Ex Vivo and In Vivo Analysis of a Novel Porcine Aortic Patch for Vascular Reconstruction.

(1) Background: The aim of the present study was the biocompatibility analysis of a novel xenogeneic vascular graft material (PAP) based on native collagen won from porcine aorta using the subcutaneous implantation model up to 120 days post implantationem. As a control, an already commercially available collagen-based vessel graft (XenoSure®) based on bovine pericardium was used. Another focus was to analyze the (ultra-) structure and the purification effort. (2) Methods: Established methodologies such as the histological material analysis and the conduct of the subcutaneous implantation model in Wistar rats were applied. Moreover, established methods combining histological, immunohistochemical, and histomorphometrical procedures were applied to analyze the tissue reactions to the vessel graft materials, including the induction of pro- and anti-inflammatory macrophages to test the immune response. (3) Results: The results showed that the PAP implants induced a special cellular infiltration and host tissue integration based on its three different parts based on the different layers of the donor tissue. Thereby, these material parts induced a vascularization pattern that branches to all parts of the graft and altogether a balanced immune tissue reaction in contrast to the control material. (4) Conclusions: PAP implants seemed to be advantageous in many aspects: (i) cellular infiltration and host tissue integration, (ii) vascularization pattern that branches to all parts of the graft, and (iii) balanced immune tissue reaction that can result in less scar tissue and enhanced integrative healing patterns. Moreover, the unique trans-implant vascularization can provide unprecedented anti-infection properties that can avoid material-related bacterial infections.

A Murine Ommaya Xenograft Model to Study Direct-Targeted Therapy of Leptomeningeal Disease.

Leptomeningeal disease (LMD) is an uncommon type of central nervous system (CNS) metastasis to the cerebral spinal fluid (CSF). The most common cancers that cause LMD are breast and lung cancers and melanoma. Patients diagnosed with LMD have a very poor prognosis and generally survive for only a few weeks or months. One possible reason for the lack of efficacy of systemic therapy against LMD is the failure to achieve therapeutically effective concentrations of drug in the CSF because of an intact and relatively impermeable blood-brain barrier (BBB) or blood-CSF barrier across the choroid plexus. Therefore, directly administering drugs intrathecally or intraventricularly may overcome these barriers. This group has developed a model that allows for the effective delivery of therapeutics (i.e., drugs, antibodies, and cellular therapies) chronically and the repeated sampling of CSF to determine drug concentrations and target modulation in the CSF (when the tumor microenvironment is targeted in mice). The model is the murine equivalent of a magnetic resonance imaging-compatible Ommaya reservoir, which is used clinically. This model, which is affixed to the skull, has been designated as the "Murine Ommaya." As a therapeutic proof of concept, human epidermal growth factor receptor 2 antibodies (clone 7.16.4) were delivered into the CSF via the Murine Ommaya to treat mice with LMD from human epidermal growth factor receptor 2-positive breast cancer. The Murine Ommaya increases the efficiency of drug delivery using a miniature access port and prevents the wastage of excess drug; it does not interfere with CSF sampling for molecular and immunological studies. The Murine Ommaya is useful for testing novel therapeutics in experimental models of LMD.

A systematic review of decellularized allograft and xenograft-derived scaffolds in bone tissue regeneration.

Bone substitutes are used in nearly half of all musculoskeletal surgeries. The "gold standard" graft is an autograft that is limited by supply and site morbidity. Therefore, allograft sources are the current alternative for clinical practice with some side effects, such as immune responses and risk of disease transmission. In this paper, we have systematically reviewed the development and characterization of decellularized allograft or xenograft-derived scaffolds as bone graft substitutes. The databases of PubMed, Cochrane, Scopus, and Web of Science were searched for experimental studies that investigated the potential of acellular allograft or xenograft-derived scaffold for bone regeneration. The search was finalized on 14 September 2020. The initial electronic database search resulted in a total of 484 studies. During the screening process, 416 studies were excluded due to not meeting the inclusion criteria. Finally, a total of 68 articles were included, in which human or animal tissues have been decellularized for bone tissue generation purposes. Although in most studies, a decellularized bone was used for the generation of a bone scaffold, other decellularized tissues, such as the human amniotic membrane or human adipose tissue, were also used in some researches for this purpose. In 42 studies out of the 68, decellularized bone scaffolds were implanted into in vivo animal models. 8 studies used animal bone tissues as an allograft. 12 studies used human tissues as a xenograft. The studies have shown that decellularized allograft or xenograft scaffolds have high biocompatibility with little or no host response, and can enhance new bone formation. Overall, the results of this study suggest that the decellularized xenograft-derived cancellous bone scaffolds can be considered as alternatives to the autologous bone graft. This systematic review might affect future research directions and the preoperative planning of graft selection.

Ridge preservation of compromised extraction sockets applying a soft cortical membrane: A canine proof-of-principle evaluation.

OBJECTIVES: To explore whether placement of a soft cortical membrane can restore and regenerate the original alveolar ridge contour in deficient sockets. MATERIALS AND METHODS: One Beagle dog was used in this proof-of-principle evaluation. In a first intervention, a standardized buccal dehiscence defect was artificially created at the distal roots of the 3rd and 4th mandibular premolars. Four weeks later, following endodontic treatment of the mesial roots, teeth were hemisected and the distal roots were extracted without raising a flap. A cortical membrane (Lamina®, Osteobiol) was placed outside of the bony envelope of the extraction socket to rebuild the buccal bone contour. Afterwards, sockets were filled with a collagen-modified porcine bone graft material (Gen-Os®, Osteobiol) to the level of the surrounding bone height. The socket orifice was closed with a porcine dermal matrix (Derma®). After four months, block specimens containing the socket-sites and remaining roots were retrieved, histologically processed and analyzed. RESULTS: Surgery and post-operative healing were uneventful. Histologically, bone formation under the membrane was found, i.e. bony protrusions and ossicles by osteoblasts could be identified. Concomitantly, the membrane showed clear signs of degradation. Bone substitute was well integrated in newly formed bone and resorption of particles was found. CONCLUSION: Three major observations were made in the present proof-of-principle study: (i) regeneration of a compromised socket seems possible when applying the presented approach, (ii) the soft cortical membrane was sufficiently stable to allow for the establishment of the contour and to inhibit soft tissue invasion and (iii) the applied xenogenic graft material was undergoing remodelling processes while allowing adequate bone regeneration.

Developmental Potential and Plasticity of Olfactory Epithelium Stem Cells Revealed by Heterotopic Grafting in the Adult Brain.

The neural stem cells (NSCs) residing in the olfactory epithelium (OE) regenerate damaged olfactory sensory neurons throughout adulthood. The accessibility and availability of these NSCs in living individuals, including humans, makes them a promising candidate for harvesting their potential for cell replacement therapies. However, this requires an in-depth understanding of their developmental potential after grafting. Here, we investigated the developmental potential and plasticity of mouse OE-derived NSCs after grafting into the adult subventricular zone (SVZ) neurogenic niche. Our results showed that OE-derived NSCs integrate and proliferate just like endogenous SVZ stem cells, migrate with similar dynamics as endogenous neuroblasts toward the olfactory bulb, and mature and acquire similar electrophysiological properties as endogenous adult-born bulbar interneurons. These results reveal the developmental potential and plasticity of OE-derived NSCs in vivo and show that they can respond to heterotopic neurogenic cues to adapt their phenotype and become functional neurons in ectopic brain regions.

Developing a xenograft model of human vasculature in the mouse ear pinna.

Humanised xenograft models allow for the analysis of human tissue within a physiological environment in vivo. However, current models often rely on the angiogenesis and ingrowth of recipient vasculature to perfuse tissues, preventing analysis of biological processes and diseases involving human blood vessels. This limits the effectiveness of xenografts in replicating human physiology and may lead to issues with translating findings into human research. We have designed a xenograft model of human vasculature to address this issue. Human subcutaneous fat was cultured in vitro to promote blood vessel outgrowth prior to implantation into immunocompromised mice. We demonstrate that implants survived, retained human vasculature and anastomosed with the circulatory system of the recipient mouse. Significantly, by performing transplants into the ear pinna, this system enabled intravital observation of xenografts by multiphoton microscopy, allowing us to visualise the steps leading to vascular cytoadherence of erythrocytes infected with the human parasite Plasmodium falciparum. This model represents a useful tool for imaging the interactions that occur within human tissues in vivo and permits visualization of blood flow and cellular recruitment in a system which is amenable to intervention for various studies in basic biology together with drug evaluation and mechanism of action studies.

Long-term survival of full-thickness corneal xenografts from α1,3-galactosyltransferase gene-knockout miniature pigs in non-human primates.

BACKGROUND: We aimed to investigate (a) the long-term survival of corneal grafts from α1,3-galactosyltransferase gene-knockout miniature (GTKOm) pigs in non-human primates as a primary outcome and (b) the effect of anti-CD20 antibody on the survival of corneal grafts from GTKOm pigs as a secondary outcome. METHODS: Nine rhesus macaques undergoing full-thickness corneal xenotransplantation using GTKOm pigs were systemically administered steroid, basiliximab, intravenous immunoglobulin, and tacrolimus with (CD20 group) or without (control group) anti-CD20 antibody. RESULTS: Graft survival was significantly longer (P = .008) in the CD20 group (>375, >187, >187, >83 days) than control group (165, 91, 72, 55, 37 days). When we compared the graft survival time between older (>7- month-old) and younger (≤7-month-old) aged donor recipients, there was no significant difference. Activated B cells were lower in the CD20 group than control group (P = .026). Aqueous humor complement C3a was increased in the control group at last examination (P = .043) and was higher than that in the CD20 group (P = .014). Anti-αGal IgG/M levels were unchanged in both groups. At last examination, anti-non-Gal IgG was increased in the control group alone (P = .013). CONCLUSIONS: The GTKOm pig corneal graft achieved long-term survival when combined with anti-CD20 antibody treatment. Inhibition of activated B cells and complement is imperative even when using GTKO pig corneas.

Randomized study to prove the quality of human ovarian tissue cryopreservation by xenotransplantation into mice.

PURPOSE: To study the quality of our human ovarian tissue cryopreservation technique as performed in the first official "International Fertility Protection Centre" in China in patients with certain cancer types using a mouse model, and to find the best site for tissue transplantation in the mouse. METHODS: Thirty-six BALB/C female nude mice were randomly divided into 3 groups, group 1: control group; group 2: ovariectomized group; group 3: ovarian tissue transplantation group. Seventy-two pieces obtained from six ovarian tissue samples from each of three cancer patients were transplanted into the ovarian bursa cavity (OBC), the subcutaneous thigh (TS) and the subcutaneous neck (NS) and removed after 1.5 and 2.5 months, respectively. Follicular growth rate (FGR), total follicle surviving rate (TFSR), tissue recovery rate (TRR), antral follicles (AF), follicle stimulating hormone (FSH), estradiol (E2) and anti-Mullerian hormone (AMH) levels were measured. RESULTS: No significant differences in FGR, OBC, NS (p > 0.05); TFSR was 100% in OBC, NS and TS. No significant differences in TRR (p > 0.05); AF were found only in OBC; TFSR was 100% after transplantation; significantly higher FGR in the 2.5 months compared to the 1.5 months-group (p < 0.05). AMH- and E2-level in group 1 and 3 were significantly higher than in group 2 (p < 0.05); in contrast, FSH was significantly lower. CONCLUSIONS: After transplantation in the mice, the thawed ovarian tissue survived and follicles developed. The ovarian fossa site was the best site for transplantation. Our animal experiments can verify that our human ovarian tissue cryopreservation technique can preserve the quality of ovarian tissue. This is the essential precondition for successful re-transplantation into the patients after performing chemo/radiotherapy to protect ovarian function and fertility.

Ultrasound-Guided Approaches to Improve Orthotopic Mouse Xenograft Models for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide. While curative approaches for early stage HCC exist, effective treatment options for advanced HCC are lacking. Furthermore, there are no efficient chemopreventive strategies to limit HCC development once cirrhosis is established. One challenge for drug development is unsatisfactory animal models. In this article, we describe an orthotopic xenograft mouse model of human liver cancer cell lines through image-guided injection into the liver. This technique provides a less invasive yet highly efficient approach to engraft human HCC into mouse liver. Similarly, image-guided injections are used to deliver chemotherapeutics locally, enabling reduction in potential systemic adverse effects, while reducing the required dose for a therapeutic effect. In summary, this image-guided strategy provides a novel and convenient approach to improve current HCC mouse models. © 2019 The Authors. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Human Xenograft Model.

Human-SCID grafting is a commonly used technique for the long-term investigation of rheumatoid arthritis (RA) explants. To establish a chimeric immunological system in NOD/SCID mice, RA patient-derived pannus tissue from the synovial membrane, articular cartilage, and bone can be transplanted subcutaneously. The same patient-derived peripheral blood mononuclear cell chimerism can be successfully achieved by intraperitoneal engraftment. This xenograft model is able to be used for initial screening of human target-specified biologics.

Tumor xenograft animal models for esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal cancer worldwide and highly prevalent in less developed regions. Management of ESCC is challenging and involves multimodal treatments. Patient prognosis is generally poor especially for those diagnosed in advanced disease stage. One factor contributing to this clinical dismal is the incomplete understanding of disease mechanism, for which this situation is further compounded by the presence of other limiting factors for disease diagnosis, patient prognosis and treatments. Tumor xenograft animal models including subcutaneous tumor xenograft model, orthotopic tumor xenograft model and patient-derived tumor xenograft model are vital tools for ESCC research. Establishment of tumor xenograft models involves the implantation of human ESCC cells/xenografts/tissues into immunodeficient animals, in which mice are most commonly used. Different tumor xenograft models have their own advantages and limitations, and these features serve as key factors to determine the use of these models at different stages of research. Apart from their routine use on basic research to understand disease mechanism of ESCC, tumor xenograft models are actively employed for undertaking preclinical drug screening project and biomedical imaging research.

Fate of Free Fat Grafts with or without Adipogenic Adjuncts to Enhance Graft Outcomes.

BACKGROUND: Free fat grafting is popular, but it is still unclear how it works. Although focusing on graft survival seems an obvious direction for improving clinical results, the authors' research suggests that long-term volume retention is in part attributable to new fat regeneration. Measures to facilitate adipogenesis may therefore be equally important. METHODS: To investigate the relative roles of survival and regeneration of fat grafts, the authors measured the fate of human lipoaspirate implanted into the scalps of immunodeficient mice, with and without stromal vascular fraction and a porcine extracellular matrix (Adipogel). Specifically, the authors were interested in volume retention, and the composition of implanted or regenerated tissue at 6 and 12 weeks. RESULTS: Free fat grafts exhibited poor volume retention and survival. Almost all of the injected human adipocytes died, but new mouse fat formed peripheral to the encapsulated fat graft. Adipogel and stromal vascular fraction improved proliferation of murine fat and human vasculature. Human CD34 stromal cells were present but only in the periphery, and there was no evidence that these cells differentiated into adipocytes. CONCLUSIONS: In the authors' model, most of the implanted tissue died, but unresorbed dead fat accounted substantially for the long-term, reduced volume. A layer of host-derived, regenerated adipose tissue was present at the periphery. This regeneration may be driven by the presence of dying fat, and it was enhanced by addition of the authors' adipogenic adjuncts. Future research should perhaps focus not only on improving graft survival but also on enhancing the adipogenic environment conducive to fat regeneration.

Transplant Tolerance: Current Insights and Strategies for Long-Term Survival of Xenografts.

Xenotransplantation is an attractive solution to the problem of allograft shortage. However, transplants across discordant species barriers are subject to vigorous immunologic and pathobiologic hurdles, some of which might be overcome with the induction of immunologic tolerance. Several strategies have been designed to induce tolerance to a xenograft at both the central (including induction of mixed chimerism and thymic transplantation) and peripheral (including adoptive transfer of regulatory cells and blocking T cell costimulation) levels. Currently, xenograft tolerance has been well-established in rodent models, but these protocols have not yet achieved similar success in nonhuman primates. This review will discuss the major barriers that impede the establishment of immunological tolerance across xenogeneic barriers and the potential solution to these challenges, and provide a perspective on the future of the development of novel tolerance-inducing strategies.

Functional Maturation and In Vitro Differentiation of Neonatal Porcine Islet Grafts.

BACKGROUND: There is a strong rationale to pursue the use of neonatal porcine islets (NPIs) as an unlimited source of islets for clinical xenotransplantation. Because NPIs are composed of immature insulin producing beta (ß) cells and ductal precursor cells, they provide an ideal model to examine culture conditions to enhance ß cell proliferation and/or ß cell neoformation from ductal cells. In an attempt to optimize the potential of NPIs as a source of ß cell grafts, we used an in vitro differentiation protocol and measured its effect on the functional maturation and differentiation of NPIs. METHODS: Pancreata from 1- to 3-day-old neonatal pigs were digested and cultured in standard Ham's F10 media for 5 days. Each independent preparation was then further cultured in Dulbecco's modified Eagle medium nutrient mixture-F12 differentiation media containing growth factors added in a stepwise fashion, or cultured in control Ham's F10 media. After 20 days in culture, islets were assessed for insulin secretory capacity, cellular composition, gene expression, and metabolic activity after transplantation in immunodeficient mice with diabetes. RESULTS: Compared with control islets, differentiated islets exhibited a significantly higher proportion of endocrine cells, proliferating cell nuclear antigen double positive ß cells, and an enhanced glucose-stimulated insulin secretory activity. Mice transplanted with differentiated islets had significantly lower blood glucose values at weeks 18 and 20 compared with nondifferentiated controls and were shown to be more glucose tolerant. CONCLUSIONS: Culturing NPIs in a 20-day stepwise differentiation media increases the proportion of endocrine cells and augments both in vitro and in vivo function of the islets.

An Experimental Study of Femto-Laser in Assisting Xenograft Acellular Cornea Matrix Lens Transplantation.

BACKGROUND The aim of this study was to evaluate the feasibility of using a femto-laser in assisting xenograft cornea matrix lens transplantation in correcting ametropia, along with evaluating the effectiveness and predictability of this procedure. MATERIAL AND METHODS A corneal matrix pouch was prepared on the right eyes on 8 healthy New Zealand rabbits by a femto-laser that was also employed to perform small incision lenticule extraction (SMILE) on 8 bovine cornea matrix lenses (+6D). A lens was treated acellular and implanted into a right rabbit cornea matrix pouch. Surface inflammation was observed at 1, 2, 4, 8, 12, and 24 weeks after surgery. Anterior ocular segment optical coherence tomography (OCT), corneal topography, retinoscopy, and cornea endothelial cell enumeration were performed. RESULTS All the surgeries were successfully performed without any complications. The hyperopia condition of the rabbit eyes transformed into myopia status at an early stage and gradually developed hyperopia. Diopter at 24 weeks after surgery was 1/3 of that before surgery. Central corneal thickness stabilized at 4 weeks after surgery. Anterior segment OCT showed a clear lens edge at early post-operative stage, and blurred edge at 24 weeks later, indicating gradual fusion with the rabbit corneal matrix. CONCLUSIONS Femto-laser assisted xenograft corneal matrix lens transplantation is safe and effective in correcting ametropia, with satisfactory predictability, thus providing novel choice for correcting ametropia.

Action of a deproteinized xenogenic biomaterial in the process of bone repair in rats submitted to inhalation of cigarette smoke.

PURPOSE: To investigate if the inorganic bovine bone matrix changes the bone formation in rats submitted to inhalation of cigarette smoke. METHODS: Twenty Wistar rats were divided into two groups: Cigarette Clot Group (CCG), which in the inhalation chamber received the smoke of 10 cigarettes, 3 times a day, 10 minutes, for 30 days and had the surgical cavity filled by clot; Cigarette Biomaterial Group (CBG), submitted to the same inhalation technique but with the cavity filled by biomaterial. RESULTS: In CCG there was a significant difference of new bone tissue in the analyzed periods (15 and 45 days), and in 15 days, there was 4.8 ± 0.42 of bone formed and 11.73 ± 0.59 (p <0.05) in 45 days. The CBG also showed a significant difference between the periods of 15 to 45 days, being respectively 6.16 ± 0.30 and 11.60 ± 0.61. However, when the groups were compared, within the same analyzed periods, a significant difference was observed only in the period of 15 days, with the new bone percentage being greater in the CBG. CONCLUSION: The bone matrix acted as an osteoinductive biomaterial, biocompatible and aided in the repair process, mainly in the initial period of recovery.

Remaining Physiological Barriers in Porcine Kidney Xenotransplantation: Potential Pathways behind Proteinuria as well as Factors Related to Growth Discrepancies following Pig-to-Kidney Xenotransplantation.

Considerable shortages in the supply of available organs continue to plague the field of solid organ transplantation. Despite changes in allocation, as well as the utilization of extended criteria and living donors, the number of patients waiting for organs continues to grow at an alarming pace. Xenotransplantation, cross-species solid organ transplantation, offers one potential solution to this dilemma. Previous extensive research dedicated to this field has allowed for resolution of xenograft failure due to acute rejection, leaving new areas of unresolved challenges as barriers to success in large animal models. Specific to kidney xenotransplantation, recent data seems to indicate that graft compromise can occur due to discrepancies in growth between breeds of donors and significant proteinuria leading to nephrotic syndrome in the recipient. Given these potential limitations, herein, we review potential pathways behind proteinuria, as well as potential causative factors related to growth discrepancies. Control of both of these has the potential to allow xenotransplantation to become clinically applicable in an effort to resolve this organ shortage crisis.

Decellularization and characterization of a whole intervertebral disk xenograft scaffold.

Intervertebral disk (IVD) degeneration is a multifactor process that results in the physical destruction of the nucleus pulposus (NP) and annulus fibrosus (AF). This compromises IVD function and causes significant disability and economic burden. Strategies to replace the entire composite structure of the IVD are limited and most approaches do not recapitulate the heterogenous biochemical composition, microarchitecture or mechanical properties of the native tissue. Our central hypothesis was that donor IVDs which resemble the size and biochemistry of human lumbar IVDs could be successfully decellularized while retaining the tissue's structure and function with the long-term goal of creating a composite scaffold for tissue engineering the human IVD. Accordingly, we optimized a procedure to decellularize bovine tail IVDs using a combination of detergents, ultrasonication, freeze-thaw cycles, and nucleases. The resultant decellularized whole IVD xenografts retained distinct AF and NP regions which contained no visible intact cell nuclei and minimal residual bovine deoxyribose nucleic acid (DNA; 65.98 ± 4.07 and 47.12 ± 13.22 ng/mg, respectively). Moreover, the NP region of decellularized IVDs contained 313.40 ± 50.67 µg/mg glycosaminoglycan. The presence of collagen type II was confirmed via immunohistochemistry. Additionally, histological analysis of the AF region of decellularized IVDs demonstrated retention of the native angle-ply collagen microarchitecture. Unconfined compression testing demonstrated no significant differences in swelling pressure and toe-region modulus between fresh and decellularized IVDs. However, linear region moduli, peak stress and equilibrium moduli were all significantly reduced. Together, this research demonstrates a successful initial step in developing a biomimetic acellular whole IVD xenograft scaffold for use in IVD tissue engineering. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A:2412-2423, 2018.

Action of a deproteinized xenogenic biomaterial in the process of bone repair in rats submitted to inhalation of cigarette smoke

Abstract Purpose: To investigate if the inorganic bovine bone matrix changes the bone formation in rats submitted to inhalation of cigarette smoke. Methods: Twenty Wistar rats were divided into two groups: Cigarette Clot Group (CCG), which in the inhalation chamber received the smoke of 10 cigarettes, 3 times a day, 10 minutes, for 30 days and had the surgical cavity filled by clot; Cigarette Biomaterial Group (CBG), submitted to the same inhalation technique but with the cavity filled by biomaterial. Results: In CCG there was a significant difference of new bone tissue in the analyzed periods (15 and 45 days), and in 15 days, there was 4.8 ± 0.42 of bone formed and 11.73 ± 0.59 (p <0.05) in 45 days. The CBG also showed a significant difference between the periods of 15 to 45 days, being respectively 6.16 ± 0.30 and 11.60 ± 0.61. However, when the groups were compared, within the same analyzed periods, a significant difference was observed only in the period of 15 days, with the new bone percentage being greater in the CBG. Conclusion: The bone matrix acted as an osteoinductive biomaterial, biocompatible and aided in the repair process, mainly in the initial period of recovery.

Patient-derived xenograft cryopreservation and reanimation outcomes are dependent on cryoprotectant type.

Patient-derived xenografts (PDX) are being increasingly utilized in preclinical oncologic research. Maintaining large colonies of early generation tumor-bearing mice is impractical and cost-prohibitive. Optimal methods for efficient long-term cryopreservation and subsequent reanimation of PDX tumors are critical to any viable PDX program. We sought to compare the performance of "Standard" and "Specialized" cryoprotectant media on various cryopreservation and reanimation outcomes in PDX tumors. Standard (10% DMSO media) and Specialized (Cryostor®) media were compared between overall and matched PDX tumors. Primary outcome was reanimation engraftment efficiency (REE). Secondary outcomes included time to tumor formation (TTF), time to harvest (TTH), and potential loss of unique PDX lines. Overall 57 unique PDX tumors underwent 484 reanimation engraftment attempts after previous cryopreservation. There were 10 unique PDX tumors cryopreserved with Standard (71 attempts), 40 with Specialized (272 attempts), and 7 with both (141 attempts). Median frozen time of reanimated tumors was 29 weeks (max. 177). Tumor pathology, original primary PDX growth rates, frozen storage times, and number of implantations per PDX model were similar between cryoprotectant groups. Specialized media resulted in superior REE (overall: 82 vs. 39%, p < 0.0001; matched: 97 vs. 36%, p < 0.0001; >52 weeks cryostorage: 59 vs. 9%, p < 0.0001), shorter TTF (overall 24 vs. 54 days, p = 0.0051; matched 18 vs. 53 days, p = 0.0013) and shorter TTH (overall: 64 vs. 89 days, p = 0.009; matched: 47 vs. 88 days, p = 0.0005) compared to Standard. Specialized media demonstrated improved REE with extended duration cryostorage (p = 0.048) compared to Standard. Potential loss of unique PDX lines was lower with Specialized media (9 vs. 35%, p = 0.017). In conclusion, cryopreservation with a specialized cryoprotectant appears superior to traditional laboratory-based media and can be performed with reliable reanimation even after extended cryostorage.