Background. Due to organ shortage, many patients do not receive donor organs. The present novel thrombolytic technique utilizes organs from donors with uncontrolled donation after circulatory deaths (uDCD), with up to 4–5 h warm ischemia, without advanced cardiopulmonary resuscitation (aCPR) or extracorporeal circulation (EC) after death. Methods. The study group of pigs (n = 21) underwent simulated circulatory death. After 2 h, an ice slush was inserted into the abdomen. Kidneys were retrieved 4.5 h after death. Lys-plasminogen, antithrombin-III (ATIII), and alteplase (tPA) were injected through the renal arteries on the back table. Subsequent ex vivo perfusion at 15 °C was continued for 3 h, followed by 3 h with red blood cells (RBCs) at 32 °C. Perfusion outcome and histology were compared between uDCD kidneys, receiving no thrombolytic treatment (n = 8), and live donor kidneys (n = 7). The study kidneys were then transplanted into pigs as autologous grafts with a single functioning autologous kidney as the only renal support. uDCD control pigs (n = 8), receiving no ex vivo perfusion, served as controls. Results. Vascular resistance decreased to <200 mmHg/mL/min (P < 0.0023) and arterial flow increased to >100 mL/100 g/min (P < 0.00019) compared to controls. In total 13/21 study pigs survived for >10 days, while all uDCD control pigs died. Histology was preserved after reconditioning, and the creatinine level after 10 days was next to normal. Conclusions. Kidneys from extended uDCD, not receiving aCPR/EC, can be salvaged using thrombolytic treatment to remove fibrin thrombi while preserving histology and enabling transplantation with a clinically acceptable early function.

Fertility preservation methods for prepubertal women about to undergo gonadotoxic chemo and/or radiation therapy are limited. Therefore, the aim of this study was to investigate the feasibility to develop an alternative fertility preservation method based on an ex vivo perfusion platform for whole ewe ovaries. Thirteen ewe ovaries were divided into two groups (group 1 and 2) that were perfused in a bioreactor for up to 7days. Group 1 (n=3) were stimulated with human menopausal gonadotropin (hMG) administered in single daily dose, while group 2 (n=10) were stimulated continuously for 24h. The perfused ovaries in group 1 showed no significant differences in follicular density, sub-follicular morphology and oocyte quality after ischaemia and after ex vivo perfusion compared with non-perfused control ovaries. The perfused ovaries in group 2 showed a significant decrease in the follicular reserve and oocyte quality compared with the control group. In total, 16 GV-MI oocytes were retrieved from both groups. This study describes for the first time the ex vivo maintenance of viable follicles of ewe ovaries with oocyte integrity and the retrieval of oocytes after ex vivo hormonal perfusion with two different protocols for up to 7days.

Background. Using a novel thrombolytic technique, we present long-term transplant function, measured by creatinine and iohexol clearance, after utilizing kidneys from porcine donors with uncontrolled donation after circulatory deaths, with 4.5–5 h of warm ischemia. Methods. Pigs in the study group were subjected to simulated circulatory death. After 2 h, ice slush was inserted into the abdomen and 4.5 h after death, the kidneys were retrieved. Lys-plasminogen, antithrombin-III, and alteplase were injected through the renal arteries on the back table. Subsequent ex vivo perfusion was continued for 3 h at 15°C, followed by 3 h with red blood cells at 32°C, and then transplanted into pigs as an autologous graft as only renal support. Living-donor recipient pigs that did not receive ex vivo perfusion, and unilateral nephrectomized pigs served as the controls. Results. Pigs in the study group (n = 13), surviving 10 d or more were included, of which 7 survived for 3 mo. Four animals in the living-donor group (n = 6) and all 5 nephrectomized animals survived for 3 mo. Creatinine levels in the plasma and urine, neutrophil gelatinase-associated lipocalin levels, Kidney Injury Marker-1 expression, and iohexol clearance at 3 mo did not differ significantly between the study and living-donor groups. Histology and transmission electron microscopy after 3 mo showed negligible fibrosis and no other damage. Conclusions. The present method salvages kidneys from extended unontrolled donation after circulatory death using thrombolytic treatment while preserving histology and enabling transplantation after ex vivo reconditioning, with clinically acceptable late function after 3 mo, as measured by creatinine and iohexol clearance.

Biological scaffold is a popular choice for the preparation of tissue-engineered organs and has the potential to address donor shortages in clinics. However, biological scaffolds prepared by physical or chemical agents cause damage to the extracellular matrix (ECM) by potentially inducing immune responses after implantation. The current study explores the fate of the decellularized (DC) scaffolds using a cocktail of chemicals following implantation without using immunosuppressants. Using the syngeneic (Lewis male-Lewis female) and allogeneic (Brown Norway male-Lewis female) models and different tissue routes (subcutaneous vs. omentum) for implantation, we applied in-depth quantitative proteomics, genomics along with histology and quantitative image analysis tools to comprehensively describe and compare the proteins following DC and postimplantation. Our data helped to identify any alteration postdecullarization as well implantation. We could also monitor route-specific modulation of the ECM and regulation of the immune responses (macrophage and T cells) following implantation. The current approach opens up the possibility to monitor the fate of biological scaffolds in terms of the ECM and immune response against the implants. In addition, the identification of different routes helped us to identify differential immune responses against the implants. This study opens up the potential to identify the changes associated with chemical DC both pre- and postimplantation, which could further help to promote research in this direction. Impact Statement The development of a biological scaffold helps in the preparation of a functional organ in the clinics. In the current study, we develop a strategy for chemical decellularization and explored two different routes to understand the differential responses elicited postimplantation. The use of sensitive protein and genomic tools to study the changes creates a favorable environment for similar efforts to develop and characterize biological scaffolds before further trials in the clinics. The current study, which was carried out without any immunosuppressive agents, could help to establish (a) appropriate chemical strategies for preparing biological scaffolds as well as (b) identify putative implantable routes to circumvent any adverse immune reactions, which will ultimately decide the outcome for acceptance or rejection of the scaffold/implant.

The larynx is a fairly complex organ comprised of different muscles, cartilages, mucosal membrane, and nerves. Larynx cancer is generally the most common type of head and neck cancer. Treatment options are limited in patients with total or partial laryngectomy. Tissue-engineered organs have shown to be a promising alternative treatment for patients with laryngectomy. In this report we present an alternative and simple procedure to construct a whole pig larynx scaffold consisting of complete acellular structures of integrated muscle and cartilage. Larynges were decellularized (DC) using perfusion-agitation with detergents coupled with ultrasonication. DC larynges were then characterized to investigate the extracellular matrix (ECM) proteins, residual DNA, angiogenic growth factors, and morphological and ultrastructural changes to ECM fibers. After 17 decellularization cycles, no cells were observed in all areas of the larynx as confirmed by hematoxylin and eosin and DAPI (4 ',6-diamidino-2-phenylindole) staining. However, DC structures of dense thyroid and cricoid cartilage showed remnants of cells. All structures of DC larynges (epiglottis [p < 0.0001], muscle [p < 0.0001], trachea [p = 0.0045], and esophagus [p = 0.0008]) showed DNA <50 ng/mg compared with native larynx. Immunohistochemistry, Masson's trichrome staining, and Luminex analyses showed preservation of important ECM proteins and angiogenic growth factors in DC larynges. Compared with other growth factors, mostly retained growth factors in DC epiglottis, thyroid muscle, and trachea include granulocyte colony-stimulating factor, Leptin, fibroblast growth factor-1, Follistatin, hepatocyte growth factor, and vascular endothelial growth factor-A. Scanning electron microscopy and transmission electron microscopy analysis confirmed the structural arrangements of ECM fibers in larynges to be well preserved after DC. Our findings suggest that larynges can be effectively DC using detergent ultrasonication. ECM proteins and angiogenic growth factors appear to be better preserved using this method when compared with the native structures of larynges. This alternative DC method could be helpful in building scaffolds from dense tissue structures such as cartilage, tendon, larynx, or trachea for futurein vitrorecellularization studies orin vivoimplantation studies in the clinic. Impact statement This study successfully created decellularized porcine larynx using novel method of perfusion-agitation with detergents and ultrasonication, which maintained three-dimensional architecture of the larynx scaffold. Our method is devoid of harmful enzymes, which may prevent cellular repopulation or induce inflammatory response uponin vivoimplantation. We studied important aspect of preservation of extracellular matrix proteins in different structures of the larynx. Hence, our decellularization method could be used as an alternative method to decellularize various dense tissues such as cartilage or tendon.

Background: The availability of an increased proportion of organs from donors after circulatory death (DCD) could solve the organ shortage globally. We present a model of extended uncontrolled DCD (uDCD), allowing sufficient time for the consent procedure in most countries. A novel reconditioning technique allows successful transplantation after prolonged warm ischemia of kidneys from uDCD donors. Methods: Two hours after circulatory death, ice slush was inserted into the abdomen of pigs. Donor organs were then retrieved 4.5 hour after death, and treated on the back-table by injecting Lys-plasminogen, antithrombin-III (AT-III), and alteplase (tPA) through the renal arteries. The kidneys were then perfused with an albumin-rich solution at 15 °C and 20 mmHg for 3 hours. Washed erythrocytes were added to the solution after increasing temperature to 32 °C and pressure to 30 mmHg, continuing the perfusion for another three hours. Argatroban and abciximab were added to inhibit re-thromboses. Reconditioning (study group) kidneys were transplanted into pigs using a novel auto-transplant technique with a single functioning autologous kidney and observed for three months. Sham-treated pigs underwent contra-lateral transplantation of the left kidney after removing the right kidney under the same anesthesia conditions and trauma as study group pigs. Findings: During perfusion, vascular resistance decreased to <200 mmHg/ml/min, significantly lower (p < 0.0022) and arterial flow increased to >100ml/100g/min, significantly higher (p < 0.00018) than kidneys not receiving thrombotlytic treatment. At 3 months post-transplant, 4/6 study and 4/6 control pigs survived. Serum creatinine and renal histology did not differ between the groups. Interpretation: We present a novel method to salvage kidneys from extended uDCD, enabling subsequent transplantation after ex-vivo reconditioning, in a clinically acceptable manner. Funding Information: Supported by grants from IngaBritt and Arne Lundbergs Research Foundation; LUA ALF Grant; The Gelin Foundation and Hans-Gabriel and Alice Trolle Wachtmeister Foundation for Medical Research.Declaration of Interests: MO is the inventor of several patents. None of the other authors reported any potential conflicts of interestEthics Approval Statement: The Regional (Malmö/Lund) Animal Experiment Ethics Committee approved the study (Dnr 5.8.18‐13977/2018; Dnr 5.8.18‐09474/2019; Dnr 5.8.18‐09182/2020). All animals were housed and cared for in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the Institute of Laboratory Resources and published by the National Institute of Health (NIH, Publication No 86‐23, revised 1996).

Biological scaffold or implant is a popular choice for the preparation of tissue-engineered organs and has the potential to address donor shortage in the clinics. However, biological scaffolds prepared by physical or chemical agents cause damage to the extracellular matrix by potentially inducing immune responses after implantation. The current study explores an alternative route for the preparation of acellular scaffolds and explores the fate of the prepared scaffolds in a milieu of immune cells following implantation without using immunosuppressant. Using the syngeneic (Lewis male-Lewis female) and allogeneic (Brown Norway male-Lewis female) models and different tissue routes (subcutaneous vs omentum) for transplantation, normal blood vascular scaffolds were implanted which was converted to acellular vascular scaffolds by in vivo natural decellularization at the end of 2 months of observation. We also prepared chemically decellularized acellular scaffolds from normal untreated blood vascular scaffolds using a cocktail of chemicals which was also similarly placed in subcutaneous and omentum sites. Here, we applied in-depth quantitative proteomics along with histology and image analysis to comprehensively describe and compare the proteome of the natural and chemically decellularized scaffold. Our data confirm that site-specific advantages exist in modulating the ECM and regulating the immune responses (macrophage and T cells) following implantation, which possibly led to the production of an acellular scaffold (natural decellularization) under in vivo conditions. The current approach opens up the possibility to create tailor-made acellular scaffolds to build functional blood vessels. In addition, the identification of different tissue sites facilitates differential immune response against the scaffolds. This study provides a rich resource aimed toward an enhanced mechanistic understanding to study immune responses under similar settings in the field of transplantation and regenerative medicine. Impact statement The development of a scaffold helps in the preparation of a functional organ in the clinics. In the current study, we prepared an acellular vascular scaffold by utilizing site specific tissue changes and vis-à-vis compared with a conventionally chemically prepared biological scaffold at genomic and protein level, which helped us to identify immunological trigger following implantation. The current study which was carried out without any immunosuppressive agents could help to establish (a) alternative strategies for preparing biological scaffolds as well as (b) implantable sites as potential bioreactors to circumvent any adverse immune reactions for acceptance of the scaffold/implant post implantation.

Large animal studies of long-term ischemia reperfusion are hampered by the use of immunosuppressive drugs to inhibit the influence of the allogeneic response. In small animals, this can be controlled by using inbred strains of the animal. For obvious reasons, this is not possible in large animals such as pigs. Since studies in pigs usually are the last step before first-in-man studies, this remains a problem trying to resemble a clinical situation. In the following short paper, we describe a novel auto kidney transplantation model that can be used for long term ischemia reperfusion studies. We also suggest a control setting to balance out the possible influence of an increased surgical trauma.

The immunogenicity of the extracellular matrix (ECM) from genetically similar (syngeneic) and dissimilar (allogeneic and xenogeneic) species has puzzled the scientific community for many years. After implantation, the literature describes an absorption of ECM material since it is biodegradable. However, no clear insight really exists to substantiate how the underlying immune and biological responses result in absorption of ECM materials. In this context, it is important to characterize infiltrating cells and identify dominant cell populations in the infiltrate. We have studied the immune response in mice after implantation of decellularized (DC) cardiac scaffolds derived from pig and mouse. The polymorphism of the infiltrate into the implanted material signifies the importance of the adaptive immune response that is distinct for xenoimplants and alloimplants. Matrix resorption takes place mainly through phagocytic cells such as mast cells, dendritic cells, and macrophages. Histochemical observations show that innate CD8+ T cells develop immune tolerance, whereas proteomic analysis predicts the different T cell progenies for alloscaffolds and xenoscaffolds. The amalgamation of graft tolerance and involvement of both B and T cell populations in the vicinity of the graft could be decisive in wound remodeling and survival of the graft. This challenging area presents potential targets for the development of immune-privileged biomaterials, immune tolerant cells, and therapeutic agents in the future. Impact statement In this study, we have characterized the allogeneic and xenogeneic immune responses for decellularized (DC) cardiac scaffolds. We postulate that although the T cells are important players for immune tolerance of DC graft, the mechanism of their differentiation inside the host is donor specific. In this study, we have reported the distinct immune responses for syngeneic DC scaffolds than allogeneic and xenogeneic scaffolds. This distinct response provides the bases for the different immune responses reported for DC homografts in the literature. This study can provide the greater insight for modification of postimplant strategies to achieve host acceptance of donor extracellular matrix scaffolds.

Decellularization of esophagus was studied using three different protocols. The sodium deoxycholate/DNase-I (SDC/DNase-I) method was the most successful as evidenced by histology and DNA quantification of the acellular scaffolds. Acellular scaffolds were further analyzed and compared with native tissue by histology, quantitative analysis of DNA, and extracellular matrix (ECM) proteins. Histologically, the SDC/DNase-I protocol effectively produced scaffold with preserved structural architecture similar to native tissue architecture devoid of any cell nucleus. ECM proteins, such as collagen, elastin, and glycosaminoglycans were present even after detergent-enzymatic decellularization. Immunohistochemical analysis of acellular scaffold showed weak expression of Gal 1, 3 Gal epitope compared with native tissue. For performing recellularization, human amnion-derived mesenchymal stem cells (MSCs) and epithelial cells were seeded onto acellular esophagus in a perfusion-rotation bioreactor. In recellularized esophagus, immunohistochemistry showed infiltration of MSCs from adventitia into the muscularis externa and differentiation of MSCs into the smooth muscle actin and few endothelial cells (CD31). Our study demonstrates successful preparation and characterization of a decellularized esophagus with reduced load of Gal 1, 3 Gal epitope with preserved architecture and ECM proteins similar to native tissue. Upon subsequent recellularization, xenogeneic acellular esophagus also supported stem cell growth and partial differentiation of stem cells. Hence, the current study offers the hope for preparing a tissue-engineered esophagus in vitro which can be transplanted further into pigs for further in vivo evaluation.

STEM CELLS TRANSLATIONAL MEDICINE 2013;2:307-315; http://dx.doi.org/10.5966/sctm.2012-0108 The above-referenced article published on March 13, 2013 in Stem Cells Translational Medicine has been retracted by agreement between the Journal Editors and co-publishers, AlphaMed Press and Wiley Periodicals, Inc. The retraction has been agreed to with acknowledgment of problems with Figure 3, which we believe make some of the data unreliable.

MUC1 is a transmembrane mucin highly expressed in the stomach. Although extensive research has uncovered many of its roles in cancer, knowledge about the functions of MUC1 in normal tissues is limited. In the present study, we showed that acetylsalicylic acid (ASA; aspirin) up-regulated MUC1/Muc1 expression in the gastric mucosa of humans and wild-type (WT) mice. ASA induced mucosal injury in all mice to a similar extent; however, WT animals and those chimaeras with Muc1 on the epithelia recovered faster than Muc1-knockout (KO) mice and chimaeras carrying Muc1 on haemopoietic but not epithelial cells. MUC1 enhanced proliferation and migration of the human gastric cell line MKN-7 and increased resistance to apoptosis. The repeated treatment regime used caused a reduction in cyclo-oxygenase-1 (Cox-1) expression, though WT animals returned faster towards pre-treatment levels and had increased Cox-2 and vascular endothelial growth factor levels during recovery. Thus we found that epithelial Muc1 is more important for the healing process than haemopoietic Muc1 and Muc1/MUC1 facilitates wound healing by enhancing cell migration and proliferation, protecting against apoptosis and mediating expression of mucosal modulators. Thus MUC1 plays essential roles during wound healing and development of treatment modalities targeting enhanced expression of MUC1 may be beneficial to treat mucosal wounds.