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.

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.

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.

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.

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.

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.

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.

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.

Shipping and Logistics Considerations for Regenerative Medicine Therapies.

Advances in regenerative medicine manufacturing continue to be a priority for achieving the full commercial potential of important breakthrough therapies. Equally important will be the establishment of distribution chains that support the transport of live cells and engineered tissues and organs resulting from these advanced biomanufacturing processes. The importance of a well-managed distribution chain for products requiring specialized handling procedures was highlighted during the COVID-19 pandemic and serves as a reminder of the critical role of logistics and distribution in the success of breakthrough therapies. This perspective article will provide insight into current practices and future considerations for creating global distribution chains that facilitate the successful deployment of regenerative medicine therapies to the vast number of patients that would benefit from them worldwide.

Ice Control during Cryopreservation of Heart Valves and Maintenance of Post-Warming Cell Viability.

Heart valve cryopreservation was employed as a model for the development of complex tissue preservation methods based upon vitrification and nanowarming. Porcine heart valves were loaded with cryoprotectant formulations step wise and vitrified in 1−30 mL cryoprotectant formulations ± Fe nanoparticles ± 0.6 M disaccharides, cooled to −100 °C, and stored at −135 °C. Nanowarming was performed in a single ~100 s step by inductive heating within a magnetic field. Controls consisted of fresh and convection-warmed vitrified heart valves without nanoparticles. After washing, cell viability was assessed by metabolic assay. The nanowarmed leaflets were well preserved, with a viability similar to untreated fresh leaflets over several days post warming. The convection-warmed leaflet viability was not significantly different than that of the nanowarmed leaflets immediately after rewarming; however, a significantly higher nanowarmed leaflet viability (p < 0.05) was observed over time in vitro. In contrast, the associated artery and fibrous cardiac muscle were at best 75% viable, and viability decreased over time in vitro. Supplementation of lower concentration cryoprotectant formulations with disaccharides promoted viability. Thicker tissues benefited from longer-duration cryoprotectant loading steps. The best outcomes included a post-warming incubation step with α-tocopherol and an apoptosis inhibitor, Q-VD-OPH. This work demonstrates progress in the control of ice formation and cytotoxicity hurdles for the preservation of complex tissues.

Gluconate-Lactobionate-Dextran Perfusion Solutions Attenuate Ischemic Injury and Improve Function in a Murine Cardiac Transplant Model.

Static cold storage is the cheapest and easiest method and current gold standard to store and preserve donor organs. This study aimed to compare the preservative capacity of gluconate-lactobionate-dextran (Unisol) solutions to histidine-tryptophan-ketoglutarate (HTK) solution. Murine syngeneic heterotopic heart transplantations (Balb/c-Balb/c) were carried out after 18 h of static cold storage. Cardiac grafts were either flushed and stored with Unisol-based solutions with high-(UHK) and low-potassium (ULK) ± glutathione, or HTK. Cardiac grafts were assessed for rebeating and functionality, histomorphologic alterations, and cytokine expression. Unisol-based solutions demonstrated a faster rebeating time (UHK 56 s, UHK + Glut 44 s, ULK 45 s, ULK + Glut 47 s) compared to HTK (119.5 s) along with a better contractility early after reperfusion and at the endpoint on POD 3. Ischemic injury led to a significantly increased leukocyte recruitment, with similar degrees of tissue damage and inflammatory infiltrate in all groups, yet the number of apoptotic cells tended to be lower in ULK compared to HTK. In UHK- and ULK-treated animals, a trend toward decreased expression of proinflammatory markers was seen when compared to HTK. Unisol-based solutions showed an improved preservative capacity compared with the gold standard HTK early after cardiac transplantation. Supplemented glutathione did not further improve tissue-protective properties.

Development of a Vitrification Preservation Process for Bioengineered Epithelial Constructs.

The demand for human bioengineered tissue constructs is growing in response to the worldwide movement away from the use of animals for testing of new chemicals, drug screening and household products. Presently, constructs are manufactured and delivered just in time, resulting in delays and high costs of manufacturing. Cryopreservation and banking would speed up delivery times and permit cost reduction due to larger scale manufacturing. Our objective in these studies was development of ice-free vitrification formulations and protocols using human bioengineered epithelial constructs that could be scaled up from individual constructs to 24-well plates. Initial experiments using single EpiDerm constructs in vials demonstrated viability >80% of untreated control, significantly higher than our best freezing strategy. Further studies focused on optimization and evaluation of ice-free vitrification strategies. Vitrification experiments with 55% (VS55) and 70% (VS70) cryoprotectant (CPA) formulations produced constructs with good viability shortly after rewarming, but viability decreased in the next days, post-rewarming in vitro. Protocol changes contributed to improved outcomes over time in vitro. We then transitioned from using glass vials with 1 construct to deep-well plates holding up to 24 individual constructs. Construct viability was maintained at >80% post-warming viability and >70% viability on days 1−3 in vitro. Similar viability was demonstrated for other related tissue constructs. Furthermore, we demonstrated maintenance of viability after 2−7 months of storage below −135 °C.

Vitrification of Heart Valve Tissues.

Application of the original vitrification protocol used for pieces of heart valves to intact heart valves has evolved over time. Ice-free cryopreservation by Protocol 1 using VS55 is limited to small samples (1-3 mL total volume) where relatively rapid cooling and warming rates are possible. VS55 cryopreservation typically provides extracellular matrix preservation with approximately 80% cell viability and tissue function compared with fresh untreated tissues. In contrast, ice-free cryopreservation using VS83, Protocols 2 and 3, permits preservation of large samples (80-100 mL total volume) with several advantages over conventional cryopreservation methods and VS55 preservation, including long-term preservation capability at -80 °C; better matrix preservation than freezing with retention of material properties; very low cell viability, reducing the risks of an immune reaction in vivo; reduced risks of microbial contamination associated with use of liquid nitrogen; improved in vivo functions; no significant recipient allogeneic immune response; simplified manufacturing process; increased operator safety because liquid nitrogen is not used; and reduced manufacturing costs. More recently, we have developed Protocol 4 in which VS55 is supplemented with sugars resulting in reduced concerns regarding nucleation during cooling and warming. This method can be used for large samples resulting in retention of cell viability and permits short-term exposure to -80 °C with long-term storage preferred at or below -135 °C.

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.

Improved long-term durability of allogeneic heart valves in the orthotopic sheep model.

OBJECTIVES: Frozen cryopreservation (FC) with the vapour phase of liquid nitrogen storage (-135°C) is a standard biobank technique to preserve allogeneic heart valves to enable a preferable allograft valve replacement in clinical settings. However, their long-term function is limited by immune responses, inflammation and structural degeneration. Ice-free cryopreserved (IFC) valves with warmer storage possibilities at -80°C showed better matrix preservation and decreased immunological response in preliminary short-term in vivo studies. Our study aimed to assess the prolonged performance of IFC allografts in an orthotopic pulmonary sheep model. METHODS: FC (n = 6) and IFC (n = 6) allografts were transplanted into juvenile Merino sheep. After 12 months of implantation, functionality testing via 2-dimensional echocardiography and histological analyses was performed. In addition, multiphoton autofluorescence imaging and Raman microspectroscopy analysis were applied to qualitatively and quantitatively assess the matrix integrity of the leaflets. RESULTS: Six animals from the FC group and 5 animals from the IFC group were included in the analysis. Histological explant analysis showed early inflammation in the FC valves, whereas sustainable, fully functional, devitalized acellular IFC grafts were obtained. IFC valves showed excellent haemodynamic data with fewer gradients, no pulmonary regurgitation, no calcification and acellularity. Structural remodelling of the leaflet matrix structure was only detected in FC-treated tissue, whereas IFC valves maintained matrix integrity comparable to that of native controls. The collagen crimp period and amplitude and elastin structure were significantly different in the FC valve cusps compared to IFC and native cusps. Collagen fibres in the FC valves were less aligned and straightened. CONCLUSIONS: IFC heart valves with good haemodynamic function, reduced immunogenicity and preserved matrix structures have the potential to overcome the known limitations of the clinically applied FC valve.

Measurement of Specific Heat and Crystallization in VS55, DP6, and M22 Cryoprotectant Systems With and Without Sucrose.

Cryopreservation represents one if not the only long-term option for tissue and perhaps future organ banking. In one particular approach, cryopreservation is achieved by completely avoiding ice formation (or crystallization) through a process called vitrification. This "ice-free" approach to tissue banking requires a combination of high-concentration cryoprotective additives such as M22 (9.4 M), VS55 (8.4 M), or DP6 (6 M) and sufficiently fast rates of cooling and warming to avoid crystallization. In this article, we report the temperature-dependent specific heat capacity of the above-mentioned cryoprotective additives in small volumes (10 mg sample pans) at rates of 5°C/min and 10°C/min using a commercially available differential scanning calorimetry (TA Instruments Q1000), in the temperature range of -150°C to 30°C. This data can be utilized in heat-transfer models to predict thermal histories in a cryopreservation protocol. More specifically, the effects of temperature dependence of specific heat due to the presence of three different phases (liquid, ice, and vitreous phase) can dramatically impact the thermal history and therefore the outcome of the cryopreservation procedure. The crystallization potential of these cryoprotectants was also investigated by studying cases of maximal and minimal crystallization in VS55 and DP6, where M22 did not crystallize under any rates tested. To further reduce crystallization in VS55 and DP6, a stabilizing sugar (sucrose) was added in varying concentrations (0.15 M and 0.6 M) and was shown to further reduce crystallization, particularly in VS55, at modest rates of cooling (1°C/min, 5°C/min, and 10°C/min).

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.

Impact of T-cell-mediated immune response on xenogeneic heart valve transplantation: short-term success and mid-term failure.

OBJECTIVES: Allogeneic frozen cryopreserved heart valves (allografts or homografts) are commonly used in clinical practice. A major obstacle for their application is the limited availability in particular for paediatrics. Allogeneic large animal studies revealed that alternative ice-free cryopreservation (IFC) results in better matrix preservation and reduced immunogenicity. The objective of this study was to evaluate xenogeneic (porcine) compared with allogeneic (ovine) IFC heart valves in a large animal study. METHODS: IFC xenografts and allografts were transplanted in 12 juvenile merino sheep for 1-12 weeks. Immunohistochemistry, ex vivo computed tomography scans and transforming growth factor-ß release profiles were analysed to evaluate postimplantation immunopathology. In addition, near-infrared multiphoton imaging and Raman spectroscopy were employed to evaluate matrix integrity of the leaflets. RESULTS: Acellular leaflets were observed in both groups 1 week after implantation. Allogeneic leaflets remained acellular throughout the entire study. In contrast, xenogeneic valves were infiltrated with abundant T-cells and severely thickened over time. No collagen or elastin changes could be detected in either group using multiphoton imaging. Raman spectroscopy with principal component analysis focusing on matrix-specific peaks confirmed no significant differences for explanted allografts. However, xenografts demonstrated clear matrix changes, enabling detection of distinct inflammatory-driven changes but without variations in the level of transforming growth factor-ß. CONCLUSIONS: Despite short-term success, mid-term failure of xenogeneic IFC grafts due to a T-cell-mediated extracellular matrix-triggered immune response was shown.

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.