WO2024020404A1 - Procédé et appareil de préparation ultra-rapide de cornées pour vitrification - Google Patents

Procédé et appareil de préparation ultra-rapide de cornées pour vitrification Download PDF

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WO2024020404A1
WO2024020404A1 PCT/US2023/070435 US2023070435W WO2024020404A1 WO 2024020404 A1 WO2024020404 A1 WO 2024020404A1 US 2023070435 W US2023070435 W US 2023070435W WO 2024020404 A1 WO2024020404 A1 WO 2024020404A1
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cornea
corneas
ring
cryoprotectant
container
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PCT/US2023/070435
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English (en)
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Xian GE
Jun Wu
Brian Wowk
Gregory M. Fahy
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21St Century Medicine, Inc.
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents

Definitions

  • This invention relates to the preservation of human, animal, and bio-artificialcorneas by vitrification, including methods and apparatus for same.
  • Vitrification has the potential to positively affect most of the corneas transplanted annually in the United States and to enable the export of tens of thousands of natural corneas and unlimited numbers of bioartificial corneas from the United States to countries where high quality corneas are needed.
  • 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), although only 57% are suitable for transplantation after 4 weeks, and only 53% retain an "excellent” rating after 2 weeks or less [9], According to Redmond et al. (Redmond, Armitage et al.
  • corneas were thicker and more opalescent than corneas that had been stored at 4°C [and] grafts cleared relatively slowly, taking 3-4 weeks to achieve good transparency.
  • corneas slowly swell and must be "deswollen" in a concentrated polymer solution such as dextran T500 for about two days before they are suitable for transplantation (Thuret, Chiquet et al. 2003).
  • Circa 2000 corneas available for research cost about $2000 for a fresh, transplant grade cornea, $600 for a cornea less than 4 days old, $350 for a cornea less than six days old, and $200 for a cornea more than six days old.
  • Corneas that are exported for transplantation because of dwindling shelf life were commonly marked down to $500 (Campbell 2000). Immediate vitrification of corneas at their time of arrival at eye banks could have therefore provided a $1400-$1800 value per cornea for many or most corneas in 2000 dollars.
  • Corneas cryopreserved by the K-C method and by other clinical freezing methods are usually able to remain clear postoperatively after cryopreservation, but up to 15% of such corneas fail immediately upon 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) versus 78% for non-cryopreserved grafts in one study (Ing, Ing et al. 1998). Freezing inevitably results in variable degrees of corneal endothelial cell loss before and after transplantation (Van Horn and Schultz 1974, Ehlers, Sperling et al.
  • Linner and Goosey claimed apparatus and methods for "cryofixation in less than one second" (col. 2, lines 26-27) by means of ultra-rapid cooling without cryoprotective agents (Linner and Goosey 1986).
  • the allegedly (but almost certainly not actually) "vitrified" cornea is then dehydrated by sublimation of water near -130°C under high vacuum to achieve the benefits of freeze-drying without the formation of ice crystals.
  • No documentation of long-term function after transplantation or of any in vitro functional or viability testing was provided, and there has been no adoption of this method by ocular surgeons since it was introduced in this 1986 patent to the knowledge of the present inventors. The method bears no resemblance in concept or in practice to the presently claimed invention.
  • a third patent entitled “Corneal vitrification, methods and devices to produce corneal vitrification and methods of use thereof” is not about corneal cryopreservation by vitrification.
  • This patent is instead directed for the formation of a glassy state in corneas in situ using optical means for the purpose of improving vision. In this process, the cornea remains warm at all times, and cryoprotective agents are never used to induce the vitreous state by cooling to temperatures below -100°C.
  • the present invention is a method for achieving nearly damage-free preservation of animal, human, and bio-artificial corneas by vitrification not by achieving cryoprotectant and osmotic equilibration of the cornea as attempted and as called for by all prior art methods but by the opposite approach of deliberately inducing extreme nonequilibration corneal dehydration to foster vitrification at very low cooling rates, prevent devitrification at very low warming rates, and prevent cryoprotectant toxicity.
  • the invention method is preferably carried out using specially designed equipment to enable appropriate handling, storage, and rewarming of the cornea while preventing corneal mechanical damage.
  • the invention provides conditions of cryoprotectant exposure that dramatically increase the glass transition temperature of the cornea by means of extreme dehydration and somehow surprisingly block cryoprotectant toxicity during prolonged exposure above 0°C.
  • the invention includes rapid introduction and washout of highly vitrifiable concentrations of cryoprotectants of elevated theoretical tonicity under practical isothermal conditions that are readily achievable in ordinary eye banks.
  • the present invention in addition to enabling truly elective timing of the use of both locally-procured and globally-shared corneas, eliminating corneal deterioration with time, eliminating seasonal fluctuations in the availability of corneas for transplantation, ensuring availability of corneas on an emergency basis, reducing logistic costs, and eliminating corneal outdating, will also have the potential to reduce rejection as a cause of graft failure by allowing tissue matching between donor and recipient during the cornea storage period.
  • the disclosure provides a method for cryopreserving a human, animal, or bioartificial cornea, comprising:
  • the method is preferably carried out under nominally isothermal conditions between 0 and 10°, and wherein
  • the cornea may be cooled and warmed at rates as low as 5°C/min or less between 0 and -100°C without ice formation, and wherein
  • a different carrier solution may be used for said reducing of the concentration of said cryoprotectant than for said increasing the concentration of the said cryoprotectant, and wherein
  • the cornea is either manipulated during the method by means of a manipulation means comprising a ring and a suspension means for the ring or the cornea is maintained immobile in the ring of the manipulation means while the concentration of cryoprotectant in contact with the cornea is changed continuously without manipulating the cornea.
  • Another embodiment of the invention comprises a manipulation means for the cornea, comprising a ring for supporting the cornea and a means for positioning said ring above the floor of any container in which the cornea may be placed so as to prevent contact between the cornea and said container floor, wherein
  • said means for positioning said ring above the floor of any container in which the cornea may be placed may consist of a stand extending from the ring downwards to the floor of the container or
  • said means for positioning said ring above the floor of any container in which the cornea may be placed may consist of a handle attached to said ring that can be grasped and held manually or by means of a positioning device, wherein
  • said means for positioning said ring above the floor of any container in which the cornea may be placed may comprise both a stand and a handle to facilitate movement of the ring and the stand and/or [00039] said means for said positioning of said ring may comprise a vertical member extending upward from the ring and attaching to a movable lid for said container in which the cornea may be placed, wherein said vertical member positions the cornea above the floor of said container without contact between the cornea and said container floor when said movable lid for said container is used to close said container, and
  • said means for said positioning of said ring may comprise in addition to said vertical member extending upward from the ring and attaching to a movable lid for said container in which the cornea may be placed a graspable handle located above the lid to further facilitate movement of the lid, vertical member, ring, and cornea within the ring.
  • FIG. 1 illustrates one embodiment of apparatus useful for carrying out the process of the invention.
  • FIG. 2 demonstrates nearly 100% cell survival (A) and 100% retention of cornea endothelial cells within the endothelial cell layer of the cornea (B) after vitrification under conditions of extreme corneal dehydration (two-step loading to 9.45M cryoprotectant) followed by rewarming and VS washout.
  • FIG. 3 compares two cryoprotectant addition and removal protocols effective in the invention.
  • FIG. 4 shows survival of vitrified/rewarmed/xenografted human corneas based on gross and microscopic examination.
  • FIG. 5 demonstrates elevated glass transition temperatures and immunity to ice formation in human corneas during rewarming at 5°C/min after previous vitrification according to protocol 2.
  • FIG. 6 shows typical cooling and warming curves, which greatly exceed the rates needed to prevent ice formation.
  • FIG. 7 describes the persistence of ECD in control and vitrified human corneas over 4 months following transplantation into monkeys.
  • FIG. 8 demonstrates the ability of vitrified human corneas to thin over time after transplantation into monkeys to the same degree and at the same rate as the best control cornea.
  • FIG. 9 displays normal human corneal morphology based on both light and scanning electron microscopy 4 months after transplantation into monkeys.
  • the invention comprises a method for cryopreserving a human, animal, or bioartificial cornea by inducing a state of extreme dehydration of the said cornea by immersion of the said cornea in a concentrated cryoprotectant solution for a time long enough to induce major exosmosis of water from the cornea but insufficient to allow corneal equilibration with the cryoprotectant solution.
  • Extreme in this context denotes dehydration sufficient to raise the TG of the cornea by at least 7°C relative to the TG of the vitrification solution employed to vitrify the cornea, reduce the change in heat capacity at the glass transition by at least 40% relative to the change in heat capacity at the glass transition of the vitrification solution employed to vitrify the cornea, and reduce the critical cooling and warming rate of the cornea between 0°C and -100°C to 5°C/min or less. Specific means for achieving these endpoints, which can be reliably applied without the necessity of measuring the achieved endpoints, are described in the Examples that follow.
  • the process comprises a loading process in which the cryoprotectant concentration is increased from 0% VS to 100% VS in preferably less than 20 min and either continuously or in less than 4 steps, and preferably when said increase occurs over 8-12 min and either continuously or in 2-3 steps.
  • the process further comprises an unloading process in which the VS is removed in preferably less than 20 min and either continuously or in 2-3 steps, and preferably in 8-15 min and continuously or in 2-3 steps.
  • the process is also carried out preferably under nominally isothermal conditions between 0 and 10°C.
  • the method further comprises exposure of the cornea, after the process of increasing the concentration to 100% VS, to 100% VS for 10-30 min before vitrification, and most preferably for 20-25 min before vitrification.
  • One preferred embodiment of the invention uses LM5 or a similar carrier optimized for vitrification as the carrier solution for addition of cryoprotective agent (Fahy, Wowk et al. 2004, Fahy 2005) and CPTES plus 2.5% chondroitin sulfate (Taylor and Hunt 1989) or another carrier optimized for stabilizing cornea viability as the carrier used for washout of the CPA. Solutions optimized to promote vitrification or for use on organs are not necessarily ideal for corneas, so reducing the exposure time to LM5 or other carriers not optimized for use on corneas is considered beneficial although it is optional in the invention.
  • the method of the invention is best carried out using a device consisting of at least one ring for supporting the cornea and one means for positioning said ring above the floor of any container in which the cornea may be placed so as to prevent contact between the cornea and said container floor.
  • Said means for positioning said ring may consist of a stand extending from the ring downwards to the floor of the container.
  • Said means for positioning said ring may alternatively consist of a handle attached to said ring that can be grasped and held manually or by means of a positioning device.
  • Said means for positioning said ring may alternatively consist of a combination of a stand and a handle to facilitate movement of the ring and the stand.
  • Said means for positioning said ring may alternatively comprise a vertical member extending upward from the ring and attaching to a movable lid for said container in which the cornea may be placed, wherein said vertical member positions the cornea above the floor of said container without contact between the cornea and said container floor when said movable lid for said container is used to close said container.
  • Said means for positioning said ring may alternatively comprise a vertical member extending upward from the ring and attaching to a movable lid for said container in which the cornea may be placed, wherein said vertical member positions the cornea above the floor of said container without contact between the cornea and said container floor when said movable lid for said container is used to close said container, wherein said movable lid includes as well a graspable handle located above the lid to further facilitate movement of the lid, vertical member, ring, and cornea within the ring .
  • the method of the invention is also best carried out using either a series of containers bearing different concentrations of cryoprotectant into which the cornea on the said positioning ring is successively transferred or using continuous cryoprotectant concentration gradients provided by gradient forming means, wherein the cornea is positioned in a bath whose concentration is continuously changed by said gradient forming means.
  • the method of the invention is also optionally carried out by transferring the cornea on its ring into a pre-cooled storage container located in a cold environment, which may be an environment whose temperature is below the TG of the cornea vitrification solution, and thereby vitrifying the cornea in a gaseous environment wherein the cooling rate may be as low as less than 5°C/min between 0°C and -100°C, and sealing said pre-cooled storage container.
  • a cold environment which may be an environment whose temperature is below the TG of the cornea vitrification solution, and thereby vitrifying the cornea in a gaseous environment wherein the cooling rate may be as low as less than 5°C/min between 0°C and -100°C
  • the invention may be carried out by transferring the cornea on its ring into a cold environment, which may be an environment whose temperature is below the TG of the cornea vitrification solution, thereby vitrifying the cornea in a gaseous environment, wherein the cooling rate may be as low as less than 5°C/min between 0°C and -100°C, and thereafter transferring the cornea on its ring into a pre-cooled storage container and sealing said precooled storage container.
  • a cold environment which may be an environment whose temperature is below the TG of the cornea vitrification solution, thereby vitrifying the cornea in a gaseous environment, wherein the cooling rate may be as low as less than 5°C/min between 0°C and -100°C
  • cryoprotectants also known as “cryoprotective agents” or “CPAs,” are molecules that reduce or prevent freezing injury by reducing or preventing the formation of ice below 0°C.
  • cryoprotectants are well known in the art (Meryman 1971, Fuller 2004, Fahy 2005, Abazari, Meimetis et al. 2015, Elliott, Wang et al. 2017). They consist of penetrating cryoprotectants (pCPAs) and non-penetrating cryoprotectants (npCPAs). The use of pCPAs and npCPAs in the invention is described in more detail below.
  • cryoprotectant or "CPA” as used herein may refer to one or to multiple cryoprotectants, and to either pCPA alone or to a mixture of pCPA and npCPA.
  • adding or washing out the solution known as IVI22 (Fahy, Wowk et al. 2004), which is a preferred solution for use in the present invention, or related solutions (Fahy 2005) can be referred to as adding or washing out "the cryoprotectant” or "the CPA” even though these solutions are composed of multiple pCPAs and multiple npCPAs.
  • a “carrier” solution is the component of a cryoprotectant solution other than the cryoprotectant (Fahy 2005, Fahy and Wowk 2021). It consists of solutes necessary for the support of cellular viability and health in the presence or absence of a cryoprotectant.
  • 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.
  • TG is the glass transition temperature, which marks the temperature range for the transition from the liquid state to the glassy state on cooling or from the glassy state to the liquid state on warming (Fahy and Wowk 2021).
  • 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 virtually lacks the translational molecular motions of a liquid.
  • vitrification is also a method of cryopreservation without ice formation.
  • a "vitrification solution” or "VS” is a cryoprotectant solution that is sufficiently concentrated to preclude ice formation during cooling to TG at the cooling rate employed to preserve the living system that is to be preserved (Fahy, MacFarlane et al. 1984, Fahy and Wowk 2021).
  • Osmotic equilibrium is considered to have been reached when the cornea is restored to its original volume by equilibration of the cryoprotectant concentration between the cornea and the cryoprotectant medium to which it has been exposed after previous osmotic volume changes of the cornea induced by changing the pCPA concentration of the medium surrounding the cornea.
  • Vsr (Vi-b) x ni/nf + b
  • Vsr the stable reduced volume of the cornea
  • Vi is the volume of the cornea in ordinary isotonic medium and in the absence of cryoprotectant
  • b is the non-osmotic volume of the cornea (a constant)
  • ni is the osmolality of the isotonic carrier solution not containing the npCPA
  • nf is the osmolality of the CPA carrier solution plus the osmolality of the npCPA.
  • Exosmosis is the movement of water out of the cornea due to immersion of the cornea in a solution having an osmolality higher than the osmolality of the cornea prior to the time of immersion in said solution.
  • Rewarming'' is the return of the temperature of a vitrified system to a temperature near 0°C or above.
  • compositions, apparatus, systems or methods provided herein can be combined with one or more of any of the other compositions, apparatus, systems or methods provided herein.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 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.
  • Example 1 Human Cornea Handling Apparatus
  • corneas tend to be damaged if not handled correctly.
  • Superfusion systems such as those previously used at the Bourne lab at the Mayo Clinic (Brunette, Nelson et al. 1989, Bourne, Shearer et al. 1994) hold corneas in a secure fashion but cannot be used to vitrify and rewarm corneas due to their large mass.
  • corneas have simply been placed on the floors of small containers, endothelial side up, presumably using forceps, to transfer them from one cryoprotectant solution to another, or to other environments.
  • this technique causes epithelial damage due to contact between the epithelium and the container floor.
  • the inventors were able to prevent this damage initially by supporting and transferring the corneas, endothelial side up, through a series of 10 ml solutions at 4°C on a square plastic O- ring that suspended the bottom of the cornea above the floor of the container when the O-ring was placed on the floor of the container. This was helpful for introducing and removing cryoprotectant but did not allow facile vitrification and rewarming and did not allow for free diffusion of cryoprotectant around the epithelial surface of the cornea.
  • the O-ring was therefore modified to the rounded (torus shaped) ring A of FIG. 1.
  • the preferred inner diameter of the O-ring is 12 mm, but this may vary from 11-13 mm, and the preferred thickness of the O-ring is 1 mm, but may vary from 0.5-4 mm.
  • the O-ring was also attached to the bottom of vertical member (wand) B of FIG. 1 to allow easy transfers from solution to solution, from VS to liquid nitrogen vapor, and from liquid nitrogen vapor to VS for rapid warming. This enabled short term experiments but did not include the ability to protect the cornea from potential damage from mechanical contact with the storage container during long term storage or transport. This latter problem was solved by incorporating the cornea loop and wand into the lid C of the long-term storage container F of FIG. 1.
  • This system enables a) transfer of the cornea from container to container without the need to manually hold or otherwise secure the wand, b) the ability to protect each solution from contamination by using the cornea transfer device A+B+C as a lid for each container during addition and removal of cryoprotectant, c) the ability to prevent contact between the cornea and the floor of each cryoprotectant container and to allow free diffusion around the epithelial side of the cornea during contact with the cryoprotectant, d) the ability to vitrify the cornea by placing the cornea handling device onto and then securing it onto the pre-cooled cornea storage container F and allowing the cornea to vitrify passively within the atmosphere of container F while being protected from contact with condensed water vapor surrounding F or, e) the ability to transfer the cornea to a different cold environment (liquid nitrogen vapor, cold air, or a combination of liquid nitrogen vapor and cold air) for vitrification and then to transfer the vitrified cornea to its storage container F by screwing or snapping the cornea handling device A+B+C onto said storage container, and finally f
  • an extension D of wand B can penetrate through lid C and, if desired, can be embellished with additional member E for easier gripping by hand or by an appropriate gripping device.
  • Example 2 Human Corneas Survive Extreme Dehydration followed by Vitrification: In Vitro Results (Loading/Unloading Method One)
  • Corneas For the experiments of Example 2, a total of 75 human corneas were obtained from the Lone Star Lions Eye Bank in Manor, Texas, or the Doheny Eye & Tissue Transplant Bank in Los Angeles, California after storage in Optisol-GS for 2-9 days at 4 9 C. These corneas were deemed unsuitable for clinical transplantation because of arcus or donor systemic disease but retained an intact endothelium and were therefore suitable for our purposes.
  • M22 has a total effective molarity, based on equating the osmotic contributions of its polymeric components to molarities (milliosmole for millimole) in the absence of other cryoprotectants (Fahy, Wowk et al. 2004), of 9.45M.
  • the melting point of M22 is about -54.9°C (Fahy, Wowk et al. 2004).
  • M22 may be said to be about 100 times as concentrated as plasma on an osmotic basis.
  • FIG. 2A and B respectively show the vital staining and ECD results of this protocol.
  • Syto 13 stains the nuclei of viable cells green (Yang, Acker et al. 1998) and ethidium homodimer-1 stains non-viable cell nuclei red.
  • selective Syto-13 binding to the nucleus provides a convenient way of identifying and therefore of counting individual corneal endothelial cells for calculation of the ECD.
  • Corneas were stained with Syto 13 and ethidium homodimer-1 in D-PBS for 40 minutes at room temperature, and a 7.5 mm button was then trephined from the central region of the cornea for observation and ECD determination.
  • the endothelium was viewed under a fluorescence microscope (Olympus, IMT-2 Inverted research microscope). As shown in FIG. 2A, virtually all corneal endothelial cells (CECs, corresponding to the pale spots in the figure) stained green, with very few red cells apparent after vitrification and M22 washout. [00091 ] In addition, very few cells were lost from the corneal endothelial monolayer (FIG. 2B). We tested the accuracy of our own ability to determine the ECD [using the Karnama et al.
  • ECD 2 ICM ECDEB (0% cell loss at our institution), where ECD 2 ICM is the corneal endothelial cell density as measured at our institution and ECDEB is the corneal endothelial cell density as measured at the eye bank.
  • a vitrified cornea showed an ECD of just over 2000 vs an expected ECD of around 2900
  • a control cornea showed an ECD of about 1900 whereas the same cornea at the eye bank had an ECD of about 2750 cells/mm 2 , indicating that a loss of ECD is at least as likely in untouched control corneas as in vitrified corneas, again demonstrating no difference in ECD between vitrified and control corneas overall.
  • even the ECD of just over 2000 in the vitrified outlier cornea lies above the cutoff line (horizontal line at 2000) for suitability for transplantation.
  • corneas immersed in 3M dimethyl sulfoxide (Me2SO; about 15.8 x isotonic) were severely damaged, whereas corneas exposed to the same concentration by transfer first into IM and then into 2M Me2SO before exposure to 3M recovered (Taylor and Hunt 1989), presumably largely due to the avoidance of osmotic (shrinkage) stress.
  • the inventors found that corneas held in M22 for only 20 min before cooling formed ice on cooing, whereas holding in M22 for 25 min precluded ice formation, implying that water efflux from the corneas continues between 20 and 25 min of M22 contact for the described protocol.
  • the most preferred holding time in M22 is 21-25 min, and especially 23-25 min, for this protocol.
  • CPA toxicity proceeds most rapidly at the highest concentration used, and in this case, exposure to full M22 lasted 2.5 times longer than exposure to either the loading solution or the unloading solution, which is another unique feature of the present method.
  • Prolonged exposure to M22, or to the highest concentration used in the process if this is not M22, is believed to be beneficial to ensure extraction of sufficient water from the cornea by osmosis to preclude ice formation during cooling and warming, the dilution tolerance evidence above and the DSC evidence described below indicating that it is this dehydration rather than M22 uptake into the cornea that is primarily responsible for stabilization of the cornea against ice formation.
  • M22 is so named because it is intended to be brought into contact with living cells only at temperatures close to -22°C to preclude toxicity at higher temperatures, but our corneas tolerated prolonged exposure to it at 4°C when treated with M22 as described.
  • cryoprotectant uptake inhibition is independent of the nature of the cryoprotectant provided a sufficiently high concentration is introduced sufficiently rapidly.
  • prevention of cellular CPA uptake will also be facilitated by the presence of npCPAs sufficient to elevate the tonicity of the CPA carrier solution appreciably.
  • CPTES which is a carrier used in past corneal studies, contains 2.5% chondroitin sulfate (CS), a negatively charged glycosaminoglycan, which may be regarded as a non-penetrating CPA.
  • the osmotic 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.
  • the exemplary M22 solution contains an npCPA polymer mixture that raises the tonicity of its carrier solution to about 1.5 times isotonic, and the present invention can be usefully practiced with the optional but beneficial inclusion of VS npCPAs that raise the tonicity of the carrier solution to 1.2-1.7 times isotonic [as calculated in the absence of cryoprotectants (Fahy, Wowk et al. 2004)] or above for inhibition of CPA uptake.
  • Example 3 Human Corneas Survive Extreme Dehydration followed by Vitrification: In Vivo Results with Human-to-Rabbit Xenografts (Loading/Unloading Method Two)
  • the carrier of M22 addition was LM5 (as always, minus calcium and magnesium) and the carrier for M22 subtraction was CPTES plus 2.5% chondroitin sulfate, and all steps were once again carried out at 4°C.
  • the two protocols shown in FIG. 3 have in common the fact that the time spent going from no CPA to 100% of full VS concentration is 10 minutes in each case, which is unheard of in the art. Similarly, with both methods, the time taken to proceed from 100% VS to 0% VS is 10 minutes, which is also unheard of in the art, especially without the use of osmotic buffers, which neither method employed. Vitrified corneas that were transplanted were stored at -145°C for 2-174 days (mean and standard deviation: 35 ⁇ 47 days) before rewarming and transplantation. No effect of storage time was apparent.
  • the globe was exposed and immobilized, and a 7.5 mm diameter central corneal disk was removed from the recipient cornea and replaced with a 7.5mm diameter corneal button from the vitrified/rewarmed human cornea.
  • the anterior chamber was allowed to reform spontaneously, and atropine eye drops were applied at the end of the procedure.
  • the postoperative care required was occasional atropine drops when the pupil was miotic.
  • Antibiotics such as gentamicin, neomycin/polymixin B/Dexamethasone ointment, and steroids such as prednisone acetate eye drops were used when needed. All grafts were observed and photographed daily for graft clarity, wound dehiscence, infection, etc.
  • grafted corneas were removed at 10 ⁇ 3 days (mean ⁇ standard deviation) postoperatively. Corneas were fixed in 2.5% glutaraldehyde and divided for examination by light microscopy (histopathology), scanning electron microscopy (SEM), and transmission electron microscopy (TEM), and all cornea evaluations were performed by a consulting ocular pathologist located at a different institution in blind fashion.
  • FIG. 4C were seen to be preserved similarly to those of control FIG.grafts (not shown).
  • FIG. Endothelial cells were flat and appeared to be intact, with easily identified borders. Transmission electron microcopy of vitrified and xenografted human corneas showed a relatively normal endothelium with some alterations consistent with a continuation of good function in the absence of a xenograft reaction and with appropriate attachment of the endothelium to Descemet's membrane (results not shown).
  • thermograms showed no evidence of freezing or melting, indicating that the cornea tissue remained perfectly ice free during cooling and rewarming at all examined rates, and that the corneas were undoubtedly also stable against ice formation even at cooling and warming rates of less than 5°C/min.
  • the only observable event was a glass transition at a temperature of -110 9 C in one cornea and -97 9 C in a second cornea.
  • a third cornea (16.2 mg) was cooled at 100°C/min to -150°C and then rewarmed at 5, 10, 20, 40, or 80°C/min. No ice melting peak was seen at any warming rate in this cornea as well, signifying that this cornea, like the others, was undoubtedly stable against ice formation even at warming rates of less than 5°C/min.
  • the glass transition temperatures observed in the three samples are all dramatically higher than the glass transition temperature of the pure M22 cryoprotectant solution that was used to prevent the corneas from freezing, which is - 124°C. It is extraordinary for the glass transition temperature of tissue to differ substantially from the cryoprotectant solution it is permeated with, and the difference can only be explained by water extraction from the cornea out of proportion to the water content of M22, which will have the effect of increasing corneal protein concentration and thereby raising TG (Rasmussen 1969).
  • This osmotic and chemical disequilibrium between the cornea and its M22 environment creates a greater fluid viscosity in the cornea than in other tissues prepared for vitrification, causing corneas to solidify at much higher temperatures than other tissues would and to achieve extraordinarily strong resistance to ice formation on either cooling or warming.
  • Cooling and Warming Methods Used. Cooling and warming were done in the same way for both Method One and Method Two corneas. Vitrification was done on or near a 14.6 cm tall platform placed in an MVE E-l cylindrical dewar containing liquid nitrogen to a depth of 13-14 cm (nominal 1.6-0.6 cm separation between the liquid nitrogen surface and the platform). A thin-walled small plastic screw-cap container (capacity, 20 ml) was positioned on the platform for pre-cooling to -170 to -190°C.
  • the cornea positioned endothelium side up on its O-ring, was placed into the atmosphere in the dewar adjacent to the container and over a pre-cooled solid surface that accelerated cooling (later found to be unnecessary) and reduced frost contact with the cornea, but the cornea could also be placed directly into the container as described above, without allowing contact between the cornea and the container floor.
  • the "bowl” of the cornea was deliberately not emptied of VS, and contained "'0.5-1 ml M22, which was sufficient to shield the endothelium from atmospheric ice and enable an appropriate cooling rate but insufficient to support potentially damaging fracturing of the M22.
  • the cornea was allowed to cool passively to -134°C (a typical cooling profile is shown in FIG. 6A) and then transferred in its sealed container to a constant-temperature storage system for storage at -145°C.
  • Vitrification in a container is preferred for reducing or preventing contamination of the cornea and avoiding direct contact between the cornea and droplets of liquid nitrogen but may result in a slower cooling rate than exposure of the cornea to the dewar atmosphere outside of a container, which could in theory be important for difficult-to-vitrify corneas.
  • the cornea was pre-warmed to -135°C to relieve accumulated stresses and prevent cracking upon fast warming and then transferred on its ring into 100-250 ml of 4°C M22 and gently agitated until the solid M22 solution within the cornea liquified.
  • the average warming rates (see FIG. 6B) were far higher than the rates required to avoid devitrification according to DSC measurements (see below). Corneas rewarmed in this way avoided most visible devitrification and did not crack.
  • Example 4 Human Corneas Survive Extreme Dehydration followed by Vitrification: In Vivo Results with Long-Term (4-Month) Human-to-Monkey Xenografts (Loading/Unloading Method
  • ECD result was the mean of measurements made by counting endothelial cells in different photographic fields of known area from each cornea. ECDs and corneal thicknesses were measured in blind fashion. At the end of the follow-up period for each monkey, BSF personnel euthanized the monkeys, removed and fixed the corneas, and sent them to our consulting ocular pathologist, who evaluated them in blind fashion for general condition and standardized corneal grading using a semi-quantitative 5-point system in which 5 is a perfect score and 1 represents widespread destruction.
  • FIG. 7 A comparison of changes in ECD between control and vitrified corneas is displayed in FIG. 7.
  • Panel A of FIG. FIG. 7 records ECD decline in the three evaluable control corneas
  • panel B records ECD changes in the six evaluable vitrified corneas.
  • the control corneas only one maintained ECD well after transplantation (stars), whereas 6 of 6 evaluable vitrified corneas did so.
  • Panel C shows the mean results (+1 SEM) for the vitrified corneas (white points) and compares them to the one good quality control cornea result (stars), showing that the average quality vitrified cornea is just as good as the best possible control, implying that vitrification has caused no change in the ECD decline expected for pristine controls.
  • Panel D presents the same data normalized to the ECD before transplantation (+ 1 SE). Vitrified corneas (white points) retain on average over 90% of the ECD over 4 months in a non-human host, as does the one good control cornea (stars), whereas the other two controls show ECD declines of more than 60%.
  • Corneal thickness is a fundamental measure of corneal health, and corneal thickness measurements are presented in FIG. 8. Both vitrified corneas (upper graph) and control corneas (lower graph, black points) tended to be thicker than normal for the first 1-2 months after transplantation, but ultimately recovered, with the average vitrified cornea (white points in the lower graph) behaving very much like the high-ECD control cornea (designated by boxes in the lower graph) and like control corneas in general, again suggesting the normalcy of the vitrified corneas after transplantation.
  • FIG. 9 displays the structural findings for endothelial cell morphology in representative control (upper left) and vitrified (upper right) human-to-monkey transplanted corneas as imaged by the ConfoScan 3 after 153 and 126 days in vivo, respectively. Morphology appeared normal in both, but as can be seen by these equal magnification images, control corneas tended to have larger cells in keeping with their lower ECDs. FIG. 9 also shows control (bottom left image) and vitrified (bottom right image) central SEM findings, which do not indicate any defects in endothelial layer structure in the vitrified and transplanted corneas.

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Abstract

L'invention concerne un procédé de manipulation, de traitement et de stockage de cornées qui permet la vitrification dans de l'air et/ou de la vapeur d'azote liquide et le réchauffage sans perte de cellule, mort cellulaire ou déficience fonctionnelle notables après transplantation par comparaison avec des cornées de contrôle non traitées. Le procédé fait intervenir un retrait de cornée extrême du fait d'un déséquilibre osmotique et d'une déshydratation cornéenne, ce qui permet d'éviter une toxicité du cryoprotecteur malgré une exposition prolongée à des concentrations de pic (vitrifiables) de cryoprotecteur au-dessus de 0 °C et un lavage par cryoprotecteur sans mise en tampon osmotique. L'appareil de cornée permet une manipulation et un stockage idéaux ainsi qu'un refroidissement à l'air à l'intérieur ou à l'extérieur du récipient utilisé pour le stockage.
PCT/US2023/070435 2022-07-18 2023-07-18 Procédé et appareil de préparation ultra-rapide de cornées pour vitrification WO2024020404A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4619257A (en) * 1984-11-30 1986-10-28 Board Of Regents, The University Of Texas System Apparatus and method for cryopreparing corneal tissue for surgical procedures
US5257128A (en) * 1988-06-22 1993-10-26 Board Of Regents, The University Of Texas System Freezing/perfusion microscope stage
US20050100876A1 (en) * 1999-04-13 2005-05-12 Organ Recovery Systems, Inc. Method of cryopreservation of tissues by vitrification
US20100151570A1 (en) * 2008-06-18 2010-06-17 The Cleveland Clinic Foundation Systems and methods for vitrifying tissue
US20170295777A1 (en) * 2015-10-14 2017-10-19 X-Therma, Inc. Compositions and Methods for Reducing Ice Crystal Formation
WO2019099922A1 (fr) * 2017-11-16 2019-05-23 X-Therma, Inc. Oligomères et polymères contenant des cavités efficaces en cryoconservation
US20210144990A1 (en) * 2018-04-05 2021-05-20 Xu Han Improved ultra-fast cooling system and methods of use

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4619257A (en) * 1984-11-30 1986-10-28 Board Of Regents, The University Of Texas System Apparatus and method for cryopreparing corneal tissue for surgical procedures
US5257128A (en) * 1988-06-22 1993-10-26 Board Of Regents, The University Of Texas System Freezing/perfusion microscope stage
US20050100876A1 (en) * 1999-04-13 2005-05-12 Organ Recovery Systems, Inc. Method of cryopreservation of tissues by vitrification
US20100151570A1 (en) * 2008-06-18 2010-06-17 The Cleveland Clinic Foundation Systems and methods for vitrifying tissue
US20170295777A1 (en) * 2015-10-14 2017-10-19 X-Therma, Inc. Compositions and Methods for Reducing Ice Crystal Formation
WO2019099922A1 (fr) * 2017-11-16 2019-05-23 X-Therma, Inc. Oligomères et polymères contenant des cavités efficaces en cryoconservation
US20210144990A1 (en) * 2018-04-05 2021-05-20 Xu Han Improved ultra-fast cooling system and methods of use

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