Porcine Heart Valve Research Articles

Optimization of Enzymatic and Chemical Decellularization of Native Porcine Heart Valves for the Generation of Decellularized Xenografts.

One of the most important medical interventions for individuals with heart valvular disease is heart valve replacement, which is not without substantial challenges, particularly for pediatric patients. Due to their biological properties and biocompatibility, natural tissue-originated scaffolds derived from human or animal sources are one type of scaffold that is widely used in tissue engineering. However, they are known for their high potential for immunogenicity. Being free of cells and genetic material, decellularized xenografts, consequently, have low immunogenicity and, thus, are expected to be tolerated by the recipient's immune system. The scaffold ultrastructure and ECM composition can be affected by cell removal agents. Therefore, applying an appropriate method that preserves intact the structure of the ECM plays a critical role in the final result. So far, there has not been an effective decellularization technique that preserves the integrity of the heart valve's ultrastructure while securing the least amount of genetic material left. This study demonstrates a new protocol with untraceable cells and residual DNA, thereby maximally reducing any chance of immunogenicity. The mechanical and biochemical properties of the ECM resemble those of native heart valves. Results from this study strongly indicate that different critical factors, such as ionic detergent omission, the substitution of Triton X-100 with Tergitol, and using a lower concentration of trypsin and a higher concentration of DNase and RNase, play a significant role in maintaining intact the ultrastructure and function of the ECM.

The Young Investigator Committee of the International Xenotransplantation Association-Perspective of advancements in the field in 2023.

The 2023 IXA conference, hosted in San Diego, CA, brimmed with excitement against the backdrop of recent innovations in both the pre-clinical and clinical realms with several first-in-human applications of xenotransplantation. The theme, "Pigs are flying," alluded to the adage that xenotransplantation would only become a clinical reality "when pigs fly," suggesting a day that might never come. The event witnessed significant attendance, with 600 participants-the highest in the history of an IXA-IPITA joint congress. Among the attendees were members of the Food and Drug Administration (FDA), the National Institutes of Health (NIH), and corporate sponsors deeply engaged in the field. We summarize the latest topics from the congress, ranging from the pros/cons of decedent models of xenotransplantation and genetic engineering of porcine heart valves, solid organs, and cells for clinical translation and their regulatory and ethical landscape.

Strengthened Decellularized Porcine Valves via Polyvinyl Alcohol as a Template Improving Processability.

The heart valve is crucial for the human body, which directly affects the efficiency of blood transport and the normal functioning of all organs. Generally, decellularization is one method of tissue-engineered heart valve (TEHV), which can deteriorate the mechanical properties and eliminate allograft immunogenicity. In this study, removable polyvinyl alcohol (PVA) is used to encapsulate decellularized porcine heart valves (DHVs) as a dynamic template to improve the processability of DHVs, such as suturing. Mechanical tests show that the strength and elastic modulus of DHVs treated with different concentrations of PVA significantly improve. Without the PVA layer, the valve would shift during suture puncture and not achieve the desired suture result. The in vitro results indicate that decellularized valves treated with PVA can sustain the adhesion and growth of human umbilical vein endothelial cells (HUVECs). All results above show that the DHVs treated with water-soluble PVA have good mechanical properties and cytocompatibility to ensure post-treatment. On this basis, the improved processability of DHV treated with PVA enables a new paradigm for the manufacturing of scaffolds, making it easy to apply.

316.2: Long term study of decellularized and glycan-modified GGTA1-KO porcine heart valves implanted into baboons

316.2: Long term study of decellularized and glycan-modified GGTA1-KO porcine heart valves implanted into baboons

Morphology and Morphometry of Aortic Valve in Humans and Porcines – A Comparative Study

Background: The aortic valve is a semilunar valve with three leaflets that are in the left ventricle’s outflow tract and only permits unidirectional blood flow away from the ventricle. The valve leaflet can tolerate extremely high mechanical and hemodynamic forces resulting from the great complexity and complicated cellular and molecular functions. Aims: The aim of the study is to provide an add-on to the understanding of the morphological and morphometrical aspects of human and porcine aortic heart valves on comparative grounds so that they can be used as a guide in the construction of three-dimensional (3D)-printed mechanical valve. Materials and Methods: Twenty swine and twenty human hearts were compared and examined in this investigation. The porcine and human hearts weighing in between 300 g and 450 g were considered, and any variation from the normal and any mechanical damage rendered during procurement were all excluded from the study. Results: The length of the attached margin of the right coronary cusp, left coronary cusp, and noncoronary cusp of human and porcine were 45.15 mm, 36.47 mm, and 41.53 mm and 36.55 mm, 43.14 mm, and 36.10 mm, respectively. The length of the free margin and the height of the cusps from nadir to the sinotubular junction and the effective height of the cusp from nadir to the free margin of the cusps of human and porcine have been compared. Conclusion: The human and swine aortic valves share morphological and morphometrical characteristics, which can be used to create 3D valves and tissue engineering procedures with greater biocompatibility and biofunctionality.

Electrospun Elastomeric Leaflet Substrates that Structurally and Mechanically Mimic Human Aortic Valve Leaflets

Creating leaflet substrates that accurately mimic the trilayered structure, anisotropic tensile characteristics, and elastomeric features of native human heart valve leaflets is essential to the success of heart valve tissue engineering. Earlier leaflet substrates have successfully replicated the mechanical properties of animal (porcine) heart valve leaflets. However, not one of them has been able to simultaneously replicate the trilayered structure and the highly anisotropic and elastomeric mechanical properties of human aortic heart valve leaflets. In this study, we employed a combination of polymers – polycaprolactone (PCL), poly(trimethylene carbonate-co-l-lactide) (PTMC-LA), and poly(trimethylene carbonate-co-caprolactone) (PTMC-CL) – in an electrospinning system to create elastomeric trilayer PCL/PTMC leaflet substrates that possess mechanical, flexural, and anisotropic properties similar to those of human native heart valve leaflets. We then compared these substrates with non-elastomeric trilayer PCL leaflet substrates to determine their effect on leaflet tissue engineering. Porcine valvular interstitial cells (PVICs) were cultured on these substrates for 1 month in static conditions to develop trilayer PCL and PCL/PTMC cell-cultured constructs. The PCL/PTMC substrates exhibited lower crystallinity and hydrophobicity than the PCL substrate, resulting in better cell adhesion and infiltration. The PCL/PTMC substrates demonstrated significantly greater cell proliferation, extracellular matrix production, and preferable gene and protein expression than the PCL substrates. Furthermore, in an osteogenic environment, the PCL/PTMC substrates showed superior resistance to calcification compared to the PCL substrates. The development of trilayer PCL/PTMC substrates with mechanical and structural characteristics resembling native tissue enhances leaflet tissue engineering.

Comparative Morphological and Morphometrical Analysis of Atrio-Ventricular Valves of Human and Porcine

There is increased incidence of valvular heart diseases in recent years due to life style modifications. The mortality rates in valvular diseases are kept in pace using various modalities of treatments. One such lifesaving treatment is valve replacement surgeries. These are done by using mechanical valve prosthesis or tissue grafts. The tissue valves prosthesis, harvested from porcine heart are called as xenograft and are increasingly used in valve repair and replacement surgeries. In the present scenario, there is a smaller number of systematically analysed literatures available on the comparative anatomy of human and porcine heart valves. Hence this study was carried out to acquire knowledge and to put forth some points to future research works on heart valves. In this study, 20 formalin fixed porcine and human hearts were procured from slaughter house and cadavers respectively. The morphology and morphometry of tricuspid valve and mitral valve was observed and analysed using spss software 20 version. All the dependent variables were compared using student t test and independent sample test. The results were tabulated and compared. It was observed that the tricuspid and the mitral valve of the porcine resembles the corresponding human heart valves in morphology and morphometry and their values were coinciding to their maximum. The porcine valve resembles human heart valves in morphology and it can be used in designing valve substitutes in replacement surgeries. Porcine valve can also be used as bio-prosthesis by matching the morphometry and by reducing the geometrical difference to their minimum by using any interventional radiology. KEY WORDS: Tissue Graft, Porcine, Tricuspid Valve, Mitral Valve, Morphology And Morphometry.

Effect of promethazine on biofilms of gram-positive cocci associated with infectious endocarditis

This study evaluated the antimicrobial activity of promethazine against Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus mutans and its effect on the antimicrobial susceptibility of biofilms grown in vitro and ex vivo on porcine heart valves. Promethazine was evaluated alone and in combination with vancomycin and oxacillin against Staphylococcus spp. and vancomycin and ceftriaxone against S. mutans in planktonic form and biofilms grown in vitro and ex vivo. Promethazine minimum inhibitory concentration range was 24.4-95.31 μg/mL and minimum biofilm eradication concentration range was 781.25-3.125 μg/mL. Promethazine interacted synergistically with vancomycin, oxacillin and ceftriaxone against biofilms in vitro. Promethazine alone reduced (p < 0.05) the CFU-counts of biofilms grown on heart valves for Staphylococcus spp., but not for S. mutans, and increased (p < 0.05) the activity of vancomycin, oxacillin and ceftriaxone against biofilms of Gram-positive cocci grown ex vivo. These findings bring perspectives for repurposing promethazine as adjuvant in the treatment of infective endocarditis.

Prevention by the CXCR2 antagonist SCH527123 of the calcification of porcine heart valve cusps implanted subcutaneously in rats.

Calcification is a main cause of bioprosthetic heart valves failure. It may be promoted by the inflammation developed in the glutaraldehyde (GA)-fixed cusps of the bioprosthesis. We tested the hypothesis that antagonizing the C-X-C chemokines receptor 2 (CXCR2) may prevent the calcification of GA-fixed porcine aortic valves. Four-week-old Sprague Dawley males were transplanted with 2 aortic valve cusps isolated from independent pigs and implanted into the dorsal wall. Four groups of 6 rats were compared: rats transplanted with GA-free or GA-fixed cusps and rats transplanted with GA-fixed cusps and treated with 1 mg/kg/day SCH5217123 (a CXCR2 antagonist) intraperitoneally (IP) or subcutaneously (SC) around the xenograft, for 14 days. Then, rats underwent blood count before xenografts have been explanted for histology and biochemistry analyses. A strong calcification of the xenografts was induced by GA pre-incubation. However, we observed a significant decrease in this effect in rats treated with SCH527123 IP or SC. Implantation of GA-fixed cusps was associated with a significant increase in the white blood cell count, an effect that was significantly prevented by SCH527123. In addition, the expression of the CD3, CD68 and CXCR2 markers was reduced in the GA-fixed cusps explanted from rats treated with SCH527123 as compared to those explanted from non-treated rats. The calcification of GA-fixed porcine aortic valve cusps implanted subcutaneously in rats was significantly prevented by antagonizing CXCR2 with SCH527123. This effect may partly result from an inhibition of the GA-induced infiltration of T-cells and macrophages into the xenograft.

Engineering Heart Valve Interfaces Using Melt Electrowriting: Biomimetic Design Strategies from Multi-Modal Imaging.

Interfaces within biological tissues not only connect different regions but also contribute to the overall functionality of the tissue. This is especially true in the case of the aortic heart valve. Here, melt electrowriting (MEW) is used to engineer complex, user-defined, interfaces for heart valve scaffolds. First, a multi-modal imaging investigation into the interfacial regions of the valve reveals differences in collagen orientation, density, and recruitment in previously unexplored regions including the commissure and inter-leaflet triangle. Overlapping, suturing, and continuous printing methods for interfacing MEW scaffolds are then investigated for their morphological, tensile, and flexural properties, demonstrating the superior performance of continuous interfaces. G-codes for MEW scaffolds with complex interfaces are designed and generated using a novel software and graphical user interface. Finally, a singular MEW scaffold for the interfacial region of the aortic heart valve is presented incorporating continuous interfaces, gradient porosities, variable layer numbers across regions, and tailored fiber orientations inspired by the collagen distribution and orientation from the multi-modal imaging study. The scaffold exhibits similar yield strain, hysteresis, and relaxation behavior to porcine heart valves. This work demonstrates the ability of a bioinspired approach for MEW scaffold design to address the functional complexity of biological tissues.

Hybrid heart valves with VEGF-loaded zwitterionic hydrogel coating for improved anti-calcification and re-endothelialization.

With the aging of the population in worldwide, valvular heart disease has become one of the most prominent life-threatening diseases in human health, and heart valve replacement surgery is one of the therapeutic methods for valvular heart disease. Currently, commercial bioprosthetic heart valves (BHVs) for clinical application are prepared with xenograft heart valves or pericardium crosslinked by glutaraldehyde. Due to the residual cell toxicity from glutaraldehyde, heterologous antigens, and immune response, there are still some drawbacks related to the limited lifespan of bioprosthetic heart valves, such as thrombosis, calcification, degeneration, and defectiveness of re-endothelialization. Therefore, the problems of calcification, defectiveness of re-endothelialization, and poor biocompatibility from the use of bioprosthetic heart valve need to be solved. In this study, hydrogel hybrid heart valves with improved anti-calcification and re-endothelialization were prepared by taking decellularized porcine heart valves as scaffolds following grafting with double bonds. Then, the anti-biofouling zwitterionic monomers 2-methacryloyloxyethyl phosphorylcholine (MPC) and vascular endothelial growth factor (VEGF) were utilized to obtain a hydrogel-coated hybrid heart valve (PEGDA-MPC-DHVs@VEGF). The results showed that fewer platelets and thrombi were observed on the surface of the PEGDA-MPC-DHVs@VEGF. Additionally, the PEGDA-MPC-DHVs@VEGF exhibited excellent collagen stability, biocompatibility and re-endothelialization potential. Moreover, less calcification deposition and a lower immune response were observed in the PEGDA-MPC-DHVs@VEGF compared to the glutaraldehyde-crosslinked DHVs (Glu-DHVs) after subcutaneous implantation in rats for 30 days. These studies demonstrated that the strategy of zwitterionic hydrogels loaded with VEGF may be an effective approach to improving the biocompatibility, anti-calcification and re-endothelialization of bioprosthetic heart valves.

Valve interstitial cells under impact load, a mechanobiology study

Understanding the relationship between mechanobiology and the biosynthetic activities of the valve interstitial cells (VICs) in health and disease under severe dynamic loading conditions is of particular interest. The purpose of this study is to further understand the mechanobiology of heart valve leaflet tissue and the VICs under impact forces. Two novel computational and experimental platforms were developed to study the effect of impact load on the VICs to monitor for apoptosis. The first objective was to design and develop an apparatus to experimentally study viability (apoptosis) of the porcine heart valve leaflet tissue VICs in the aortic position under controlled impact forces. Apoptosis was assessed based on terminal transferase dUTP nick end-labelling (TUNEL) assay. The second objective was to develop a computational platform to estimate the stress and strain fields in the vicinity of VICs when the tissue experiences impact forces. A nonlinear finite element (FE) model with an anisotropic, hyperelastic and heterogeneous material model for the matrix and cells was developed. Preliminary results confirm that interstitial cells are successfully resistant to impact loads up to 30 times more than normal physiological conditions. Additionally, the structure and composition of heart valve leaflet tissue provides a mechanical shield for VICs protecting them from excessive mechanical forces such as impact loads. Although, the entire tissue may experience excessive stresses, which may lead to structural damage, the stresses around and near VICs remain consistency low. Results of this study may be used for heart valve leaflet tissue-engineering, as well as further understanding the mechanobiology of the VICs in health and disease.

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.

Aortic Leaflet Stresses Are Substantially Lower Using Pulmonary Visceral Pleura Than Pericardial Tissue.

Background: Porcine heart and bovine pericardium valves, which are collagen-based with relatively little elastin, have been broadly utilized to construct bioprosthetic heart valves (BHVs). With a larger proportion of elastin, the pulmonary visceral pleura (PVP) has greater elasticity and could potentially serve as an advantageous biomaterial for the construction/repair of BHVs. The question of how the aortic valve’s performance is affected by its bending rigidity has not been well studied. Methods: Based on the stress–strain relationships of the pericardium and PVP determined by planar uni-axial tests, a three-dimensional (3D) computational fluid–structure interaction (FSI) framework is employed to numerically investigate the aortic valve’s performance by considering three different cases with Young’s modulus as follows: , , and kPa, respectively. Results: The stroke volumes are 112, 99.6, and 91.4 ml as Young’s modulus increases from 375 to 750 and 1500 kPa, respectively. Peak geometric opening area (GOA) values are 2.3, 2.2, and 2.0 cm2 for , 750, and 1500 kPa, respectively. The maximum value of the aortic leaflet stress is about 271 kPa for kPa, and it increases to about 383 and 540 kPa for and 1500 kPa in the belly region at the peak systole, while it reduces from 550 kPa to 450 and 400 kPa for , 750, and 1500 kPa, respectively, at the instant of peak “water-hammer”. Conclusion: A more compliant PVP aortic leaflet valve with a smaller Young’s modulus, , has a higher cardiac output, larger GOA, and lower hemodynamic resistance. Most importantly, the aortic leaflet stresses are substantially lower in the belly region within the higher compliance PVP aortic valve tissue during the systole phase, even though some stress increase is also found during the fast-closing phase due to the “water-hammer” effect similar to that in the pericardial tissue. Future clinical studies will be conducted to test the hypothesis that the PVP-based valve leaflets with higher compliance will have lower fatigue or calcification rates due to the overall lower stress.

Prevention of the calcification of porcine heart valve leaflets implanted subcutaneously in rats by an interleukin-8 receptors antagonist (SCH527123)

Bioprosthetic heart valve calcification may result from interactions between the host's immune system and the glutaraldehyde (GA)-fixed tissue that makes up the leaflets. We hypothesized that antagonizing interleukin-8 (IL-8) receptors, CXCR-1 and CXCR-2, may prevent the calcification of GA-fixed porcine aortic valves implanted subcutaneously in rats. Three-week-old Sprague Dawlay males were transplanted with 2 aortic valve leaflets isolated from pigs and implanted into the dorsal wall. Four groups of 3–4 rats (6–8 independent leaflets each time) were compared: glutaraldehyde (GA)-free leaflets, GA-fixed leaflets (0.6% GA for 24 h before implantation), GA-fixed leaflets and treatment of rats with 1 mg/kg SCH5217123 (a CXCR1/2 antagonist) injected once a day locally subcutaneously (SC) or intraperitoneally (IP). Fourteen days later, rats underwent blood leukocyte count and CT-scan, before porcine leaflets explantation and histology and biochemistry analyses. As expected, strong calcification of ectopic valves was induced by the GA. However, we observed a significant about 40% decrease in this effect when rats were treated with the SCH527123. These effects were confirmed by CT-scan acquisitions and Alizarin red staining of valves. Besides, GA pre-incubation of valves significantly increased leukocytes count in rat blood, an effect that is significantly prevented by IP SCH527123 (6.9, 16.6 and 6.8 × 10 9 /L in the GA-free, GA and GA + IP SCH527123 groups respectively, P = 0.0129). Immunohistochemistry of ectopic valves highlighted higher infiltration of CD3 (lymphocytes) and CD68 (macrophages) cells in the GA group compared to the GA-free and GA + SCH527123 groups. Therefore, the calcification of GA-fixed porcine aortic valve leaflets, implanted subcutaneously in rats, is significantly prevented by antagonizing IL-8 receptors with SCH527123. This effect might partly result from an inhibition of the GA-induced leukocytes activation and recruitment into the graft tissue.

Surgical reconstruction of the right ventricular outflow tract-The clock is still ticking.

Surgical reconstruction of the right ventricular outflow tract-The clock is still ticking.

Chemokine-Induced PBMC and Subsequent MSC Migration Toward Decellularized Heart Valve Tissue.

Enhancing the recellularization of a decellularized heart valve in situ may lead to an improved or ideal heart valve replacement. A promising approach is leveraging the immune response for inflammation-mediated recellularization. However, this mechanism has not been previously demonstrated in vitro. This study investigated loading the chemokine MCP-1 into decellularized porcine heart valve tissue and measured the migration of human peripheral blood mononuclear cells (PBMCs) and mesenchymal stem cells (MSCs) toward the chemokine loaded valve tissue. The results of this study demonstrate that MCP-1-loaded tissues increase PBMC migration compared to non-loaded tissues. Additionally, we demonstrate MCP-1-loaded tissues that have recruited PBMCs lead to increased migration of MSCs compared to decellularized tissue alone. The results of this study provide evidence for the inflammation-mediated recellularization mechanism. Furthermore, the results support the use of such an approach for enhancing the recellularization of a decellularized heart valve.

Elimination of macrophages reduces glutaraldehyde-fixed porcine heart valve degeneration in mice subdermal model.

Glutaraldehyde‐fixed porcine heart valve (GPHV) calcify and deteriorate over time. The aim of this study was to explore the roles macrophages play in mediating calcification and degeneration of the valve's connective tissue matrix. GPHV were implanted subcutaneously in the abdomens of C57BL/6 mice. The mice were equally divided into two study groups: (a) GPHV +phosphate buffered saline (PBS) liposomes, and (b) GPHV +clodronate liposomes. GPHV were collected for further analyses at 4 weeks post implant. Macrophages were almost depleted from the spleens of mice injected with clodronate liposomes as indicated by immunohistochemical staining. Furthermore, the expression of matrix metalloproteinase‐2 (MMP‐2), MMP‐9, and proinflammatory cytokines like IL‐1β, IL‐6, MCP‐1, MIP‐1a, MIP‐1b, were downregulated in the GPHV +Clodronate liposomal group compared with the GPHV+PBS liposomal group. Clodronate liposomal treatment led to significant decreases in the expression of RUNX2, ALP and OPN as well as less calcium deposits in GPHVs compared with PBS liposomal treatment. This finding indicated that infiltrating macrophages are critically involved in the development of calcification and deterioration in GPHVs. Macrophage depletion by clodronate liposomes decreased the extent of GPHV’s calcification and deterioration.

Dot blots of solubilized extracellular matrix allow quantification of human antibodies bound to epitopes present in decellularized porcine pulmonary heart valves.

The present study reports the development of a sensitive dot blot protocol for determining the level of preformed antibodies against porcine heart valve tissue derived from wild-type (WT) and α-Gal-KO (GGTA1-KO) pigs in human sera. The assay uses decellularized and solubilized heart valve tissue; antibody binding found in this dot blot assay could be correlated with antibody titers of preformed anti-α-Gal and anti-Neu5Gc antibodies detected by a sensitive ELISA. The ultimate protocol had an inter-assay variance of 9.5% and an intra-assay variance of 9.2%, showing that the test is reliable and highly reproducible. With the aid of this dot blot assay, we found significant variation with regard to antibody contents among twelve human sera. Binding of preformed antibodies to WT tissue was significantly higher than to GGTA1-KO tissue. The dot blot assay described herein could be a valuable tool to measure preformed antibody levels in human sera against unknown epitopes on decellularized tissue prior to implantation. Ultimately, this prescreening may allow a matching of the porcine xenograft with the respective human recipients in demand and thus may become an important tool for graft long-term survival similar to current allotransplantation settings.

Freeze-Drying of Decellularized Heart Valves for Off-the-Shelf Availability.

Malfunctioning heart valves can cause severe health problems, which if left untreated can lead to death. One of the treatment options is to replace a diseased heart valve with a decellularized valve construct prepared from human or animal material. Decellularized tissue scaffolds closely resemble properties of native tissue, while lacking immunogenic factors of cellular components. After transplantation, circulating stem and progenitor cells of the patient adhere to the scaffold resulting in in vivo tissue regeneration of the valve. Decellularized heart valve scaffold implants need to be stored to be readily available whenever needed, which can be done by freeze-drying. The advantage of freeze-drying is that it does not require bulky and energy-consuming freezing equipment for storage and allows easy transport. This chapter outlines the entire process from decellularization to freeze-drying to obtain dry decellularized heart valves, which after a simple rehydration step, can be used as implants. The protocol is described for porcine heart valves, but procedures can easily be adapted for material obtained from other species.