Apparatus and method for organ preservation

Abstract

The present invention relates to a method and device for long-term cryopreservation of a biological sample, including a vascularized tissue, innervated tissue, or both, such as but not limited to a limb.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 066,925, filed August 18, 2020, entitled “DEVICE AND A METHOD FOR ORGAN PRESERVATION,” the contents of which are incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present invention relates to the controlled freezing of biological material, such as tissue, and more particularly to a device suitable for this purpose and a method of using the same. BACKGROUND

[0004] The main principle guiding modern medicine reconstruction is to replace “like tissue” with “like tissue.” Thus, a wounded or deformed body part is replaced with a similar, unwounded counterpart. Despite significant progress in this field over the last few decades, reconstruction is still limited by the quantity and complexity of available autologous (i.e., “self”) replacement parts. This limitation is exacerbated during major trauma, with the size and complexity of the wounded tissue further limiting the availability of donor sites. Injuries affecting complex defects composed of several different tissues, such as limb amputations or large-scale facial injuries, cannot be replaced with autologous tissue and require the use of allogeneic (i.e., “foreign”) donated tissue, as recently demonstrated in the rapidly growing field of vascular composite allografts (VCAs). Despite impressive successes in limb and face transplantation, the VCA field is hampered by a strong immune rejection response to transplanted composite tissue (higher than in any other transplantation field), requiring a large dose of a lifelong immunosuppression regimen to ensure the survival of the allogeneic transplant, which can lead to serious, even life-threatening adverse outcomes.

[0005] It would be highly beneficial to use amputated tissue (i.e., nerve, tendon, and vascularized flaps and bone grafts) for autologous limb replantation or microsurgical reconstruction immediately after trauma, and even possibly obviate the need for VCAs, eliminating the need for mandatory immunosuppression. However, immediate replantation of autologous limbs or tissue is not possible, as acute rescue and lifesaving procedures take precedence according to the “life before limb” strategy, and the limb ischemia window is short (4-6 hours). The time limitation is further intensified under combat surgical conditions, making complex microsurgical replantation or microsurgical reconstruction (which requires specific operating room equipment and a trained microsurgical team) practically impossible.

[0006] One approach that allows for reconstruction using autologous tissue is to preserve its viability until the patient's condition allows for reconstructive surgery. In most modern trauma and combat injuries, such as contaminated wounds, the first 12 hours are dedicated to damage control and the most urgent vascular and orthopedic interventions. Within one to two days after injury, some selected patients can attempt some reconstructive work, but most of these complex reconstructive surgeries can be attempted only after several days to weeks after injury. Technological modifications, such as ex vivo perfusion and subzero preservation, can help extend the time of solid organ harvesting and transplantation. However, the optimistic range of extending graft survival time using these technologies has not exceeded 24 hours and, for liver grafts, extended to 96 hours. To date, the only approach suggested for long-term (days to months) organ or tissue preservation is low subzero cryopreservation through either directed freezing (DF) or vitrification, both of which are based on cryopreservation of living tissue at temperatures below (-80°C). Directed freezing has been successfully used to freeze a variety of tissues, including sheep ovaries, human ovaries, rat and pig whole livers, and rat hearts. Vitrification has been used to successfully freeze rabbit kidneys, blood vessels, corneas, and valves. There have been recent reports of successful transplantation of intact rat hindlimbs after 7 days of preservation by DF and vitrification, but the survival time of the limbs was only 72 hours.

[0007] The main challenge of cryopreservation is to prevent the damage caused by the formation of intracellular and extracellular ice crystals generated by conventional cryopreservation methods.

[0008] Vitrification is the solidification of a liquid into a solid glassy phase. Tissue vitrification is achieved by rapid cooling and rapid warming of the tissue in the presence of high concentrations of cryoprotective agents (CPAs). The probability of vitrification = CR or WR x μ x 1 / V; the probability of vitrification increases with increasing cooling / warming rate (CR or WR) and increasing viscosity (μ) and decreasing volume (V). These conditions allow the cryopreservation of cells and tissues without ice formation prior to transformation into a glassy state, thereby greatly reducing freeze damage. Despite the great potential of vitrification in cryopreservation, it has three major drawbacks that have prevented its use to date for large tissues / organs: (1) rapid and uniform cooling and warming, which is required for minimal ice nuclei and growth, is very challenging for large tissues; (2) high concentrations of CPAs, which are required for successful tissue vitrification, can cause irreversible tissue toxicity and osmotic damage if not rapidly introduced and removed; and (3) rapid cooling beyond the glass transition temperature (Tg) causes tissue breakage. The fastest cooling method available is through liquid nitrogen (LN) slush (-204°C to -210°C). To achieve LN slush, LN needs to be cooled to near its freezing point (-210°C). LN slush is created by applying a negative pressure, thereby reducing the temperature of LN to between -205°C and -210°C. LN slush is then formed, and the cooling rate increases dramatically. The cooling rate is particularly enhanced in the first phase of cooling when going from room temperature to 0°C. The cooling rate is increased by 2 to 6 times compared to quenching to LN (-196°C).

[0009] Increasing CR or WR and μ increases the probability of vitrification, but also increases the probability of breakage. This is because increasing viscosity (μ) also increases Tg, which increases CR or WR due to the increase in ΔT. To date, vitrification has only been successful in the cryopreservation of cells and small tissues, oocytes, sperm, embryos and ovarian sections, blood vessels, corneas, valves, and rabbit kidneys.

[0010] There remains a great need for a vitrification device and / or method that would allow long-term cryopreservation of large tissues, such as complex vascularized tissues, e.g., limbs and organs, that would enable their long-term survival after syngeneic retransplantation. SUMMARY

[0011] In some embodiments, the present invention relates to a two-step method for cryopreserving a biological sample, such as a tissue, comprising a rapid and brief deep-freezing step followed by a gradual longer freezing step.

[0012] The present invention is based in part on the surprising finding that tissues vitrified according to the methods disclosed herein provide long-term survival rates after syngeneic retransplantation.

[0013] In some embodiments, the methods of the present application include a first, second-long (e.g., rapid) freezing step that brings the biological sample to a temperature below zero degrees, followed by a second, minute-long (e.g., gradual) freezing step that brings the biological sample to the glass transition point temperature (Tg') of the sample. As described herein, the increased long-term survival of the biological sample after syngeneic reimplantation indicates that, for optimal long-term preservation, the biological sample should be maintained at the Tg', rather than at the lower temperatures of liquid nitrogen or its vapor.

[0014] In some embodiments, the present application relates to an apparatus comprising a first container adapted to hold a sample; an actuator configured to adjust the position of the container through a passageway along a cooling axis; a heating element in contact with the container; and a control unit in operable communication with the actuator and the heating element and configured to provide a predetermined temperature to the sample.

[0015] According to a first aspect, there is provided a method for cryopreserving a biological sample, comprising the steps of: (a) contacting the biological sample with a vitrification solution (VS) at a volume / volume (v / v) ratio ranging from 50: 1 to 1: 1; (b) subjecting the biological sample of step (a) to a temperature ranging from -210°C to -197°C for a time period of 3-10 seconds; and (c) subjecting the biological sample of step (b) to a temperature equal to or higher than the glass transition point temperature (Tg) of the VS and ranging from -140°C to -100°C for a time period of at least 3 minutes; thereby cryopreserving the biological sample.

[0016] According to another aspect, there is provided a method of preparing a biological sample for implantation into a subject in need thereof, comprising the steps of: (a) contacting the biological sample with a VS at a volume / volume (v / v) ratio ranging from 50: 1 to 1: 1; (b) subjecting the biological sample of step (a) to a temperature ranging from -210°C to -197°C for a time period of 3-10 seconds; and (c) subjecting the biological sample of step (b) to a temperature equal to or higher than the Tg of the VS and ranging from -140°C to -100°C for a time period of at least 3 minutes; thereby preparing the biological sample for implantation into a subject in need thereof.

[0017] According to another aspect, there is provided a method for cryopreserving a limb, preparing a limb for transplantation to a subject in need thereof, or both, comprising the steps of: (a) contacting a biological sample comprising a limb with a VS at a volume / volume (v / v) ratio ranging from 50: 1 to 1: 1; (b) subjecting the biological sample comprising a limb in step (a) to a temperature ranging from -210°C to -197°C for a time period of 3-10 seconds; and (c) subjecting the biological sample comprising a limb in step (b) to a temperature equal to or higher than the Tg’ of said VS and ranging from -140°C to -100°C for a time period of at least 3 minutes; thereby cryopreserving a limb, preparing a limb for transplantation to a subject in need thereof, or both.

[0018] According to another aspect, there is provided a method of transplanting a biological sample in a subject in need thereof, comprising the steps of: (a) cryopreserving a biological sample according to the method of the present application; and (b) thawing or heating and transplanting the cryopreserved biological sample to the subject.

[0019] According to another aspect, there is provided a device comprising: a first container configured to hold a sample; an actuator configured to adjust the position of said first container by passage along a cooling axis; a heating element in contact with the first container; and a control unit in operable communication with the actuator and the heating element, and configured to control the temperature of the sample by commanding: (a) the configuration of the actuator such that the position of said first container is adjusted by passage along a cooling axis; (b) the heating element to apply a predetermined amount of heat to the first container; or (a) and (b).

[0020] In some embodiments, the biological sample comprises a tissue or an organ that is vascularized, innervated, or both.

[0021] In some embodiments, the biological sample comprises a limb.

[0022] In some embodiments, the method further comprises preserving the biological sample after the second subjecting, comprising further cooling the biological sample to a temperature below the Tg or between -197°C to -150°C at a rate ranging from -1°C / min to -10°C / min.

[0023] In some embodiments, the biological sample obtained after step (a) is submerged in, covered by, or both, said VS.

[0024] In some embodiments, the contacting is under vacuum conditions.

[0025] In some embodiments, the contacting is in an elastic bag.

[0026] In some embodiments, the biological sample obtained after step (a) is substantially free of air.

[0027] In some embodiments, the biological sample obtained after step (b) comprises an outer surface in contact with the VS, wherein the outer surface is characterized by having a temperature ranging from -20°C to 0°C.

[0028] In some embodiments, the first exposure is to freezing at a rate of -330°C / min to -500°C / min.

[0029] In some embodiments, the first exposure is in a slurry of frozen liquid or under freezing conditions of -220°C to -150°C.

[0030] In some embodiments, the second exposure is to freezing at a rate of -3°C / min to -10°C / min.

[0031] In some embodiments, the second exposure is in: (a) a vapor of a frozen liquid; or (b) cooling conditions providing a temperature ranging from -85°C to -75°C, followed by a vapor of a frozen liquid.

[0032] In some embodiments, the cooling conditions providing a temperature ranging from -85°C to -75°C comprise a cooling liquid having a temperature ranging from -85°C to -75°C.

[0033] In some embodiments, the period of at least 3 minutes in step (c) is at least 30 minutes.

[0034] In some embodiments, the method further comprises a step prior to step (a) comprising perfusing the biological sample with the VS.

[0035] In some embodiments, the perfusing is at room temperature for a period of up to 5 minutes.

[0036] In some embodiments, the VS comprises at least one of: a penetrating cryoprotectant, a non-penetrating cryoprotectant, or a combination thereof, and optionally further comprises: a macromolecule, an antioxidant, a medium, or any combination thereof.

[0037] In some embodiments, the VS comprises: 15-25% (w / v) ethylene glycol, 15-25% (v / v) dimethyl sulfoxide, 15-25 (v / v) fetal bovine serum, and 0.3-0.7 M trehalose, all in a Wisconsin static preservation solution.

[0038] In some embodiments, the method further comprises step (d) comprising storing the cryopreserved biological sample for a period of at least 24 hours.

[0039] In some embodiments, the subject is in need of an organ replacement.

[0040] In some embodiments, the method further comprises the step of washing and perfusing the cryopreserved biological sample, wherein the step is prior to transplanting the cryopreserved biological sample to the subject.

[0041] In some embodiments, the transplant is an autologous transplant or an allogenic transplant.

[0042] In some embodiments, the axis is parallel to a longitudinal axis of the channel.

[0043] In some embodiments, the heating element is positioned at the top of the first container and at least partially overlaps therewith.

[0044] In some embodiments, the device further comprises at least one temperature sensor.

[0045] In some embodiments, the at least one temperature sensor is coupled to the first container such that the temperature of the first container is measurable by the temperature sensor.

[0046] In some embodiments, the control unit is in operable communication with the at least one temperature sensor.

[0047] In some embodiments, the device further comprises a second container configured to hold a cryogenic liquid, a slush thereof, a vapor thereof, or any combination thereof.

[0048] In some embodiments, the cryogenic liquid comprises liquid nitrogen (LN2).

[0049] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the application, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0050] Further embodiments and full applicable scope of the present application will become apparent from the detailed description that follows. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figures 1A-1FMicrographs and graphs showing long-term survival of vascularized inguinal flaps after long-term cryopreservation at -80°C. (1A) Vascularized inguinal flaps were perfused with cryoprotectant (CPA) at room temperature, immersed in liquid nitrogen (LN) slush for the indicated times, transferred to LN vapor for 2 minutes, warmed, re- grafted into syngeneic recipients and photographed. (1B) Vascularized inguinal flaps were cryopreserved according to the protocol and stored at -80°C for 24 hours (1C) or 9 days (1D) before being grafted in a syngeneic manner and followed up and photographed at the indicated times or removed for histological examination at postoperative day 12 (POD12). Flaps stored at -80°C for 24 hours (1 Ea-1 Ec) or 9 days (1 Ed-1 Ef) were sectioned for histological staining with hematoxylin and eosin (H&E) and photographed. Vascularized inguinal flaps were perfused with CPA, immersed in LN slush for 3 seconds and then transferred to LN vapor for 30 minutes and the temperature within the flap was measured (1F).

[0052] Figures 2A-2C Graphs, schematics and micrographs showing long-term survival of vascularized inguinal flaps after cryopreservation at Tg'. (2AI) Tg' of vitrification solution (VS) was determined by differential scanning calorimetry (DSC) analysis. (2AII) Vascularized inguinal flaps were perfused with CPA at room temperature and then transferred to LN vapor. The temperature within the flap was measured until Tg' was reached. (2B and 2CI) Vascularized inguinal flaps were perfused with CPA at room temperature, incubated in LN vapor for 30 minutes, warmed, re-grafted and photographed at the indicated times. (2CII) Vascularized inguinal flaps cryopreserved and grafted as described in 2B were removed at POD12 for histological analysis. Slides were stained with H&E and photographed.

[0053] Figures 3A-3C Schematics, graphs showing long-term stability of hindlimbs after cryopreservation. (3A) Protocol of the two-stage limb cryopreservation protocol. (3BI) Limbs were perfused with cryoprotectant (CPA) at room temperature, immersed in LN slush for 4 seconds and then transferred to LN vapor. (3BII) Limbs were perfused with CPA at room temperature, immersed in LN slush for 4 seconds, transferred to LN vapor for 10 minutes, warmed, re- grafted and photographed at the indicated times. Perfusion of the peripheral limb was demonstrated by inducing bleeding by needle pricking at POD10 and POD32. At POD32, the limb was removed for histological analysis; sections were stained with H&E (3C). Normal skin and muscle structure is shown.

[0054] Figures 4A-4EIncludes schematic diagrams and micrographs showing the long-term survival of the hind limb after cryopreservation under Tg'. The limb was perfused with cryoprotectant (CPA) at room temperature, immersed in LN thawed plasma for 4 seconds, transferred to LN vapor for 2 hours, heated, and then transplanted (4A), with photographs taken at designated times (4B). (4C) Perfusion of the peripheral limb was confirmed by needle-induced bleeding at POD28. At POD32, the limb was removed for histological analysis, and photographs were taken of the front (4DI) and back (4DII) of the harvested limb; sections were stained with H&E. Normal skin, fat, and muscle structures were shown (4E).

[0055] Figures 5A-5C Includes schematic diagrams and photomicrographs showing neural regeneration and functional sensation in re-transplanted cryopreserved limbs. (5AI) Figure 3A The two-stage cryopreservation protocol described herein involves cryopreserving a limb and then transplanting it as a fifth limb into the lumbar region of a recipient rat. (5AII) At POD12, following subknee amputation of the recipient limb, the transplanted cryopreserved limb was moved from the lumbar position to the subknee position without severing its blood supply to the recipient's femoral vessels and sutured. The recipient's tibial nerve was connected to the cryopreserved limb to allow nerve regeneration and restoration of sensation. (5B) Twenty-two days after subknee transplantation, the rats were able to walk using the cryopreserved limb. (5CI) Twenty-three days after subknee transplantation, the tibial nerve was removed for histological analysis, and sections proximal and distal to the nerve anastomosis were stained for neurofilament (NF (total axon)) and choline acetyltransferase (ChAT (motor axon)) proteins. Nuclear staining was also performed using DAPI. (5CII) A graph showing quantitative fiber counting performed using a digital system is displayed.

[0056] Figures 6A-6C This includes histological micrographs and graphs showing that transplanted cryopreserved limbs and fresh limbs exhibit similar muscle integrity. Below-knee limb transplants were perfused with saline or CPA, washed, and immediately transplanted as fresh controls (saline and CPA controls, respectively), and based on... Figure 3A and 4AThe protocol shown was compared to similar grafts cooled to ~ -80°C or Tg, respectively. At >12 postoperative days (POD), the limbs were removed and fixed, and sections were prepared from different parts of the limbs, stained with hematoxylin and eosin, and documented. Muscle mass was scored and compared by an unaware pathologist. (6A) Histological micrographs showing selected presentations of muscle sections from different control and experimental groups. (6BI) Comparative statistical analysis graph showing muscle scores of control, experimental limbs. (6BII) Vertical bar graph showing no significant difference between muscle mass of fresh control and experimental limbs (P = 0.6). (6C) Vertical bar graph showing comparison of long-term limb survival between groups of limbs frozen using directional freezing (DF), limbs vitrified to temperatures below Tg, and limbs vitrified to temperatures > Tg.

[0057] Figure 7 Simplified diagrammatic view of a first container comprising a device according to some embodiments of the application.

[0058] Figure 8 Simplified diagrammatic view of a rear view of a control unit comprising a device according to some embodiments of the application.

[0059] Figure 9 Simplified diagrammatic view of a total view of a control unit panel comprising a device according to some embodiments of the application.

[0060] Figure 10 Simplified diagrammatic view of a total view of a second container control comprising a device according to some embodiments of the application.

[0061] Figure 11 Simplified diagrammatic view of an upper close-up of a first container comprising a device according to some embodiments of the application.

[0062] Figure 12 Simplified diagrammatic view of a total view of a control unit panel comprising a device according to some embodiments of the application.

[0063] Figure 13 Simplified diagrammatic view of a total view of a second container control comprising a device according to some embodiments of the application.

[0064] Figure 14 Simplified diagrammatic view of a total view of an actuator comprising a device according to some embodiments of the application.

[0065] Figure 15 Simplified diagrammatic view of a total view of a second container comprising a device according to some embodiments of the application. DETAILED DESCRIPTION

[0066] In some embodiments, the present application relates to a vitrification device and method providing long-term survival of cryopreserved vascularized and / or innervated tissue.

[0067] Method

[0068] According to some embodiments, a method for cryopreserving a biological sample is provided.

[0069] According to some embodiments, a method of preparing a biological sample for transplantation to, for example, a subject in need thereof is provided.

[0070] According to some embodiments, a method for cryopreserving a limb, for preparing a limb for transplantation to a subject in need thereof, or both is provided.

[0071] In some embodiments, the method comprises the steps of: (a) contacting a biological sample with a vitrification solution (VS) at a volume / volume (v / v) ratio ranging from 50: 1 to 1: 1; (b) subjecting the biological sample to a temperature ranging from -210°C to -197°C for a time period of 3-10 seconds; and (b) subjecting the biological sample of step (a) to a temperature equal to or higher than the glass transition temperature (Tg) of the VS and ranging from -140°C to -100°C for a time period of at least 3 minutes.

[0072] In some embodiments, the method further comprises preserving the biological sample after the second subjecting, comprising further cooling the sample to a temperature lower than the glass transition point temperature of the VS or between -197°C to -150°C at a rate ranging from -1°C / min to -10°C / min.

[0073] As used herein, the terms "preserving" and "storing" can be interchangeable.

[0074] In some embodiments, the cryopreserved biological sample is stored at a temperature that is the Tg of the VS. In some embodiments, the cryopreserved biological sample is stored at a temperature that is lower than the Tg of the VS. In some embodiments, the cryopreserved biological sample is stored at a temperature that is higher than the Tg of the VS.

[0075] In some embodiments, the biological sample comprises a tissue or an organ.

[0076] In some embodiments, the tissue or organ comprises a vasculature. In some embodiments, the tissue or organ comprises at least one nerve, neuron, axon, dendrite, ganglion, or any combination thereof. In some embodiments, the tissue or organ is vascularized, innervated, or both.

[0077] In some embodiments, the tissue or organ is selected from the group consisting of: a limb, a kidney, a heart, a liver, a lung, a pancreas, an intestinal tract, a skin, or a tissue fragment derived therefrom.

[0078] In some embodiments, the biological sample comprises a limb.

[0079] In some embodiments, the limb comprises an upper limb, a lower limb, or a plurality of limbs comprising an upper limb, a lower limb, or a combination thereof.

[0080] As used herein, the terms "subjecting", "cooling", "chilling", and "freezing" can be used interchangeably.

[0081] In some embodiments, the (v / v) ratio of the biological sample and the VS ranges from 50:1 to 1:1, 100:1 to 1:1, 250:1 to 1:1, 40:1 to 10:1, 25:1 to 5:1, or 45:1 to 6:1. Each possibility represents a separate embodiment of the application.

[0082] In some embodiments, the biological sample obtained after step (a) is submerged in the VS, is covered by the VS, or both.

[0083] In some embodiments, the biological sample obtained after step (a) is at least partially submerged in the VS, is at least partially covered by the VS, or both.

[0084] In some embodiments, the contacting is under vacuum conditions.

[0085] In some embodiments, the method comprises placing the biological sample in an elastic bag. In some embodiments, the method comprises the step of providing the biological sample in an elastic bag.

[0086] In some embodiments, the contacting is in an elastic bag. In some embodiments, the elastic bag comprises a plastic bag. In some embodiments, the elastic bag comprises a sealable elastic bag. In some embodiments, the vacuum is applied while the biological sample is in the elastic bag. In some embodiments, the elastic bag is sealed after the vacuum is applied to the biological sample in contact with the VS.

[0087] In some embodiments, the biological sample is contacted with the VS in an elastic bag at a (v / v) ratio of 50:1 to 1:1, 100:1 to 1:1, 250:1 to 1:1, 40:1 to 10:1, 25:1 to 5:1, or 45:1 to 6:1. Each possibility represents a separate embodiment of the application.

[0088] In some embodiments, the application of vacuum is paused, stopped or interrupted (or any combination and equivalent thereof) after the flexible bag comprising the biological sample and the VS is free of air or substantially free of air.

[0089] As used herein, the term "vacuum" includes the application of any pressure lower than atmospheric pressure.

[0090] In some embodiments, the biological sample obtained after step (a) is free of air. In some embodiments, the biological sample obtained after step (a) is substantially free of air.

[0091] In some embodiments, the biological sample obtained after step (b), e.g. resulting from the application of vacuum, comprises an outer surface in contact with the VS. In some embodiments, the outer surface of the biological sample is characterized by having a temperature in the range of 0°C to -20°C, -1°C to -15°C, -3°C to -19°C or 0°C to -17°C. Each possibility represents a separate embodiment of the application.

[0092] In some embodiments, the first exposure is under conditions suitable to reduce the temperature of the biological sample below zero degrees Celsius for at most 7 minutes, at most 6 minutes, at most 5 minutes, at most 4 minutes, at most 3 minutes, at most 2 minutes or at most 1 minute or any value and range therebetween. Each possibility represents a separate embodiment of the application.

[0093] In some embodiments, the conditions suitable to reduce the temperature of the biological sample below zero degrees Celsius comprise any cooling rate equal to or higher than -5°C / min

[0094] In some embodiments, the first exposure comprises freezing at a rate of -10°C / min to -20°C / min, -5°C / min to -40°C / min, -30°C / min to -70°C / min, -60°C / min to -120°C / min, -130°C / min to -270°C / min, -230°C / min to -350°C / min, -330°C / min to -370°C / min, -310°C / min to -390°C / min, -300°C / min to -385°C / min, -295°C / min to -365°C / min, -280°C / min to -395°C / min or -315°C / min to -375°C / min. Each possibility represents a separate embodiment of the application.

[0095] In some embodiments, the first exposure is in a slush of a frozen liquid. In some embodiments, the first exposure is at a freezing condition of -220 °C to -190 °C. In some embodiments, the first exposure is in a vapor of a frozen liquid. In some embodiments, the first exposure is in a frozen liquid, or at a freezing condition of -150 °C to -90 °C.

[0096] In some embodiments, the second exposure is frozen at a rate of -6 °C / min to -10 °C / min, -3 °C / min to -15 °C / min, -2 °C / min to -13 °C / min, -4 °C / min to -11 °C / min, -5 °C / min to -17 °C / min, -6 °C / min to -12 °C / min, -6 °C / min to -8 °C / min. Each possibility represents a separate embodiment of the application.

[0097] In some embodiments, the second exposure is in: (a) a vapor of a frozen liquid; or (b) a cooled liquid having a temperature ranging from -120 °C to -40 °C, and then in a vapor of a frozen liquid. In some embodiments, the second exposure is in: (a) a vapor of a frozen liquid; or (b) a cooled condition providing a temperature ranging from -85 °C to -75 °C, and then in a vapor of a frozen liquid.

[0098] In some embodiments, the cooled condition providing a temperature ranging from -85 °C to -75 °C comprises a cooled liquid having a temperature ranging from -85 °C to -75 °C.

[0099] In some embodiments, the period of at least 3 minutes comprises: at least 4 minutes, at least 5 minutes, at least 7 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 2 days, at least 7 days, at least 14 days, at least 1 month, or at least 3 months, or any value and range therebetween. Each possibility represents a separate embodiment of the application. In some embodiments, the period of at least 3 minutes comprises: 3-10 minutes, 5-25 minutes, 5-40 minutes, 10-60 minutes, 15-90 minutes, 1-3 hours, 2-12 hours, 4-18 hours, 8-24 hours, 12-36 hours, 1-3 days, 2-7 days, 5-14 days, 2-5 weeks, or 1-3 months. Each possibility represents a separate embodiment of the application.

[0100] In some embodiments, the contacting comprises perfusion of the biological sample.

[0101] Methods for perfusing a biological sample, e.g., a tissue including blood vessels, are common and will be apparent to one of ordinary skill in the art.

[0102] In some embodiments, perfusing includes perfusing at least one blood vessel of the biological sample. In some embodiments, perfusing includes perfusing at least one artery of the biological sample. In some embodiments, perfusing includes contacting at least one blood vessel of the biological sample with: (a) a solution including physiological saline, heparin, or a combination thereof; (b) a balancing solution; (c) a vitrification solution; or any combination of (a) to (c).

[0103] In some embodiments, the method further includes a step comprising perfusing the biological sample with a cryoprotective agent. In some embodiments, the method further includes a step comprising perfusing the biological sample with a solution including physiological saline, heparin, or a combination thereof. In some embodiments, the method further includes a step comprising perfusing the biological sample with a balancing solution. In some embodiments, the method further includes a step comprising perfusing the biological sample with a VS. In some embodiments, the step of perfusing the biological sample is prior to subjecting the biological sample to a temperature ranging from -210°C to -197°C for 3-10 seconds for the first time (e.g., step (a)).

[0104] In some embodiments, the term "balancing solution" includes any solution suitable for preparing or enhancing the cold tolerance or stability of the biological sample. In some embodiments, the balancing solution is provided to the biological sample after perfusing the biological sample with a solution including physiological saline, heparin, or a combination thereof. In some embodiments, the balancing solution is provided to the biological sample prior to perfusing the biological sample with a VS. In some embodiments, the balancing solution enhances or increases the cryoprotective activity or effect of the VS.

[0105] As used herein, "cold tolerance" or "cold stability" refers to the ability of a tissue or organ to withstand cryopreservation without or with only minimal tissue damage, e.g., freeze damage, hemorrhaging, etc., thereby allowing for subsequent manipulation or use of the cryopreserved tissue or organ, e.g., its warming / thawing and / or transplantation.

[0106] In one embodiment, the balancing solution includes: 5-10% (v / v) ethylene glycol (EG); 2.5-10% (v / v) DMSO; and 5-25% (v / v) fetal calf serum (FCS), all in a Wisconsin static preservation solution.

[0107] As used herein, the terms "cryoprotecting agent" and "cryoprotectant" are interchangeable and include any compound that can be used to protect a biological sample, e.g., a cell, tissue, organ, or portion thereof, or any combination thereof, from damage from ice formation, e.g., formation of ice crystals or any other cold-related damage.

[0108] In some embodiments, the perfusion is for a period of time of at least 10 seconds, at least 20 seconds, at least 30 seconds, at least 40 seconds, at least 50 seconds, at least 60 seconds, at least 2 minutes, at least 3 minutes, at least 4 minutes, or up to 5 minutes, or any value and range therebetween. Each possibility represents a separate embodiment of the present application. In some embodiments, the perfusion is for a period of time of 5 to 60 seconds, 1 to 3 minutes, 1 to 4 minutes, 1 to 5 minutes, 2 to 3 minutes, 2 to 4 minutes, 2 to 5 minutes, 3 to 4 minutes, 3 to 5 minutes, or 4 to 5 minutes. Each possibility represents a separate embodiment of the present application.

[0109] In some embodiments, the contacting, perfusion, or both are performed at room temperature. In some embodiments, the contacting, perfusion, or both are performed at ambient temperature. In some embodiments, the contacting, perfusion, or both are performed at a temperature of 15 °C to 28 °C.

[0110] It is common and apparent to one of ordinary skill in the art to perfuse a biological sample and / or tissue with a supplemental cryoprotectant. Non-limiting examples of cryoprotectants include, but are not limited to, DMSO, glycerol, ethylene glycol, propylene glycol, sucrose, trehalose, 2-methyl-2,4-pentanediol (MPD), and / or others.

[0111] In some embodiments, the VS comprises a permeating cryoprotectant, a non- permeating cryoprotectant, or a combination thereof. In some embodiments, the VS further comprises: a macromolecule, an antioxidant, a medium, or any combination thereof. In some embodiments, the VS comprises a permeating cryoprotectant, a non-permeating cryoprotectant, or a combination thereof, and optionally further comprises: a macromolecule, an antioxidant, a medium, or any combination thereof. In some embodiments, the VS comprises a permeating cryoprotectant, a non-permeating cryoprotectant, a macromolecule, an antioxidant, a medium, or a combination thereof.

[0112] In some embodiments, the permeating cryoprotectant is selected from: DMSO, ethylene glycol, propylene glycol (PG), glycerol (GLY), or any combination thereof.

[0113] In some embodiments, the non-permeant cryoprotectant includes a disaccharide, an oligosaccharide, a polysaccharide, or any combination thereof. In some embodiments, the non-permeant cryoprotectant is selected from the group consisting of: sucrose, trehalose, dextran, or any combination thereof.

[0114] As used herein, the term "macromolecule" includes fetal bovine serum, human serum albumin, polysucrose, any combination thereof, or any equivalent thereof.

[0115] In some embodiments, the antioxidant includes Epigallocatechin gallate (EGCG), astaxanthin, or a combination thereof.

[0116] In some embodiments, the culture medium includes any growth medium suitable for cell and / or organ culture. In some embodiments, the culture medium includes PBS, HDMI, or a combination thereof.

[0117] In some embodiments, the VS includes: 15-25% (w / v) of ethylene glycol, 15-25% (v / v) of dimethyl sulfoxide, 15-25 (v / v) of fetal bovine serum, and 0.3-0.7 M of trehalose, all in a Wisconsin static preservation solution.

[0118] In some embodiments, the method further includes a step of storing the cryopreserved sample for at least: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 1 month, or 3 months, or any value and range therebetween. Each possibility represents a separate embodiment of the present application. In some embodiments, the method further includes a step of storing the cryopreserved sample for 1 to 3 days, 2 to 5 days, 1 to 7 days, 3 to 12 days, 6 to 14 days, 1 to 3 weeks, 2 to 5 weeks, 3 to 6 weeks, or 1 to 4 months, 1 to 100 years. Each possibility represents a separate embodiment of the present application.

[0119] In some embodiments, the storage period includes any time period as long as the survival time of the tissue after re-implantation is maintained in the range of 7-28 days. In some embodiments, the storage period includes any time period as long as the survival time of the tissue after re-implantation is maintained in at least: 7 days, 14 days, 21 days, 28 days, or any value and range therebetween. Each possibility represents a separate embodiment of the present application.

[0120] In some embodiments, the storage is at the glass transition temperature (Tg) ± 1 °C, ± 2 °C, ± 3 °C, ± 4 °C, ± 5 °C, ± 6 °C, ± 7 °C, ± 8 °C, ± 9 °C, or ± 10 °C, or any value and range therebetween, of the frozen sample. Each possibility represents a separate embodiment of the present application.

[0121] In some embodiments, the Tg comprises or consists of the Tg of the VS.

[0122] As used herein, the terms "Tg" and "Tg initial (Tg')" are interchangeable.

[0123] In some embodiments, the biological sample comprises a tissue or organ having a volume of at least: 1 cm 3 , 15 cm 3 , 50 cm 3 , 100 cm 3 , 150 cm 3 , 200 cm 3 , 300 cm 3 , 500 cm 3 , 1,000 cm 3 , 2,000 cm 3 , 3,000 cm 3 , 5,000 cm 3 , 7,000 cm 3 , 8,500 cm 3 , 10,000 cm 3 , or any value and range therebetween. Each possibility represents a separate embodiment of the present application. In some embodiments, the biological sample comprises a tissue or organ having a volume of 3-50 cm 3 , 30-250 cm 3 , 25-500 cm 3 , 55-650 cm 3 , 200-1,500 cm 3 , 300-5,000 cm 3 , 450-6,500 cm 3 , 300-7,500 cm 3 , 1,000-9,500 cm 3 , 2,000-8,500 cm 3 , 3,000-10,000 cm 3 , or 1,000-12,000 cm 3 . Each possibility represents a separate embodiment of the present application.

[0124] In some embodiments, a method of transplanting a biological sample in a subject in need thereof is provided, comprising the steps of: (a) cryopreserving a biological sample according to the methods disclosed herein; and (b) thawing and / or warming and transplanting the cryopreserved sample to the subject.

[0125] The terms "thawing" and "warming" are used interchangeably herein.

[0126] In some embodiments, a method of transplanting a biological sample in a subject in need thereof is provided, comprising the steps of: (a) cryopreserving a biological sample according to the methods disclosed herein; and (b) transplanting the cryopreserved sample to the subject.

[0127] In some embodiments, the method further comprises the step of thawing and / or warming the cryopreserved sample prior to transplanting the cryopreserved sample to the subject.

[0128] In some embodiments, the subject is a mammalian subject, for example, but not limited to, a human subject.

[0129] In some embodiments, the subject is in need of an organ replacement.

[0130] In some embodiments, the organ replacement is due to organ damage or disease. In some embodiments, the organ damage comprises damage-induced or associated organ damage.

[0131] In some embodiments, the damage comprises trauma.

[0132] In some embodiments, the subject is suffering from or has suffered from trauma.

[0133] In some embodiments, the trauma comprises amputation.

[0134] In some embodiments, the method further comprises the step of washing out, eliminating, expelling, discarding, or any combination thereof, the cryoprotectant after thawing the cryopreserved sample. In some embodiments, the method further comprises the step of washing out, eliminating, expelling, discarding, or any combination thereof, the cryoprotectant prior to transplanting the cryopreserved sample.

[0135] In some embodiments, the method further comprises the step of perfusing the cryopreserved biological sample. In some embodiments, the method further comprises the step of perfusing the cryopreserved biological sample prior to transplanting the cryopreserved biological sample.

[0136] In some embodiments, the washing and / or perfusion comprises using a solution comprising sucrose. In some embodiments, the washing and / or perfusion comprises using any solution known to those skilled in the art that is suitable for the current procedure described herein. In some embodiments, the washing and / or perfusion comprises using a solution comprising sucrose. In some embodiments, the solution comprises sucrose at a concentration of: at least 100 mM, at least 125 mM, at least 250 mM, at least 500 mM, or at least 1,000 mM (1 M), or any number and range therebetween. Each possibility represents a separate embodiment of the application. In some embodiments, the solution comprises sucrose at a concentration of 100 mM to 500 mM, 250 mM to 750 mM, or 100 mM to 1,000 mM. Each possibility represents a separate embodiment of the application.

[0137] In some embodiments, the transplant is an autologous transplant.

[0138] In some embodiments, the transplant is an allogeneic transplant.

[0139] Apparatus

[0140] According to some embodiments, there is provided an apparatus comprising: a first container 102 configured to hold a sample 104; an actuator 300 configured to adjust a vertical position of the first container 102 through a passage along a cooling axis; a heating element 152 in contact with the first container 102; and a control unit 200 in operable communication with the actuator 300 and the heating element 152 and configured to control a temperature of the sample 104.

[0141] In some embodiments, the cooling axis is parallel to a longitudinal axis of the passage.

[0142] In some embodiments, the apparatus further comprises at least one temperature sensor. As used herein, the terms "temperature sensor" and "thermocouple" are interchangeable.

[0143] In some embodiments, the at least one temperature sensor is coupled to the first container 102 such that a temperature of the first container 102 is measurable by the temperature sensor.

[0144] In some embodiments, the control unit 200 is in operable communication with the at least one temperature sensor.

[0145] In some embodiments, the control unit 200 is configured to control or is in communication with the heating element such that the biological sample is cryopreserved according to the methods disclosed herein. In some embodiments, the control unit activates or controls the heating element such that the biological sample of step (a) of the methods disclosed herein is subjected to a temperature ranging from -210 °C to -197 °C for a time period of 3-10 seconds.

[0146] In some embodiments, the control unit 200 activates or controls the heating element such that the biological sample of step (b) of the method disclosed herein is subjected to a temperature ranging from -140°C to -100°C for a period of at least 3 minutes.

[0147] In some embodiments, the device further comprises a second container 126 configured to contain the cryogenic liquid 190, its slush, its vapor, or any combination thereof.

[0148] In some embodiments, the cryogenic liquid comprises liquid nitrogen (LN2). In some embodiments, the cryogenic liquid comprises LN2 slush. In some embodiments, the cryogenic liquid comprises LN2 vapor.

[0149] Referring to FIG. 5, which is a simplified pictorial diagram of an overview of some components of a device according to some embodiments of the present application, including: a first container 102 configured to contain a sample 104; an in situ limit switch 106; a control unit 200; a tray 204; a first container cable 110; a limit switch blocker 112; a first container support cable (width side) 114; a first container support cable 116; a first container cover fastening screw (width side) 118; a heating element spring cable 120; a thermocouple spring cable 122; a first container cover fastening screw (length side) 124; a second container configured to contain a cryogenic liquid 126; and a thermocouple 128.

[0150] Referring to FIG. 6, which is a simplified pictorial diagram of a back view of a control unit 200 of a device according to some embodiments of the present application, including: a vacuum pump power outlet 402; and a main power outlet and fuse switch 202.

[0151] Referring to FIG. 7, which is a simplified pictorial diagram of a front view of a control unit 200 of a device according to some embodiments of the present application, including: a vacuum pump power outlet 402; and a main power outlet and fuse switch 202. Figure 7 Referring to FIG. 8, which is a simplified pictorial diagram of an overview of a device according to some embodiments of the present application, including: a control unit 200; a first container 102; a housing of the device 500; a vacuum tubing 404; a vacuum pump 406; a cryogenic liquid / slush 190; a second container configured to contain a cryogenic liquid 126; and a thermocouple 128.

[0152] Referring to FIG. 9, which is a simplified pictorial diagram of an overview of a device according to some embodiments of the present application, including: a control unit 200; a first container 102; a housing of the device 500; a vacuum tubing 404; a vacuum pump 406; a cryogenic liquid / slush 190; a second container configured to contain a cryogenic liquid 126; and a thermocouple 128. Figure 8, which is a full view simplified schematic of a first container of an apparatus according to some embodiments of the present application, including: a thermocouple socket 132 of a thermocouple 128; a thermocouple socket 134; a thermocouple plug 130 of the thermocouple 128; a thermocouple spring cable 134; a thermocouple cable 136 of the thermocouple 128; a thermocouple plug 138; a heating element socket 140; a limit switch bracket 142; a heating element spring cable 120; a first container support cable (width side) 114; a first container lid fastening screw (length side) 124; a first container hanging hook 144; a limit switch 146; a first container hanging metal ring 148; a first container support cable 114; a heating element cover plate 150; a heating element 152; a sample 104; and a first container lid fastening screw (width side) 118.

[0153] Referring to Figure 9 , which is an upper close-up simplified schematic of a top portion of an apparatus according to some embodiments of the present application, including: a limit switch bracket 142; a limit switch 144; a thermocouple plug 130 of a thermocouple 128; a heating element socket 140; a heating element plug 152; a control unit 200; a tray 204; a first container cable 110; a limit switch baffle 112; a first container hanging hook 144; and a first container hanging metal ring 148.

[0154] Referring to Figure 10 , which is a full view simplified schematic of a control unit 200 panel of an apparatus according to some embodiments of the present application, including: a control unit front panel 206; a vacuum pump stop / start switch 208; a start process switch 210; a programmable logic controller (PLC) liquid crystal display (LCD) 212; an alarm red indicator light 214; a vacuum pump on green indicator light 216; and a cooling process in progress green indicator light 218.

[0155] Referring to Figure 11 , which is a full view simplified schematic of a second container control of an apparatus according to some embodiments of the present application, including: a vacuum valve manifold 154; a vacuum pressure relief valve 156; a vacuum supply valve 158; a vacuum pressure gauge 160; a second container lid O-ring 162; a second container 126; a thermocouple through hole 164; a second container lid 166; and a thermocouple 128.

[0156] Referring to Figure 12 , which is a full view simplified schematic of an actuator 300 of an apparatus according to some embodiments of the present application, including: a thermocouple socket 130 of a thermocouple 128; a heating element power socket 140; a thermocouple socket 134; a vacuum pump power socket 402; a cable collection wheel 302; a first container cable 110; a stepper motor bracket panel 304; a stepper motor with integrated driver 306; and a main power socket and fuse switch 202.

[0157] Referring to Figure 13 FIG. 2 is a simplified schematic diagram of a second container of an apparatus according to some embodiments of the application, including: a vacuum valve manifold 154; a vacuum relief valve 156; a vacuum supply valve 158; a vacuum pressure gauge 160; a thermocouple 128; a frozen liquid / slush 190; and a second container 126.

[0158] In some embodiments, the second container has a longitudinal axis. In some embodiments, the longitudinal axis is parallel to the cooling axis. In some embodiments, the actuator 300 is configured to adjust the position of the first container 102 through the passage along the cooling axis.

[0159] In some embodiments, the heating element 152 is in contact with the first container 102. In some embodiments, the heating element 152 is positioned on top of the first container 102 and at least partially overlaps it. In some embodiments, the heating element 152 is configured to heat the first container, thereby increasing the temperature of the sample.

[0160] In some embodiments, the control unit 200 is in operable communication with the actuator 300 and the heating element 152. In some embodiments, the control unit 200 is configured to control the temperature of the sample 104 by commanding: (a) the configuration of the actuator 300 such that the first container 102 moves through the passage along the cooling axis; (b) the heating element 152 to apply a predetermined amount of heat to the first container 102; or (a) and (b). In some embodiments, the actuator 300 is configured to adjust the position of the first container along the cooling axis. In some embodiments, the actuator 300 adjusts the position of the first container to the lower portion of the second container, thereby decreasing the temperature of the sample. In some embodiments, the actuator 300 adjusts the position of the first container to the top portion of the second container, thereby increasing the temperature of the sample. In some embodiments, the actuator 300 adjusts the position of the first container such that the position of the first container does not exceed 1 cm, 2 cm, 5 cm, 7 cm, 10 cm, 15 cm, 20 cm, or 25 cm above the top portion of the second container, or any value and range therebetween. Each possibility represents a separate embodiment of the application.

[0161] In some embodiments, the actuator 300 adjusts the position of the first container such that the position of the first container does not exceed 1 cm, 2 cm, 5 cm, 7 cm, 10 cm, 15 cm, 20 cm, or 25 cm above the surface of the frozen liquid stored or contained within the second container, or any value and range therebetween. Each possibility represents a separate embodiment of the application.

[0162] In some embodiments, the temperature sensor can be a thermocouple. In some embodiments, the temperature sensor can be a wireless temperature sensor. In some embodiments, the temperature sensor can be an infrared sensor. In some embodiments, the device comprises at least one temperature sensor positioned at the first container configured to measure the temperature of the sample. In some embodiments, the device comprises at least one temperature sensor positioned at the second container configured to measure the temperature of the LN2 slush. In some embodiments, the temperature sensor is in operable communication with the control unit.

[0163] In some embodiments, the device further comprises a non-thermal conductive portion, e.g., an insulating portion. In some embodiments, the non-conductive portion comprises a non-thermal conductive material, e.g., an insulating material.

[0164] Any non-thermal conductive material known in the art can be used. In some embodiments, the non-thermal conductive material is selected from the group consisting of butane, hydrazine, chloroform, hydrazine, 1,1,2-trichloro-trifluoroethane, 1,2-dichlorotetrafluoroethane, tetrafluoroethane, argon, carbon dioxide, diethyl ether, isobutane, pentane, perfluorocyclobutane, propane, tetrafluoromethane, CFC-11, HCFC-141b, methanol, ethanol, glycerol, diethyl ether, acetone, ethylene glycol, non-thermally conductive polyoxyethylene fluid containing glass such as fiberglass or glass beads, propylene glycol, acrylic glass, asphalt, cement, clay, concrete, ceramic-filled Corian, cork, cotton insulation, diatomaceous earth, epoxy, fiberglass, foam glass, glass beads or marbles, glass wool, gypsum, magnesite, magnesium oxide insulation, mineral insulation, nylon, perlite, plastic foam insulation, expanded polystyrene, polyurethane, porcelain, PTFE, PVC, Pyrex glass, sand, vermiculite aerogel, styrofoam, polyurethane foam, vermiculite, vinyl ester, and combinations thereof.

[0165] In some embodiments, the non-thermal conductive portion comprises a plurality of sequential layers. In some embodiments, the non-conductive portion comprises a plurality of concentric layers.

[0166] As used herein, the term "plurality" refers to any integer of at least 2, at least 3, at least 4, at least 5, at least 7, or at least 10, or any value and range therebetween. Each possibility represents a separate embodiment of the present application. In some embodiments, the plurality comprises 2-5, 2-7, 2-10, 3-8, or 3-10. Each possibility represents a separate embodiment of the present application.

[0167] In some embodiments, each layer of the plurality of sequential layers is in contact with at least one other layer. In some embodiments, the layers of the plurality of sequential layers are not in contact with one another. In some embodiments, the voids between the layers of the plurality of sequential layers that are not in contact with one another are filled with air or an inert gas. In some embodiments, the voids between the layers of the plurality of sequential layers that are not in contact with one another are free of any liquid or gas. In some embodiments, the voids between the layers of the plurality of sequential layers that are not in contact with one another are free of air.

[0168] General Terms

[0169] As used herein, the term "about" means ± 10%.

[0170] The terms "comprise," "comprising," "include," "including," "have," "having," and "contain," "containing," and the like mean "including but not limited to."

[0171] The term "consisting of means "including and limited to," as in the phrase "consisting of only A and B."

[0172] The term "consisting essentially of means the composition, method or structure can include additional ingredients, steps and / or components, but only if the additional ingredients, steps and / or components do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0173] As used herein, "exemplary" means "serving as an example, instance, or illustration." Any implementation described as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations.

[0174] The word "optionally" as used herein means "may be present or absent." Any particular embodiment of the application can include a plurality of optional features, unless such features conflict.

[0175] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0176] Throughout this application, various embodiments of the application can be presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and that one of skill in the art will understand that the described ranges are to be interpreted as "provided any number within that range." Thus, although a range including an end value may be closed, e.g. 1 to 6, other embodiments could also fall outside the specified range, e.g. 7. Thus, the ranges should be read to include both the stated range and any sub-ranges therefrom.

[0177] Whenever a numerical range is indicated herein, it is meant to include any cited number (fractional or integral) within the indicated range. The phrases "ranging / range between" a first indicate number and a second indicate number and "ranging / ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicate number and all the fractional and integral numbers therebetween.

[0178] The term "method" as used herein refers to manners, means, techniques and procedures for accomplishing a specified task or tasks. It is contemplated that the methods described herein could be implemented in a variety of ways, and that the application should be understood to be directed to each such manner, combination of manners, and each such way and combination of ways of accomplishing the specified tasks.

[0179] It is to be understood that certain features that are, individually, described as being part of a single embodiment can also be provided separately or in any suitable combination. Conversely, certain features that are individually described as part of separate embodiments can also be provided in combination, or in any suitable sub-combination, or in any other described embodiment of the application. It is not intended that the described embodiments be limited to the described combination of features. Certain features described in the context of various embodiments are not to be regarded as essential features of those embodiments, unless the absence of those elements renders the embodiment non-functional.

[0180] The description of the various embodiments of the application is intended for purposes of illustration, of explanation, and not limitation, of the scope of the present application. Many modifications, variations, and changes in detail can be practiced with the elements and combinations of elements as disclosed herein without departing from the scope of the described embodiments. The terminology used in the description presented herein is intended to be interpreted in its broadest reasonable manner, even though it is used in conjunction with a detailed description of the preferred embodiments of the application. Although specific terms are employed herein, they are used only in the generic and descriptive sense, and not for purposes of limitation.

[0181] The various embodiments and aspects of the application as described above and claimed in the claims find experimental support in the following examples.

[0182] Examples

[0183] Generally, the nomenclature used herein and the laboratory procedures utilized in the present application include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980).New York (1980); see also, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" vols. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications" Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all incorporated herein by reference. Additional general references are provided within the present document.

[0184] Materials and Methods

[0185] Animals

[0186] Male brown Norway rats, 12-16 weeks old, were raised under regular conditions and used as both donors and recipients of limb allotransplantation. All animal experiments were approved by the Tel Aviv Sourasky Medical Center Institutional Animal Care and Use Committee.

[0187] Surgical Procedure

[0188] limb harvesting

[0189] Anesthesia was induced in the induction cage with 2% isoflurane and oxygen at a flow rate of 5 L / min. Anesthesia was maintained with 1-2% isoflurane inhalant. The donor's surgical site was prepared and a skin incision was made around the circumference of the right hind limb, 1 cm above the knee. First, the skin of the upper thigh was mobilized to expose the vastus medialis and adductor muscle group on the medial thigh. All of the superficial muscle groups from the thigh but across the knee joint were transected around the knee joint to expose the knee joint. The tendon of the vastus quadriceps was transected above the iliac ligament. The gastrocnemius and soleus muscles were excised, but the Achilles tendon was left intact. After opening the knee joint capsule, the knee joint was completely disarticulated by transecting all ligaments. These muscles and ligaments left sufficient margins of residual tissue for secondary repair. Second, the sciatic nerve was found posterior to the tarsal joint to expose the tibial and peroneal nerves. After placement of a 10-0 nylon suture tag, the tibial nerve was transected in the middle of the calf and the peroneal nerve was transected above the fibulae capitulum. These nerves left mesoneural tissue intact. Third, the femoral vessels were transected at the level of the inguinal ligament takeoff. The femoral vessels were then clamped and transected above the previously placed suture tag. Using a 22 gauge angiocatheter, approximately 5 mL of 0.9% warm saline and 50 U of heparin were manually infused through the femoral artery until the venous outflow was clear. Once transected, the donor limb (graft) was wrapped in moist gauze until it received vitrification. The donor animal was euthanized.

[0190] limb transplantation

[0191] After freezing and thawing, the recipient animal was anesthetized and prepared for surgery. A diagonal incision was made in the hip, approximately 1.5 cm long, slightly above the limb, further distally to expose and transect the femoral vessels to leave sufficient length similar to the donor limb. The donor limb was transplanted by performing vascular anastomosis with simple interrupted sutures of 10-0 nylon. The muscle groups were approximated to the abdominal wall and 6-0 prolene lactated Ringer's solution (5 mL, IP) was administered. The skin was closed with interrupted 4-0 nylon sutures.

[0192] limb transplantation repositioning to below the knee

[0193] below the knee amputation of the recipient limb

[0194] The recipient rat is anesthetized and the right hind limb and small back of the animal are shaved. An incision is made from the waist to the medial region of the thigh. The femoral artery and vein are isolated above the knee joint for anastomosis. The vitrified limb is first fixed in order to remain stable during the anastomosis. The anastomosed blood vessels are kept under appropriate tension.

[0195] Positioning and attachment of the vitrified limb below the knee

[0196] Ten to fourteen days after the transplantation of the vitrified limb as a fifth limb, an incision is made in the recipient limb on the same side of the limb as the skin flap, in the anterior tibial region of the lower leg, to expose the superficial muscles. The recipient limb is then amputated below the knee, while preserving the recipient's gastrocnemius muscle, soleus muscle, Achilles tendon and tibial nerve portion, ending approximately 1 cm above the ankle. The scar of the vitrified limb is incised along the original incision and the subcutaneous fascia and fibrous cord are separated until the complete absence of vascular occlusion. The vitrified limb is then moved below the amputated knee. The corresponding vitrification is performed below the knee: first, the posterior capsule of the knee is sutured and attached. Second, the knee ligaments are repaired; 1) the cruciate ligament of the knee; 2) the collateral ligaments on both sides; 3) the anterior capsule of the knee and the left and right capsules. Third, the patellar ligament and Achilles tendon are sutured. Fourth, the recipient and donor tibial nerves are anastomosed 1 cm above the ankle. Fifth, the superficial muscles (vastus medialis, vastus lateralis, biceps femoris, semitendinosus, semimememus and adductor group) are repaired. Sixth, the skin of the adjusted recipient area is sutured to the skin of the vitrified limb.

[0197] Vascularized inguinal skin flap

[0198] Flap harvesting

[0199] In the donor, a 3 x 4 cm flap is harvested from the groin, based on the femoral artery and vein, including the underlying cornea of the plane directly above the abdominal muscle and the inguinal adipose tissue. After harvesting, the flap is perfused with heparinized Ringer lactate solution (50 U of heparin in 10 ml of Ringer lactate) until the venous effluent becomes clear.

[0200] Flap transplantation

[0201] A defect of the same size was created on the right side of the recipient rat. After freezing and thawing the flap, the femoral arteries of the recipient and donor were anastomosed end-to-end using standard microsurgical techniques under a surgical microscope with 10 / 0 nylon sutures. The flap was then sutured to the defect using 4 / 0 absorbable sutures (Vicryl, Ethicon, Inc.).

[0202] Vitrification of limbs

[0203] Manually infuse each harvested limb with 5 ml of the following solution for 0.5 minutes:

[0204] 1. Equilibrium solution (ES): Wisconsin static preservation solution (SPS-1, organ preservation system) consisting of 7.5% ethylene glycol (EG) + 7.5% DMSO + 20% fetal bovine serum (FCS).

[0205] 2. Vitrification solution (VS): SPS-1 solution of 20% EG + 20% DMSO + 20% FCS + 0.5M trehalose.

[0206] After the infusion is completed, the limb is inserted into an empty cryoprotectant bag. Add 5 ml of VS to a freezer bag (Miltenyi Biotec, Bergisch Gladbach, Germany), and vacuum and heat-seal the bag. Then, using a VitMaster device (IMT, Nes-Ziona, Israel), insert the bag into the LN slurry (between -205 and -210°C) for 4 seconds, and then place it in LN vapor 1 cm above the LN surface for 2 minutes or up to 2 hours, as instructed.

[0207] Limb heating program

[0208] Insert the cryopreservation bag into a water bath heated to 38°C until the limb thaws (approximately 2-3 minutes). Open the bag, remove the limb, and insert it into SPS-1 supplemented with 1M sucrose for 2 minutes, then insert it into physiological saline for another 2 minutes.

[0209] Then manually irrigate the limb with the following washing solutions (1 ml / 0.5 min for each solution). Washing solution 1 (WS1): SPS-1 solution of 1M sucrose and 20% FBS; WS2: 1 / 1 dilution of WSI in SPS-1; WS3: 1 / 1 dilution of WS2 in SPS-1; WS4: 1 / 1 dilution of WS3 in SPS-1. After WS4, rinse the limb with 10–20 ml of physiological saline.

[0210] Vascularized groin flap vitrification procedure

[0211] Harvested flaps were manually perfused for 0.5 min with 1 ml each of the following solutions:

[0212] 1. Equilibration solution (E.S): SPS-1 (Organ Preservation Solutions) of 7.5% ethylene glycol (EG) + 7.5% DMSO + 20% fetal calf serum (FCS).

[0213] 2. Vitrification solution (V.S): SPS-1 solution of 20% EG + 20% DMSO + 20% FCS + 0.5 M trehalose.

[0214] After the perfusion, the flaps were inserted into an empty cryovial (CryoVials®, Miltenyi Biotec, Bergisch Gladbach, Germany), 1 ml of V.S was added, and the vial was evacuated and heat-sealed. The vial was then inserted into the LN slush (between -205 and -210°C) for 3 seconds using a VitMaster device (IMT, Nes-Ziona, Israel), then it was placed 1 cm from the LN surface in the LN vapor for 2 minutes, and then transferred to a -80°C freezer for 24 hours or 7-9 days. Alternatively, immediately before warming, the flap-bearing vial was placed in the LN vapor for 30 minutes.

[0215] Flap warming procedure

[0216] The cryovial was inserted into a water bath heated to 38°C until the limb was thawed (about 30 seconds). The vial was opened, and the flap was removed and inserted into SPS-1 supplemented with 1 M sucrose for 2 minutes, and then into physiological saline for another 2 minutes.

[0217] The limb was then manually perfused with the following washing solutions (1 ml of each solution per 0.5 min). Wash solution 1 (WS1): SPS-1 solution of 1 M sucrose and 20% FBS, WS2: 1 / 1 dilution of WSI in SPS-1, WS3: 1 / 1 dilution of WS2 in SPS-1, WS4: 1 / 1 dilution of WS3 in SPS-1. After WS4, the limb was washed with 10-20 ml of physiological saline.

[0218] Post-transplantation treatment

[0219] All transplanted rats wore an Elizabeth collar from post-surgery day 2 (POD2) to POD30 and were treated daily with intraperitoneal injections of Clexane (75 IU / kg) and Baytril (5 mg / kg) until POD7.

[0220] Histological analysis ​

[0221] After sacrifice of the animals, the limbs and flap samples were harvested and paraffin sections were prepared. Sections were stained with hematoxylin and eosin (H&E) and images were acquired by Philips Image Management System 3.3.1 HF1.

[0222] Immunohistochemical staining of nerve regeneration

[0223] The tibial nerve including the proximal and distal anastomosis sites was resected and fixed in 2.5% paraformaldehyde (PFA). The nerve sample was dissected into two segments, the proximal nerve anastomosis (recipient part) and 0.5 cm distal to the nerve anastomosis (donor cryopreserved part). The tissue was then embedded in paraffin blocks and sectioned at a thickness of approximately 5 pm. Sections were stained with anti-choline acetyltransferase antibody (ChAT) as a marker for motor neuron fibers (goat anti-CHAT AB144 Millipore) and anti-neurofilament (NF) antibody as a marker for general neuron fibers (CD-NB300-133 Novus). DAPI was used for non-specific nuclear staining. Both stainings (ChAT and NF) were performed separately in order to assess the number of intact motor fibers compared to the total number of neuron fibers. Stained sections were examined and photographed using a fluorescence OR microscope (Eclipse Ni-U; Nikon, Tokyo, Japan) equipped with Plan Fluor objectives (6x, 20x) connected to a camera (DS-Qi1, NiKon). Blind digital morphometric analysis was performed by a pathologist and cell quantification counts were performed using a digital system. Digital images were collected and analyzed using Image Pro software. Images were assembled using Adobe Photoshop (Adobe Systems, San Jose, CA).

[0224] Example 1

[0225] Long-term survival of cryopreserved vascularized inguinal flaps after transplantation

[0226] The main parameters of the large tissue vitrification protocol were calibrated using a rat model of a vascularized inguinal flap, which mimics the vascularized flaps used in routine plastic surgery practice. Calibration of the cryoprotectant perfusion time showed that long perfusion times (>5 min) resulted in flap death within the first 3 days after transplantation, while perfusion times shorter than 5 min allowed long-term survival of the inguinal flap (data not shown). To further reduce CPA exposure, the inguinal flap was rapidly cooled to subzero temperatures using LN slush. To minimize tissue cracking / breakage from exposure to LN slush, the inguinal flap was cooled in LN slush for increasingly longer time intervals and the damage was assessed. After up to 7 seconds of slush exposure and 2 min of LN vapor freezing, blood reperfusion of the vascularized inguinal flap was observed and there were no signs of bleeding or damage. In contrast, in flaps exposed to LN slush for 10 seconds followed by 2 min of LN vapor, multiple bleeding points were noted, indicating damaged blood vessels Figure 1A ). Further extension of the flap exposure to LN slush to 20 seconds resulted in complete vessel rupture, precluding flap transplantation after creep Figure 1A ). Following the next phase, the flap was perfused, briefly cooled in LN slush (3 seconds), cooled to approximately -60°C in LN vapor for 5 min, and immediately transferred to a -80°C freezer for long-term cooling for 24 hours Figure 1C ) or 9 days Figure 1D ). After cryopreservation, the flaps were warmed, rinsed, retransplanted onto a syngeneic recipient, and followed for survival. It was seen that flaps cryopreserved for 24 hours or 9 days exhibited long-term viability, including hair growth starting at POD 21. Interestingly, all cryopreserved flaps showed a white "milky" skin color, which was sometimes accompanied by white hair growth. The integrity of the different skin layers and subcutaneous fat tissue was further confirmed in histological analysis of flaps cryopreserved for 24 hours Figure 1E a-1Ec) or 9 days Figure 1E d-1Ef) at POD 12. Despite good survival, flaps cryopreserved for 9 days showed increased clinical signs compared to flaps cryopreserved for 24 hours, indicating that, as can be expected, the storage of living tissue at temperatures above the Tg' of the cryoprotectant is not stable.

[0227] To achieve a more reliable long-term cryopreservation technique, the inventors evaluated the effect of cooling the flap to a more stable vitrification solution Tg' which was determined to be approximately -121°C Figure 2A I). Due to the relatively thin nature of the flap and the rapid cooling in LN vapor, we removed the LN slush freezing phase by incubating the flap in LN vapor for 30 min, allowing it to cool at a rate of approximately 80°C / min, freezing it entirely to Tg'Figure 2A II), which is comparable to other successful tissue cryopreservation reports ( Figure 2B Cooling the flap to the Tg' of the vitrification solution was successful, as evidenced by its long-term survival after transplantation. Figure 2C Normal appearance of all skin layers and subcutaneous fat in histological evaluation on day I and 12 postoperative day (POD12). Figure 2C As indicated in II), rapid cooling to Tg' and long-term storage at Tg' can provide an effective alternative to rapid cooling and cryopreservation of LN slurry.

[0228] Example 2

[0229] Long-term survival of cryopreserved vascularized hindlimbs after transplantation

[0230] Next, the novel cryopreservation protocol was applied to larger and more complex hind limb models. Due to the larger volume of the limb compared to the groin flap, a brief (4-second) LN molten plasma cooling phase was included before the more progressive LN vaporization phase. Figure 2A This two-stage cooling strategy allows for rapid cooling to sub-zero temperatures (-4°C), followed by more gradual cooling (7°C / min) to -70°C, without tissue damage or dehiscence, thus contributing to long-term survival (30 days) of the transplanted limb, normal hair growth, and peripheral blood perfusion. Figures 2B-2C As indicated by ) Importantly, in addition to normal skin histology, there were abnormalities during freezing, transplantation (POD 0), and up to 30 days post-transplantation ( Figure 2B II) A large portion of the limb muscles showed normal structure, indicating the feasibility of limb function recovery once nerve reinnervation is applied.

[0231] Example 3

[0232] Long-term limb survival after cryopreservation at Tg'

[0233] As indicated regarding skin flaps, the inventors next investigated the viability of limbs when frozen to a more stable Tg', a temperature that ensures long-term survival when stored in LN vapor or any other cryogenic device capable of maintaining this temperature. The limbs underwent vitrification (…). Figure 3A The limb underwent a brief (4-second) LN molten plasma cooling phase followed by 2 hours in the LN vapor phase, sufficient to lower the limb temperature to Tg'. Following transplantation, the cryopreserved limbs survived until the end of the POD28 experiment, demonstrating hair regeneration. Figure 3B ) and good peripheral blood perfusion ( Figure 3C). As can be seen in FIG. 3D, the majority of the limb that was buried subcutaneously during the transplantation procedure showed a normal well-perfused appearance of the subcutaneous muscle region. Sacrifice biopsies showed normal skin architecture (FIG. 3E). Since muscle survival is critical for the functional recovery of the limb after reinnervation, and muscle is considered the most sensitive limb tissue, muscle survival and recovery after cryopreservation was evaluated and compared to the muscle of the non-cryopreserved transplantation control. Biopsies were taken from different limb sections of 9 limbs and evaluated after H&E staining of the histological sections. As can be seen in FIG. 3F, there was no significant difference between the muscle state of the cryopreserved limbs and the fresh limbs perfused with either saline or CPA before transplantation Figure 6A and B, the muscle state of the cryopreserved limbs at about -80°C or Tg was not significantly different from the muscle state of the fresh limbs perfused with either saline or CPA before transplantation Figures 6A-6B ). All samples showed normal skin, subcutaneous, and fascial aspects. Most muscle fascia showed normal architecture and preserved muscle fibers with peripheral nuclei. Few fascia showed signs of necrosis and regeneration with central nuclei. Some fascia showed edema and perimuscular / intermuscular fasciitis inflammation infiltrates composed of macrophages and lymphocytes. Some fascia showed zonal nerve root atrophy. Finally, some fascia was replaced by adipose tissue. The fresh control group and the cryopreserved limbs showed similar features after transplantation and no significant differences were found, indicating the efficacy of our cryopreservation method in preserving muscle integrity. In agreement with the histological findings, long-term survival of the limbs cryopreserved by this protocol was significantly better than the limbs cryopreserved by DF or cooling to temperatures below the Tg of the vitrification solution Figure 6C

[0234] Example 4

[0235] Transplantation of the cryopreserved limbs demonstrated nerve regeneration and functional sensation

[0236] To demonstrate the ability of the cryopreserved limbs to regain their functionality, a new two-stage transplantation model was developed in which the cryopreserved limb was first transplanted as a fifth limb until it recovered from the transplantation procedure (~POD 12), and then transferred to replace the below-knee amputation Figure 4A Importantly, the limb was still connected to blood vessels when it was transferred from the rat's waist (where it was initially transplanted) to the below-knee position, ruling out the need for vascular anastomosis and the consequent limb ischemia. After repositioning and connection to the recipient knee, the recipient donor tibial nerve was anastomosed near the ankle of the transplanted limb. On day 21 after the limb was repositioned to the below-knee position, the rats were able to walk freely on their transplanted limb, and the limb was viable Figure 4B ​). While ambulation with the cryopreserved limb was primarily supported by the recipient's muscle, this demonstrated the ability of the limb to support the rat's body weight and functional ambulation. Furthermore, use of the limb can be important for improving nerve regeneration, similar to physical therapy of patients after hand transplantation. Importantly, pain sensation was restored in the proximal paw region at day 21 after nerve reconnection (data not shown). Immunohistochemical analysis further demonstrated nerve regeneration, which showed staining for neurofilament and motor neurons at about 0.5 cm distal to the nerve anastomosis ( Figure 4C ).

[0237] DISCUSSION

[0238] Successful cryopreservation of large tissues, limbs, or organs has the potential to revolutionize the field of medical reconstruction and organ transplantation. The present inventors recently reported short-term (3 days) successful survival of rat hindlimbs after cryopreservation by directed freezing or vitrification. The current work demonstrates for the first time that vascularized groin flaps and hindlimbs successfully survive long-term after cryopreservation and transplantation in a rat model. This was achieved using a new cryopreservation protocol that modified three important parameters of the existing vitrification protocol. First, it involved rapid cryoprotectant perfusion at room temperature. Second, freezing was performed in two stages, rapid cooling to subzero temperature (about 360 °C / min), followed by more gradual cooling (about -8 °C / min) to Tg' (-121 °C). Finally, the temperature for long-term storage was at the Tg' of the solution, rather than at lower liquid nitrogen (-180 °C) or low liquid nitrogen vapor freezing conditions (< -150 °C), which exist in LN-based dry shippers containers. At POD 28-32, the viability of the limb was demonstrated by hair growth, peripheral blood perfusion, and normal skin, fat, and muscle histology analysis. Furthermore, using a novel below-knee transplantation model that included nerve reinnervation of the limb, nerve regeneration and restoration of functional sensation of the cryopreserved limb were demonstrated. Overall, these findings provide a solid foundation for future development of cryopreservation protocols for large tissues, limbs, and organs.

[0239] One of the advantages of the cryopreservation of vascularized tissue / limb / organ is that the mass transfer limitations of the cryoprotectant can be addressed by arterial perfusion, allowing for its rapid and efficient distribution compared to non-vascularized tissue. Due to the toxicity of high concentrations of cryoprotectant (40% V / V in the vitrification solution disclosed herein), this work evaluated the effectiveness of cryoprotectant perfusion at room temperature, minimizing the perfusion time. This decision was based on preliminary experiments that showed that only flaps that were rapidly perfused (<5 minutes) could retain their viability after transplantation, most likely due to reduced damage to their blood vessels by the cryoprotectant. To further reduce the toxicity of the cryoprotectant and the freeze damage, a cooling step involving LN slush was introduced, and the limb was provided with a heat conduction of more than 200°C / min, reaching subzero temperatures in 4 seconds.

[0240] To date, the only practical use of vitrification is for the cryopreservation of small tissues and cells (i.e., oocytes, sperm, embryos) that benefit from rapid cooling and heating, have little ice crystal growth, and are cryopreserved without damage using relatively low concentrations of cryoprotectant. In this case, the growth of ice crystals is inhibited by rapid heat transfer in the presence of cryoprotectant. However, this rapid cooling and heating is currently not possible in large tissues. The present invention provides a two-stage cooling protocol, with a short-term LN slush immersion for rapid subzero temperatures, followed by a slower cooling (7°C / min) in LN vapor. In fact, the inventors attempted to rapidly freeze the limb to approximately -80°C by LN slush immersion (~400°C / min) without success compared to the successful two-stage gradual freezing (~7°C / min) protocol disclosed herein, resulting in the limb's death at POD2 due to blood clot formation (data not shown).

[0241] Repeated attempts to store the limb or flap for short periods in the MVE dry shipper at -186°C were unsuccessful, resulting in its rapid death after transplantation. The results presented show that cryopreservation is most successful when the tissue is kept at temperatures close to Tg'. To standardize the proposed two-step vitrification procedure, the inventors developed a new vitrification device that can control the rapid cooling in LN slush for a predetermined period of time and then raise the temperature to Tg' for long-term storage (Figures 6-14). The simplicity of the proposed protocol and device will make immediate cryopreservation of amputations and tissues in hospitals and combat environments a simple method.

[0242] Modern reconstructive surgery relies mainly on autologous parts from donor sites (e.g. vascularized flaps, bone and nerves) where they are harvested without causing disability. Despite its great success, reconstruction is still limited when the injury is large and even more so when it occurs in complex structures such as a limb or face. The recently emerged VCA expanded the range of reconstruction by utilizing composite transplantation from foreign brain-dead donors. However, despite its success, the routine clinical use of VCA was delayed due to the massive immune rejection response of the recipient to the foreign graft, the need for life-long use of immunosuppressive drugs to enable the graft to survive, often leading to severe adverse reactions and sometimes even death. The present inventors suggest that immediate post-trauma cryopreservation of amputated and vascularized tissues will enable autologous reconstruction / re-transplantation to be delayed until the patient is stable or until conditions allow for such a complex surgery (skilled microsurgical team + microsurgical operating room and use of contaminated tissues during trauma). Furthermore, unlike the shortage of solid organs, the smaller demand of limbs can be stored / banked for future use. Such a bank will push the field of VCA by increasing the HLA match between donor and recipient, which will reduce the risk of allograft rejection. It will also eliminate the strict time limit of limited viability of the limb after harvesting (up to 6 hours) and allow for transplantation surgery to be performed in a planned manner. Furthermore, unlike solid organs, limb matching also requires aesthetic matching, which can be improved by a limb bank.

[0243] According to this method, the current study takes another step in the clinical use of cryopreservation to augment complex tissue reconstruction after trauma. Using the cryopreservation protocol disclosed herein, long-term survival of limbs and vascularized flaps was achieved after freezing to about Tg' temperature (< -100°C) and transplantation. This first ever achieved long-term survival of cryopreserved tissue lays the foundation for preservation times of large living tissues beyond 72 hours, expanding the window of lifesaving procedures. Importantly, beneficial reconstruction does not necessarily have to rely on whole limb replantation, but can also be performed with smaller tissue components (vascularized flaps and bone grafts, nerves, skin grafts, etc.) that are often lacking after large scale trauma and preclude morbidity at the donor site. Importantly, in addition to limb survival only, histological analysis was able to demonstrate viable muscle after cryopreservation and long-term transplantation, indicating that functional cryopreserved limb restoration is feasible, which depends on muscle restoration. Although the whole muscle bundle was well preserved, histological analysis at >POD10 also showed regeneration of muscle cells, which can be a response to cryoproduction-induced damage or muscle denervation due to nerve disturbance during amputation. The feasibility of limb functional restoration after cryopreservation was further achieved using our novel two-stage transplantation model, which allowed us to show regeneration of nerves and limb sensation after knee-down retransplantation and nerve anastomosis. Taken together, the presented findings demonstrate the great potential of our cryopreservation method to improve the feasibility of functional limb retransplantation or cryobanking of limb allotransplantation after trauma.

[0244] While the application has been described in connection with specific embodiments thereof, it will be readily appreciated by those skilled in the art that numerous alternatives, modifications and variations can be made thereto without departing from the spirit and scope of the application as set forth in the appended claims.

[0245] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present application. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

1. A method for cryopreserving a limb, preparing a limb for transplantation to a subject in need thereof, or both, comprising the steps of: a. contacting a biological sample comprising the limb with a vitrification solution (VS) at a volume / volume (v / v) ratio ranging from 50: 1 to 1: 1; b. subjecting the biological sample comprising the limb in step (a) to a temperature ranging from -210°C to -197°C for a time period of 3-10 seconds in a slush of a freezing liquid or under freezing conditions; and c. subjecting the biological sample comprising the limb in step (b) to a temperature equal to or higher than the Tg’ of the VS and ranging from -140°C to -100°C for a time period of at least 3 minutes, either: (a) in a vapor of a freezing liquid; or (b) under cooling conditions providing a temperature ranging from -85°C to -75°C, followed by a vapor of a freezing liquid; thereby cryopreserving a limb, preparing a limb for transplantation to a subject in need thereof, or both, wherein the cryopreserved limb is characterized by long-term survival after syngeneic retransplantation.

2. The method of claim 1, further comprising preserving the biological sample after the second subjecting, comprising further cooling the biological sample to a temperature lower than the Tg or between -197°C to -150°C at a rate ranging from -1°C / min to -10°C / min.

3. The method of claim 1, wherein the biological sample comprising the limb obtained after step (a) is submerged in, covered by, or both, the VS.

4. The method of claim 1, wherein the contacting is under vacuum conditions.

5. The method of claim 1, wherein the contacting is in an elastic bag.

6. The method of claim 1, wherein the biological sample comprising the limb obtained after step (b) comprises an outer surface in contact with the VS, wherein the outer surface is characterized by having a temperature ranging from -20°C to 0°C.

7. The method of claim 1, wherein the first subjecting is frozen at a rate of -330°C / min to -500°C / min.

8. The method of claim 1, wherein the slush of a freezing liquid or freezing conditions are at a temperature of -220°C to -150°C.

9. The method of claim 1, wherein the second subjecting is frozen at a rate of -3°C / min to -10°C / min.

10. The method of claim 1, wherein the cooling conditions providing a temperature ranging from -85°C to -75°C comprise a cooling liquid having a temperature ranging from -85°C to -75°C.

11. The method of claim 1, wherein the time period of at least 3 minutes in step (c) is at least 30 minutes.

12. The method of claim 1, further comprising a step prior to step (a), comprising perfusing the biological sample comprising the limb with the VS.

13. The method of claim 12, wherein the perfusion is at room temperature for a time period of up to 5 minutes.

14. The method of claim 1, wherein the VS comprises at least one of: a permeating cryoprotectant, a non-permeating cryoprotectant, or a combination thereof, and optionally further comprises: macromolecules, antioxidants, media, or any combination thereof.

15. The method of claim 1, wherein the VS comprises: 15-25% (w / v) ethylene glycol, 15-25% (v / v) dimethyl sulfoxide, 15-25 (v / v) fetal bovine serum, and 0.3-0.7 M trehalose, all in a Wisconsin static cryopreservation solution.

16. The method of claim 1, further comprising step (d) comprising preserving the cryopreserved biological sample comprising the limb for a time period of at least 24 hours.

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