Tissue preservation system

Abstract

The present invention provides methods and compositions for storage and preservation of tissues, for example peripheral nerve allografts, for extended periods of time at room temperature in a sterile tissue culture container or bag.

Description

[0001] TITLE OF THE INVENTION

[0002] TISSUE PRESERVATION SYSTEM

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. provisional application Serial No. 63 / 620,549, filed on January 12, 2024, which is herein incorporated by reference in its entirety.

[0005] FIELD OF THE INVENTION

[0006] The invention relates to the field of tissue storage and preservation, for example autograft or allograft tissue, and more specifically to the field of long-term tissue storage and preservation, for example peripheral nerve tissue storage and preservation.

[0007] BACKGROUND OF THE INVENTION

[0008] Allograft or other tissue samples are used to treat many diseases and / or defects. These grafts are procured from organ donors and must be stored to allow for viral and bacterial testing for safety prior to shipping to surgical centers for implantation into patients. Based on studies looking at viability of the cells in the grafts, recommendations have been given for implanting tissues as soon after recovery from donors as possible in order to maximize success. Safety testing takes a minimum of 7 days and more often 14-30 days for final clearance. Storage of tissue, such as allograft tissue, for transplantation or other scientific or medical purposes allows time for medical testing, recipient patient preparation, or to preserve tissues for other purposes. Storage conditions for allograft or other tissue samples may influence tissue viability, integrity, and / or sterility.

[0009] Improved tissue preservation methods and compositions that would allow for longer storage of tissues while maintaining viability would represent a significant advance in the field.

[0010] SUMMARY OF THE INVENTION

[0011] Briefly described, embodiments of this disclosure provide methods, compositions and an apparatus for tissue preservation. The present disclosure provides a process for peripheral nerve tissue preservation comprising storing the peripheral nerve tissue at room temperature in a tissue storage container or bag comprising serum-free culture medium for from at least 7 to 70 days prior to implantation, wherein at least 50%, at least 60% or at least 70% of the cells of said peripheral nerve tissue remain viable after said storing compared to the viability of the cells of the peripheral nerve tissue at day 0 (time of recovery from the tissue donor). In certain embodiments, the process comprises testing the tissue for viability at least once prior to implantation in a patient. Tn some embodiments, testing for viability comprises assaying the medium withdrawn from the container or bag. In other embodiments, testing for viability comprises adding a resazurin solution to the medium and determining the fluorescence level, wherein increased fluorescence indicates higher cell viability.

[0012] In further embodiments, the process comprises changing the medium at least once during the storing. In other embodiments, the process comprises changing the medium about once every two weeks during the storing.

[0013] In certain embodiments, the medium comprises Dulbecco's Modified Eagle Medium (DMEM), insulin, transferrin, selenium, at least a first antibiotic compound, at least a first antimycotic compound, L-glutamine, non-essential amino acids, ascorbic acid, dexamethasone and vitamin B 12. In some embodiments, the at least a first antibiotic compound comprises penicillin and streptomycin. In other embodiments, the at least a first antimycotic compound is amphotericin B. In yet other embodiments, the medium comprises penicillin, streptomycin and amphotericin B. In still other embodiments, the medium is serum-free.

[0014] In various embodiments, the peripheral nerve tissue is sensory nerve tissue (which carry sensory information from the body's sensory receptors (such as those for touch, temperature, and pain) to the central nervous system (CNS)), motor nerve tissue (which transmit signals from the CNS to the muscles and glands, allowing for voluntary and involuntary muscle movements and control of glandular secretions) or mixed nerve tissue (many peripheral nerves contain a mixture of both sensory and motor fibers, enabling them to transmit signals in both directions). In some embodiments, the peripheral nerve tissue is sciatic, peroneal, radial, ulnar or median nerve tissue. In certain embodiments, the peripheral nerve tissue comprises an allograft or an autograft. In other embodiments, the peripheral nerve tissue comprises an allograft or autograft, the process further comprising lavaging of the peripheral nerve tissue in isotonic solution prior to the storing. In yet other embodiments, the process further comprises implanting the tissue in a subject in need thereof following the storing. In certain embodiments, the room temperature is between about 19°C and about 27°C. In other embodiments, room temperature is about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, or about 27°C.

[0015] The present disclosure also provides a composition comprising Dulbecco's Modified Eagle Medium (DMEM), insulin, transferrin, selenium, at least a first antibiotic compound, at least a first antimycotic compound, L-glutamine, non- essential amino acids, ascorbic acid, dexamethasone and vitamin B-12. In certain embodiments the composition comprises penicillin, streptomycin and amphotericin B.

[0016] The foregoing and other aspects of the invention will become more apparent from the following detailed description.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows the cell viability of rat sciatic nerve allografts at day 0, day 30 and day 60. *p<0.05. The viability levels are: 0 - 0-9%; 1 - 10-19%; 2 - 20-29%; 3 - 30-39%; 4 - 40-49%; 5 - 50-59%; 6 - 60-69%; 7 - 70-79%; 8 - 80-89%; 9 - 90- 99%; 10 - 100%.

[0019] FIG. 2 shows the cell viability of canine sciatic nerve allografts at day 0, day 30 and day 60. *p<0.05. The viability levels are: 0 - 0-9%; 1 - 10-19%; 2 - 20-29%; 3 - 30-39%; 4 - 40-49%; 5 - 50-59%; 6 - 60-69%; 7 - 70-79%; 8 - 80-89%; 9 - 90- 99%; 10 - 100%.

[0020] FIG. 3 shows the viable cell density of the three MOPS® solutions at 0 days, 30 days, 60 days and 90 days. *p<0.05, **p<0.01, ***p<0.001.

[0021] FIG. 4 shows the viable cell density of the three MOPS® solutions at 30 days, 60 days and 90 days. **p<0.01, ***p<0.001.

[0022] FIG. 5. Apparatus for measurement of sciatic function index.

[0023] FIG. 6. Results of sciatic function index (gait) based on average of three pawprints. * p<.05, inter-group comparison. # p<05, comparison to pre-operative value.

[0024] FIG. 7. Apparatus for measurement of muscle coordination. FIG. 8. Results of muscle coordination based on measuring best of three trials of Rotarod walk time. * p<.05, inter-group comparison. # p<.05, comparison to pre-operative value.

[0025] FIG. 9. Apparatus for measurement of grip strength (muscle strength).

[0026] FIG. 10. Results of muscle strength based on measuring average of two trials of bilateral hind limb grip strength. * p<.05, inter-group comparison. # p<.05, comparison to pre-operative value.

[0027] FIG. 11 . Ratio of left (operated) to right (non-operated) gastrocnemius mass. * p<.05, inter-group comparison.

[0028] DETAILED DESCRIPTION

[0029] The following definitions and methods are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0030] The present disclosure provides a process and apparatus for tissue preservation. The process includes, in one embodiment, removing viable tissue, such as allograft or autograft tissue, from a donor, testing of the tissue for infectious diseases and / or mechanical and / or biochemical activity for viability, placement of the viable tissue into a sterile tissue container or bag as described herein with a culture medium capable of maintaining the viability and sterility of the tissue, and storing the tissue for extended periods of time prior to implantation into a recipient. As used herein, the term “allograft” refers to a tissue graft from a donor of the same species as die recipient but not genetically identical, whereas the term “autograft” refers to a tissue graft wherein the donor and the recipient are the same individual.

[0031] Allograft tissue can be removed from a donor by techniques known in the art. For instance, general aseptic surgical methods or other physical intervention of an allograft may include but are not limited to excision, resection, amputation, transplantation, microsurgery, general surgery, laser surgery, robotic surgery, or autopsy, among others.

[0032] Tissue or allograft sources may be peripheral nervous system tissue from all types of organisms, including, but not limited to human, porcine, ovine, bovine, canine, equine, and others. In one embodiment, the source of the tissue or allograft is human. Although the description herein may refer to allograft tissue, one of skill in the art appreciates that other tissues find use in the method.

[0033] Once removed from the donor, the allograft is stored within the sterile tissue culture chamber including, but not limited to, the chamber described herein, for an extended period of time. In one embodiment, the allograft is stored at room temperature in culture media. In specific embodiments, the room temperature is between about 19°C and 27°C, including about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, or about 27°C. In another embodiment, the allograft is stored at a temperature that is not less than about 12°C and not more than about 30°C.

[0034] As used herein, the term “culture media” refers to liquid, semi-solid, or solid media used to support tissue growth and / or preservation and / or development in a non-native environment. Further, by culture media is meant a sterile solution that is capable of stabilizing and preserving the tissue in order to maintain its biological activity, cell viability, and sterility. Suitable tissue culture media are known to one of skill in the art, as discussed in detail subsequently. The media components can be obtained from suppliers other than those identified herein and can be optimized for use by those of skill in the art according to their requirements. Culture media components are well-known to one of skill in the art and concentrations and / or components may be altered as desired or needed. The media-to-tissue ratio within the sterile tissue culture chamber may be about 10-50:1 per volume.

[0035] An unexpected benefit of the present procedure is that tissue samples can be maintained viable and sterile for an extended period of time relative to methods of the prior art. For instance, typically in the prior art, upon removal of an allograft from a donor, the tissue was stored on ice or at around 4°C. Tissues prepared according to this method tended to remain suitably viable for around 7-28 days. However, the procedure described herein provides for a surprising and unexpected increase in viability of allograft tissue. Tissues prepared and stored according to the procedure described herein remain viable for an extended period of time relative to storage at 4°C. By an extended period is meant at least between about 7-120 days, at least between about 20-90 days, or at least between about 20-70, about 40-70, about 50-90 or about 60-120 days. In one embodiment an extended period is meant up to at least about 70 days. It has been found that long-term storage of tissue may be facilitated by replacement of initial culture medium with fresh, sterile medium. The present disclosure provides a system and device that allows for just this. Thus, media can be conveniently changed as necessary. In one embodiment, the medium is changed at least once, twice, or three times during storage. The media may be changed, in specific embodiments, about once every other day, at least once a week, at least once every two weeks, or at least about once a month during storage.

[0036] The extended storage period allows for examination or testing of the allograft and / or culture media for a number of factors, such as viability, biological activity, blood type compatibility, HLA typing, genotyping, SNP detection, and / or infection with diseases. Compounds that may be detected or tested may be obtained from culture media withdrawn from the container or bag such as, but not limited to, bacterial or virus infections, nitric oxide, prostaglandin E2, matrix metalloproteinase (MMP)-2, MMP-3, MMP-9, and MMP-13, vascular endothelial growth factor (VEGF), interleukin (IL)-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-15, and IL-18, granulocyte macrophage colony-stimulating factor (GM-CSF), Interferon gammainduced protein (IP)- 10, IFNy, keratinocyte chemoattractant (KC), MCP-1 , and TNFa. Tissue may be tested using methods known in the art, such as by diagnostic PCR or with antibodies against biomarkers such as, but not limited to, those described above. The viability may also be monitored during storage by adding a resazurin solution to the media at a final concentration of about 10 pg / ml and incubated at room temperature for 18-24 hours. During the incubation, resazurin is converted to resorufin by viable cells. A 200 pl sample of the media can be taken and the fluorescence level determined using a fluorescence reader (540-570 nm excitation, 580-610 nm emission). Increased fluorescence is indicative of higher cell viability. Higher viability samples typically have a fluorescence reading of -800-1200 units using a Synergy HT set at a sensitivity of 25 on the reader.

[0037] In view of the above, the process provides for preservation of at least 50%, at least 60% or at least 70% of the peripheral nervous allograft tissue after storage at room temperature for at least 21 , at least 28, at least 35, at least 42 or at least 45 days. In an embodiment, at least 50%, at least 60% or at least 70%, up to at least around 99%, including about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% or greater of the tissue is preserved when stored for 21 days, 28 days, 35 days, 42 days, 45 days, 60 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 105 days, 110 days, 115 days, or 120 days or more.

[0038] In one embodiment, the process includes storing the tissue in a tissue storage container or bag, for example an FDA-approved sterile tissue storage container or bag. In another embodiment, the process further includes implanting the tissue in a subject in need thereof following the storing.

[0039] Having now generally described the present disclosure, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present disclosure, unless specified. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the present disclosure. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

[0040] EXAMPLES

[0041] Example 1 - Preservation of Peripheral Nerve Tissue in MOPS®

[0042] The present disclosure provides a system comprising a container and a solution for preserving human neural tissue (peripheral nerve, spinal cord) from tissue donors for subsequent use in allograft transplants. The Missouri Osteochondral Preservation System (MOPS®) has proven safe and effective for accomplishing similar goals for osteochondral and meniscal allograft transplantation, and is currently in widespread clinical use across North America. Therefore, MOPS® provides an ideal platform from which to optimize peripheral nerve allograft tissue preservation for transplantation. The container is preferably made of medical grade materials, suitable for transport and shelf-stable storage, and sealable. The solution is comprised of a cell or tissue culture media, such as Dulbecco’s Modified Eagle Medium (DMEM), high or low concentrations of glucose, antibiotic compounds, antimycotic compounds, dexamethasone, and one or more of the following: neurotrophic factor(s) such as glial cell-derived neurotrophic factor, cobalamin, neurotrophin-3, neurotrophin-4, epidermal growth factor, fibroblast growth factor, hydroxytyrosol, immunomodulator(s) such as FK506, etanercept, adalimumab, infliximab, ustekinumab, cyclosporin A, and / or anti-fibrosis factors such as nintedanib, pirfenidone, hyaluronan - among other chemicals and compounds, and may or may not contain serum. The neural tissue can be preserved and stored at 10-40°C for up to 120 days after recovery from the tissue donor such that at least 50% of the tissue preserved by this method remain viable.

[0043] Development and validation of the present system that can preserve fresh peripheral nerve tissue to allow for clinical accessibility and feasibility for consistently successful large-gap peripheral nerve transplants revolutionizes clinical practice and enhances donor tissue use and impact. The present tissue preservation method that enhances the ability to implement PEGf into clinical practice and mitigate the need for autograft nerve tissue, eliminating the donor site morbidity and functional inadequacies associated with peripheral nerve autografts, greatly increases the number of functional digits, hands, feet, and extremities that can be saved. The presently described MOPS® system, which does not require refrigeration, allows for use in settings without refrigeration capabilities, such as wartime environments and developing world communities.

[0044] The present study was designed to assess shelf-stable preservation of peripheral nerve tissue in MOPS® (500 ml DMEM, 5 ml ITS (insulin (10 mg / L), transferrin 55 mg / L), selenium (6.7 pg / L)), 5 ml total penicillin (100 U / ml), streptomycin (100 pg / ml), amphotericin B (2.5 pg / ml), 5 ml L-glutamine (2 mM), 5 ml non-essential amino acids (0.1 mM), 1 ml ascorbic acid (50 ng / ml), and 5.46 ml dexamethasone 0.1 pM)).

[0045] Sciatic nerve was recovered from canine specimens. Each nerve was preserved in MOPS® for a total duration of 56 days. At Day 0, Day 14, Day 28, and Day 56, longitudinal and cross sections of each nerve were analyzed for cell viability and histology. To assess cell viability, sections of the preserved nerve at each time point were stained in phosphate buffered saline containing the live cell stain calcein AM (1 mg / mL) and the dead stain Ethidium homodimer-1 (1 pM; Thermo Fisher Scientific) for 30 minutes at room temperature. After staining, tissues were rinsed with phosphate buffered saline to remove excess stain, and full-thickness images of stained neural tissue were obtained at 4x using a BX51 fluorescence microscope (Olympus). Images were obtained using an F-View II 12-bit B&W CCD camera (Olympus). In this assay, viable cells appear green and nonviable cells appear red. Viability was assessed subjectively for cell distribution at each time point. For histological evaluation, nerves were stained with hematoxylin and eosin (H&E) and luxol fast blue counterstained with the periodic acid-Schiff (LFB-PAS). These visual analyses demonstrate cell viability through Day 56. This data shows that standard MOPS® can preserve neural tissue viability for sufficient duration to allow for clinical application.

[0046] Another study was conducted using canine and rat sciatic nerve allografts. 18 sciatic nerve allografts were obtained from canine, and 24 sciatic nerve allografts were obtained from rat. The canine and rat sciatic nerve allografts were each split into three groups. One group was stored for 0 days (baseline), one group was stored for 30 days at room temperature (21°C), and one group was stored for 60 days at room temperature (21 °C). Cell viability was assessed using the following scoring scale: Score 0 - 0-9% viability level, Score 1 - 10-19% viability level, Score 2 - 20- 29% viability level, Score 3 - 30-39% viability level, Score 4 - 40-49% viability level, Score 5 - 50-59% viability level, Score 6 - 60-69% viability level, Score 7 - 70-79% viability level, Score 8 - 80-89% viability level, Score 9 - 90-99% viability level, Score 10 - 100% viability level.

[0047] To assess tissue viability, sections of the preserved nerve at each time point was stained in phosphate buffered saline containing the live cell stain calcein AM (1 mg / mL) and the dead stain SYTOX Blue (1.25 mM; Thermo Fisher Scientific) for 30 minutes at room temperature in a dark room. After staining, tissues were rinsed with phosphate buffered saline to remove excess stain, and full-thickness images of stained neural tissue were obtained at 4x using a BX51 fluorescence microscope (Olympus). Images were obtained using an F-View II 12-bit B&W CCD camera (Olympus). In this assay, viable cells appear green and nonviable cells appear red. Viability was assessed subjectively based on comparison to day-0 tissues.

[0048] After routine histologic processing, sectioning, and staining, H&E and LFB- PAS -stained slides were used for evaluation using a standardized in-house scoring system for peripheral nerve histopathology for which higher scores indicate more severe pathology (Table 1).

[0049] Table 1

[0050]

[0051] The cell viability results for the canine sciatic nerve allografts preserved in MOPS® at day 0, day 14, day 28 and day 56 demonstrated excellent cell viability through Day 56. Mild axonal atrophy with some degeneration of neural support tissue was demonstrated, which was most apparent at Day 56 in canine nerves preserved MOPS®. Cell viability results for rat sciatic nerve allografts are shown in FIG. 1, and the cell viability results for the canine sciatic nerve allografts are shown in FIG. 2. The cell viability staining results for the rat sciatic nerve allografts and canine sciatic nerve allografts at day 0, day 30 and day 60 again show excellent cell viability. Histological analysis of the rat and canine sciatic nerve allografts confirmed the excellent cell viability at day 30 and day 60.

[0052] The results showed that MOPS® maintained sufficient nerve allograft viability at both 30 days and 60 days. This is a significant improvement over previous storage methods that see a steep decline at 30 days to less than 50% viability.

[0053] Example 2 - Optimization of a Shelf-Stable Preservation System for Fresh (Viable) Nerve Allograft Tissue for Treatment of Peripheral Nerve Injuries (PNI)

[0054] The annual incidence of traumatic peripheral nerve injuries (PNIs) in the U.S. is 2% to 5% of patients admitted to a Level I trauma center. Unlike other tissues, regeneration of nerves is slow (roughly 1 mm per day) once sprouting occurs. As a result, suboptimal outcomes with large defects or more proximal injuries are common as the motor end plates degrade prior to the regenerating nerve reaching its target. Further, sprouting nerves are susceptible to local factors that prevent functional regeneration. Following immediate loss of axonal integrity, cessation of action potentials ensues. Within 1-3 days, irreversible Wallerian degeneration affects axons distal to the site of injury. It is possible for unaided regeneration to occur from the surviving proximal stump, but many times this process is not expeditious enough to result in any meaningful recovery of the affected digit(s) or extremity. When large gaps occur, the situation is more challenging. Despite being the gold standard for large nerve defects (>4 cm), small sensory nerve autografts generally produce limited functional recovery at the recipient nerve site at best with sustained sensory deprivation at the donor nerve site. When using such autografts for motor or mixed motor / sensory nerve repair, there is typically a size and axon count mismatch in addition to function type mismatch. Thus, the ability to use nerve allografts that are size-, axon count- and orientation-, as well as function-matched is appealing. Presently, only decellularized nerve allografts are available for clinical use due to the limitations with immune response to fresh (viable) nerve allografts. Decellularized nerve allografts are also limited as these provide a scaffold by which to guide nerve regeneration but are still subject to the hurdles associated with Wallerian degeneration.

[0055] When achieved successfully, the technique of polyethylene glycol-mediated nerve fusion (PEGf) has been shown to prevent severed axons from undergoing Wallerian degeneration. This results in enhanced functional recovery over a shorter duration. Successful PEGf has been described in animal models for primary nerve repair as well as nerve reconstruction with allografts, and there is a case report of successful nerve repair using PEGf in a human patient. Recent data have also demonstrated that PEGf results in a degree of immunoprotective effect, which has historically been one of the primary limitations for successful fresh nerve allograft transplantation. Successfully translating the PEGf peripheral nerve allograft transplantation technique to human clinical use for PNIs with large gaps not amenable to primary repair is dependent on the ability to preserve viability and function in donor nerve tissue for a duration that allows for recovery, processing, disease testing, scheduling, distribution, and subsequent implantation. Presently, there is no such system validated for clinical use. The Missouri Osteochondral Preservation System (MOPS®) has proven safe and effective for accomplishing similar goals for osteochondral and meniscal allograft transplantation, and is currently in widespread clinical use across North America. Therefore, MOPS® provides an ideal platform from which to optimize peripheral nerve allograft tissue preservation for transplantation. Based on the storage conditions and success in preserving other fresh tissues for sufficient duration and quality for clinical success, the MOPS® methodology has the potential to address current limitations to consistently and successfully treat large-gap peripheral nerve injuries.

[0056] The present study was designed to assess shelf-stable preservation of peripheral nerve tissue in MOPS® (500 ml DMEM, 5 ml ITS (insulin, transferrin, selenium), 5 ml total penicillin, streptomycin, amphotericin B, 5 ml L-glutamine, 5 ml non-essential amino acids, 1 ml ascorbic acid, and 5.46 ml dexamethasone), MOPS®- N1 (MOPS® + glial cell-derived neurotrophic factor (GDNF; 20 pg in 500 ml MOPS® media)), and MOPS®-N2 (MOPS® + 1 g Cobalamin (vitamin B-12; 1.47 mM in 500 ml MOPS® media)) in an ex vivo setting. While the data shown above in Example 1 supports the use of standard MOPS® for peripheral nerve allograft preservation, a neural tissue-specific formulation may further enhance functional preservation of peripheral nerve allografts. Glial cell-derived neurotrophic factor (GDNF) is a promising addition to MOPS® as it has been reported to promote survival and axonal regeneration when added to sensory nerve grafts prior to use for reconstruction of motor neurons. The addition of cobalamin to MOPS® is attractive as Vitamin B complex is known to help alleviate degeneration in the nervous system by acting as a scavenger of reactive oxygen species and by providing neuroprotective function with anti-apoptotic and anti-necrotic effects on neurons. Each of these additives provides a different compelling and innovative mechanism for potential optimization of MOPS® for neural tissue preservation while also being considerate of a feasible pathway to clinical use.

[0057] Using viable cell density data, a one-way ANOVA power analysis was performed, which determined that a sample size of 5 rat sciatic nerve explants per group / time point would be sufficient to reach the desired power of 0.8 with alpha = 0.05. The end points 0, 30, 60, and 90 days after recovery were based on tissue bank logistics and practical application towards clinical use. The outcome measures, tissue viability, histological score, and compound action potentials, were selected to best inform likelihood for functional success in vivo.

[0058] To assess tissue viability, sections of the preserved nerve at each time point was stained in phosphate buffered saline containing the live cell stain calcein AM (1 mg / mL) and the dead stain Ethidium homodimer- 1 (1 pM; Thermo Fisher Scientific) for 30 minutes at room temperature in a dark room. After staining, tissues were rinsed with phosphate buffered saline to remove excess stain, and full-thickness images of stained neural tissue were obtained at 4x using a BX51 fluorescence microscope (Olympus). Images were obtained using an F-View II 12-bit B&W CCD camera (Olympus). In this assay, viable cells appear green and nonviable cells appear red. Viability was assessed subjectively based on comparison to day-0 tissues to determine percentage of viable (green) cell density (%VCD) in each group at each time point.

[0059] After routine histologic processing, sectioning, and staining, H&E and LFB- P AS -stained slides were used for evaluation using a standardized in-house scoring system for peripheral nerve histopathology for which higher scores indicate more severe pathology (Table 1, above).

[0060] Compound action potentials (CAPs) were assessed as a method of evaluating electrical viability and functionality of the nerve tissue. CAPs were extracellularly stimulated in vitro and recorded by placing stimulating and recording electrodes at least 1 cm apart across a section of nerve allograft. The amplitude was measured from the baseline to the peak of the CAP.

[0061] Statistical Analyses: Statistically significant (p<0.05) differences among groups were assessed using one-way ANOVA (%VCD, CAP) or ANOVA on ranks (histopathology score). Significant differences within groups over time were assessed using repeated measures ANOVA (%VCD, CAP) or repeated measures ANOVA on ranks (histopathology score).

[0062] The results of the studies are shown below in Table 2 (MOPS®), Table 3 (MOPS® + GDNF) and Table 4 (MOPS® + B-12).

[0063]

[0064] Table 3

[0065] Table 4

[0066] FIG. 3 shows the results of the viable cell density of the three MOPS® solutions at 0 days, 30 days, 60 days and 90 days. FIG. 4 shows the results of the viable cell density of the three MOPS® solutions at 30 days, 60 days and 90 days.

[0067] Example 3 - MOPS®-Preserved Peripheral Nerve Allograft Transplantation using PEG-fusion (PEGf) Repair in a Validated Rodent Model of Sciatic Nerve Injury

[0068] In this study, a rat sciatic nerve gap model using PEGf was utilized to compare control peroneal autograft reconstruction (Group 0), PEG fusion + plus cellularized sciatic nerve allograft stored in MOPS®, MOPS®-N1, or MOPS®-N2 at room temperature for 30 or 60 days after recovery (Group la and Group lb), or PEG fusion + plus viable sciatic nerve allograft stored in Normosol at 4°C for 30 or 60 days after recovery (Group 2a and Group 2b). The use of MOPS®, MOPS®-N1, or MOPS®-N2 preserved nerves was informed based on the data collected in Example 2, above.

[0069] The PEG fusion technique to be used for each group was that previously described (Ghergherehchi et al., J. Neurosci. Meth. 314:1-12, 2019). Adult Sprague Dawley rats were used for this study. Animals were handled for acclimation and trained in the testing procedure for at least one week prior to surgery. Rats were housed at room temperature, kept on a reverse 12:12 light cycle, and given food and water ad libitum. On the day of surgery, the lateral aspect of the left hindlimb was trimmed of fur and disinfected with alternating swabs of 10% iodine / povidone and 70% ethyl alcohol. The right hindlimb served serve as the intact control. Rats were anesthetized using a small mammalian anesthetic device. Animals were induced to anesthesia with 4% inhaled isoflurane and kept at 2-2.5% for the duration of the surgery. An oxygen flow rate of 0.5-1 L / min was maintained. Ophthalmic ointment was applied to each eye to prevent drying. A 2-3.5 cm incision was made through the skin and the left biceps femoris muscle was reflected to expose the sciatic nerve. Connective tissue around the sciatic nerve was trimmed with microscissors. Complete sciatic nerve transections were made in calcium-containing isotonic extracellular fluid and / or sterile isotonic Lactated Ringers by fine-dissection scissors to completely sever all axons as well as their endo-, peri-, and epineural sheaths. A 6-8 mm segment was ablated in mid-thigh, leaving an 8-10 mm gap between cut axonal ends. Because intact nerves are under tension, an ablation produces a gap that is several mm longer than the removed segment. Nerve allograft 1-3 mm longer than the gap created was procured to produce tension-free neurorrhaphies at each repair site. Based on assigned group, a donor peripheral nerve allograft that matches the diameter of the host nerve was selected. Identical procedures for neurorrhaphy and PEG-fusion were performed for the proximal and distal ends of all nerve co-optation sites. The graft nerve and host sciatic nerves were washed with hypotonic Plasmalyte A and 1% methylene blue (Acros Organics). All axonal ends will be trimmed to provide smooth cut ends whose flat planes can be very closely apposed with at least four 10-0 microsutures through the epineurium. Nerves then receive a sterile hypotonic solution of 50% w / w 3.35 kDa PEG (Sigma- Aldrich) in distilled water directly applied for 1-2 min to the lesion sites to nonspecifically repair / fuse closely apposed cut axonal ends. After neurorrhaphy, lesion sites of PEG-fused nerves were washed several times with sterile isotonic Lactated Ringers containing calcium to repair any remaining axolemmal holes with calcium-induced vesicles or other membrane-bound structures. After assaying for CAPs and / or CMAPs (see below), muscle and skin incision were closed with 5-0 sutures and / or wound clips. Animals recovered from surgery on heated pads and were returned to standard housing. Animals received a 5 mg / kg subcutaneous injection of carprofen.

[0070] Recording of CAPs and compound muscle action potentials (CMAPs) was performed across the intact native nerve, across the nerve after resection, and again across the reconstruction site to confirm successful PEG fusion. Stimulating and recording wire-hook electrodes were placed at 2-4 mm proximal and 2-4 mm distal, respectively, to the implanted allograft nerve. CAPs and CMAPs were digitally recorded with a Powerlab data acquisition system (AD Instruments) to assess whether axonal continuity was reestablished through the graft across both coaptation sites. CAP conduction across a successful PEG-fused graft evoked twitches of muscles innervated distal to the graft. Before sciatic nerve transection, after transection, and after PEG-fusion, CAPs and CMAPs were recorded to assess electrophysiological continuity as evidence of restored axonal integrity. Through-conduction confirms successful PEG-fusion of axons having axolemmal and cytoplasmic continuity restored at both coaptation sites. In this setting, the CAPs and CMAPs are regarded as a binary yes / no measure of PEG-fusion success. Sciatic functional index (SFI) was used as a functional outcome measure during the postoperative period. The SFI is a common and reliable test to assess function and return of behaviors mediated by the sciatic nerve in rats and mice. The SFI score is highly dependent upon foot flexion and toe spread, movements controlled by lower leg and foot muscles innervated by the sciatic nerve, e.g., tibialis anterior, soleus, or gastrocnemius muscles. Any recovery is typically associated with gradual decreases in the absolute value of negative SFI scores as axons regenerating by outgrowth from severed surviving proximal axonal stumps that increasingly reinnervate more denervated distal muscle fibers.

[0071] For SFI tests, both hind paws were marked with red or blue ink, right side unoperated and left injured, respectively. Rats were placed upon an inclined 100 mm wide board, 5 ft long, lined with paper strips. After a rat runs back to its home cage across the paper-lined board, footprints were measured by testers blinded to treatment. Three variables were measured for both normal and experimental footprints: footprint length, total toe spread, and intermediate toe spread. The average of the two runs was recorded as the SFI score for the day. Animals were first tested 3d after surgery, then weekly for at least 42 d post-operatively (PO). Data were recorded in spreadsheets and graphs prepared in Microsoft Excel. In order to obtain reliable results, the criteria outlined by Ghergherehchi et al. supra) were used. Animals must run for three consecutive steps for each hindlimb without stops or hesitation, which can create artificially long footprints. If the animal stepped with the dorsum of its paw, this was excluded as it creates an artificially short footprint. It has also been observed that the contralateral hindlimb sometimes compensates for the injured limb by increasing weight bearing, which increases the print length of the unoperated limb. This compensation rarely significantly affects the SFI score. Animals were excluded from the study if they exhibited signs of autotomy, which results in the complete removal of digits in the affected hind limb. When these specific selection criteria are followed, the SFI is a reliable measure of functional return of sciatic mediated behaviors.

[0072] At the 6 weeks postoperative time point, the reconstructed nerve was recovered and assessed for graft viability and histological scoring as described in Example 2, above. Masson’s Trichrome stained slides were used to assess area of fibrosis using Clemex Vision PE software version 8.0 (Clemex Technologies Inc, Longueuil, Quebec). Immunohistochemical analysis to assess for mature axon count (using ncurofilamcnt heavy chain [NF-H]), immature axon count (with Growth Associated Protein 43 [GAP- 43]), and vascularization (using cluster of differentiation 31 [CD-31]) was performed on sections of both the transplanted nerve and controls. In all fields, NF-H, GAP-43 and CD-31 immunopositive cells were identified and scored from 0 to 5 points (0: no positive cells, 1: fair, 2: fair to moderate, 3: moderate, 4: moderate to intensive, 5: intensive antibody staining). All fields’ means will be calculated.

[0073] Statistical analyses: Statistically significant (p<0.05) differences among groups were assessed using one-way ANOVA (%VCD, CAP, CMAP, SFI) or ANOVA on ranks (histopathology score). Significant differences within groups over time were assessed using repeated measures ANOVA (%VCD, CAP, CMAP, SFI) or repeated measures ANOVA on ranks (histopathology score).

[0074] Example 4 - Studies Comparing MOPS®-N and Normosol® in Rats

[0075] A variety of studies were performed on rats to compare the effectiveness of rat sciatic nerve allografts stored for 28 or 56 days in MOPS®-N or normosol®. Studies were also performed on autografts stored without PEGf.

[0076] Surgical Procedure. A 2-3.5 cm incision was made through the skin and the left biceps femoris muscle was reflected to expose the sciatic nerve. Connective tissue around the sciatic nerve was trimmed with microscissors. Complete sciatic nerve transections were made in calcium-containing isotonic extracellular fluid by fine-dissection scissors to completely sever all axons as well as their endo-, peri-, and epineural sheaths. A 6-8 mm segment was ablated in mid-thigh, leaving an 8-10 mm gap between cut axonal ends.

[0077] Based on assigned group, a donor peripheral nerve allograft that matched the diameter of the host nerve was selected. For the autograft group, the sciatic nerve was identified, explanted, rotated 180 degrees, and reimplanted. Identical procedures for neurorrhaphy and PEG-fusion were performed for the proximal and distal ends of all nerve co-optation sites.

[0078] The graft nerve and host sciatic nerves were washed with hypotonic Plasmalyte A and 1% methylene blue (Acros Organics). All axonal ends were trimmed to provide smooth cut ends whose flat planes can be very closely apposed with at least three 10-0 microsuturcs through the cpincurium. Nerves then receive a sterile hypotonic solution of 50% w / w 3.35 kDa PEG (Sigma-Aldrich) in distilled water will be directly applied for 1-2 min to the lesion sites to nonspecifically repair / fuse closely apposed cut axonal ends. After neurorrhaphy, lesion sites of PEG-fused nerves were washed several times with sterile isotonic Lactated Ringers containing calcium to repair any remaining axolemmal holes with calcium- induced vesicles or other membrane-bound structures. Muscle and skin incision were closed with 5-0 sutures and / or tissue glue.

[0079] Outcome measures. Functional outcome assessments were performed prior to surgical procedures, then weekly for 6 weeks post-operatively.

[0080] Sciatic Function Index (SFI). For SFI tests, the apparatus as previously described (Fricker et. al., Neural Regen. Res. 11:829-834, 2016) was utilized (FIG. 5). Rats were placed on an inclined 100 mm wide, 5 foot long board. Rats traveled up the board for a total of three trials. Images from each trial were taken and processed in ImageJ. Three variables were measured for both normal and experimental footprints: footprint length, total toe spread, and intermediate toe spread. The average of the three runs was recorded as the SFI score for the timepoint. The results are shown in FIG. 6.

[0081] Rotarod (Muscle Coordination). The rotarod motor coordination used a 5 cm diameter metal rod covered in grey rubber foam placed in a walking lane 5-6 cm wide (FIG. 7). The rod was set to increase rotation from 8 to 29 rpm over 60 seconds. The rat was lowered down onto the rod rotating at 8 rpm. The time until the rat falls off of the rod was recorded along with the reason for trial end (fall, jump, passive rotation). Procedure is repeated for a total of 3 trials separated by 10 min intertrial intervals and the average latency of the three tests was recorded as the latency to fall for the timepoint. The results are shown in FIG. 8).

[0082] Grip Strength. For grip strength test a grip strength meter is positioned horizontally. Rat test subjects were held by the tail / scruff of the neck and lowered towards the apparatus (FIG. 9). The animal was allowed to grab the T-bar and then pulled backwards in the horizontal plane. The force applied to the grid / bar just before the rat lost its grip was recorded as the peak tension. The average of two runs was recorded as the grip strength for the timepoint. The results are shown in FIG. 10. Muscle Mass Ratio. Muscle mass was evaluated by dissecting the gastrocnemius muscles from both the surgically treated (affected) and the contralateral non- surgical (unaffected) limbs of the rats at the conclusion of the experiment. Upon removal, each muscle was immediately weighed using a precision scale. The muscle mass ratio was calculated by dividing the mass of the gastrocnemius muscle from the surgical limb by the mass of the corresponding muscle from the non-surgical limb. The results are shown in FIG. 11.

[0083] In all studies after 6 weeks the results using the rat sciatic nerve tissue stored in MOPS®-N was superior to the results using the rat sciatic nerve tissue stored in normosol®, and in most instances superior to the results on autografts stored without PEGf.

[0084] Example 5 - MOPS®-Preserved Human Peripheral Nerve Allograft Transplantation

[0085] Source of the product including how the human cells, tissues, and cellular and tissue-based products are recovered. To prepare the tissues for preservation, peripheral nerves (10 cm sections), including sciatic, peroneal, radial, ulnar and median, will be aseptically recovered, using minimal blunt and sharp dissection, from qualified organ and tissue donors at certified organ procurement organizations or AATB accredited tissue banks as part of the standard organ and tissue recovery process.

[0086] Clear, step-by-step description of how the product is processed, packaged (including the range of product sizes, when applicable), and stored. After aseptic recovery, each peripheral nerve allograft is sampled for disease testing, and then oriented so as to maintain proximal-distal orientation on a sterile tissue platen and placed in a sealed tissue bag or container filled with 100 ml of tissue storage media (MOPS®-N) consisting of DMEM with insulin lOmg / L, transferrin 55mg / L, sodium selenite 6.7pg / L, penicillin, streptomycin, amphotericin B (100 U / ml penicillin / 100 ug / ml streptomycin / 2.5 pg / ml amphotericin B), L-glutamine (2 mM, non-essential amino acids (O.lmM), L-ascorbic acid (50 ng / ml ascorbic acid, dexamethasone (O.lpM), vitamin B- 12 (1.47mM), which allows for preservation of cell and tissue viability such that it can perform the same basic functions as the native nerve following storage and transplantation as an allograft. The bags / containers are stored in a clean room at ambient temperature and humidity at a certified tissue bank until disease testing is completed to allow for packaging for distribution to point of care, where the same storage conditions will be followed until used for peripheral nerve repair, reconstruction, replacement, or supplementation in an indicated patient.

[0087] Way the product is to be used and product labeling, including package inserts and any instructions for use documents. The MOPS®-Nerve Allograft Preservation System will be labeled for use as a peripheral nerve processing and storage system that allows for preservation of donor tissue function during recovery, processing, and point-of-care storage for allograft transplantation in the homologous-use repair, reconstruction, replacement, or supplementation of a recipient's peripheral nerve to perform the same basic function in the recipient as in the donor.

[0088] How the product meets all of the criteria in 21 C.F.R. § 1271.10(a). The product is a human tissue (peripheral nerve) intended for allograft transplantation in the homologous-use repair, reconstruction, replacement, or supplementation of a recipient's peripheral nerve to perform the same basic function in the recipient as in the donor.

[0089] The processing of the product does not alter the original relevant characteristics of the peripheral nerve tissue relating to the tissue's utility for peripheral reconstruction, repair, or replacement; and does not alter the relevant biological characteristics of cells or tissue.

[0090] In contrast to fresh-frozen, cryopreserved, and decellularized peripheral nerves, the tissues processed and preserved using the MOPS®-Nerve Allograft Preservation System are not altered such that they lose cell viability, tissue morphology, or biologic characteristics that allow for native nerve function.

[0091] Features described with respect to certain exemplary embodiments can be combined and sub-combined in and / or with features described with respect to various other exemplary embodiments, even if such combinations and / or sub-combinations are not specifically described herein. Also, different aspects and / or elements of cxcmplary embodiments, as disclosed herein, can be combined and sub-combined in a similar manner as well. Further, some exemplary embodiments, whether individually and / or collectively, can be components of a larger system, wherein other procedures can take precedence over and / or otherwise modify their application. Additionally, a number of steps can be required before, after, and / or concurrently with exemplary embodiments, as disclosed herein. Note that any and / or all methods and / or processes, at least as disclosed herein, can be at least partially performed via at least one entity in any manner.

[0092] Various terminology used herein is for describing particular exemplary embodiments and is not intended to be necessarily limiting of this disclosure. As used herein, various singular forms "a," "an" and "the" are intended to include various plural forms as well, unless a context clearly indicates otherwise. Various terms "comprises," “includes,” "comprising" and / or “including” when used in this specification specify a presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence and / or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0093] As used herein, a term "or" is intended to mean an inclusive "or" rather than an exclusive "or." That is, unless specified otherwise, or clear from context, "X employs A or B" is intended to mean any of a set of natural inclusive permutations. That is, if X employs A; X employs B; or X employs both A and B, then "X employs A or B" is satisfied under any of the foregoing instances.

[0094] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in an art to which this disclosure belongs. Various terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with a meaning in a context of a relevant art and should not be interpreted in an idealized and / or overly formal sense unless expressly so defined herein.

[0095] As used herein, a term "about" and / or "substantially" refers to a + / - 10% variation from a nominal value / term. Such variation is always included in any given value / term provided herein, whether or not such variation is specifically referred thereto.

[0096] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that the present disclosure is capable of further modifications by one of skill in the art. It is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible. The present disclosure is therefore intended to encompass any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth.

[0097] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. For example, all of the disclosed components of the preferred and alternative embodiments are interchangeable providing disclosure herein of many systems having combinations of all the preferred and alternative embodiment components. This application is therefore intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this disclosure pertains and which fall within the limits of the appended claims. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

What is claimed is:

1. A process for peripheral nerve tissue preservation comprising storing the peripheral nerve tissue at room temperature in a container comprising serum-free culture medium for from at least 7 to 120 days prior to implantation, wherein at least 50% of the cells of said peripheral nerve tissue remain viable after said storing compared to the viability of the cells of the peripheral nerve tissue at day 0.

2. The process of claim 1, comprising testing the tissue for viability at least once prior to implantation in a patient.

3. The process of claim 2, wherein testing for viability comprises assaying the medium withdrawn from said container.

4. The process of claim 2, wherein testing for viability comprises adding a resazurin solution to the medium and determining the fluorescence level, wherein increased fluorescence indicates higher cell viability.

5. The process of claim 1 comprising changing said medium at least once during the storing.

6. The process of claim 5, comprising changing the medium about once every two weeks during the storing.

7. The process of claim 1, wherein the medium comprises Dulbccco's Modified Eagle Medium (DMEM), insulin, transferrin, selenium, at least a first antibiotic compound, at least a first antimycotic compound, L-glutamine, non-essential amino acids, ascorbic acid, dexamethasone and vitamin B-12.

8. The process of claim 7, wherein the at least a first antibiotic compound comprises penicillin and streptomycin.

9. The process of claim 7, wherein the at least a first antimycotic compound is amphotericin B.

10. The process of claim 7, wherein the medium comprises penicillin, streptomycin and amphotericin B.

11. The process of claim 1 , wherein the peripheral nerve tissue is sensory nerve tissue, motor nerve tissue or mixed nerve tissue.

12. The process of claim 1, wherein the peripheral nerve tissue comprises an autograft or an allograft.

13. The process of claim 1, wherein the peripheral nerve tissue comprises autograft or an allograft, the process further comprising lavaging of the peripheral nerve tissue in isotonic solution prior to said storing.

14. The process of claim 1, further comprising implanting the tissue in a subject in need thereof following said storing.

15. The process of claim 1, wherein the room temperature is between about 19°C and about 27°C.

16. A composition comprising Dulbecco's Modified Eagle Medium (DMEM), insulin, transferrin, selenium, at least a first antibiotic compound, at least a first antimycotic compound, L-glutamine, non-essential amino acids, ascorbic acid, dexamethasone and vitamin B- 12.

17. The composition of claim 16, wherein the composition comprises penicillin, streptomycin and amphotericin B .

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