Patent Publication Number: US-2022211923-A1

Title: Cryoprotective Compositions, Surgical Kits, and Methods for Protection of a Surgical Site During Cryosurgery

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     The present application is a continuation-in-part of U.S. patent application Ser. No. 17/482,898, filed Sep. 23, 2021, which claims the benefit of U.S. Provisional Patent Application No. 63/082,790, filed Sep. 24, 2020, the disclosures of each of which are hereby incorporated by reference in their entireties. 
    
    
     BACKGROUND 
     Technical Field 
     The present disclosure relates to cryoprotective compositions comprising cryoprotectant agents (CPAs) of low toxicity and sufficient viscosity to remain within body spaces, for example surrounding organs and nerve structures, for sufficient duration for performing certain treatments or cryosurgical procedures. Also included are surgical kits for making cryoprotective compositions for use during surgical procedures and methods for protecting surgical sites during cryosurgery using the cryoprotective compositions. 
     Background 
     Prostate cancer is the second-most common cancer among men (after skin cancer) and the second leading cause of cancer death for men in the United States and most Western European countries. Prostate cancer can often be treated successfully. However, many of the current treatment options and procedures have associated risks and common complications that affect quality of life of survivors following treatment. Radical therapy (e.g., radical prostatectomy and radiotherapy) has been shown to have excellent outcomes in terms of prostate cancer specific survival for localized prostate cancer. However, such procedures can have undesirable side effects, such as urinary incontinence or erectile dysfunction. 
     Cryoablation has been shown to be an effective therapy for all stages of prostate cancer, serving as both a primary treatment option and a salvage treatment option. Minimally invasive cryoablation therapy has similar 5 year and 10 year outcomes for low and intermediate risk prostate cancer (D&#39;Amico risk classification). Minimally invasive cryoablation therapy also has encouraging outcomes for high risk prostate cancer compared to other therapies, including radical prostatectomy, radiation (conformal and external beam), and brachytherapy. In recent decades, technological advancements for prostate cryosurgery have been focused on improving the efficacy of targeted freezing and protection of the adjacent extra-prostatic tissues. 
     Despite such advances, a major complication of cryotherapy continues to be the injury to adjacent normal tissues, which can cause erectile dysfunction. In cryotherapy treatment of prostate cancer, for example, a significant side effect is injury to the neurovascular bundle, which contributes to erectile dysfunction, because these nerves can be directly exposed to lethal levels of cold temperature during cryotherapy. This detrimental side effect is extremely common because injury to these nerves occurs in 80% to 90% of performed procedures in the case of whole gland treatment. In focal prostate cryotherapy, the incidence is lower with 40%. This high incident rate of injury to these nerves is due to the close proximity of the neurovascular bundle to the prostate. While a significant number of patients may experience full or partial recovery within about twelve months following therapy, some patients are left to seek alternative therapies aimed at restoration of potency. 
     Efforts have been made to spare the neurovascular bundle and increase quality of life for patients by, for example, active heating of tissues in proximity to the treatment area and focal cryotherapy under image guidance. However, such approaches may suffer from a lack of precision in positioning and/or an increase in the possibility of cancer reoccurrence. 
     Active heating by placement of a heated needle to the surgical site creates a “divot” of warming to counteract effects of tissue freezing. However, challenges of this procedure relating to probe placement and lack of precision in temperature control reduce reliability of this procedure, which has not been widely accepted. 
     For focal therapy, only a quadrant or section of the prostate that contains the cancer is treated. Focal cryotherapy was developed to minimize injuries to adjacent structures, such as the urethral sphincter, rectum, and nerve tissues, thereby reducing the risk of urinary incontinence, rectal injury, and to help preserve erectile function. However, focal cryotherapy has succeeded only in exchange for an increase in the possibility of cancer reoccurrence. Moreover, focal cryotherapy is reserved for a select group of patients whose probability of cancer reoccurrence in another section of the prostate is minimal. 
     In view of these challenges, there is a need in the art for cryoprotective composition(s) and surgical method(s) that provide sufficient protection for the entire periprostatic space, while allowing for treatment of the prostate without risks and complications often associated with cryotherapies. Also, cryosurgical method(s) are needed to reduce instances of cancer reoccurrence, which can occur from focal therapy. 
     More generally, there is a need in the art for cryoprotective composition(s) for use within the body and/or externally to protect body tissue from the effects of cold temperatures. Also, there is a need in the art for cryoprotective composition(s) for use with plants to protect the plants from the effects of cold temperatures. 
     SUMMARY 
     According to an example of the disclosure, a surgical kit for providing a cryoprotective composition configured to be applied during cryotreatment of a patient includes: at least one first container containing a predetermined amount or volume of at least one biodegradable and/or bioerodible fluid agent; and at least one second container containing a predetermined amount or volume of at least one non-toxic cryoprotectant agent. The predetermined amount or volume of the of at least one biodegradable and/or bioerodible fluid agent and the predetermined amount or volume of at least one non-toxic cryoprotectant agent are configured to be mixed together to form the cryoprotective composition in which a therapeutically effective amount of the cryoprotective composition deposited in a body space of the patient in proximity to the cryotreatment remains within at least a portion of the body space for a duration of the cryotreatment and at least a portion of a body tissue proximate to the body space is viable after the cryotreatment. 
     According to another example of the disclosure, a method of making a cryroprotective composition for use during a surgical procedure using the previously described kit includes: transferring the predetermined amount or volume of at least one biodegradable and/or bioerodible fluid agent from the at least one first container to the at least one second container; and mixing the predetermined amount or volume of at least one biodegradable and/or bioerodible fluid agent with the predetermined volume of the at least one non-toxic cryoprotectant agent in the at least one second container to form the cryoprotective composition. 
     According to another example of the disclosure, a kit for making a cryoprotective composition includes: a first container containing about 10 mL to about 50 mL of a solution comprising from about 1.0 weight % to about 3.0 weight % hyaluronic acid in a buffer solution; a second container containing about 10 mL to about 50 mL of 5 weight % to about 15 weight % of Dimethyl sulfoxide (DMSO) in water; and a third container containing from about 2000 mg to 5000 mg trehalose powder. 
     According to another example of the disclosure, a cryoprotective composition for use in cryosurgery includes: at least one biodegradable and/or bioerodible fluid agent comprising a gel of from about 1 weight percent to about 10 weight percent of the composition; a sugar cryoprotectant agent comprising from about 5 weight percent to about 10 weight percent of the composition; Dimethyl sulfoxide (DMSO) comprising from about 1 weight percent to about 5 weight percent of the cryoprotective composition; and a saline buffer solution. The cryoprotective composition does not cause substantial nerve damage when positioned in a body space for a duration of a cryosurgical procedure. 
     Non-limiting examples of the present invention will now be described in the following numbered clauses: 
     Clause 1: A surgical kit for providing a cryoprotective composition configured to be applied during cryotreatment of a patient, the kit comprising: at least one first container containing a predetermined amount or volume of at least one biodegradable and/or bioerodible fluid agent; and at least one second container containing a predetermined amount or volume of at least one non-toxic cryoprotectant agent, wherein the predetermined amount or volume of the of at least one biodegradable and/or bioerodible fluid agent and the predetermined amount or volume of at least one non-toxic cryoprotectant agent are configured to be mixed together to form the cryoprotective composition in which a therapeutically effective amount of the cryoprotective composition deposited in a body space of the patient in proximity to the cryotreatment remains within at least a portion of the body space for a duration of the cryotreatment and at least a portion of a body tissue proximate to the body space is viable after the cryotreatment. 
     Clause 2: The kit of clause 1, wherein the cryotreatment of the patient comprises a cryosurgical procedure. 
     Clause 3: The kit of clause 1 or clause 2, wherein the body space comprises a periprostatic space. 
     Clause 4: The kit of any of clauses 1-3, wherein the predetermined amount or volume of the at least one non-toxic cryoprotectant agent has an osmolality of about 200 mOsm/kg to about 400 mOsm/kg and a pH of about 6 to 8. 
     Clause 5: The kit of any of clauses 1-4, wherein the at least one first container comprises a prefilled syringe containing the predetermined amount or volume of at least one biodegradable and/or bioerodible fluid agent. 
     Clause 6: The kit of clause 5, wherein the at least one first container contains from about 10 mL to about 50 mL of a solution comprising from about 1.0 weight % to about 3.0 weight % hyaluronic acid or from about 3.0 weight % to about 5.0 weight % hydroxyethyl cellulose. 
     Clause 7: The kit of clause 5 or clause 6, wherein the solution contained in the first container further comprises phosphate-based saline (PBS) solution. 
     Clause 8: The kit of any of clauses 1-7, wherein the at least one second container comprises a sealed vial containing the at least one non-toxic cryoprotectant agent. 
     Clause 9: The kit of any of clauses 1-8, wherein the at least one non-toxic cryoprotectant agent comprises from about 5 weight percent to about 15 weight percent of the cryoprotectant agent in solution, and wherein the predetermined amount or volume of the at least one non-toxic cryoprotectant agent in the at least one second container is from 10 mL to 50 mL of the solution of the at least one non-toxic cryoprotectant agent. 
     Clause 10: The kit of any of clauses 1-9, wherein the at least one non-toxic cryoprotectant agent comprises about 5 weight % to about 15 weight % of Dimethyl sulfoxide (DMSO) in water contained in the at least one second container. 
     Clause 11: The kit of any of clauses 1-10, wherein the at least one non-toxic cryoprotectant agent comprises at least one of alcohol(s), saccharide(s), polysaccharide(s), polymer(s), sulfoxide(s), amide(s), or amine(s) contained in the second container. 
     Clause 12: The kit of any of clauses 1-11, wherein the at least one second container comprises a first sealed vial containing Dimethyl sulfoxide (DMSO) and a second sealed vial containing at least one of alcohol(s), saccharide(s), or polysaccharide(s). 
     Clause 13: The kit of clause 12, wherein the at least one second vial contains a lyophilized or freeze-dried powder comprising the alcohol(s), saccharide(s), polysaccharide(s), polymer(s), sulfoxide(s), amide(s), or amine(s). 
     Clause 14: The kit of any of clauses 1-13, wherein the first at least one first container comprises a prefilled syringe containing the predetermined amount or volume of the at least one biodegradable and/or bioerodible fluid agent, the kit further comprising a surgical injection syringe to be used for delivery of the cryoprotective composition to the body space of the patient. 
     Clause 15: The kit of clause 14, further comprising a plurality of pen needles configured to be mounted to the prefilled syringe for transferring the at least one biodegradable and/or bioerodible fluid agent from the prefilled syringe to an interior of the at least one second container and/or for transferring the prepared cryoprotective composition from the interior of the at least one second container to an interior of the injection syringe. 
     Clause 16: The kit of clause 15, wherein the plurality of pen needles comprise about an 18 gauge needle to about a 22 gauge needle for mixing, aspiration, and/or therapeutic use. 
     Clause 17: The kit of any of clauses 1-16, wherein the at least one first container comprises a prefilled syringe containing at least one of hyaluronic acid or hydroxyethyl cellulose, and the at least one second container comprises a first sealed vial containing Dimethyl sulfoxide (DMSO) and a second sealed vial containing trehalose, the kit further comprising a surgical injection syringe to be used for delivery of the cryoprotective composition to the body space of the patient, and a plurality of pen needles configured to be mounted to the prefilled syringes for transferring the at least one biodegradable and/or bioerodible fluid between the prefilled syringe and the sealed vials to produce the cryoprotective composition and/or to the injection syringe for delivering the cryoprotective composition to the body space of the patient. 
     Clause 18: The kit of any of clauses 1-17, wherein the cryoprotective composition formed from the predetermined volume of at least one biodegradable and/or bioerodible fluid agent and the predetermined volume of at least one non-toxic cryoprotectant agent has a final fluid volume of about 25 mL to about 75 mL. 
     Clause 19: A method of making a cryroprotective composition for use during a surgical procedure using the kit of any of clauses 1-18, the method comprising: transferring the predetermined amount or volume of at least one biodegradable and/or bioerodible fluid agent from the at least one first container to the at least one second container; and mixing the predetermined amount or volume of at least one biodegradable and/or bioerodible fluid agent with the predetermined volume of the at least one non-toxic cryoprotectant agent in the at least one second container to form the cryoprotective composition. 
     Clause 20: The method of clause 19, further comprising drawing the cryoprotective composition from an interior of the at least one second container to at least one surgical injection syringe of the kit. 
     Clause 21: The method of clause 20, further comprising protecting a surgical site during prostate cryosurgery by injecting the therapeutically effective amount of the cryoprotective composition from the syringe into the body space of the patient, wherein the body space is a periprostatic space of a patient. 
     Clause 22: A kit for making a cryoprotective composition, comprising: a first container containing about 10 mL to about 50 mL of a solution comprising from about 1.0 weight % to about 3.0 weight % hyaluronic acid in a buffer solution; a second container containing about 10 mL to about 50 mL of 5 weight % to about 15 weight % of Dimethyl sulfoxide (DMSO) in water; and a third container containing from about 2000 mg to 5000 mg trehalose powder. 
     Clause 23: The kit of clause 22, wherein the first container comprises a prefilled syringe containing the hyaluronic acid in the buffer solution and the second and third containers comprise sealed vials. 
     Clause 24: The kit of clause 22 or clause 23, wherein the buffer solution comprises PBS solution. 
     Clause 25: A cryoprotective composition for use in cryosurgery comprising: at least one biodegradable and/or bioerodible fluid agent comprising a gel of from about 1 weight percent to about 10 weight percent of the composition; a sugar cryoprotectant agent comprising from about 5 weight percent to about 10 weight percent of the composition; Dimethyl sulfoxide (DMSO) comprising from about 1 weight percent to about 5 weight percent of the cryoprotective composition; and a saline buffer solution, wherein the cryoprotective composition does not cause substantial nerve damage when positioned in a body space for a duration of a cryosurgical procedure. 
     Clause 26: The cryoprotective composition of clause 25, wherein the at least one biodegradable and/or bioerodible fluid agent comprises hydroxyethyl cellulose or hyaluronic acid. 
     Clause 27: The cryoprotective composition of clause 26, wherein the at least one biodegradable and/or bioerodible fluid agent comprises less than about 5 weight percent hydroxyethyl cellulose. 
     Clause 28: The cryoprotective composition of clause 26 or clause 27, wherein the at least one biodegradable and/or bioerodible fluid agent comprises less than about 2 weight percent hyaluronic acid. 
     Clause 29: The cryoprotective composition of any of clauses 25-28, wherein the sugar cryoprotectant agent comprises trehalose. 
     Clause 30: The cryoprotective composition of clause 29, wherein the trehalose comprises from 7 weight percent to 8 weight percent of the composition. 
     Clause 31: The cryoprotective composition of any of clauses 25-30, wherein the saline buffer solution comprises PBS solution. 
     Clause 32: The cryoprotective composition of clause 31, wherein the composition comprises up to about 95 weight percent PBS solution. 
     Clause 33: The cryoprotective composition of any of clauses 25-32, wherein: the at least one biodegradable and/or bioerodible fluid agent comprises less than about 2 weight percent hyaluronic acid, the sugar cryoprotectant agent comprises about 7 weight percent to about 8 weight percent trehalose, and the saline buffer solution comprises PBS solution. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other features and characteristics of the present disclosure, as well as the methods of operation, use, and functions of the related elements of structures and the combination of parts and economies of manufacture, will become more apparent upon consideration of the following description and the appended claims with reference to the accompanying drawings, all of which form a part of this specification, wherein like reference numerals designate corresponding parts in the various figures. It is to be expressly understood, however, that the drawings are for the purpose of illustration and description only and are not intended as a definition of the limit of the invention. 
       Further features and other examples and advantages will become apparent from the following detailed description made with reference to the drawings in which: 
         FIG. 1A  is a schematic drawing showing a surgical kit for making a cryoprotective composition for use during a cryosurgical procedure, according to an aspect of the disclosure; 
         FIG. 1B  is a flow chart showing steps for making a cryoprotective composition using the surgical kit of  FIG. 1A , according to an aspect of the present disclosure; 
         FIGS. 2A and 2B  are schematic drawings of a patient showing an injection site on the perineum for injecting cryoprotective composition(s) of the present disclosure to the periprostatic space; 
         FIGS. 2C and 2D  are schematic drawings of anatomical structures of the periprostatic space showing insertion of the needle for injecting the cryoprotective composition(s) of the present disclosure; 
         FIGS. 3A and 3B  are schematic drawings of the periprostatic space showing an amount of the cryoprotective composition(s) dispersed between the prostate and rectum near the base of the prostate; 
         FIGS. 4A and 4B  are additional schematic drawings of the periprostatic space showing an additional amount of the cryoprotective composition(s) applied laterally about the prostate creating additional space between the prostate and rectum; 
         FIG. 5  is a flow chart showing a surgical method using the cryoprotective composition(s) of the present disclosure; 
         FIGS. 6A and 6B  are examples of 3D prostate models created from image data of prostates with prostate cancer, according to an example of the present disclosure; 
         FIGS. 7A-7D  are temperature profiles for cross sections of a prostate after cryoprobes are sequentially turned off, which can be used to create a model of prostate temperature during cryosurgery, according to an example of the present disclosure; 
         FIG. 7E  is a graph showing timing of turning cryoprobes on and off for cryoprobes used during a cryosurgical procedure, according to an example of the present disclosure; 
         FIG. 8A  is a graph showing a temperature of the neurovascular bundle determined from a computer-generated model for a cryosurgical procedure performed for the prostate; 
         FIG. 8B  is a temperature profile applied by a cryostage device during ex vivo testing of nerves used for the examples of the present disclosure; 
         FIG. 9  is a photograph of a nerve and stimulation and measurement leads for testing nerves during examples of the present disclosure; 
         FIG. 10  is a photograph showing a cryostage device for freezing nerves used during examples of the present disclosure; 
         FIG. 11  is a graph showing action potentials measured during testing of nerves; 
         FIG. 12  is a schematic drawing showing an experimental set-up for testing nerves including a frozen section used for examples of the present disclosure; 
         FIGS. 13A-13F  are graphs showing experimental results for testing of a cryoprotectant agent on phrenic nerves of a swine; 
         FIGS. 14A-14F  are graphs showing experimental results for testing of a cryoprotectant agent on sciatic nerves of a rat; 
         FIGS. 15A-15C  are graphs showing additional experimental results for testing of a cryoprotectant agent on phrenic nerves of a swine; 
         FIGS. 16A-16C  are graphs showing additional experimental results for testing of a cryoprotectant agent on sciatic nerves of a rat; 
         FIGS. 17A-17D  are graphs showing experimental results for toxicity testing of hydroxyethyl cellulose; 
         FIG. 18  is a graph showing experimental results for toxicity and freezing testing of a cryoprotective composition including cellulose and a cryoprotectant agent, according to an example of the present disclosure; 
         FIGS. 19A and 19B  are graphs showing experimental results for toxicity testing of sodium hyaluronate; 
         FIG. 19C  is a graph showing experimental results for toxicity testing of a composition of sodium hyaluronate, DMSO, and trehalose; and 
         FIGS. 20A-20F  are graphs showing experimental results for testing of a composition comprising PBS solution and a cryoprotectant agent on phrenic nerves of a swine. 
     
    
    
     DETAILED DESCRIPTION 
     As used herein, the singular form of “a”, “an”, and “the” include plural referents unless the context clearly states otherwise. 
     As used herein, the terms “right”, “left”, “top”, and derivatives thereof shall relate to the invention as it is oriented in the drawing figures. For a device, system, or surgical tool, the term “proximal” refers to a portion of the device, system, or tool nearest to the access site through which the device, system, or tool enters the body. The term “proximal” can also refer to the portion of the tool being held or manipulated by a user. For example, a handle of a tool may be located at the “proximal end” of the tool. The term “distal” refers to the opposite end of a device, system, or tool from the proximal end and, for example, to the portion of the device or system that is inserted farthest into the patient&#39;s body (e.g., farthest into the periprostatic space). The term “distal” can also refer to an end or portion of a tool farthest from the portion of the tool held or manipulated by the user. However, it is to be understood that the invention can assume various alternative orientations and, accordingly, such terms are not to be considered as limiting. Also, it is to be understood that the invention can assume various alternative variations and stage sequences, except where expressly specified to the contrary. It is also to be understood that the specific devices and processes illustrated in the attached drawings, and described in the following specification, are examples. Hence, specific dimensions and other physical characteristics related to the embodiments disclosed herein are not to be considered as limiting. 
     For the purposes of this specification, unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, dimensions, physical characteristics, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. 
     Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Numerical values may inherently contain certain errors resulting from a standard deviation found in their respective testing measurements. 
     Also, it should be understood that any numerical range recited herein is intended to include all sub-ranges subsumed therein. For example, a range of “1 to 10” is intended to include any and all sub-ranges between and including the recited minimum value of 1 and the recited maximum value of 10, that is, all subranges beginning with a minimum value equal to or greater than 1 and ending with a maximum value equal to or less than 10, and all subranges in between, e.g., 1 to 6.3, or 5.5 to 10, or 2.7 to 6.1. 
     The present disclosure is directed to a cryoprotective composition(s) that protects internal or external body tissues of patients or tissues and surfaces of plants from exposure to cold temperatures, which may result in damage to the body tissue. As used herein, “body tissue” of the patient refers to arrangements of cells grouped together by structure and/or function. Non-limiting examples of body tissue may include muscle tissues, epithelial tissues, connective tissues, and/or nervous tissues. Body tissue can also include internal organs, such as interstitial tissues of the prostate, rectum, liver, lung, heart, kidney, brain, thyroid, or any other organ that may be exposed to cold temperatures during, for example, a cryosurgical treatment or procedure. Body tissue can also include external tissues, such as skin. Body tissue can also include tissues of plants including, for example, parenchyma, collenchyma, and sclerenchyma tissues of a plant, as well as vascular tissues and dermal tissues of plants. 
     Protecting body tissue means that the cryoprotective composition(s) preserves the body tissue so that it can function normally or substantially normally following exposure to a damaging condition. For example, the cryoprotective composition may preserve nerve cells so that after freezing and thawing, the nerve cells are able to generate an action potential and/or an action potential is able to translate along a length of the nerve cells, as could occur prior to the freezing and thawing. 
     As used herein, the “patient” can be any species of the human or animal kingdom having body tissues, such as nerve cells, that can be damaged by exposure to cold or freezing temperatures. Non-limiting examples of patients include mammal(s), such as human(s) and/or non-mammalian animal(s). Non-limiting examples of mammal(s) include primate(s) and/or non-primate(s). Primate(s) include human(s) and non-human primate(s), including but not limited to male(s), female(s), adult(s), and children. Non-limiting examples of non-human primate(s) include monkey(s) and/or ape(s), for example, chimpanzee(s). Non-limiting examples of non-primate(s) include cattle (such as cow(s), bull(s) and/or calves), pig(s), camel(s), llama(s), alpaca(s), horse(s), donkey(s), goat(s), rabbit(s), sheep, hamster(s), guinea pig(s), cat(s), dog(s), rat(s), mice, lion(s), whale(s), and/or dolphin(s). Non-limiting examples of non-mammalian animal(s) include bird(s) (e.g., duck(s) or geese), reptile(s) (e.g., lizard(s), snake(s), or alligator(s)), amphibian(s) (e.g., frog(s)), and/or fish. In some examples, the animals can be zoological animals, human pets, and/or wild animals. 
     As used herein, “cold temperature” for patients means that a temperature of a material or object contacting the body tissue that should be preserved or protected is below about 0° C., or about −10° C., or about −30° C. Non-limiting examples of the objects or materials that may contact the body tissue can include cryoprobes used for surgical procedures, other frozen and/or super-cooled medical devices, or fluids capable of being cooled to below 0° C. injected to the patient for certain cryotherapy treatments and procedures. The “material” can also be air if, for example, the patient&#39;s skin is exposed to cold temperatures. 
     For plants, “cold temperature” means that the temperature of a material contacting exposed surfaces or portions of the plant is below about 0° C., or about −5° C., or about −15° C. The material contacting the body tissue can be in gaseous, liquid, and/or solid form, or any combination thereof. Non-limiting examples of materials that can contact exposed surfaces of plants includes air (when air temperature is at or below freezing) or precipitation (e.g., ice or snow). For plants, non-limiting examples of surfaces or portions of a plant that can be protected by the cryoprotective composition can include ground, vascular, and/or dermal tissue of the plant. Non-limiting examples of ground tissue include parenchyma, collenchyma, and sclerenchyma. Non-limiting examples of vascular tissue include tracheids and vessel elements, sieve tubes, and companion cells. Non-limiting examples of dermal tissue include epidermal cell, stomata and trichromes. Each plant organ (roots, stems, leaves) contains all three types of tissue. Dermal tissue covers and protects the plant, and controls gas exchange and water absorption (in the roots). 
     During treatments and/or surgical procedures of patients, cryoprotectant agents can be used to prevent freezing or damage to body tissues during or after the treatment and/or surgery. As discussed previously, there are many treatments and surgical procedures that involve treatment with cold temperatures to prevent pain, lessen swelling, or destroy or treat defective or injured tissue, such as cancer cells, including, for example, cryosurgical treatment for prostate cancer, liver cancer, cervical cancer, thyroid cancer, and other conditions. It may be desirable to protect adjacent tissue which is not the target of such treatment or surgery from the effects of the cold temperatures or freezing. 
     The cryoprotective composition(s) desirably remain in place at an injection or deposition site for a duration of a treatment or surgical procedure. After the completion of the procedure, the cryoprotective composition(s) can begin to degrade or erode and can be absorbed by or passed through the body. Further, the cryoprotective composition(s) desirably has low toxicity and does not appreciably damage nerves or body tissue in proximity to the injection or deposition site for the cryoprotective composition(s). 
     In some examples, the cryoprotective composition(s) of the present disclosure are intended to preserve body tissues and nerves from damage during prostate cryotherapy treatment. While the inventive cryoprotective composition(s) will be discussed first with reference to prostate cryotherapy treatment, one of ordinary skill in the art will appreciate that these cryoprotective composition(s) can be useful in any treatment, surgical, or cryosurgical applications to inhibit damage to adjacent tissues and organs, as desired. 
     In other examples, the cryoprotective composition(s) of the present disclosure can be used to diminish side effects in focal cryotherapy with regards to erectile function. The cryoprotective composition(s) of the present disclosure can also be used to increase surgical safety with regard to damage to surrounding organs, namely the intestines. The cryoprotective composition(s) of the present disclosure can also be used for other cryo-applications in the body including, for example, metastases of the liver, lung, or skeleton. It is believed that the cryoprotective composition(s) of the present disclosure may become safer and more efficient allowing for protection of surrounding structures, such as vessels and nerves. 
     In other examples, the cryoprotective composition(s) of the present disclosure can be used for any surgery involving neuronal tissue where tissue cooling is involved. In such cases, it is believed that a cryoprotective composition(s) comprising a gel may protect non-targeted areas during, for example, brain surgery or spinal surgery, from negative effects caused by cooling and/or exposure to cold temperatures. 
     In some other examples, the cryoprotective composition(s) of the present disclosure can be used to protect body tissues during other types of cryo-treatments as are presently performed or as may be identified in the future. For example, it may be possible that cryotherapy could be used as an alternative treatment for presently-performed neuromodulation therapy (e.g., neuromodulation for treatment of hyperactive bladder, intestinal disorders, and chronic pain). If cryotherapy is used to treat such conditions, then the cryoprotective composition(s) of the present disclosure may be used to protect body tissues surrounding treatment sites during such procedures. 
     In some examples, the cryoprotective composition(s) can be used in topical protective composition(s) and skin creams or locations for protecting external portions of the body tissue, such as the skin, from exposure to cold temperatures. 
     In some examples, the cryoprotective composition(s) can be used to protect surfaces, external tissues, and/or internal tissues of plants from exposure to cold temperatures, which might result in damage to the tissue. For example, the cryoprotective composition(s) of the present disclosure can protect surfaces of plants from freezing and/or exposure to freezing temperatures, which would otherwise damage portions of the plant. The cryoprotective composition(s) of the present disclosure can also be used for protecting other internal and/or external tissue and organs of plants including, for example, ground, vascular, and/or dermal tissues of plants. Non-limiting examples of ground tissues include parenchyma, collenchyma and sclerenchyma. Non-limiting examples of vascular tissues include tracheids and vessel elements, sieve tubes and companion cells. Non-limiting examples of dermal tissues include epidermal cell, stomata and trichomes. Each plant organ (roots, stems, leaves) contains all three types of tissue: Dermal tissue covers and protects the plant, and controls gas exchange and water absorption (in the roots). 
     Cryosurgery Examples 
     While current prostate cryotherapy procedures have low morbidity for urinary incontinence with 0-1%, as previously discussed, complications related to erectile dysfunction occur more frequently (about 40% erectile dysfunction for focal therapy and 70% for whole gland therapy). It is believed that when using the cryoprotective composition(s) of the present disclosure, cryotherapy can become a viable treatment option for patients, while reducing the risk of complications, such as erectile dysfunction and other conditions caused by damage to adjacent tissue and organs. 
     In order to provide such protection, as described in further detail herein, the cryoprotective composition(s) can be applied during prostate cryotherapy to at least partially, or fully, surround the prostate and/or neurovascular nerve bundle. It is believed that applying the cryoprotective composition in this manner will also address other common complications associated with prostate cryotherapy procedures comprising, but not limited to, rectal injury due to the close proximity of the anterior rectum to the posterior prostate. It is believed that risks of rectal injury during cryotherapy are particularly high when the prostate has been previously irradiated, as radiation treatment causes scarring that reduces the space between the rectum and prostate. The cryoprotective composition(s) may also be used to protect other non-targeted anatomical structures during prostate cryotherapy procedures comprising, for example, the urethra, urogenital diaphragm, external urinary sphincter, and/or bladder neck smooth muscle sphincter, which may be susceptible to damage from exposure to cold and/or freezing. 
     In some examples, the cryoprotective composition(s) creates space around the prostate, which physically separates sensitive structures, such as the neurovascular bundle and the rectum, from the prostate to reduce direct and indirect effects of the cold (e.g., cryotherapy agents) on such sensitive structures. In addition to creating physical separation, the cryoprotective composition(s) are also configured to interfere with various electrochemical and/or chemical mechanisms that cause cryoinjury. For example, the injected cryoprotective composition(s) are believed to preserve an ability of the neurovascular bundle to generate action potentials, which may be inhibited when the neurovascular bundle is exposed to cold (e.g., cryotherapy agents). Further, use of the cryoprotective composition(s) of the present disclosure can confine cryoinjury to selected target zones (e.g., the entire prostate and/or select quadrants of the prostate), which protects surrounding structures from thermal damage. 
     In some examples, it is believed that the cryoprotective composition(s) can be used in clinical practice with image guidance by commonly used ultrasound, such as transrectal ultrasound, and delivered by a conventional needle to surgical sites that would otherwise be subjected to damage by exposure to cold during cryotherapy. Image guidance for delivery of the cryoprotective composition(s) can also be provided by other known medical imaging systems, such as MRI or CT. In some examples, the cryoprotective composition is delivered during a pre-treatment phase of a cryosurgical procedure. As such, it is believed that no changes may need to be made to existing cryosurgical protocols. For example, for procedures using the cryoprotective composition(s) of the present disclosure, there is no need for complicated control systems (e.g., electronic control systems for active heating devices), modifications of existing surgical equipment, or introduction of new devices, as is often needed for active heating devices and newly developed focal treatment methods. Further, since the cryoprotective composition(s) is not expected to come into contact with the target tumor, there is no risk of compromising efficacy of tumor ablation, which could increase the possibility for cancer recurrence. 
     The cryoprotective composition(s) of the present disclosure may also have numerous uses beyond use in cryotherapy of the prostate. For example, in many oncological fields, there may be an interest in treating certain tumor(s) and/or cancers by performing in situ (e.g., a tissue preserving approach) tumor treatment instead of organ removal for solid tumors or tumor metastasis. For example, the cryoprotective composition(s) may be used in surgical procedures for treatment of one or more of tumor(s) and/or lesions around an eye or eye orbit, esophageal tumor(s) and/or cancer, pancreatic tumor(s) and/or cancer, liver tumor(s) and/or cancer, brain tumor(s) and/or cancer, kidney tumor(s) and/or cancer, lung tumor(s) and/or cancer, thyroid tumor(s) and/or cancer, or breast tumor(s) and/or cancer. 
     For example, during treatment of tumors and lesions around the eye and the eye orbit by cryosurgery, vital structures such as the retina, the lacrimal duct, and the optic nerve can become damaged. The cryoprotective composition(s) can also be applied in proximity to such vital structures to avoid tissue damage and complications. 
     Cryotherapy is also used to treat Barrett&#39;s esophagus and esophageal tumor(s) and/or cancer. In such procedures, there is a potential for perforation of the esophagus, which can be a serious and life-threatening complication. The cryoprotective composition(s) can be applied adjacent to the esophagus to protect the esophagus and, in particular, to prevent perforation of esophageal structures or damage to adjacent neural tissue. 
     The cryoprotective composition(s) of the present disclosure can also be used for treatment of pancreatitis and pancreatic tumor(s) and/or cancer, to avoid serious complications caused by injuries to the bile duct during cryosurgery, and for treatment of liver tumor(s) and/or cancer. For example, during cryosurgery on the liver, where tumors are located close to large blood vessels, it is important to freeze the tumors as close to the blood vessels as possible without damaging the blood vessels themselves. The cryoprotective composition(s) may be applied in proximity to the large blood vessels, which allows the cryotherapy agent to be applied close to the vessels, while reducing risks that the blood vessels will be damaged. 
     The cryoprotective composition(s) and surgical methods described herein may also be applicable for non-oncological surgical procedures where cryotherapy is desirable and adjacent tissues need to be protected. For example, cryotherapy can be performed in order to treat cardiac arrhythmias and/or for ablation of aberrant nerves in proximity to the pulmonary artery. A serious complication of these procedures can be damage to phrenic nerves, such that the diaphragm is no longer able to contract and normal breathing is not possible. The cryoprotective composition(s) of the present disclosure may be deposited in proximity to phrenic nerves to preserve normal function for these structures. 
     The cryoprotective composition(s) can also be used during rectal surgeries, such as cryotherapy of rectal proctitis. For example, the cryoprotective composition(s) can be used to avoid complications, such as vascular necrosis, that can occur during cryotherapy of cutaneous lesions and/or perforation(s) and fistula formation. 
     Cryoprotective Composition(s) 
     In some examples, the cryoprotective composition(s) of the present disclosure is configured for use in cryosurgery. In order to provide suitable protection for body tissues surrounding and/or in proximity to a surgical site, in some examples the cryoprotective composition(s) is configured to be of a viscosity such that a therapeutically effective amount of the cryoprotective composition(s) deposited in a body space of a patient in proximity to a cryotreatment site or a surgical site remains within or approximately within at least a portion of the body space for a duration of the cryotreatment or surgical procedure. Also, in some examples, at least a portion of a body tissue(s) proximate to the body space of the patient in proximity to a cryotreatment site or a surgical site is or remains viable after the cryotreatment or surgical procedure. Also, in some examples, the cryoprotective composition(s) does not cause substantial nerve and/or tissue damage when positioned in the body space for the duration of the cryosurgical procedure. 
     As used herein, “substantial nerve damage” can refer to nerves that do not have at least about 10%, about 15%, about 30%, or about 50% action potential recovery after being frozen. Methods for measuring action potential recovery of nerves are described in Experimental Examples described elsewhere in the present disclosure. 
     The cryoprotective composition(s) may protect tissues adjacent to surgical sites during cryosurgical procedures by creating space between target tissues exposed to cryosurgical agents and surrounding body tissue. Also, the cryoprotective composition(s) have cryoprotective or antifreeze characteristics that prevent the cryoprotective composition(s) from freezing and prevent the freezing temperatures from passing from the cryotherapy injection site and/or target tissue through the cryoprotective composition(s) to surrounding tissues. In some examples, the combination of spacing and cryoprotective properties may protect surrounding tissues from being exposed to cold temperatures that might cause cellular structures of the surrounding body tissues to crystallize and/or freeze, thereby injuring and/or destroying the cellular structures of the surrounding body tissues. Further, in order to avoid causing damage to the surrounding body tissues, in some examples the cryoprotective composition(s) may comprise primarily biocompatible materials. 
     A “biocompatible material” means that the material and degradation products thereof are substantially non-toxic to cells or organisms within acceptable tolerances, for example, comprising substantially non-carcinogenic and substantially non-immunogenic substances, and can be cleared or otherwise degraded in a biological system, such as an organism (patient or plant) without substantial toxic effect. Non-limiting examples of degradation mechanisms within a biological system can comprise chemical reactions, hydrolysis reactions, and enzymatic cleavage. For example, as described in further detail herein, cryoprotectant agents of the cryoprotective composition(s) can be selected that, in concentrations used for the cryoprotective composition(s), do not appreciably affect an ability of nerve cells to generate action potentials. Methods for determining action potential recovery for nerves are described in Experimental Examples described elsewhere in the present disclosure. 
     In some examples, the cryoprotective composition(s) comprises at least one biodegradable and/or bioerodible fluid agent(s). As used herein, a “fluid agent” means a liquid, foam, suspension, mixture, aerosol, or gel configured to be deposited or injected into a predetermined area of the internal or external body of the patient or to be coated on or applied within a portion of a plant. In some examples, once deposited or injected, the fluid agent(s) remain in place for the duration of a treatment or cryosurgical procedure or for the time period in which resistance to freezing is desired. In some examples, the fluid agent comprises a biocompatible liquid. Alternatively or in addition, the fluid agent can comprise a biocompatible gel, such as a gel comprising a biocompatible polymer. The fluid agent can also comprise and/or be mixed with various thickening agents, as are commonly used in the food and cosmetic industries, as well as with selected biocides and/or preservatives. 
     For example, the cryoprotective composition can comprise one or more of the following gels and/or thickening agents: protein-based ingredients (e.g., gelatin and collagen); sugar-based ingredients (e.g., honey and syrup); polymer-based ingredients (e.g., polyvinyl alcohol and Poloxamer 407); and/or polysaccharides (e.g., hyaluronic acid). Poloxamer 407 is a triblock copolymer comprising a central hydrophobic block of polypropylene glycol flanked by two hydrophilic blocks of polyethylene glycol. Other suitable fluid agents can comprise other additives that have been generally recognized as safe (GRAS) for use in foods and medications. In some examples, the biocompatible gel can comprise cellulose, cellulose ethers, such as hydroxyethyl cellulose, hydroxypropyl cellulose, and/or hydroxypropyl methylcellulose. 
     In other examples, the thickening agent or gel can comprise hyaluronic acid, such as hyaluronic acid produced from plant, animal, or microbial sources. In some examples, the thickening agent or gel is a cross-linked hyaluronic acid as described, for example, in U.S. Pat. No. 10,117,822 entitled “Cross-linked hyaluronic acid, process for the preparation thereof and use thereof in the aesthetic field”, which is incorporated by reference herein in its entirety. 
     As described in further detail herein, in some examples, the fluid agent can be selected to permit sufficient diffusion into body tissue and/or to remain within a body space or body cavity for a duration of a treatment or surgical procedure prior to substantially degrading or eroding. Also, the fluid agent can be selected so that the cryoprotective composition, at about room temperature (e.g., between about 20° C. and about 25° C.), is of suitable viscosity to be ejected from a syringe through a needle cannula, such as a cannula of an 18 gauge needle to a 22 gauge needle. In some examples, in order to provide a composition of suitable viscosity, the fluid agent or gel can comprise up to about 10 weight percent of the cryoprotective composition, less than about 5 weight percent of the cryoprotective composition, less than about 2 weight percent of the cryoprotective composition, about 1 weight percent to about 10 weight percent of the cryoprotective composition, about 1 weight percent to about 5 weight percent of the cryoprotective composition, 1 weight perfect to 4 weight percent of the cryoprotective composition, or about 1 weight percent to about 2 weight percent of the cryoprotective composition. 
     In some examples, the fluid agent is used to produce a stable deposit in the body space to provide sufficient time for the delivered cryoprotectant agent (CPA) to diffuse into the targeted tissue without being quickly absorbed into the intracellular space and bloodstream. However, mixing the cryoprotectant agent with higher viscosity substances, such as gels having high viscosity at body temperature, may impede diffusion of the cryoprotectant agent contained in the gel. Therefore, viscosity of the fluid agent and cryoprotective composition can be selected to permit sufficient diffusion into target tissues. 
     In some examples, the fluid agent can comprise an ultrasound gel (e.g., Aquasonic 100 ultrasound gel manufactured by Parker Laboratories, Inc.). The ultrasound gel can comprise, for example: thickening agents or texture enhancers (e.g., polymers of acrylic acids, such as poly(acrylic acids) and/or acrylates/C10-30 alkyl acrylate crosspolymer); surfactants and emulsifiers (e.g., triethanolamine); preservative, antibacterial, and/or antifungal agents (methylchloroisothiazolinone and methylisothiazolinone); and deionized water. For example, an exemplary ultrasound gel can comprise: 0.3 to 1 percentage weight per volume (w/v %) of Carbomer; 0.3 to 1 w/v % of triethanolamine; 0.5 to 2 w/v % of monopropylene glycol; 0.05 to 0.15 w/v % of methylchloroisothiazolinone and methylisothiazolinone; and deionized water. 
     In some examples, a viscosity of the fluid agent and/or the cryoprotective composition(s) prior to application in or to the patient can range from about 150 cP to about 3,000 cP at a temperature of about 20° C. to about 37° C., or about 25° C. Viscosity can be measured using a viscometer or rheometer. 
     In some examples, the pH of the fluid agent and/or cryoprotective composition(s) can range from about 4 to about 8, or from about 6.5 to about 7.5 or a pH of about 7, at a temperature of about 20° C. to about 37° C., or about 25° C. 
     As used herein, “biodegradable or “bioerodible” means a material that, once deposited or injected into the body and placed in contact with bodily fluids and tissues, will degrade either partially or completely through chemical and/or biological reactions with the bodily fluids and/or tissues. Non-limiting examples of such chemical reactions can comprise acid/base reactions, hydrolysis reactions, and enzymatic cleavage. The biodegradation or erosion rate of the fluid agent may be manipulated, optimized, or otherwise adjusted so that the cryoprotective composition(s) remains in place in the body for the duration of the surgery before passing from the injection site. 
     As used herein, remaining in place (e.g., at a deposited location or an injected location) for the “duration of the treatment” or “duration of the surgery” means that the cryoprotective composition(s) has a viscosity such that a therapeutically effective amount of the cryoprotective composition(s) sufficient to protect body tissues (e.g., the neurovascular bundle, other quadrants of the prostate, nerves, organs, and other body tissues) remains in place for at least a duration of the treatment or surgical procedure. 
     As used herein, a “therapeutically effective amount” means an amount of the cryoprotective composition(s) sufficient to protect surrounding tissues from cold created by cryotherapy agents used during cryosurgery, for example, to prevent and/or inhibit freezing of cellular structures of the surrounding tissues. This prevention and/or inhibition of freezing can be a result of physical spacing between target tissues and surrounding tissues created by the injected cryoprotective composition(s) and/or cryoprotective or antifreeze properties of the cryoprotective composition(s). For example, when the cryoprotective composition(s) is used for cryosurgery of the prostate, the “therapeutically effective amount” of the cryoprotective composition may be an amount of the cryoprotective composition(s) sufficient to prevent and/or inhibit crystallization or freezing of nerve cells of the neurovascular bundle and to preserve an ability of nerves of the neurovascular bundle to generate action potentials. The “therapeutically effective amount” could also be an amount of the cryoprotective composition(s) sufficient to maintain a temperature of body structures or tissues (e.g., the neurovascular bundle, rectum, or bladder) encapsulated, covered, or in proximity to the cryoprotective composition(s) at a temperature ranging from about 30° C. to about 36° C., or at least about 30° C. as cryotherapy agents are applied to the surgical site and target tissue (e.g., to the prostate and/or to tumor(s) or cancerous tissue to be destroyed). It is believed that nerve tissues might continue to function even after being exposed to temperatures of about negative 10° C. However, it is believed that a threshold for non-recoverable injury without a cryoprotectant agent may be exposure to negative 3° C. for at least about 5 minutes. In some examples, when the cryoprotective composition is applied to the neurovascular bundle, the threshold for non-recoverable injury may be improved to be from about negative 10° C. to about negative 15° C. for at least about 10 minutes. 
     Desirably, the cryoprotective composition passes, deteriorates, or is eliminated from the body shortly after completion of the treatment or surgical procedure. For example, the duration of the treatment or surgical procedure (e.g., duration that the cryoprotective composition remains at its deposited or injection location) can be, for example, from about 10 minutes to about 5 hours, from about 20 minutes to about 2 hours, or from about 30 minutes to about 2 hours. It is believed that most cryosurgical treatments or procedures can be performed within a period of about 10 minute to about 5 hours. 
     As previously discussed, the “body space” can refer to any cavity or space surrounding a body tissue or organ to be treated by a cryosurgical treatment or procedure. When the cryoprotective composition(s) is used for prostate surgery, the body space can refer to the periprostatic space, which is the space surrounding the prostate and/or a space defined by the prostate, bladder, and rectum. The periprostatic space refers to a space that is limited posteriorly by the rectum, anteriorly by fat tissue and the pubic bone (symphysis), and laterally by the periprostatic fat tissue and/or pelvic floor muscles. Anatomical structures of the periprostatic space are shown in the schematic drawings of  FIGS. 2C-4B , and are described in further detail in the discussion of surgical methods using the cryoprotective composition(s) disclosed herein. 
     In other examples, the body space can refer to spaces surrounding a solid mass, such as a primary or metastasized tumor. In other examples, the body space can refer to spaces surrounding other organs, such as the liver, heart, lungs, eyes, brain, or spine. 
     The cryoprotective composition(s) further comprises at least one non-toxic cryoprotectant agent that does not cause substantial nerve damage when positioned in the body space for the duration of the treatment or cryosurgical procedure, or as applied externally. In some examples, the at least one non-toxic cryoprotectant agent can comprise up to about 20 weight percent, or about 0.01 to about 20 weight percent, or about 1 to about 20 weight percent, or about 2 to about 20 weight percent, or about 5 to about 10 weight percent, or about 10 to about 20 weight percent, or about 10 to about 15 weight percent, or about 5 weight percent, or less than about 5 weight percent of the cryoprotective composition(s). 
     In some examples, the cryoprotectant agent can be relatively transparent to ultrasound, so as not to interfere with ultrasonic imaging (e.g., images from a transrectal ultrasound probe) used to guide injection of the cryoprotective composition. As used herein, “relatively transparent to ultrasound” means a material having a relative transmissivity of at least about 88% or ranging from about 88% to about 100% for a frequency range of from about 4 MHz to about 10 MHz using the method for determining relative transmissivity in Poltawski, L., &amp; Watson, T. (2007),  Relative transmissivity of ultrasound coupling agents commonly used by therapists in the UK, Ultrasound in medicine  &amp;  biology,  33(1), 120-128. 
     The cryoprotective composition(s) comprising the cryoprotectant agent(s) can have a thermal conductivity similar to water. For example, the thermal conductivity can be less than about 150% of water, which is 598.03 mW/mK at 20° C., 555.75 mW/mK at 0.01° C. For example, thermal conductivity of the cryoprotective composition(s) can be less than about 900 mW/mK between 0.01 and 20° C., as measured by ASTM standard D7896 (Transient Hot Wire Liquid Thermal Conductivity Method). 
     The cryoprotectant agent is configured to be mixed with the fluid agent prior to or during application of the cryoprotective composition(s) to the body space of the patient. As used herein, “non-toxic” means that the cryoprotectant agent should be of limited cellular and neurological toxicity to a patient so that it does not appreciably damage body tissues, such as skin, nerves, organs, and/or nerve bundles for patients or plant tissues for plants, when deposited or injected into the body or applied to external portions of the body. Cellular toxicity (i.e., toxicity to cellular or body tissues) is measured by the viability of cells after exposure to the cryoprotectant agent (under specific time and temperature). The viability is measured using membrane integrity dye (e.g., Trypan Blue) and metabolic activity assay (e.g., CCK8 assay). As used herein, for body tissues, a “non-toxic” cryoprotectant agent means a cryoprotectant agent having no more than a 50% decrease of “viability” and/or that at least 50% of a tissue sample treated with the cryoprotectant agent remains viable compared to a sample treated under the same conditions with saline (e.g., phosphate-buffered saline (PBS)). As used herein, maintaining “viability” or “viable” means that cells, tissues, or nerves retain or regain an ability to perform an intended function following removal of a damaging condition, such as removal of exposure to cold or freezing temperature. Neurological toxicity is measured with the electrically evoked compound action potential (eCAP) methods. As used herein, for nerves, a “non-toxic” cryoprotectant agent refers to a cryoprotectant agent having no more than a 50% decrease of action potential amplitude (compared to a sample treated under the same conditions with Krebs solution). Thus, nerve tissues are or remain viable when nerves of the tissue sample show no more than a 50% decrease of action potential amplitude (compared to a sample treated under the same conditions with Krebs solution). Exposure condition for evaluating toxicity of nerve tissue is defined as 30 min at 37° C. 
     Various cryoprotectant agents that can be used with the cryoprotective composition(s) of the present disclosure are described in Elliott, G. D., Wang, S., &amp; Fuller, B. J. (2017). Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology, 76, 74-91, which is hereby incorporated by reference. Non-limiting examples of cryoprotectant agents can comprise at least one of: alcohol(s) and/or derivative(s) thereof (e.g., methanol, ethanol, glycerol, propylene glycol, and/or ethylene glycol); sugar(s) and/or sugar alcohol(s), (e.g., monosaccharides and/or disaccharides, for example, glucose, galactose, lactose, fructose, sucrose, trehalose, raffinose, mannitol, and/or sorbitol); polymer(s) (e.g., polyethylene glycol, polyvinyl pyrrolidone, milk proteins, serum proteins, and/or peptones); polysaccharides (e.g., dextrans, Ficoll, and/or hydroxyethyl starch); sulfoxides (e.g., Dimethyl sulfoxide (DMSO)); amides (e.g., acetamide, formamide, and/or Dimethylacetamide); and amines (e.g., proline, glutamine, and/or betaine). 
     In some examples, the cryoprotective composition(s) can comprise solutions or mixtures comprising, but not limited to: 3-OMG (3-O-methyl-D-glucose); DP6 (3.0M dimethylsulfoxide (Me 2 SO)+3.0M 1,2-propanediol (DP)); VS55 (a vitrification solution comprising DMSO, formamide, and propylene glycol); and/or M22. M22 is a cryoprotectant agent solution comprising: Dimethyl sulfoxide (22.305 w/v %); formamide (12.858 w/v %); ethylene glycol (16.837 w/v %); N-methylformamide (3 w/v %); 3-methoxy-1,2-propanediol (4 w/v %); polyvinyl pyrrolidone K12 (2.8 w/v %); X-1000 ice blocker (1 w/v %); and Z-1000 ice blocker (2 w/v %). 
     In some examples, the cryoprotective composition(s) can comprise less than 20%, less than 10%, less than 5%, or less than 1% by weight of Dimethyl sulfoxide (DMSO) and/or ethylene glycol, or about 1 to about 20 weight percent, or about 5 to about 15 weight percent, or less than about 10 weight percent of DMSO and/or ethylene glycol, or is free of DMSO and/or ethylene glycol to avoid nerve damage. 
     In some examples, the cryoprotectant agent can comprise molecule(s), compound(s) and/or polymer(s) that resist freezing below a temperature of about negative 15° C. to about 0° C., or about negative 10° C. As used herein, “polymers” or “polymer compositions” comprise, without limitation, homopolymers, heteropolymers, co-polymers, block polymers, block co-polymers, and can be both natural and/or synthetic. A polymer “comprises” or is “derived from” a stated monomer if that monomer is incorporated into the polymer. Thus, the incorporated monomer (monomer residue) that the polymer comprises is not the same as the monomer prior to incorporation into a polymer, in that at the very least, certain groups are missing and/or modified when incorporated into the polymer backbone. For example, the cryoprotectant agent comprising a polymer can comprise one or more of polyethylene glycol, polyvinyl pyrrolidone, milk proteins, serum proteins, peptones, dextrans, Ficoll, or hydroxyethyl starch. 
     In some examples, the cryoprotective composition(s) can further comprise additional materials to adapt the cryoprotective composition(s) for particular uses. For example, the cryoprotective composition(s) can comprise Krebs phosphate solution (e.g., Krebs-Henseleit buffer solution) or PBS solution for improving distribution of the cryoprotectant agent through the other components of the cryoprotective composition(s). In some examples, the amount of Krebs phosphate solution or PBS solution in the cryoprotective composition(s) can range from about 1.0 weight percent to about 95 weight percent, or about 5.0 weight percent to about 90 weight percent of the cryoprotective composition(s). 
     In some examples, the cryoprotective composition(s) can comprise multiple cryoprotectant agents. For example, the cryoprotective composition(s) can comprise 5.0 weight percent DMSO+0.2M trehalose in Krebs solution or PBS solution, 5.0 weight percent DMSO+0.4M trehalose in Krebs solution or PBS solution, or 10 weight percent DMSO+0.2M trehalose in Krebs solution or PBS solution. 
     As discussed previously, in some other examples, the cryoprotective composition(s) comprises a topical cryoprotective composition configured to be applied to skin. In such examples, the cryoprotective composition can comprise the biodegradable and/or bioerodible fluid agent and the non-toxic cryoprotectant agent. The topical cryoprotective composition(s) can be configured to protect skin from damage caused by environmental conditions, such as exposure to cold or freezing, for example, by exposure to cryotherapy agents. In some examples, the cryoprotective composition(s) can be configured to protect the skin from frostbite. As used herein, “substantial damage” to the skin means frostbite, such as frostnip, superficial frostbite, and/or deep frostbite, and can comprise damage to the epidermis, dermis, and/or subcutaneous tissue, redness, skin whitening, blisters, and/or numbness. The topical composition can also comprise other components and compositions commonly used for lotions, balms, solutions, and compositions that are applied to skin including, for example, water, oil(s), alcohol(s), propylene glycol(s), wax(es), preservatives, emulsifiers, antimicrobials, absorption promoters, and/or fragrances. 
     As previously discussed, in some examples, the cryoprotective composition(s) can also be applied to plants by coating, spraying, immersion, or any other application method. The cryoprotective composition(s) for plants can be configured to protect surfaces of the plants from environmental conditions, such as exposure to cold or freezing temperatures or frozen materials, such as precipitation (e.g., ice or snow). The cryoprotective composition(s) for plants can also comprise other components and compositions commonly applied to plants including, for example, micronutrients, macronutrients, pesticides, insecticides, herbicides, rodenticides, fungicides, biocides, plant growth regulators, fertilizers, microbes, soil additives, adhesion promoting-agents, and/or surfactants. 
     Premixed Components of Cryoprotective Compositions 
     The cryoprotectant agents and fluid agents of the present disclosure, which can be used for protecting body tissues during a cryosurgical procedure, as well as for protecting tissues of plants from freezing when atmospheric temperatures drop below freezing, can be provided as a ready-for-use premixed composition. Alternatively, various combinations of separate ingredients or pre-mixtures can be provided to a user, which can be mixed together immediately prior to use to form the cryoprotective composition. The pre-mixed cryoprotective composition and/or ingredients of a cryoprotective composition intended to be mixed together immediately prior to use can be provided in different types of sealed or unsealed medical containers. As used herein, a “container” refers to a hollow or partially hollow member comprising a chamber or reservoir having an interior volume sufficient to contain a predetermined amount or volume of the premixed composition or ingredient(s) of the composition. In some examples, the “container” can be a syringe (e.g., a prefilled syringe), syringe barrel, cartridge (e.g., an injector cartridge), disposable pouch, laboratory beaker, sealed vial, test tube, or other types of medical containers, which can be filled with the predetermined amount or volume of the premixed cryoprotective composition or ingredient(s). The container can also be a multi-chamber (e.g., two or more chambers) syringe, such as a dual chamber syringe used for reconstitution of a drug prior to delivery to a patient. The multi-chamber syringe can comprise different chambers containing components of the cryoprotective composition, which are mixed together as the components are ejected from the chambers of the syringe. 
     In some examples, cryoprotectant compositions can be mixed during manufacturing by, for example, adding a desired amount of the one or more cryoprotectant agents to a solution of the fluid agent or gel. The resulting solution can be mixed to distribute the cryoprotectant agent through the fluid agent by, for example, stirring, shaking (e.g., shaking a container containing the solution), inverting the container, or other mixing methods. In some examples, components of the cryoprotective composition can be provided as fluids or liquids. For example, the fluid agent can comprise a gelling agent (e.g., hydroxyethyl cellulose or hyaluronic acid) in saline solution or in a buffer solution, such as Krebs solution (e.g., Krebs-Henseleit buffer) or PBS solution. For example, a saline solution can comprise 0.9% normal saline. Sodium chloride injection (USP) solution can comprise 9 g/L sodium chloride. USP has an osmolarity of 308 mOsmol/L (calc). USP contains 154 mEq/L sodium and 154 mEq/L chloride. 
     Phosphate-buffered saline (“PBS solution”) is a buffer solution having a pH ˜7.4, which is used in biological research. PBS solution is a water-based salt solution comprising one or more of disodium hydrogen phosphate, sodium chloride and, in some formulations, potassium chloride and potassium dihydrogen phosphate. PBS solution helps to maintain a constant pH. Generally, the osmolarity and ion concentrations of the PBS solutions match those of the human body. An exemplary PBS solution is PBS Solution tampon saline (tamponnée phosphate) by Bichsel Laboratorium Dr. G. Bichsel AG 3800 Interlaken. The PBS Solution tampon saline solution comprises: 0.68 g/100 mL Natrii chloridum Ph. Eur; 0.04 g/100 mL Kalii dihydrogenophosphas Ph. Eur.; 0.15 g/100 mL Natrii dihydrogenosphosphas dihydricus Ph. Eur.; and Aqua ad inject Ph. Eur. ad sol. The solution has a pH of about 7.1 to about 7.3. 
     In order to provide a cryoprotective composition of suitable viscosity for use during cryosurgical procedures, the fluid agent or gel can be up to about 10 weight percent of the cryoprotective composition, less than about 5 weight percent of the cryoprotective composition, less than about 2 weight percent of the cryoprotective composition, about 1 weight percent to about 10 weight percent of the cryoprotective composition, about 1 weight percent to about 5 weight percent of the cryoprotective composition, 1 weight perfect to 4 weight percent of the cryoprotective composition, or about 1 weight percent to about 2 weight percent of the cryoprotective composition. 
     The cryoprotectant agent may also be provided as a fluid in solution, such as a solution of the cryoprotectant agent in water. The solution can be added to the fluid agent to form the cryoprotective composition. In some examples, the cryoprotective composition comprises one type of cryoprotectant agent. For example, the cryoprotective composition can comprise less than 20%, less than 10%, less than 5%, or less than 1% by weight of a cryoprotectant agent, such as Dimethyl sulfoxide (DMSO) and/or ethylene glycol, or about 1 to about 20 weight percent, or about 5 to about 15 weight percent, or less than about 10 weight percent of DMSO and/or ethylene glycol. In other examples, the composition comprises two or more types of cryoprotectant agents, such as DMSO and a sugar, such as trehalose. For example, the cryoprotective composition can comprise 5.0 weight percent DMSO+0.2M trehalose in Krebs solution or PBS solution, 5.0 weight percent DMSO+0.4M trehalose in Krebs solution or PBS solution, or 10 weight percent DMSO+0.2M trehalose in Krebs solution or PBS solution. 
     In other examples, the cryoprotectant agent can be provided as a predetermined amount of a powder, such as a freeze-dried powder. The powder can be mixed with a solution of another type of cryoprotectant agent, a buffer solution, and/or the fluid agent to form the cryoprotective composition. For example, about 1000 mg to about 5000 mg, or about 2000 mg to about 4000 mg, or, preferably, about 3750 mg of the sugar, such as trehalose powder, can be added to a solution of the fluid agent and/or other types of cryoprotectant agents in order to make the cryoprotective composition. As previously described, the solid or liquid cryoprotectant agents can be mixed with the fluid agent during manufacturing to produce the cryoprotective composition. For example, the cryoprotectant agent(s) can be mixed with the fluid agent by introducing the cryoprotectant agent into a container or vessel that contains the fluid agent and mixing the agents together by stirring, shaking the container, or other mixing methods. 
     Following mixing, a predetermined volume of the mixed cryoprotective composition can be filled into a container for transport to a storage location or medical facility. For example, the predetermined volume of the cryoprotective composition can be a volume suitable for use during a cryosurgical procedure or a volume of cryoprotective composition sufficient for protecting portions of a predetermined number of plants from freezing. In some examples, the container may be filled with a single-use amount of cryoprotective composition, such as from about 5 mL to 100 mL of the composition, or about 20 mL to 80 mL of the composition, or about 25 mL to 75 mL of the composition, or preferably about 50 mL of the cryoprotective composition. The container can be disposable, so that once the cryoprotective composition is expelled from the container, the container can be thrown away, recycled, or discarded. 
     In other examples, ingredients of the cryoprotective composition can be provided separately and mixed together immediately prior to use. For example, the fluid agent can be provided as a separate solution isolated from and intended to be mixed with the cryoprotective agent to form the cryoprotective composition. In some examples, the fluid agent can be provided as a fluid contained in a container. For example, a gelling agent (e.g., hydroxyethyl cellulose or hyaluronic acid) can be provided in the container along with a buffer solution, such as Krebs solution or PBS solution. In other examples, the gelling agent can be provided in a separate container and can be mixed with the buffer solution immediately prior to use to form the fluid agent. As previously described, the fluid agent can then be mixed with the cryoprotectant agent to form the cryoprotective composition. 
     The cryoprotectant agent(s) can be provided in one or multiple containers. For example, different types of cryoprotectant agents can be provided together in a single container, such as a single container that contains a solution of DMSO and trehalose in water. In other examples, the cryoprotectant agents can be provided in separate containers. For example, a first container can contain a solution of DMSO. A second container can contain a solution of another type of cryoprotectant agent, such as an alcohol(s), saccharide(s), polysaccharide(s), polymer(s), sulfoxide(s), amide(s), or amine(s). The solutions of the different types of cryoprotectant agents can be mixed together prior to use. The resulting mixed cryoprotectant solution can then be mixed with the fluid agent or gel to produce the cryoprotective composition of the present disclosure. 
     As discussed in further detail herein, solutions can be mixed by stirring, agitation, or other mixing techniques to produce the cryoprotective composition. Once the ingredients of the cryoprotective composition are mixed together, the formed cryoprotective composition can be drawn into a delivery syringe or another suitable container for use during a cryosurgical procedure. For example, as previously discussed, a volume of from 1 mL to 100 mL, or from 20 mL to 80 mL, or from 25 mL to 75 mL, or preferably about 50 mL can be drawn into the syringe or to another container to provide a filled syringe or container with a sufficient volume of the cryoprotective composition for use during a cryosurgical procedure. 
     In some examples, ingredients for the cryoprotective compositions of the present disclosure are selected to control electrolyte concentration and/or osmolality of the final cryoprotective composition. As used herein, “osmolality” can refer to a measure of an electrolyte and water balance of a solution. In particular, osmolality can be defined as a number of osmoles (Osm) of solute per kilogram of solvent (Osm/kg), such that a larger osmolality number indicates a greater concentration of solutes and electrolytes in solution. Osmolality can be measured by an osmometer, such as a freezing point osmometer or a vapor pressure osmometer. For example, osmometer measurements can be obtained from the OsmoTECH® XT Osmometer, by Advanced Instruments, Norwood, Mass., USA. 
     In some examples, chemical interactions between a sugar cryoprotectant agent, such as trehalose, and a composition containing saline may affect cryoprotective properties of the composition. Negative effects of the saline on the sugar may become more significant as the electrolyte concentration or osmolality of a composition comprising trehalose increases. Therefore, desirably, osmolality of a solution containing the trehalose, water, and a saline solution (e.g., PBS solution) can be limited to preserve cryoprotectant properties of the sugar cryoprotectant agent. In some examples, osmolality of a composition comprising the sugar cryoprotectant agent (e.g., trehalose), water, and the saline solution (e.g., PBS solution) ranges from about 200 mOsm/kg to about 400 mOsm/kg, or about 250 mOsm/kg to about 350 mOsm/kg, or about 270 mOsm/kg to about 330 mOsm/kg. In some examples, acidity (pH) of the cryoprotective composition can range from about 6.0 to about 8.0, or about 6.7 to about 7.5, or, about 7.0. 
     Sugar cryoprotectant agents, such as trehalose, may promote bacteria growth and contamination of the cryoprotective composition over time. Therefore, in some examples, the sugar concentration of the cryoprotective composition can be limited to avoid contamination. For example, as previously described, the cryoprotective composition can comprise less than about 20 weight percent, or about 0.01 to about 20 weight percent, or about 1 to about 20 weight percent, or about 2 to about 10 weight percent, or about 5 to about 10 weight percent, or about 7.5 weight percent, or less than about 7.5 weight percent of the sugar cryoprotectant agent (e.g., trehalose). Also, as described in further detail herein, ingredients of the cryoprotective composition can be provided as a kit in separate containers and mixed together shortly before use. The sugar cryoprotectant agent (e.g., trehalose) can also be provided as a powder to avoid microbial growth and contamination. 
     Surgical Kits for Making Cryoprotective Compositions 
     In order to simplify processes for making the cryoprotective compositions of the present disclosure, ingredients and/or components of the cryoprotective composition, along with instruments or tools for making the composition, can be provided together as a surgical kit, such as the exemplary surgical kit  210  shown in  FIG. 1A . 
     In some examples, the surgical kit  210  for making and using the cryoprotective composition comprises a first container(s) containing a predetermined amount or volume of a biodegradable and/or bioerodible fluid agent and a second container(s) containing a predetermined amount or volume of a non-toxic cryoprotectant agent. As previously described, the container(s) can be any type of medical container used for storing medical fluids and compositions including, for example, a syringe (e.g., a prefilled syringe or multi-chamber syringe), syringe barrel, cartridge (e.g., an injector cartridge), disposable pouch, laboratory beaker, sealed vial, test tube, or other types of medical containers. 
     The predetermined amounts or volumes of the biodegradable and/or bioerodible fluid agent(s) and the non-toxic cryoprotectant agent(s) are selected so that, when mixed together, the resulting cryoprotective composition is suitable for use in any of the previously described cryosurgical procedures. For example, the resulting composition can be used during treatment of prostate cancer and/or destruction and removal of other cancers and/or tumors of the prostate. In such cases, the cryoprotective composition can be injected to a periprostatic space of the patient. The cryoprotective compositions of the present disclosure are configured such that a therapeutically effective amount of the cryoprotective composition deposited in a body space of the patient in proximity to the cryotreatment remains within the body space for a duration of the cryotreatment, such as for about 10 minutes to about 5 hours. The cryoprotective compositions can also be used to protect plant tissue from freezing due to atmospheric temperature. 
     In some examples, the therapeutically effective amount can be an amount of the cryoprotective composition sufficient to inhibit freezing and/or crystallization of the body tissue proximate to the body space when cryotherapy agents are used for the cryosurgical procedure. Further, the therapeutically effective amount of the composition can be an amount sufficient to maintain a temperature of surrounding body tissues of at least about 30° C. Also, at least a portion of a body tissue proximate to the body space desirably remains viable after the cryotreatment is completed. The therapeutically effective amount can also be an amount of the composition sufficient to inhibit freezing and/or crystallization of plant tissue when the plant tissue is exposed to normal and/or expected temperatures for a particular patent growing region. 
     In some examples, the fluid agent and the cryoprotectant agent are selected to limit electrolyte concentration and acidity of the formed composition. For example, as previously described, a solution used as an ingredient of the cryoprotectant composition comprising the sugar cryoprotectant agent (e.g., trehalose), water, and a saline solution (e.g., PBS solution) can have an osmolality ranging from about 200 mOsm/kg to about 400 mOsm/kg, or about 250 mOsm/kg to about 350 mOsm/kg, or about 270 mOsm/kg to about 330 mOsm/kg. In addition, acidity (pH) of the cryoprotective composition desirably ranges from about 6.0 to about 8.0, or about 6.7 to about 7.5, or, about 7.0. 
     In some examples, as shown in  FIG. 1A , the first container can be a pre-filled syringe  212  containing a predetermined volume of the biodegradable and/or bioerodible fluid agent in amount(s) and type(s) disclosed herein. In some examples, the pre-filled syringe  212  can comprise a needle or needle cannula permanently attached to an open distal end of the syringe for expelling the fluid agent from the prefilled syringe  212 . In other examples, as described in further detail herein, the kit  210  can comprise removable needles, such as pen needles, that can be attached to a port or nozzle of the syringe  212  prior to use. The pre-filled syringe  212  can be a commercially available syringe used for transferring medical fluids from a vial to a fluid line or for fluid delivery from a barrel of the syringe  212  to a patient through a removable needle, such as a pen needle. Exemplary syringes which can be filed with the predetermined volume of the biodegradable and/or bioerodible fluid agent of the present disclosure are commercially available from many different manufacturers. For example, the prefilled syringe  212  can be a Luer-Lok™ syringe by Becton, Dickinson and Co. 
     The pre-filled syringe  212  can contain the predetermined amount or volume of the biodegradable and/or bioerodible fluid agent. In some examples, the biodegradable and/or bioerodible fluid agent is a gel or gelling agent, such as a predetermined volume of about 1 mL to about 50 mL or, preferably about 25 mL of the fluid agent. For example, the prefilled syringe  212  can contain a solution comprising from 3.0 weight % to about 5.0 weight % hydroxyethyl cellulose in a phosphate-based saline (PBS) buffer solution. In other examples, the fluid agent in the prefilled syringe  212  can comprise from 1.0 mL to 50 mL or, preferably about 25 mL of a hyaluronic acid solution. For example, the prefilled syringe  212  can contain a solution of about 1.0 weight % to about 3.0 weight % hyaluronic acid in the PBS solution. 
     In some examples, the second container(s) of the kit  210 , which contains the predetermined amount or volume of the non-toxic cryoprotectant agent, can be a seal vial(s)  214 ,  216 . For example, the sealed vial(s)  214 ,  216  can be a Type 1 (borosilicate glass) vial comprising an open top sealed by a pierceable elastomeric septum. In some examples, the sealed vial(s)  214 ,  216  contain about 10 mL to about 100 mL, or about 20 mL to about 50 mL, or preferably, about 25 mL of the non-toxic cryoprotectant agent. For example, the kit  210  can comprise a sealed glass vial  214  containing about 5 weight % to about 15 weight % of Dimethyl sulfoxide (DMSO) in water. 
     Alternatively or in addition, the kit  210  can also comprise another sealed glass vial  216  containing other cryoprotectant agents including alcohol(s), saccharide(s), polysaccharide(s), polymer(s), sulfoxide(s), amide(s), or amine(s) contained in the second container. In some examples, the alcohol(s), saccharide(s), polysaccharide(s), polymer(s), sulfoxide(s), amide(s), or amine(s) contained in the sealed vial  216  can be provided in solution, such a solution having a concentration of about 1M to about 3M. In other examples, some cryoprotectant agents can be provided as a powder, such as a powder formed by lyophilization or freeze drying. In some examples, the kit  210  comprises a sealed vial  216  containing trehalose. For example, the sealed vial  216  can contain about 1000 mg to about 5000 mg, or about 2000 mg to about 4000 mg, or, preferably, about 3750 mg of the sugar, such as trehalose powder. 
     The kit  210  can also comprise surgical devices or tools for preparing the cryoprotective composition and for delivering the cryoprotective composition to the body space of the patient during the cryosurgical procedure. For example, the kit  210  can comprise pen needles  218  configured to be mounted to syringes, such as the prefilled syringe  212  containing the fluid agent, for transferring fluids between containers of the kit  210 . The kit  210  can also comprise one or more therapeutic needles  220  configured to be mounted to a fluid delivery or injection syringe for administering the cryoprotective composition to the body space of the patient. In some examples, the pen needles  218  for transferring fluids between the containers can be about 18 gauge needle to about 22 gauge needles. The pen needles  218  for therapeutic use may also be from about 18 gauge needle to about 22 gauge needles. The therapeutic pen needles  220  may be longer in length than the administration pen needles  218  so that the cryoprotective composition can be injected into body spaces that are spaced apart from the skin surface by a substantial distance or depth. 
     The kit  210  can also comprise an empty fluid delivery or injection syringe  222  for injecting the formed cryoprotective composition to the body space of the patient. The injection syringe  222  can be a standard fluid delivery syringe comprising a syringe barrel that contains between 25 mL and 100 mL, preferably, about 50 mL of fluid. In other examples, the injection syringe  222  may be smaller (e.g., may contain a lower fluid volume) for certain medical applications requiring highly precise positioning of the cryoprotective composition, such as brain surgery. The injection syringe  222  can comprise a nozzle, such as a luer tip, configured to be connected to the therapeutic needle  220  sized for delivering the cryoprotective composition to the body space of the patient. As shown in  FIG. 1A , the injection syringe  222  is initially empty. 
     As described in further detail herein, the cryoprotective composition can be drawn from one of the fluid containers, such as one of the sealed vials  214 ,  216 , into the barrel of the injection syringe  222 . For example, the cryoprotective composition made from the predetermined volume of the fluid agent and the predetermined volume of the cryoprotectant agent can have a total volume of from about 10 mL to about 100 mL, or about 25 mL to about 75 mL, preferably about 50 mL. The cryoprotective composition can then be administered to the body space of the patient by pushing a plunger rod and stopper through the syringe barrel of the injection syringe  212  to expel the formed cryoprotective composition from the barrel through the therapeutic pen needle  220  to the patient. 
     Methods of Making Cryoprotective Composition from Surgical Kits 
     A flow chart showing steps for using the surgical kit  210  to make the cryoprotective composition is shown in  FIG. 1B . The method can be performed by a trained medical practitioner, such as a physician, surgeon, physician&#39;s assistant, medical resident, or similarly training medical professional, at a surgical site immediately prior to performing a cryosurgical procedure. 
     As previously described, the surgical kit  210  can comprise the first container, such as the prefilled syringe  212 , containing the predetermined volume of the biodegradable and/or bioerodible fluid agent. The kit  210  can also comprises the second container(s), such as the sealed vial(s)  214 ,  216 , containing the predetermined amounts or volumes of the non-toxic cryoprotectant agent. The kit  210  can also comprise the empty injection syringe  222 , such as a 50 mL syringe comprising a nozzle or luer tip configured to be connected to a therapeutic needle, as well as other pen needles  218 ,  220 , for transferring fluids between the containers. 
     At step  310 , the method optionally, initially comprises attaching a pen needle  218  to the prefilled syringe  212  containing the fluid agent. Alternatively, the prefilled syringe  212  can include a permanently attached needle, which can be covered by a cap, sleeve, or shield. In that case, the practitioner can remove the cap or retract the sleeve or shield to expose the needle. At step  312 , after the pen needle  218  is attached to the syringe  212  or a needle of the syringe  212  is exposed, the method comprises inserting the pen needle  218  through a septum of a sealed vial  214  containing the cryoprotectant agent, such as a vial  214  containing the Dimethyl sulfoxide (DMSO), and injecting the fluid agent from a barrel of the prefilled syringe  212  into the vial  214  through the pen needle  218 . Once the fluid agent is expelled into the vial  214 , at step  314 , the practitioner mixes the fluid agent with the DMSO by, for example, shaking or repeatedly inverting the vial  214  in order to mix the fluid components contained therein. 
     After the fluid agent and cryoprotectant agent (e.g., DMSO) are mixed, at step  316 , the practitioner may withdraw the mixed fluid from the vial  214  back into the syringe  212  and then, optionally, transfer the fluid to a second sealed vial  216 , such as a vial  216  containing trehalose. For example, the practitioner can insert the needle  218  through the septum of the sealed vial  216  and expel the fluid, which can comprise both the fluid agent and the DMSO, into the vial  216  containing the trehalose. As previously described, in some examples, the trehalose can be provided as a powder, such as a lyophilized or freeze dried powder. Alternatively, the trehalose can be provided as a solution, such as a solution of trehalose in water, phosphate buffered saline (PBS) solution, or Krebs solution. Once the fluid agent and DMSO are expelled into the vial  216 , the needle  218  can be removed from the vial  216 . 
     At step  318 , the practitioner next shakes, agitates, and/or repeatedly inverts the vial  216  to mix the fluid agent, DMSO, and trehalose in order to form the cryoprotective composition. Next, at step  320 , the practitioner can optionally attach a therapeutic pen needle  220  to the empty or injection syringe  222  and insert the needle  220  into the vial  216  containing the mixed cryoprotective composition. At step  324 , the practitioner then withdraws the mixed cryoprotective composition from the vial  216  into a barrel of the injection syringe  222  by, for example, retracting a plunger rod and stopper of the syringe  222  through the syringe barrel. Once the injection syringe  222  is filled by the mixed cryoprotective composition, the practitioner can use the prepared cryoprotective composition during a cryosurgical procedure, using any of the surgical methods and procedures described herein. 
     Methods for Performing Cryotherapy Procedures 
     As previously described, the cryoprotective composition(s) can be used in various cryosurgical procedures comprising, for examples, treatment of prostate cancer and/or for destruction and removal of other cancers and/or tumors. In some examples, a method for preparing a surgical site, for protecting the surgical site during cryosurgery, and/or for performing cryosurgery comprises injecting a therapeutically effective amount of any of the previously described cryoprotective compositions into a body space, such as into a periprostatic space  10  (shown in  FIGS. 2C-4B ) surrounding the prostate of a patient. In some examples, the protective composition(s) is injected after the cryotherapy probes are positioned in the target tissue(s). Alternatively, the protective composition(s) may be applied prior to introducing the cryotherapy probes to the target tissue(s). As shown in  FIGS. 2C-4B , the periprostatic space  10  is defined by or comprises the prostate  12 , rectum  14 , neurovascular bundle  16 , and bladder  18 . As described in further detail herein, therapeutically effective amounts (shown generally by reference numbers  28  and  36 ) of the cryoprotective composition(s) are injected into the periprostatic space  10  to protect the prostate  12  and neurovascular bundle  16  during cryosurgical procedures. 
     As previously discussed, the cryoprotective composition(s) comprises, for example, the at least one biodegradable and/or bioerodible fluid agent and the at least one non-toxic cryoprotectant agent. The cryoprotective composition is desirably of sufficient viscosity to remain within a body space, such as the periprostatic space  10 , for a duration sufficient for performing a cryosurgical procedure in proximity to the body space. Desirably, the cryoprotective composition does not cause substantial nerve damage when positioned in the body space for the duration of the cryosurgical procedure. 
     As previously discussed, as used herein, a “therapeutically effective amount” means an amount of the cryoprotective composition(s) sufficient to protect surrounding tissues from cold created by cryotherapy agents used during cryosurgery, for example, to prevent and/or inhibit freezing of cellular structures of the surrounding tissues. This prevention and/or inhibition of freezing can be a result of physical spacing between target tissues and surrounding tissues created by the injected cryoprotective composition(s) and/or cryoprotective or antifreeze properties of the cryoprotective composition(s). For example, when the cryoprotective composition(s) is used for cryosurgery of the prostate, the “therapeutically effective amount” of the cryoprotective composition may be an amount of the cryoprotective composition(s) sufficient to preserve an ability of nerves of the neurovascular bundle  16  to generate action potentials. For example, for a cryosurgical procedure for the prostate  12 , about 1.0 mL to about 100 mL, or about 5.0 mL to about 75 mL, or about 10 mL to about 50 mL, or more of the cryoprotective composition(s) may be needed to provide appropriate protection for the prostate  12  and neurovascular bundle  16 . One of ordinary skill in the art understands that the amount of the cryoprotective composition(s) injected to the surgical site can vary based upon the size of the area/volume to be protected. 
     With reference to  FIG. 5 , in some examples, the injection of the cryoprotective composition comprises, at step  110 , inserting a tip  20  of a syringe needle  22  (shown in  FIGS. 2C and 2D ) through a perineum region  2  of the patient at, for example, the midline position (shown by dashed line  4 ) extending from the anus  6  to the scrotum  8 , as shown in  FIGS. 2A and 2B . The proposed injection site is shown by reference number  34  in  FIGS. 2A and 2B . As previously discussed, in some examples, the cryoprotective composition(s) is applied after the cryotherapy probes are in place. Alternatively, the cryoprotective composition(s) can be applied prior to insertion of the cryotherapy probes to the target tissues of the prostate  12 . For cryosurgical procedures for the prostate  12 , the patient is in the lithotomy position. As used herein, the “lithotomy position” refers to a position with the patient lying on his back with the legs flexed 90 degrees at the hips. The patient&#39;s knees are bent at 70 to 90 degrees and padded footrests attached to the table support the patient&#39;s legs. A size of the needle  22  can, for example, range from an 18 gauge needle to a 22 gauge needle, though one of ordinary skill in the art may select an appropriately needle size depending upon, for example, the type of surgical procedure being performed, the amount of the cryoprotective composition(s) needed to provide sufficient protection for surrounding tissues and structures, and viscosity and other characteristics of the cryoprotective composition(s) being injected. 
     At step  112 , under ultrasound guidance, the method comprises advancing the tip  20  of the needle  22  from the insertion site  34  in the perineum region  2  to perforate the Denonvillier&#39;s fascia extending between the prostate  12  and rectum  14  (shown generally by reference number  24 ) of the periprostatic space  10  distal to the prostate  12 . Ultrasound guidance can be provided, for example, by a transrectal ultrasound probe  26 , as shown in  FIGS. 2C and 2D , inserted into the rectum  14  of the patient. 
     At step  114 , the method further comprises injecting a first amount  28  of the cryoprotective composition(s) to a portion of the periprostatic space  10  between the prostate  12  and the rectum  14  near a base  30  of the prostate  12 . The first amount  28  of the cryoprotective composition(s) is shown schematically in  FIGS. 3A and 3B . At step  116 , the method further comprises continuing to inject the first amount  28  of the cryoprotective composition(s) from the tip  20  of the needle  22  while retracting the needle  22  from the base  30  of the prostate  12  towards an apex  32  of the prostate  12 . It is believed that the space created by this initial injection of the cryoprotective composition(s) (e.g., the first amount  28  of the cryoprotective composition shown in  FIGS. 3A and 3B ) allows for easy movement of the needle  22  in the periprostatic space  10 , thereby enabling further injection laterally to regions surrounding the prostate  12  and neurovascular bundle  16 . For example, the first amount  28  of the cryoprotective composition can create a space or distance D1 (shown in  FIG. 4A ) between the lower surface of the prostate  12  and an upper surface of the rectum  14 . In order to provide suitable protection for tissues of the rectum  14  during cryosurgery and/or to permit lateral movement of the needle  22  towards the neurovascular bundle  16 , the distance D1 can be from about 1.0 mm to about 50 mm or from about 5.0 mm to about 20 mm. 
     When the first amount  28  of the cryoprotective composition is released into the periprostatic space  10 , the cryoprotective composition covers spaces between the base  30  and an apex  32  of the prostate  12 . At step  118 , the method can further comprise moving the tip  20  of the needle  22  laterally while injecting a second amount (shown generally be reference number  36  in  FIGS. 4A and 4B ) of the cryoprotective composition to cover additional portions of the prostate  12  and to more fully surround portions of the neurovascular bundle  16 . In addition to moving the needle  22  laterally, the needle  22  may also be inserted or retracted to obtain desired coverage of the prostate  12 . Once the first amount  28  and the second amount  36  of the protective composition(s) is applied to the periprostatic space  10  and the prostate  12 , the cryoprotective composition may substantially or fully envelop the prostate  12 , providing suitable protection for structures surrounding the prostate  12 . In other examples, the cryoprotective composition may only be applied in proximity to selected areas or portions of the prostate  12  dependent upon the type of localized procedure being performed. For many surgical procedures, following injection of the first amount  28  and the second amount  36 , the protective composition(s) will cover from about 10% to about 90%, or about 50% to about 75%, or about 60% to about 70% of the surface of the prostate  12 . 
     As discussed previously, the cryoprotective composition comprises the biodegradable and/or bioerodible fluid agent. The fluid agent is configured and/or selected to remain in place within the periprostatic space  10  for a duration of the cryosurgical procedure. For example, the duration of the surgical procedure (e.g., duration that the cryoprotective composition remains at its injection location) can be, for example, from about 10 minutes to about 5 hours, from about 20 minutes to about 2 hours, or from about 30 minutes to about 2 hours. 
     At step  120 , a method for cryosurgery further comprises, following injection of the therapeutically effective amount of the cryoprotective composition to the selected portions of the periprostatic space  10 , performing a cryosurgical procedure while the cryoprotective composition is positioned in the periprostatic space  10 . As discussed herein, the cryosurgical procedure can be a cryosurgical procedure for removal or destruction of cancerous tissue in the prostate, such as tumor ablation or any cryosurgical procedure. 
     As discussed herein, the cryoprotective composition(s), method(s) of preparing a surgical site, and surgical method(s) of the present disclosure can also be used for treatment of conditions and/or for use with other surgical procedures in addition to treatment of prostate cancer and/or removal of tumor(s) from the prostate. For example, the cryoprotective composition(s) can be used with surgical methods for destruction or removal of solid masses comprising, for example, tumor(s) and/or cancer (e.g., metastasized tumor(s) and/or primary tumor(s)) located in the lungs, liver, kidney, adrenals, breast, and/or skin of the patient. In some examples, the cryoprotective composition(s) can be used with cryosurgical methods for treating one or more of cardiac arrhythmia, ablation of aberrant nerves in proximity to a pulmonary artery, tumor(s) and/or lesions around an eye or eye orbit, esophageal tumor(s) and/or cancer, pancreatitis, pancreatic tumor(s) and/or cancer, rectal proctitis, liver tumor(s) and/or cancer, brain tumor(s) and/or cancer, cryolipolysis, chronic pain caused by trigeminal neuralgia, neurologic pain at various regions in the body, and/or organ transplantation. 
     In some examples, the protective composition(s) can be applied to external portions of the body tissue, such as all or a portion of the skin of a patient. For example, the protective composition(s) can be applied before or during, for example, cryolipolysis in order to preserve the appearance of the skin. Specifically, in cryolipolysis, the outer appearance of the skin should be preserved while freezing fatty tissue below the skin. It is believed that the protective composition(s) can provide such protection for outer layers of skin without interfering with freezing of fat cells in proximity to the skin. The protective composition may also be added to skin creams or lotions and applied, for example, to reduce risks of exposure to cold and to avoid developing frostbite. 
     Methods of Use for Topical Cryoprotective Compositions 
     In some examples, as previously described, the cryoprotective composition is a topical composition that is applied to skin to protect the skin from exposure to environmental conditions, such as freezing temperatures or wind. The topical composition can be applied to skin in a manner similar to how other topical compositions and locations are applied to skin. For example, a user or patient can obtain a container, such as a bottle, tube, vial, or another suitable container holding the composition. The user can apply the cryoprotective composition directly to the skin by, for example, squeezing the composition from a tube or pouring the composition from a bottle onto the skin. The user or patient can then rub or otherwise disperse the composition over a surface of the skin to ensure appropriate coverage for any areas of skin exposed to harsh environmental conditions. In order to ensure proper protection, the cryoprotective composition may be periodically reapplied, such as one ever few hours. 
     Methods for Applying a Cryoprotective Composition to Plants 
     As previously described, in some examples, the cryoprotective composition(s) can be used to protect internal or external tissues of plants from exposure to cold temperatures. In some examples, a method for applying the cryoprotective composition to a plant to protect the plant from frost and/or freezing can comprise coating at least a portion of a surface of the plant with the cryoprotective composition. The plant can be, for example, a fruit tree, citrus tree, grape vine, flowering tree, flowering bush, grasses, hay, grains, corn, or any other plant that may be exposed to freezing temperatures. Applying the cryoprotective composition to the surface of the plant can include applying the cryoprotective composition to, for example, leaves, stems, buds, flowers, or fruit of the plant. In some examples, the composition can also be applied to roots of the plant. Portions of the applied composition in proximity to the roots can be absorbed by the roots, thereby introducing the cryoprotectant agent into internal structures or tissues of the plant. Once absorbed by the plant, the cryoprotectant agent can protect internal tissues of the plant from cold temperatures, freezing, or frost. For some plants, components of the cryoprotective composition can also be absorbed by leaves, stems, or flowers of the plant, or by any other portion of the plant capable of absorbing liquids, emulsions, or solutions. 
     In some examples, applying the cryoprotective composition to surfaces of the plant can include coating surfaces of the plant with the composition by, for example, spraying the composition over the surface of the plant, pouring the composition over the surface of the plant, or immersing a portion of the plant in a bath of the cryoprotective composition. For example, roots of the plant may be immersed in a bath of the cryoprotective composition so that the composition can be absorbed into interior tissues of the plant. 
     EXPERIMENTAL EXAMPLES 
     To evaluate cryoinjury of the nerves and cryoprotection effectiveness of the cryoprotective composition(s) disclosed herein, the present inventors have performed the following examples. 
     Example 1: Establishment of the NVB Temperature Profile and Models for Cryoprotectant Agent Loading 
     3D models of the prostates were created from image data showing prostate cancer (PCa). Tumor locations and cryoprobes were placed in analogy to clinical cases based on surgical experience. Freezing cycles were then performed that mirror clinical processes. The 3D models were generated from a heat transfer finite element model (FEM) used to derive a spatial-temporal temperature distribution across the prostate and adjacent tissue. The temperature histories of the neurovascular bundle (NVB) under various cryosurgery scenarios were then created according to the generated models. The created temperature histories were then used for testing of different cryoprotectant agents (in Example 2) in order to model effectiveness and toxicity of the different cryoprotectant agents based in realistic clinical applications. 
     More specifically, in order to produce the 3D models of the prostate, MR images were imported using native Digital Imaging and Communications in Medicine (DICOM) data files that were converted to 3D renditions. The tumor regions within the prostate and the placement of cryoprobes were annotated in analogy to clinical cases based on a surgeon&#39;s experience. The DICOM files were viewed in 3D Slicer as segmented prostate regions. The 3D prostate model was converted to Standard Triangle Language (STL) format and imported into COMSOL Multiphysics® simulation software, which is a proprietary finite element analysis and modeling software program produced by COMSOL Inc. (Stockholm, Sweden). The size, shape, and location of the tumors were marked in the 3D prostate model, as well as the actual locations and real-world geometries of the cryoprobes. A urethral warming catheter, through which warm saline continuously flows during a surgical procedure, was also included in the model. The topographical anatomy and distribution of the NVB were modeled by computerized planimetry and diffusion tensor imaging (DTI). Examples of a PCa cryosurgery model reconstructed based on data for the created image files are shown in  FIGS. 6A and 6B . 
     It is recognized that characterization of the cryoprobe is necessary for accurately recreating the cryosurgical process in the model. Therefore, in order to accurately model boundary conditions, the distributed cryogen temperature and convective coefficient were adjusted to match the temperatures at various locations around the cryoprobes and the locations of the 0° C., −20° C., and −40° C. isotherms using known methods, as described in Etheridge, et al., “Methods for Characterizing Convective Cryoprobe Heat Transfer in Ultrasound Gel Phantoms”,  J Biomech Eng ., February 2013, 135(2), and as shown in  FIGS. 7A-7D  for one specific cryoprobe model. 
     A heat transfer finite element method (FEM) was then used to derive the spatial-temporal temperature distribution across the prostate and adjacent tissue. In the simulation, the cryoprobes of a selection of models were turned “on” and “off” based on the timings from the surgical records. The timings for the cryoprobes are shown schematically in  FIG. 7E . Pennes&#39; bioheat equation was used as the governing equation for the analytical solution of the temperature distribution. The phase changes occurring during the freezing process were modeled based on an enthalpy method and the thermal properties were derived from the literature. 
     It is believed that the simulations described hereinabove for recreating PCa cryosurgeries simulate conditions that the NVB is subjected to during surgery, which provides a temperature history of the NVB throughout the surgical process. Accordingly, the generated model was used to determine a time-dependent graph of temperatures to which the NVB is exposed to during prostate surgery. An exemplary generated time-dependent graph created from the 3D model is shown in  FIG. 8A . As shown in  FIG. 8A , in the modeled example, it was determined that the temperature of the NVB was as low as −10° C. with a cooling rate of about −10° C./min. These simulated NVB temperature histories were validated against temperature records collected during cryosurgery procedures. The time-dependent graph was used to control temperatures applied to nerves during ex vivo testing, as described hereinafter. Specifically, a graph showing temperatures on the nerves applied by a cryostage device over a testing cycle is shown in  FIG. 8B . 
     Example 2: Assessment of Acute Nerve Cryoinjury and Recovery with the Cryoprotectant Agent 
     Ex vivo models were used to determine the cryoinjury threshold and to quantify the extent of cryoinjury mitigation provided by the cryoprotective composition. Specifically, as described in further detail herein, fresh phrenic nerves procured from swine and fresh sciatic nerves from rats were dissected and verified for function. 
     Fresh nerves from swine and rats were selected by the inventors for ex vivo testing because such nerves are believed to be comparable to human anatomy. Several papers have investigated and compared the anatomy of nerves in humans to pigs, rats, or mice. For example, the following papers have addressed these issues: Stakenborg, et al., “Comparison between the cervical and abdominal vagus nerves in mice, pigs and humans,”  Neurogastroenterology  &amp;  Motility  (2020): 32; Ding, et al., “Anatomical anomalies of the laryngeal branches of the vagus nerve in pigs,”  Laboratory Animals  (2012) 46:338-340; and Pelot, “Quantified Morphology of the cervical and subdiaphragmatic Vagus nerves of Human, Pig, and Rat,”  Frontiers in Neuroscience  (2020) 14. Most of these studies focus on the vagus nerve because vagal nerve stimulation is evaluated as a novel approach to treat immune-mediated disorders, such as Crohn&#39;s disease. Nevertheless, the present inventors believe that these studies show that human nerves are comparable to porcine phrenic nerves and sciatic nerves of rats in terms of myelinated and unmyelinated fibers. In particular, the nerve composition is believed to be particularly comparable between humans and pigs leading to the conclusion that porcine nerves and nerves obtained from rats are a valuable substitute to human nerves. In particular, the Pelot article studied the morphology of nerve fibers in human, pigs, and rats in detail. Pelot observed a greater variability in human nerve morphology than pigs and rats. Porcine nerves were generally found to be slightly smaller in size than human nerves, but there were ten times more fascicles in the porcine nerve for the same size. The rat nerves were ten times smaller and had a smaller protective perineurium. 
     For these reasons, the present inventors consider porcine nerves as a valuable substitute for the nerve study. The fact that there are ten times more fascicles in the same nerve may also render it more suitable as a substitute of the human erectile nerves. It is believed that rat nerves are more vulnerable as they are of smaller size (10×) and less protected by the perineurium. 
     A compound action potential recording system coupled with an oxygenated bath was set up to assess the changes in nerve function due to injury and recovery over a period of 3 hours. A cryostage with precise control of the surface temperature and its rate of change was used to simulate the thermal conditions experienced by the neurovascular bundle during cryosurgery. The cryostage was configured to apply temperatures according to the temperature profile in  FIG. 8B . During ex vivo testing, the phrenic and sciatic nerves were exposed to one of the following conditions: (i) cryoprotectant agent perfusion, (ii) freezing conditions, or (iii) both the cryoprotectant agent perfusion and the freezing conditions. The action potential of the nerves following exposure to the cryoprotectant agent and/or freezing was then measured. A series of cryoprotectant agents were evaluated in terms of toxicity and effectiveness in preserving action potentials of the neurovascular bundle using the ex vivo nerves. 
     More specifically, in order to assess the extent of cryoinjury through ex vivo models, a bipolar compound action potential (AP) stimulation and recording system was constructed. The compound action potential recording system was a bipolar compound action potential stimulation and recording system based on well-established experiments from McGill University. See  The McGill Physiology Virtual Lab, Compound Action Potential , http://www.medicine.mcgill.ca/physio/vlab/CAP/prep.htm. The system comprises: a series of electrodes in contact with the nerves; stimulation leads for evoking an action potential that travels down the nerves; and two pairs of collection leads for recording the action potential proximal and distal to the freezing site. Both the stimulation and recording of the action potential are synchronized with a computer. A procedure for preparation of the nerves is described in Goff, R. P., Bersie, S. M., &amp; Iaizzo, P. A. (2014),  In vitro assessment of induced phrenic nerve cryothermal injury. Heart rhythm,  11(10), 1779-1784. Specifically, two phrenic nerves were dissected from swine and placed in modified Krebs-Henseleit buffer (i.e., “Krebs solution”), after which fatty sheaths and connective tissue surrounding the nerves were removed. The prepared nerves were then placed in a nerve recording chamber. The nerves were exposed to a constant temperature of 37° C. and a constant supply of oxygen once placed in the chamber. A photograph of the system is shown in  FIG. 9 . 
     In the freezing setup, the nerves were laid on a cryostage with freezing and warming stages programmed to provide the temperature profile shown in  FIG. 8B . A photograph of the freezing setup used for exposing the nerves to freezing conditions is shown  FIG. 10 . It was determined that a 5 mm section of the nerves that were in contact with the cooling surface of the cryostage was susceptible to the freezing conditions. Further, temperatures measured by thermocouples or collection leads positioned in proximity to this 5 mm section of the nerves were found to be in good agreement with a temperature indicator on the cryostage. In neural conduction experiments, a frozen region of the nerves was marked and placed between the proximal and distal recording leads Immediately after a freezing step, the nerves were placed in oxygenated Krebs solution at 37° C., and changes in action potential were monitored every 30 minutes for 3 hours. This example aimed to approximate behavior of nerves following acute injury and fast recovery. 
     A schematic diagram showing the experimental setup for testing action potential of the nerves after freezing is shown in  FIG. 12 . The present inventors believe that impacts of freezing can be quantified by comparing the action potential proximal and distal to the freezing site. In particular, upon stimulation of the nerves, the evoked action potential propagates along the nerves. The action potential can be measured at the proximal leads before reaching the site of injury. Depending on the extent of injury and its recovery, the conduction of the nerves may be completely lost so that no action potential is recorded at the distal leads. However, nerves that are only partially injured or has partially recovered from injury still retains conduction capability, and the action potential can travel past the injury site and be recorded at the distal leads. An example of this comparison is shown by comparing the graphs of  FIG. 11 , in which the dotted line A indicates the time at which the stimulus was applied, the magnitude B and latency peak C indicate an AP proximal to the freezing site, and the distance D represents the distance traveled by the generated signal through the freezing site. The waveform spike following the distance D indicate the AP following the freezing site. 
     In the experiments for determining the injury threshold, the nerves were subjected to a variety of freezing conditions consisting of three key parameters—the end temperature, cooling rate, and warming rate. The action potential recovery was measured for each example. Ranges for these three parameters were determined from a series of simulations that cover a wide range of situations during cryosurgery. 
     Action potential measurements for each example are reported herein as a normalized action potential amplitude. The normalized action potential amplitude (magnitude of the AP at distal leads/magnitude of the AP at proximal leads) is believed to be a key indicator of the continuity of action potential transduction, with 0 indicating complete blockage of the evoked action potential at the freezing site. In addition to the action potential magnitude, an increase in latency (slower conduction speed) and a decrease in voltage gradient (slower depolarization and repolarization of the cells) may also be indicators of injury to the nerves. 
     Dissected porcine phrenic nerves and rat sciatic nerves were exposed to a cryoprotectant agent (CPA) containing modified Krebs solution and incubated for 30 min After exposure to the CPA, both porcine phrenic nerves and rat sciatic nerves were subject to the freezing and warming conditions shown in  FIG. 8B . After both exposure to the CPA and freezing, recovery of the evoked AP in the nerves is monitored for 3 hours. 
     Four categories of cryoprotectant agents were evaluated and compared: (1) conventional DMSO-based cryoprotectant agents, (2) modified DMSO-based cryoprotectant agents developed with the goal of reducing the usage of DMSO, (3) branded cryoprotectant agents used in the cryopreservation of cells, tissue, and organs, and (4) alcohol (and their derivatives) based cryoprotectant agents that are free of DMSO. Experimental results for the most promising cryoprotectant agents are reported herein and shown in  FIGS. 13A-18 . 
     Experimental results for phrenic nerve CPA toxicity testing are shown in  FIGS. 13A-13F , which are graphs showing changes in normalized action potential over time for different cryoprotectant agents (both with and without freezing).  FIGS. 13A-13C  show effects of exposing the phrenic nerves to CPA without freezing. Specifically,  FIG. 13A  shows a control, where no CPA was applied to the phrenic nerves;  FIG. 13B  shows exposure of the phrenic nerves to a CPA solution of 5% DMSO+0.4M Trehalose; and  FIG. 13C  shows exposure of the nerves to a CPA solution of 5% DMSO.  FIGS. 13D-13F  show effects of both freezing and exposure of the phrenic nerves to the CPA. Specifically,  FIG. 13D  shows a control in which the phrenic nerves are frozen without being exposed to the CPA solution;  FIG. 13E  shows effects of freezing and exposure to a CPA solution of 5% DMSO and 0.4 M Trehalose for the phrenic nerves; and  FIG. 13F  shows effects of freezing and exposure to a CPA solution of 5% DMSO. Normalized action potential values for other cryoprotectant agents are shown in Table 1, as follows. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Effect of cryoprotectant agent (CPA) on the phrenic nerves 
               
            
           
           
               
               
            
               
                   
                 # of nerves with &gt;30% AP Recovery at 180 min 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Averaged normalized 
               
               
                   
                 CPA  
                   
                 AP amplitude  
               
               
                   
                 Exposure 
                 w/Freezing 
                 at 180 min 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 5% DMSO and 0.2M 
                 6/6 
                 3/4 
                 0.48 
               
               
                 Trehalose 
                   
                   
                   
               
               
                 5% DMSO and 0.4M 
                 4/4 
                 6/7 
                 0.42 
               
               
                 Trehalose 
                   
                   
                   
               
               
                 10% ethylene glycol 
                 3/3 
                 2/4 
                 0.23 
               
               
                 (EG) and Sucrose 
                   
                   
                   
               
               
                 5% DMSO 
                 4/4 
                 1/4 
                 0.31 
               
               
                 5% M22 
                 3/3 
                 0/3 
                 0.13 
               
               
                 0.4M Trehalose 
                 4/4 
                 1/4 
                 0.25 
               
               
                 10% DMSO 
                 4/4 
                 0/4 
                 0.11 
               
               
                 10% propylene glycol 
                 3/3 
                 2/5 
                 0.1 
               
               
                 (PG) 
                   
                   
                   
               
               
                 10% ethylene glycol 
                 3/3 
                 0/4 
                 0 
               
               
                 (EG) 
               
               
                   
               
            
           
         
       
     
     Experimental results for sciatic nerve CPA toxicity testing are shown in  FIGS. 14A-14F .  FIGS. 14A-14C  show effects of exposing the sciatic nerves of a rat to CPA without freezing. Specifically,  FIG. 14A  shows a control, where no CPA was applied to the sciatic nerves;  FIG. 14B  shows exposure of the sciatic nerves to a CPA solution of 5% M22; and  FIG. 14C  shows exposure of the sciatic nerves to a CPA solution of 5% DMSO.  FIGS. 14D-14F  show effects of freezing and exposure to the CPA for the sciatic nerves. Specifically,  FIG. 14D  shows a control in which the sciatic nerves are frozen without being exposed to the CPA solution;  FIG. 14E  shows effects of freezing and exposing the sciatic nerves to a CPA solution of 5% M22; and  FIG. 14F  shows effects of freezing and exposure to a CPA solution of 5% DMSO. Normalized action potential values for other cryoprotectant agents are shown in Table 2, as follows. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Effects of cryoprotectant agent (CPA) on the sciatic nerves 
               
            
           
           
               
               
            
               
                   
                 # of nerves with &gt;30% AP Recovery at 180 min 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Averaged normalized 
               
               
                   
                 CPA  
                   
                 AP amplitude  
               
               
                   
                 Exposure 
                 w/Freezing 
                 at 180 min 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 5% M22 
                 3/4 
                 4/7 
                 0.3 
               
               
                 5% DMSO 
                 3/3 
                 1/4 
                 0.37 
               
               
                 10% DMSO 
                 3/3 
                 0/7 
                 0.11 
               
               
                 15% DMSO 
                 3/5 
                 0/3 
                 0 
               
               
                 5% DMSO + 0.2M 
                 4/4 
                 0/4 
                 0 
               
               
                 Trehalose 
               
               
                   
               
            
           
         
       
     
     As shown in  FIGS. 13D and 14D , the phrenic nerves and the sciatic nerves do not present any detectable AP after freezing. As shown by comparing  FIG. 13A  with  FIGS. 13B and 13C  (also by comparing  FIG. 14A  with  FIGS. 14B and 14C ), applying the CPA solution to the nerves reduced the action potential compared to when no CPA was present. As shown by comparing  FIG. 13D  with  FIGS. 13E and 13F  (also by comparing  FIG. 14D  with  FIGS. 14E and 14F ), applying the CPA solution to the nerves protected the nerves from freezing so that a detectable AP was observed following freezing when the nerves were exposed to the CPA composition. 
       FIGS. 15A-15C  show effects of the CPA and freezing on the phrenic nerves over time.  FIG. 15A  shows results for a toxicity screening in which the nerves were exposed to CPA without freezing. AP amplitude prior to exposure to CPA (−30 minutes), at exposure to CPA (0 minutes), and 60 minutes, 120 minutes, and 180 minutes following exposure to the CPA for the porcine phrenic nerves. The CPAs tested were: 5% DMSO+0.4M Trehalose; 5% DMSO; 0.4M Trehalose; 10% DMSO; 10% EG-Sucrose; 5% DMSO+0.2M Trehalose; and 5% M22.  FIG. 15B  shows AP amplitude over time for the phrenic nerves for a treatment screening with the nerves exposed to both the CPA solution and to freezing for the following CPA solutions: 5% DMSO+0.4M Trehalose; 5% DMSO; 0.4M Trehalose; 10% DMSO; 10% EG-Sucrose; 5% DMSO+0.2M Trehalose; and 5% M22.  FIG. 15C  shows a comparison between a control (no CPA or freezing), direct freezing, toxicity, and treatment for 5% DMSO+0.2M Trehalose. 
       FIGS. 16A and 16B  show effects of the CPA and freezing on the sciatic nerves over time.  FIG. 16A  shows AP amplitude prior to exposure to CPA (−30 minutes), at exposure to CPA (0 minutes), and 60 minutes, 120 minutes, and 180 minutes following exposure to the CPA for the rat sciatic nerves.  FIG. 16B  shows AP amplitude over time for the sciatic nerves exposed to both the CPA solution and to freezing. The CPAs tested were 5% DMSO, 7.5% DMSO, 10% DMSO, 15% DMSO, 20% DMSO, and 5% M22.  FIG. 16C  shows a comparison between a control (no CPA or freezing), direct freezing, toxicity, and treatment for 5% M22. 
     As previously described, the cryoprotective composition can comprise a fluid agent, such as a gel, that increases the viscosity of the composition. In some examples, the fluid agent comprises cellulose, such as hydroxyethyl cellulose.  FIGS. 17A-17D  show experimental results for toxicity tests for a composition comprising 1% hydroxyethyl cellulose ( FIG. 17A ), 2% hydroxyethyl cellulose ( FIG. 17B ), 3% hydroxyethyl cellulose ( FIG. 17C ), or 4% hydroxyethyl cellulose ( FIG. 17D ). In each case, an action potential was observed for the nerves following exposure to the composition comprising hydroxyethyl cellulose. 
       FIG. 18  shows effects of a composition comprising a gel (e.g., hydroxyethyl cellulose) in combination with a cryoprotectant agent. Specifically,  FIG. 18  compares the normalized action potentials over time for 2% hydroxyethyl cellulose without freezing (left side of  FIG. 18 ), 2% hydroxyethyl cellulose with freezing (middle of  FIG. 18 ), and 2% hydroxyethyl cellulose, 5% DMSO, and 0.2M Trehalose (right side of  FIG. 18 ). As shown in  FIG. 18 , each example including the 2% hydroxyethyl cellulose composition generated action potentials, indicating that 2% hydroxyethyl cellulose is not toxic for the phrenic nerve cells. Further, the averaged normalized AP amplitude over 180 minutes (as shown in following Table 3) for a composition with 2% hydroxyethyl cellulose alone was found to be 1.02. In contrast, the averaged normalized AP amplitude over 180 minutes for the composition of 2% hydroxyethyl cellulose, 5% DMSO, and 0.2M Trehalose was 1.3, indicating that addition of the cryoprotectant agent increased action potential amplitude compared to when no cryoprotectant agent was present. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Effect of gel/CPA on the phrenic nerves 
               
            
           
           
               
               
            
               
                   
                 # with &gt;50% AP Recovery at 180 min 
               
            
           
           
               
               
               
               
            
               
                   
                   
                   
                 Averaged 
               
               
                   
                 CPA 
                 With 
                 normalized AP 
               
               
                   
                 Exposure 
                 Freezing 
                 amplitude at 180 min 
               
               
                   
               
               
                 2% Hydroxyethyl cellulose 
                 2/2 
                 3/3 
                 1.02 
               
               
                 2% Hydroxyethyl  
                 3/3 
                 4/4 
                 1.3  
               
               
                 cellulose + 5% 
                   
                   
                   
               
               
                 DMSO + 0.2M Trehalose 
               
               
                   
               
            
           
         
       
     
     As previously discussed, another example of a gel-like fluid agent, which can be used with the cryoprotective composition of the present disclosure, is a hyaluronic acid composition, such as sodium hyaluronate.  FIGS. 19A and 19B  show experimental results for toxicity tests for a composition comprising 1% sodium hyaluronate ( FIG. 19A ) and 2% sodium hyaluronate ( FIG. 19B ). In each case, an action potential was observed for the nerves following exposure to the composition comprising the sodium hyaluronate.  FIG. 19C  shows experimental results for toxicity tests for a composition comprising 1% sodium hyaluronate, 5% DMSO, and 0.2M trehalose. An action potential was also observed for the nerves following exposure to this composition, which indicates that the composition has low toxicity or at least that addition of sodium hyaluronate does not substantially modify toxicity of a composition comprising 5% DMSO and 0.2M trehalose. 
     Example 3: Evaluation of Cryoprotectant Agent and Composition Osmolality and Viscosity 
     Examples were also performed to evaluate osmolality of cryoprotectant agents of the present disclosure. As previously described, in some examples, the cryoprotectant agent of the present disclosure comprising trehalose desirably has an osmolality of about 270 mOsm/kg and a pH of about 7.0. Experimental results showing osmolality for different compositions comprising cryoprotectant agents, specifically trehalose, are shown in Table 4. As previously described, Dimethyl sulfoxide (DMSO) can also be added to the composition to improve cryoprotectant characteristics of the composition. 
     The compositions listed in Table 4 were made by mixing the following components in a vial to form a solution: Dimethyl sulfoxide (DMSO) (Sigma-Aldrich™, Saint Louis, Mo., USA); D-(+)-Trehalose dihydrate (Sigma-Aldrich™, Saint Louis, Mo., USA); and Phosphate-buffered saline (PBS) (Bichsel, Interlaken, Switzerland). Specifically, the PBS buffered solution and water were first mixed at room temperature. Then the trehalose and DMSO (if present) were added and solubilized at room temperature by vortex (5 minutes). Acidity and osmolality of the resulting solutions was measured at a temperature of 25° C. Acidity (pH) of the solutions was measured by a pH meter (SevenCompact Cond meter S230, Mettler Toledo, Switzerland). Osmolality of the solutions was measured by an Osmometer (OsmoTECH® XT, Advanced Instruments, Norwood, Mass., USA). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Osmolality of cryoprotectant agents 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 Volume 
                 Volume 
                   
                   
               
               
                 DMSO 
                   
                 H 2 O 
                 PBS 
                   
                 Osmolality 
               
               
                 (mL) 
                 Trehalose 
                 (mL) 
                 (mL) 
                 pH 
                 (mOsm/Kg) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 None 
                 0.15M 
                 0.5 
                 0.5 
                 7.2 ± 0.2 
                 271 ± 8  
               
               
                 None 
                 0.2M 
                 0.5 
                 0.5 
                 7.2 ± 0.2 
                 309 ± 20 
               
               
                 None 
                 0.2M 
                 0.8 
                 0.2 
                 7.2 ± 0.2 
                 247 ± 22 
               
               
                 None 
                 0.2M 
                 None 
                 1 
                 7.3 ± 0.2 
                 415 ± 20 
               
               
                 None 
                 0.2M 
                 1.0 
                 None 
                 6.0 ± 0.2 
                 192 ± 20 
               
               
                 0.05 
                 0.2M 
                 0.475 
                 0.475 
                 7.3 ± 0.2 
                 1070 ± 149 
               
               
                   
               
            
           
         
       
     
     In some examples, an osmolality value of about 300±30 mOsm/kg can be desirable for a composition comprising trehalose. As shown by Examples 1-3 of Table 4, several trehalose solutions were made having osmolality values within the range of 300±30 mOsm/kg. In Example 6 of Table 4, adding DMSO to the composition increases osmolality above 300±30 mOsm/kg; however it is believed that an effective cryoprotective composition is obtained. Also, adding DMSO does not substantially effect acidity (pH) of the composition. 
     Formulations of hyaluronic acid and hydroxyethyl cellulose were also prepared to determine viscosity and to confirm that the cryoprotective compositions of the present disclosure are injectable. Experimental results showing viscosity and injectability for cryoprotective compositions of the present disclosure are shown in Table 5. 
     The compositions of Table 5 were made with the following components: Dimethyl sulfoxide (DMSO) (Sigma-Aldrich™, Saint Louis, Mo., USA); D-(+)-Trehalose dihydrate (Sigma-Aldrich™, Saint Louis, Mo., USA); 2-Hydroxyethyl cellulose average Mv ˜1,300,000 (Sigma-Aldrich™, Saint Louis, Mo., USA); and/or Laboratory grade hyaluronic acid sodium salts, 1.75-2.0 MDa molecular weight (MW), (Contipro, Dolní Do-brouc, Czech Republic). The hydroxyethyl cellulose or hyaluronic acid was contained in a syringe, specifically a 3 mL Syringe, Luer-Lok™ Syringe by Becton, Dickinson and Co. (Franklin Lakes, N.J.). 
     The compositions were prepared by the previously described method shown in  FIG. 1B . Specifically, both 100 mg of hyaluronic acid and 200 mg of hydroxyethyl cellulose were solubilized overnight (vortex) in 5 ml of PBS solution. Then the resulting gels were withdrawn with a 5 ml syringe. The gels were then mixed in a vial (Type I glass vial by Schott, Mainz, Germany) containing either 5 ml of DMSO (10%) or 5 ml of water. Then the mixtures of the gel/DMSO (10 ml) were withdrawn from the vials with a 10 ml syringe and mixed with a vial containing 750 mg of trehalose by manual mixing with a needle until complete dissolution of the trehalose occurred. 
     Viscosity of the composition was measured and the composition was ejected from the syringe to assess injectability of the cryoprotective composition. Experimental results for the composition are shown in Table 5. Injectability of the resulting compositions (shown in Table 5) was described as “good” by subjective testing with several people (n=6). In future testing, a texture analyzer may be used to determine quantitative values for texture of the resulting solutions. Additional test can be done with a contact free rheometer. Also, injectability forces may be evaluated using sensors to measure forces applied to an appropriate syringe and needle. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Viscosity and injectability of compositions 
               
            
           
           
               
               
               
               
               
            
               
                 DMSO  
                   
                   
                 Viscosity at 
                 Inject- 
               
               
                 (mL) 
                 Trehalose 
                 Syringe 
                 20° C. (1 s −1 ) 
                 ability 
               
               
                   
               
               
                 5 mL DMSO 
                 750 mg  
                 5 mL Hyaluronic  
                 8.97 ± 0.06  
                 Good 
               
               
                 (10%) 
                 Trehalose 
                 acid (2%) 
                 Pa · s 
                   
               
               
                 5 mL water 
                 None 
                 5 mL Hyaluronic  
                 6.79 ± 0.01  
                 Good 
               
               
                   
                   
                 acid (2%) 
                 Pa · s 
                   
               
               
                 5 mL DMSO 
                 750 mg  
                 5 mL Hydroxyethyl 
                 6.19 ± 0.01  
                 Good 
               
               
                 (10%) 
                 Trehalose 
                 cellulose (4%) 
                 Pa · s 
                   
               
               
                 5 mL water 
                 None 
                 5 mL Hydroxyethyl 
                 8.09 ± 0.15 
                 Good 
               
               
                   
                   
                 cellulose (4%) 
                 Pa · s 
               
               
                   
               
            
           
         
       
     
     Example 4: Cryoprotective Compositions Comprising PBS Solution 
     Previously described experimental Examples 1 and 2 showed toxicity and freezing effects for compositions without a saline buffer solution or for a composition comprising Krebs solution. By contrast, experimental results for phrenic nerve CPA toxicity and freezing testing for a cryoprotective composition comprising PBS solution are shown in  FIGS. 20A-20E .  FIGS. 20A-20E  are graphs showing changes in normalized action potential over time for different cryoprotective compositions (both with and without freezing).  FIGS. 20A and 20B  show effects of exposing the phrenic nerves to the composition without freezing. Specifically,  FIG. 20A  shows a control, where only PBS solution (without a CPA) was applied to the phrenic nerves; and  FIG. 20B  shows exposure of the phrenic nerves to composition comprising PBS, 5 weight % DMSO, and 7.5 weight % trehalose.  FIG. 20C  shows effects of freezing for the composition comprising PBS, 5 weight % DMSO, and 7.5 weight % trehalose.  FIGS. 20D and 20E  show toxicity and freezing effects for a composition comprising PBS, the CPA, and 1 weight percent of the hyaluronic acid (HA) gel. Specifically,  FIG. 20E  shows toxicity effects for a composition comprising PBS, 5 weight % DMSO, 7.5 weight % trehalose, and 1 weight % hyaluronic acid gel.  FIG. 20D  shows freezing effects for the composition comprising PBS, 5 weight % DMSO, 7.5 weight % trehalose, and 1 weight % hyaluronic acid gel. 
     Table 6 shows toxicity and freezing effects (e.g., normalized action potential values) for various compositions comprising the CPA and either Krebs solution (KHB) or PBS solution. The results in Table 6 show that compositions with either Krebs solution or PBS solution in combination with the CPA are capable of preserving action potential generation for the phrenic nerve cells. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Comparison of KHB and PBS buffer solutions 
               
            
           
           
               
               
            
               
                   
                 # of nerves with &gt;30% 
               
               
                   
                 AP Recovery at 180 min 
               
            
           
           
               
               
               
               
            
               
                 CPA on porcine phrenic 
                   
                   
                 Averaged normalized 
               
               
                 nerve 
                   
                   
                 AP amplitude at 180 
               
               
                 (CPA: 5% DMSO + 
                 CPA 
                   
                 min for CPA w/ 
               
               
                 7.5% Trehalose) 
                 Exposure 
                 w/Freezing 
                 freezing 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                 CPA in KHB 
                 6/6 
                 3/4 
                 0.48 
               
               
                 CPA in PBS 
                 2/2 
                 3/3 
                 0.57 
               
               
                 CPA in PBS + 1% HA 
                 3/3 
                 4/6 
                 0.29 
               
               
                 CPA in KHB + 1% HA 
                 5/5 
                  6/17 
                 0.27 
               
               
                 CPA in KHB + 2% 
                 5/5 
                 4/4 
                 1.28 
               
               
                 cellulose 
                   
                   
                   
               
               
                 2% cellulose in KHB (gel 
                 5/5 
                 3/3 
                 1.02 
               
               
                 alone) 
                   
                   
                   
               
               
                 1% HA in KHB alone (gel  
                 3/3 
                 0/3 
                 0 
               
               
                 alone) 
               
               
                   
               
            
           
         
       
     
     The preceding examples and embodiments of the invention have been described with reference to various examples. Modifications and alterations will occur to others upon reading and understanding the foregoing examples. Accordingly, the foregoing examples are not to be construed as limiting the disclosure.