water content of M22, which has the effect of increasing the corneal protein concentration and thereby raising the TG (Rasmussen 1969). Due to the osmotic and chemical imbalance (infiltrative water extraction) between the cornea and its M22 environment, the viscosity of the liquid in the cornea becomes higher than that in other tissues that are ready for vitrification, the cornea solidifies at a much higher temperature than other tissues, and very strong resistance to ice formation is obtained during both cooling and warming.

[0080] Further evidence for this conclusion comes from the fact that the magnitude of the glass transition, which reflects the water content of the tissue, was also dramatically smaller than that seen in other tissues and CPA solutions. The change in the heat capacity of the cornea was approximately 0.6 J / (g °C), while the change in the heat capacity of the CPA solution is typically close to 2 J / (g °C) through the glass transition, suggesting that the water content is reduced by approximately three-fold compared to a normal system. This is surprising considering that the water content of M22 is only about 41% of the volume of the solution. Additional evidence of dehydration is the observed stiffness of the cornea after loading with M22.

[0081] The cooling and warming methods used. Cooling and warming were performed in the same manner for both methods on the corneas. Vitrification was carried out on or near a platform with a height of 14.6 cm placed in an MVE E-1 cylindrical dewar filled with liquid nitrogen to a depth of 13 - 14 cm (the nominal separation distance between the liquid nitrogen surface and the platform was 1.6 - 0.6 cm). A small plastic screw-cap container with a thin wall (capacity 20 ml) was positioned on the platform and pre-cooled to -170 to -190 °C. The cornea was positioned on the O-ring with the endothelial side up, placed in the atmosphere within the dewar adjacent to the container, and placed on a pre-cooled solid surface that promoted cooling (later found to be unnecessary) and reduced frost contact with the cornea, but did not allow contact between the cornea and the floor of the container. The cornea could also be placed directly into the container as described above. The "bowl" of the cornea was intentionally not emptied of VS and contained approximately 0.5 - 1 ml of M22. This protected the endothelium from atmospheric ice and was sufficient to achieve an appropriate cooling rate, but was insufficient to support fragmentation that could cause damage to M22.

[0082] The cornea was cooled passively to -134 °C (a typical cooling profile is shown in Figure 6A), then placed in a sealed container, transferred to a cryopreservation system, and stored at -145 °C. Vitrification within the container is preferred to reduce or prevent corneal contamination and avoid direct contact between the cornea and liquid nitrogen droplets, but the cooling rate may be slower than exposing the cornea to the dewar atmosphere outside the container. Theoretically, this is important for corneas that are difficult to vitrify.

[0083] For warming, to relieve the accumulated stress and prevent cracking during rapid warming, the cornea was pre-warmed to -135 °C and then transferred onto its ring into 100 - 250 ml of 4 °C M22 and gently stirred until the solid M22 within the cornea liquefied. The average warming rate (see Figure 6B) was much higher than the rate required to avoid devitrification by DSC measurement (see below). The cornea re-warmed in this way almost completely avoided visible devitrification and no cracking occurred.

[0084] Example 4: Human corneas survive extreme dehydration and subsequent vitrification: In vivo results with long-term (4 months) xenografts from human to monkey (loading / unloading method 2) Human corneas stored at 4°C for 2 - 10 days in Optisol-GS were obtained from the Mann-Lion's-Eye Bank or the Doheny Eye Tissue Transplant Bank in Los Angeles. The corneas were vitrified and stored at -145°C for up to 161 days according to the above method. As before, no visible ice formation or cracks occurred during vitrification.

[0085] Whenever the inventors could arrange for the transplantation of fresh non-vitrified corneas, they brought the stored corneas (using a transporter specially designed to maintain the cornea above the temperature of liquid nitrogen and below TG during transportation) to St. Kitts Island. Since control human corneas deteriorate over time, they were transported directly from the eye bank to the St. Kitts Island Behavioral Sciences Foundation. There were no statistically significant differences between the vitrified donors and the control donors in terms of gender, postmortem time, refrigerated storage time, or age. However, most of the control corneas were affected by arcus, while the vitrified corneas were not.

[0086] A total of 8 vitrified corneas and 8 control corneas were transplanted under good conditions on velvet at the St. Kitts Island Behavioral Sciences Foundation, which has a facility approved for primate surgical research. Anesthesia was in accordance with the standard protocol of the Behavioral Sciences Foundation, and our surgical method was the same as the heterologous transplantation experiment using rabbits, except that a 6.5 mm corneal punch was transplanted into a 6 mm recipient corneal gap. On St. Kitts Island, except for Cornea 9, no cracks or devitrification occurred during warming. Cornea 9 showed some ice formation during warming, presumably because it had been stored for a long time by chance. The average storage time at -145°C was 70.6 ± 54.23 days.

[0087] All grafts were observed daily by BSF staff for graft transparency, epithelial healing, wound dehiscence, infection, and inflammation after surgery. Photographs of the grafts were taken daily for the first week and at 2 weeks, 1, 2, 3, and 4 months after transplantation. The monkeys were anesthetized and their eyes were examined by BSF staff using a Nidek ConfoScan 3 human clinical specular microscope at 2 weeks, 1, 2, 2.5, 3, and 4 months after xenotransplantation, and images were recorded and sent to us from the BSF staff. The ConfoScan 3 provides analysis of corneal endothelial cell characteristics and density, and corneal thickness. Each ECD result was the average of measurements taken by counting endothelial cells in different photographic fields of a known area of each cornea. ECD and corneal thickness were measured by a masked method. At the end of the follow-up period for each monkey, the BSF staff euthanized the monkeys, removed and fixed the corneas, and sent them to a consulting ophthalmic pathologist. There, they evaluated the general condition by a masked method and standardized the grading of the corneas using a semi-quantitative 5-point system where 5 is the highest and 1 represents extensive destruction.

[0088] Of the eight control corneas, only three corneas became at least partially transparent, and the rest remained permanently cloudy. Of the eight vitrified corneas, six became transparent, were slightly cloudy during the first few days after surgery, but remained transparent until the 169-day follow-up. One of the two cloudy corneas, Cornea 9, had devitrification occur upon rewarming (violation of temperature maintenance during storage?). On average, five of the eight control corneas could be at least partially evaluated by specular microscopy despite cloudiness, and the onset time of endothelial visualization was 64.6 days on average (range, 18 - 119 days) after transplantation. Of the eight vitrified corneas, all except Cornea 9 were sufficiently visualized for evaluation by Confocan 3 after an average onset time of 20.9 days (range, 14 - 26 days). The difference between the two groups may be partially due to the deterioration of the control corneas during transportation, which is an issue that vitrification is trying to solve. Comparing corneas transported at -145°C (n = 3) and corneas transported at -180°C (n = 5), there was no effect of the transportation temperature on the vitrified corneas.

[0089] One of the most important factors for the long-term success of corneal transplantation is the rate of decline of ECD over time after transplantation. The comparison of the changes in ECD between control corneas and vitrified corneas is shown in Figure 7. Panel A of Figure 7 records the ECD decline in three evaluable control corneas, and panel B records the ECD changes in six evaluable vitrified corneas. Among the control corneas, only one (star) maintained a good ECD after transplantation, while all six of the evaluable vitrified corneas maintained it. Panel C shows the average results (±1 SEM) of the vitrified corneas (white dots), compares them with the results of one good-quality control cornea (star), and shows that the average-quality vitrified corneas are as good as the best control possible. This means that vitrification did not cause a change in the decline of ECD expected in the original control. The regression line for the vitrified corneas is ECD = 2700 + 330*exp[-0.0117t], where t is given in days, predicting a plateau of ECD of about 2700 cells / mm2 in about one year. Panel D shows the same data normalized to the pre-transplant ECD (±1 SE). The vitrified corneas (white dots) retain more than 90% of their ECD on average over four months, similar to one good control cornea (star) in non-human hosts, while the other two controls show a decline in ECD of more than 60%.

[0090] The thickness of the cornea is a basic measure of the health of the cornea, and the measured values of the corneal thickness are shown in Figure 8. Both the vitrified corneas (upper graph) and the control corneas (lower graph, black dots) tended to be thicker than normal during the first 1-2 months after transplantation, but eventually recovered. The average vitrified corneas (white dots in the lower graph) showed behavior very similar to that of the high-ECD control corneas (designated by boxes in the lower graph) and generally to the control corneas, which also suggests the normality of the vitrified corneas after transplantation.

[0091] Figure 9 shows the structural findings of endothelial cell morphology in representative controls (upper left) and vitrified corneas transplanted from human to monkey imaged by ConfoScan 3 at 153 days and 126 days, respectively, in vivo. Both morphologies appeared normal, but as can be seen from these magnified images, the control corneas tended to have larger cells in accordance with the low ECD. Figure 9 also shows the central SEM findings of the control (lower left image) and vitrification (lower right image), which do not show defects in the endothelial layer structure in the vitrified and transplanted corneas.

[0092] The results of the blinded pathological scoring of these corneas showed that 3 out of 8 control corneas and 6 out of 8 vitrified corneas obtained better scores, which independently confirmed our in vivo results.

[0093] Example 5: Survival of Vitrified Rabbit Corneas after One-Step Introduction and Washing of M22 The inventors also demonstrated that rabbit corneas survived direct transfer from a cryoprotectant-free medium to M22 and direct transfer from M22 to a cryoprotectant-free medium based on the results of vital staining and the maintenance of excellent ECD (data not shown).

[0094] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were specifically and individually indicated to be incorporated by reference for all purposes.

[0095] Although various specific embodiments / aspects have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure.

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Claims

1. A method for cryopreserving human, animal, or bio-artificial corneas by vitrification, The cornea is placed in a cryoprotective solution, A method comprising increasing the concentration of the cryoprotective agent in the solution surrounding the cornea from 0% of the target cryoprotective agent concentration for vitrification to 100% of the target cryoprotective agent concentration for vitrification over a period of 0 to 25 minutes.

2. The method according to claim 1, wherein the concentration of the cryoprotectant is increased in 1 to 3 stages.

3. The method according to claim 1, wherein the concentration of the cryoprotectant is continuously increased.

4. The method according to claim 3, wherein the concentration of the cryoprotectant is increased over 8 to 12 minutes to 100% of the target cryoprotectant concentration for vitrification.

5. The method according to claim 3, wherein the concentration of the cryoprotectant is increased over 5 to 15 minutes to 100% of the target concentration for vitrification.

6. The method according to claim 2, wherein the concentration of the cryoprotectant is increased over 8 to 12 minutes to 100% of the target cryoprotectant concentration for vitrification.

7. The method according to claim 2, wherein the concentration of the cryoprotectant is increased over 5 to 15 minutes to 100% of the target cryoprotectant concentration for vitrification.

8. The method according to any one of claims 1 to 7, further comprising: maintaining the cornea in a solution containing 100% of the target cryoprotective agent concentration for vitrification for 10 to 30 minutes prior to vitrification.

9. Further comprising reducing the concentration of the cryoprotectant in the cryoprotectant solution surrounding the cornea from 100% of the target concentration for vitrification to 0% of the target concentration for vitrification over a period of 0 to 25 minutes, The method according to claim 1, wherein the step of reducing the concentration of the cryoprotectant in the cryoprotectant solution surrounding the cornea from 100% of the target concentration for vitrification to 0% of the target concentration for vitrification is performed continuously or in one to three steps.

10. The method according to claim 1 or 9, wherein the cornea is operated in the method by an operating means comprising a ring and a suspension means for the ring, or the cornea is kept immobile within the ring of the operating means, during which the concentration of the cryoprotectant in contact with the cornea is continuously changed without manipulating the cornea.

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