The impact of heart valve and partial heart transplant models on the development of banking methods for tissues and organs: A concise review.

Cryopreserved human heart valves fill a crucial role in the treatment for congenital cardiac anomalies, since the use of alternative mechanical and xenogeneic tissue valves have historically been limited in babies. Heart valve models have been used since 1998 to better understand the impact of cryopreservation variables on the heart valve tissue components with the ultimate goals of improving cryopreserved tissue outcomes and potentially extrapolating results with tissues to organs. Cryopreservation traditionally relies on conventional freezing, employing cryoprotective agents, and slow cooling to sub-zero centigrade temperatures; but it is plagued by the formation of ice crystals and cell damage upon thawing. Researchers have identified ice-free vitrification procedures and developed a new rapid warming method termed nanowarming. Nanowarming is an emerging method that utilizes targeted application of energy at the nanoscale level to rapidly rewarm vitrified tissues, such as heart valves, uniformly for transplantation. Vitrification and nanowarming methods hold great promise for surgery, enabling the storage and transplantation of tissues for various applications, including tissue repair and replacement. These innovations have the potential to revolutionize complex tissue and organ transplantation, including partial heart transplantation. Banking these grafts addresses organ scarcity by extending preservation duration while preserving biological activity with maintenance of structural fidelity. While ice-free vitrification and nanowarming show remarkable potential, they are still in early development. Further interdisciplinary research must be dedicated to exploring the remaining challenges that include scalability, optimizing cryoprotectant solutions, and ensuring long-term viability upon rewarming in vitro and in vivo.

Imaging the distribution of iron oxide nanoparticles in hypothermic perfused tissues.

PURPOSE: Herein, we evaluate the use of MRI as a tool for assessing iron oxide nanoparticle (IONP) distribution within IONP perfused organs and vascularized composite allografts (VCAs) (i.e., hindlimbs) prepared for cryopreservation. METHODS: Magnetic resonance imaging was performed on room-temperature organs and VCAs perfused with IONPs and were assessed at 9.4 T. Quantitative T1 mapping and T2∗ -weighted images were acquired using sweep imaging with Fourier transformation and gradient-echo sequences, respectively. Verification of IONP localization was performed through histological assessment and microcomputer tomography. RESULTS: Quantitative imaging was achieved for organs and VCAs perfused with up to 642 mMFe (36 mgFe /mL), which is above previous demonstrations of upper limit detection in agarose (35.7mMFe [2 mgFe /mL]). The stability of IONPs in the perfusate had an effect on the quality of distribution and imaging within organs or VCA. Finally, MRI provided more accurate IONP localization than Prussian blue histological staining in this system, wherein IONPs remain primarily in the vasculature. CONCLUSION: Using MRI, we were able to assess the distribution of IONPs throughout organs and VCAs varying in complexity. Additional studies are necessary to better understand this system and validate the calibration between T1 measurements and IONP concentration.

Marker-Independent In Situ Quantitative Assessment of Residual Cryoprotectants in Cardiac Tissues.

Cryomedium toxicity is a major safety concern when transplanting cryopreserved organs. Therefore, thorough removal of potentially toxic cryoprotective agents (CPAs) is required before transplantation. CPAs such as dimethyl-sulfoxide (DMSO), propylene glycol (PG), and formamide (FMD), routinely employed in ice-free cryopreservation (IFC), have advantages in long-term preservation of tissue structures compared with conventional cryopreservation employing lower CPA concentrations. This study evaluated the impact of potential residual CPAs on human cardiac valves. Raman microspectroscopy and Raman imaging were established as nondestructive marker-independent techniques for in situ quantitative assessment of CPA residues in IFC valve tissues. In detail, IFC valve leaflets and supernatants of the washing solutions were analyzed to determine the washing efficiency. A calibration model was developed according to the CPA's characteristic Raman signals to quantify DMSO, PG and FMD concentrations in the supernatants. Single point Raman measurements were performed on the intact tissues to analyze penetration properties. In addition, Raman imaging was utilized to visualize potential CPA residues. Our data showed that washing decreased the CPA concentration in the final washing solution by 99%, and no residues could be detected in the washed tissues, validating the multistep CPA removal protocol routinely used for IFC valves. Raman analysis of unwashed tissues showed different permeation characteristics depending on each CPA and their concentration. Our results demonstrate a great potential of Raman microspectroscopy and Raman imaging as marker-independent in situ tissue quality control tools with the ability to assess the presence and concentration of different chemical agents or drugs in preimplantation tissues.

Mapping Extracellular Matrix Proteins in Formalin-Fixed, Paraffin-Embedded Tissues by MALDI Imaging Mass Spectrometry.

Collagens and elastin form the fundamental framework of all tissues and organs, and their expression and post-translational processing are tightly regulated in disease and health. Because of their unique structural composition and properties, it is a recognized challenge to access these protein structures within the complex tissue microenvironment to understand how localized changes modulate tissue health. We describe a new workflow using a combination of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) with matrix metalloproteinase (MMP) enzymes to access and report on spatial localization of collagen and elastin sequences in formalin-fixed, paraffin-embedded (FFPE) tissues. The developed technology provides new access to collagens and elastin sequences localized to tissue features that were previously unattainable. This high-throughput technological advance should be applicable to any tissue regardless of disease type, tissue origin, or disease status and is thus relevant to all research: basic, translational, or clinical.

Simultaneous monitoring of different vitrification solution components permeating into tissues.

Cryopreservation can be used for long-term preservation of tissues and organs. It relies on using complex mixtures of cryoprotective agents (CPAs) to reduce the damaging effects of freezing, but care should be taken to avoid toxic effects of CPAs themselves. In order to rationally design cryopreservation strategies for tissues, it is important to precisely determine permeation kinetics of the protectants that are used to ensure maximum permeation, while minimizing the exposure time and toxicity effects. This is particularly challenging with protectant solutions consisting of multiple components each with different physical properties and diffusing at a different rate. In this study, we show that an attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) setup can be used to simultaneously monitor diffusion of multiple components in a mixture into tissues in real time. Diffusion studies were done with decellularized heart valves using a sucrose-DMSO mixture as well as vitrification solution VS83. To assess diffusion kinetics of different solutes in mixtures, the increase in specific infrared absorbance bands was monitored during diffusion through the tissue. Solute specific wavenumber ranges were selected, and the calculated area was assumed to be proportional to the CPA concentration in the tissue. A diffusion equation based on Fick's second law of diffusion fitted the experimental data quite well, and clear differences in permeation rates were observed among the different mixture components dependent on molecular size and physical properties.

The Grand Challenges of Organ Banking: Proceedings from the first global summit on complex tissue cryopreservation.

The first Organ Banking Summit was convened from Feb. 27 - March 1, 2015 in Palo Alto, CA, with events at Stanford University, NASA Research Park, and Lawrence Berkeley National Labs. Experts at the summit outlined the potential public health impact of organ banking, discussed the major remaining scientific challenges that need to be overcome in order to bank organs, and identified key opportunities to accelerate progress toward this goal. Many areas of public health could be revolutionized by the banking of organs and other complex tissues, including transplantation, oncofertility, tissue engineering, trauma medicine and emergency preparedness, basic biomedical research and drug discovery - and even space travel. Key remaining scientific sub-challenges were discussed including ice nucleation and growth, cryoprotectant and osmotic toxicities, chilling injury, thermo-mechanical stress, the need for rapid and uniform rewarming, and ischemia/reperfusion injury. A variety of opportunities to overcome these challenge areas were discussed, i.e. preconditioning for enhanced stress tolerance, nanoparticle rewarming, cyroprotectant screening strategies, and the use of cryoprotectant cocktails including ice binding agents.

Comparison and evaluation of biomechanical, electrical, and biological methods for assessment of damage to tissue collagen.

In regard to evaluating tissue banking methods used to preserve or otherwise treat (process) soft allograft tissue, current tests may not be sufficiently sensitive to detect potential damage inflicted before, during, and after processing. Using controlled parameters, we aim to examine the sensitivity of specific biomechanical, electrical, and biological tests in detecting mild damage to collagen. Fresh porcine pulmonary heart valves were treated with an enzyme, collagenase, and incubated using various times. Controls received no incubation. All valves were cryopreserved and stored at -135 °C until being rewarmed for evaluation using biomechanical, permeability, and cell viability tests. Statistically significant time dependent changes in leaflet ultimate stress, (p = 0.006), permeability (p = 0.01), and viability (p ≤ 0.02, four different days of culture) were found between heart valves subjected to 0-15 min of collagenase treatment (ANOVA). However, no statistical significance was found between the tensile modulus of treated and untreated valves (p = 0.07). Furthermore, the trends of decreasing and increasing ultimate stress and viability, respectively, were somewhat inconsistent across treatment times. These results suggest that permeability tests may offer a sensitive, quantitative assay to complement traditional biomechanical and viability tests in evaluating processing methods used for soft tissue allografts, or when making changes to current validated methods. Multiple test evaluation may also offer insight into the mechanism of potential tissue damage such as, as is the case here, reduced collagen content and increased tissue porosity.

Xeno-immunogenicity of ice-free cryopreserved porcine leaflets.

BACKGROUND: Undesirable processes of inflammation, calcification, or immune-mediated reactions are limiting factors in long-term survival of heart valves in patients. In this study, we target the modulatory effects of ice-free cryopreservation (IFC) of xenogeneic heart valve leaflet matrices, without decellularization, on the adaptive human immune responses in vitro. METHODS: We tested porcine leaflet matrices from fresh untreated, conventionally cryopreserved (CFC), and IFC pulmonary valves by culturing them with human blood mononuclear cells for 5 d in vitro. No other tissue treatment protocols to modify possible immune responses were used. Matrices alone or in addition with a low-dose second stimulus were analyzed for induction of proliferation and cytokine release by flow cytometry-based techniques. Evaluation of the α-Gal epitope expression was performed by immunohistochemistry with fluorochrome-labeled B4 isolectin. RESULTS: None of the tested leaflet treatment groups directly triggered the proliferation of immune cells. But when tested in combination with a second trigger by anti-CD3, IFC valves showed significantly reduced proliferation of T cells, especially effector memory T cells, in comparison with fresh or CFC tissue. Moreover, the cytokine levels for interferon-γ (IFNγ), tumor necrosis factor α, and interleukin-10 were reduced for the IFC-treated group being significantly different compared with the CFC group. However, no difference between treatment groups in the expression of the α-Gal antigen was observed. CONCLUSIONS: IFC of xenogeneic tissue might be an appropriate treatment method or processing step to prevent responses of the adaptive immune system.

Impact of storage solution formulation during refrigerated storage upon chondrocyte viability and cartilage matrix.

Various preservation solutions have been evaluated for longer hypothermic cartilage storage for tissue transplantation; however, the results are mixed. This research was carried out to determine whether phosphate-buffered saline (PBS) or organ preservation solutions would preserve both the extracellular matrix and chondrocytes of articular cartilage better than culture medium during refrigerated storage in the time frame that cartilage is stored for clinical use. Porcine cartilage plugs were stored, without the underlying bone, in culture medium with and without fetal bovine serum (FBS), PBS, Belzer's and Unisol solutions for 1 month at 4°C. Metabolic activity was tested using a resazurin reduction method, and matrix permeability was evaluated by measuring electrical conductivity. Storage in culture medium with 10% FBS was shown to provide good cartilage metabolic function for 7 days, decreasing to about 36% after 1 month of storage. There was no significant difference between samples stored in culture medium with and without FBS after 1 month of storage (p = 0.5005). Refrigerated storage of cartilage in PBS and two different solutions (Belzer's and Unisol) designed for optimal refrigerated tissue and organ storage results in loss of chondrocyte function and retention of matrix permeability. In contrast, the opposite, namely significantly better retention of chondrocyte function and loss of matrix permeability, was observed with culture medium. Future research should be focused on combining retention of chondrocyte function and matrix permeability by storage solution formulation.

Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions.

Expanding cryopreservation methods to include a wider range of cell types, such as those sensitive to freezing, is needed for maintaining the viability of cell-based regenerative medicine products. Conventional cryopreservation protocols, which include use of cryoprotectants such as dimethylsulfoxide (Me2SO), have not prevented ice-induced damage to cell and tissue matrices during freezing. A family of antifreeze proteins (AFPs) produced in the larvae of the beetle, Dendroides canadensis allow this insect to survive subzero temperatures as low as -26°C. This study is an assessment of the effect of the four hemolymph D. canadensis AFPs (DAFPs) on the supercooling (nucleating) temperature, ice structure patterns and viability of the A10 cell line derived from the thoracic aorta of embryonic rat. Cryoprotectant solution cocktails containing combinations of DAFPs in concentrations ranging from 0 to 3mg/mL in Unisol base mixed with 1M Me2SO were first evaluated by cryomicroscopy. Combining multiple DAFPs demonstrated significant supercooling point depressing activity (∼9°C) when compared to single DAFPs and/or conventional 1M Me2SO control solutions. Concentrations of DAFPs as low as 1 µg/mL were sufficient to trigger this effect. In addition, significantly improved A10 smooth muscle cell viability was observed in cryopreservation experiments with low DAFP-6 and DAFP-2 concentrations in combination with Me2SO. No significant improvement in viability was observed with either DAFP-1 or DAFP-4. Low and effective DAFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating DAFPs in solutions used to cryopreserve cells and tissues.

The future of partial heart transplantation.

Heart valve replacement in children is an unsolved problem in congenital cardiac surgery because state-of-the-art heart valve implants do not grow. This leads to serial repeat operations to replace outgrown heart valve implants. Partial heart transplantation is a new transplant that helps alleviate this problem by delivering growing heart valve implants. In the future, partial heart transplantation has the potential to complement conventional heart transplantation for treating children with congenital cardiac disease primarily affecting the heart valves.

Bacteria Endocarditis Caused by Mycoplasma hyorhinis in a Juvenile, Immunosuppressed Pig (Sus scrofa domesticus) following Partial Heart Transplantation.

Domestic swine (Sus scrofa domesticus) are important translational models for cardiovascular transplant studies. This can be attributed to the anatomic and physiologic similarities of their cardiovascular system to humans. Transplant studies frequently employ clinically relevant immunosuppression regimens to prevent organ rejection postoperatively. Immunosuppression can lead to opportunistic infection, including presentations that are novel or poorly described in immunocompetent hosts. In this study, we describe the first case of Mycoplasma hyorhinis-induced endocarditis affecting the pulmonary valve in a juvenile, immunosuppressed pig following a partial heart transplantation procedure. Clinical signs of infection began at 15 d postoperation, were consistent with a variety of infectious agents, including Mycoplasma hyorhinis, and included lethargy, respiratory signs, and elevated white blood cell counts. By 28 d post procedure, lameness and soft tissue swelling around the left tarsus developed. Joint fluid obtained by arthrocentesis was PCR positive for Mycoplasma hyorhinis and negative for other tested pathogens. Despite antimicrobial treatment, the transplanted pulmonary valve developed leaflet thickening, stenosis, and insufficiency starting at 30 d after the procedure. At 86 d posttransplantation, the pig reached experimental endpoints and was humanely euthanized for necropsy and histopathology. The pulmonary valve had numerous dark red vegetative expansions of all 3 leaflets. Postmortem testing of a vegetative lesion was positive for Mycoplasma hyorhinis, confirming the etiologic agent responsible for endocarditis. Mycoplasma hyorhinis-induced endocarditis of an orthotopic transplanted pulmonary valve has yet to be described in swine. This case report demonstrates that infections following immunosuppression may present with novel or undercharacterized clinical signs.

Viable Vitreous Grafts of Whole Porcine Menisci for Transplant in the Knee and Temporomandibular Joints.

The shortage of suitable donor meniscus grafts from the knee and temporomandibular joint (TMJ) impedes treatments for millions of patients. Vitrification offers a promising solution by transitioning these tissues into a vitreous state at cryogenic temperatures, protecting them from ice crystal damage using high concentrations of cryoprotectant agents (CPAs). However, vitrification's success is hindered for larger tissues (>3 mL) due to challenges in CPA penetration. Dense avascular meniscus tissues require extended CPA exposure for adequate penetration; however, prolonged exposure becomes cytotoxic. Balancing penetration and reducing cell toxicity is required. To overcome this hurdle, a simulation-based optimization approach is developed by combining computational modeling with microcomputed tomography (µCT) imaging to predict 3D CPA distributions within tissues over time accurately. This approach minimizes CPA exposure time, resulting in 85% viability in 4-mL meniscal specimens, 70% in 10-mL whole knee menisci, and 85% in 15-mL whole TMJ menisci (i.e., TMJ disc) post-vitrification, outperforming slow-freezing methods (20%-40%), in a pig model. The extracellular matrix (ECM) structure and biomechanical strength of vitreous tissues remain largely intact. Vitreous meniscus grafts demonstrate clinical-level viability (≥70%), closely resembling the material properties of native tissues, with long-term availability for transplantation. The enhanced vitrification technology opens new possibilities for other avascular grafts.

Characterization of a simplified ice-free cryopreservation method for heart valves.

The aim of the present study was to characterize the hemocompatibility of ice-free cryopreserved heart valves in anticipation of future human trials. Porcine pulmonary heart valves were infiltrated with either an 83 % cryoprotectant solution followed by rapid cooling and storage at --80 °C or with 10 % DMSO and control rate freezing to --80 °C and storage in vapor phase nitrogen as conventional frozen controls. Cryopreserved leaflets were compared with fresh, decellularized and glutaraldehyde-fixed control valve leaflets using a battery of coagulation protein assays after exposure to human blood. Von Willebrand Factor staining indicated that most of the endothelium was lost during valve processing prior to cryopreservation. Hemocompatibility, employing thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, beta-thromboglobulin and terminal complement complex SC5b-9, was preserved compared with both fresh and frozen leaflets. Hemocompatibility differences were observed for cryopreserved leaflets versus both decellularized and glutaraldehyde fixed controls. In conclusion, the hemocompatibility results support the use of ice-free cryopreservation as a simplified preservation method because no statistically significant differences in hemocompatibility were observed between the two cryopreservation methods and fresh untreated controls.

Morbidity and Mortality of Heterotopic Partial Heart Transplantation in Rodent Models.

Unrepairable congenital heart valve disease is an unsolved problem in pediatric cardiac surgery because there are no growing heart valve implants. Partial heart transplantation is a new type of transplant that aims to solve this problem. In order to study the unique transplant biology of partial heart transplantation, animal models are necessary. This study aimed to assess the morbidity and mortality of heterotopic partial heart transplantation in rodent models. This study assessed two models. The first model involved transplanting heart valves from donor animals into the abdominal aortic position in the recipient animals. The second model involved transplanting heart valve leaflets into the renal subcapsular position of the recipient animals. A total of 33 animals underwent heterotopic partial heart transplantation in the abdominal aortic position. The results of this model found a 60.61% (n = 20/33) intraoperative mortality rate and a 39.39% (n = 13/33) perioperative mortality rate. Intraoperative mortality was due to vascular complications from the procedure, and perioperative mortality was due to graft thrombosis. A total of 33 animals underwent heterotopic partial heart transplantation in the renal subcapsular position. The results of this model found a 3.03% (n = 1/33) intraoperative mortality rate, and the remaining 96.97% survived (n = 32/33). We conclude that the renal subcapsular model has a lower mortality rate and is technically more accessible than the abdominal aortic model. While the heterotopic transplantation of valves into the abdominal aortic position had significant morbidity and mortality in the rodent model, the renal subcapsular model provided evidence for successful heterotopic transplantation.

Freezing Biological Time: A Modern Perspective on Organ Preservation.

Transporting tissues and organs from the site of donation to the patient in need, while maintaining viability, is a limiting factor in transplantation medicine. One way in which the supply chain of organs for transplantation can be improved is to discover novel approaches and technologies that preserve the health of organs outside of the body. The dominant technologies that are currently in use in the supply chain for biological materials maintain tissue temperatures ranging from a controlled room temperature (+25 °C to +15 °C) to cryogenic (-120 °C to -196 °C) temperatures (reviewed in Criswell et al. Stem Cells Transl Med. 2022). However, there are many cells and tissues, as well as all major organs, that respond less robustly to preservation attempts, particularly when there is a need for transport over long distances that require more time. In this perspective article, we will highlight the current challenges and advances in biopreservation aimed at "freezing biological time," and discuss the future directions and requirements needed in the field.

Immunogenicity of Homologous Heart Valves: Mechanisms and Future Considerations.

Pediatric valvar heart disease continues to be a topic of interest due to the common and severe clinical manifestations. Problems with heart valve replacement, including lack of adaptive valve growth and accelerated structural valve degeneration, mandate morbid reoperations to serially replace valve implants. Homologous or homograft heart valves are a compelling option for valve replacement in the pediatric population but are susceptible to structural valve degeneration. The immunogenicity of homologous heart valves is not fully understood, and mechanisms explaining how implanted heart valves are attacked are unclear. It has been demonstrated that preservation methods determine homograft cell viability and there may be a direct correlation between increased cellular viability and a higher immune response. This consists of an early increase in human leukocyte antigen (HLA)-class I and II antibodies over days to months posthomograft implantation, followed by the sustained increase in HLA-class II antibodies for years after implantation. Cytotoxic T lymphocytes and T-helper lymphocytes specific to both HLA classes can infiltrate tissue almost immediately after implantation. Furthermore, increased HLA-class II mismatches result in an increased cell-mediated response and an accelerated rate of structural valve degeneration especially in younger patients. Further long-term clinical studies should be completed investigating the immunological mechanisms of heart valve rejection and their relation to structural valve degeneration as well as testing of immunosuppressant therapies to determine the needed immunosuppression for homologous heart valve implantation.

Nanowarming and ice-free cryopreservation of large sized, intact porcine articular cartilage.

Successful organ or tissue long-term preservation would revolutionize biomedicine. Cartilage cryopreservation enables prolonged shelf life of articular cartilage, posing the prospect to broaden the implementation of promising osteochondral allograft (OCA) transplantation for cartilage repair. However, cryopreserved large sized cartilage cannot be successfully warmed with the conventional convection warming approach due to its limited warming rate, blocking its clinical potential. Here, we develope a nanowarming and ice-free cryopreservation method for large sized, intact articular cartilage preservation. Our method achieves a heating rate of 76.8 °C min-1, over one order of magnitude higher than convection warming (4.8 °C min-1). Using systematic cell and tissue level tests, we demonstrate the superior performance of our method in preserving large cartilage. A depth-dependent preservation manner is also observed and recapitulated through magnetic resonance imaging and computational modeling. Finally, we show that the delivery of nanoparticles to the OCA bone side could be a feasible direction for further optimization of our method. This study pioneers the application of nanowarming and ice-free cryopreservation for large articular cartilage and provides valuable insights for future technique development, paving the way for clinical applications of cryopreserved cartilage.

Preclinical evaluation of ice-free cryopreserved arteries: structural integrity and hemocompatibility.

OBJECTIVE: Arterial allografts are routinely employed for reconstruction of infected prosthetic grafts. Usually, banked cryopreserved arteries are used; however, existing conventional freezing cryopreservation techniques applied to arteries are expensive. In contrast, a new ice-free cryopreservation technique results in processing, storage and shipping methods that are technically simpler and potentially less costly. The objective of this study was to determine whether or not ice-free cryopreservation causes tissue changes that might preclude clinical use. METHODS: Conventionally frozen cryopreserved porcine arteries were compared with ice-free cryopreserved arteries and untreated fresh controls using morphological (light, scanning electron and laser scanning microscopy), viability (alamarBlue assay) and hemocompatibility methods (blood cell adhesion, thrombin/antithrombin-III-complex, polymorphonuclear neutrophil-elastase, ß-thromboglobulin and terminal complement complex SC5b-9). RESULTS: No statistically significant structural or hemocompatibility differences between ice-free cryopreserved and frozen tissues were detectable. There were no quantitative differences observed for either autofluorescence (elastin) or second harmonic generation (collagen) measured by laser scanning microscopy. Cell viability in ice-free cryopreserved arteries was significantly reduced compared to fresh and frozen tissues (p < 0.05). CONCLUSIONS: The formation of ice in aortic artery preservation did not make a difference in histology, structure or thrombogenicity, but significantly increased viability compared with a preservation method that precludes ice formation. Reduced cell viability should not reduce in vivo performance. Therefore, ice-free cryopreservation is a potentially safe and cost-effective technique for the cryopreservation of blood vessel allografts.

Culturing with trehalose produces viable endothelial cells after cryopreservation.

Dimethylsulfoxide, the most commonly employed cryoprotectant for cells, has well documented cytotoxic effects in patients. Among the compounds available that may provide protection to cells and tissues during preservation with less cytotoxicity is trehalose. Some animals, such as brine shrimp and tardigrades, accumulate trehalose during periods of extreme environmental stress. In this study, experiments were performed to evaluate the effects of culturing a bovine endothelial cell line (ATCC #CCL-209) in the presence of trehalose prior to preservation by freezing. A number of factors were shown to contribute to cell retention of metabolic activity and proliferative potential including cell culture time with trehalose and the solution conditions during cryopreservation. Using an optimized protocol consisting of 24 h of cell culture with 0.2 M trehalose followed by cryopreservation with 0.2-0.4 M trehalose in sodium bicarbonate buffered Eagles minimum essential medium at pH 7.4 resulted in 87±4% post-preservation cell metabolic activity expressed as relative fluorescence based upon reduction of resazurin to resorufin. This new method provides an alternative preservation strategy to the more classical preservation methods employing dimethylsulfoxide available for cells and tissues.