Decellularization of native blood vessels is a promising technology to generate 3D biological scaffolds for vascular grafting. Blood vessel decellularization has been performed in previous studies under various experimental conditions, that complicates comparison and optimization of suitable protocols. The goal of this work was to systematically compare the decellularization and recellularization efficacy of 5 different protocols utilizing the detergents sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), CHAPS and TritonX-100 together with DNA-removing enzymes on porcine vena cava in a perfusion bioreactor setup. Additionally, we tested the effect of DNase on the extracellular matrix (ECM) properties. We found that all protocols could efficiently decellularize blood vessels. Mechanical strength, collagen preservation and ECM integrity were similar among all tested detergents, yet TritonX protocols required long-term DNase application for complete decellularization. However, TritonX-based protocols showed the greatest recellularization efficacy with HUVECs in vitro. Furthermore, we developed a novel protocol for TritonX which improved recellularization and reduced total process time and ECM stiffness compared to previous protocols. SDS, SDC and CHAPS based protocols had a lower recellularization potential. In conclusion, decellularization of blood vessels can be achieved with all tested reagents, but TritonX treated ECM can be most efficiently recellularized with endothelial cells.

Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.

Background In order to preserve fertility in young women with disseminated cancer, e.g. leukemia, an approach that has been suggested is to retransplant isolated small follicles within an ovarian matrix free from malignant cells and with no risk for contamination. The present study evaluates the first step to create a bioengineered ovarian construct that can act as growth-supporting tissue for isolated small follicles that are dependent on a stroma for normal follicular maturation. The present study used the intact mouse ovary to develop a mouse ovarian scaffold through various protocols of decellularization. Material and methods Potential Immunogenic DNA and intracellular components were removed from whole mouse ovaries by agitation in a 0.5% sodium dodecyl sulfate solution (Protocol 1; P1), or in a 2% sodium deoxycholate solution (P2) or by a combination of the two (P3). The remaining decelluralized ovarian extracellular matrix structure was then assessed based on the DNA- and protein content, and was further evaluated histologically by haematoxylin and eosin-, Verhoeff’s van gieson- (for elastin), Masson’s trichrome- (for collagens) and Alcian blue (for glycosaminoglycans) staining. We also evaluated the decellularization efficiency using the mild detergent Triton-X100 (1%). Results Sodium dodecyl sulfate efficiently removed DNA and intracellular components from the ovarian tissue but also significantly reduced the integrity of the remaining ovarian extracellular matrix. Sodium deoxycholate, a considerably milder detergent compared to sodium dodecyl sulfate, preserved the ovarian extracellular matrix better, evident by a more distinct staining for glycosaminoglycan, collagen and elastic fibres. Triton-X100 was found ineffective as a decellularization reagent for mouse ovaries in our settings. Conclusions The sodium dodecyl sulfate generated ovarian scaffolds contained minute amounts of DNA that may be an advantage to evade a detrimental immune response following engraftment. The sodium deoxycholate generated ovarian scaffolds had higher donor DNA content, yet, retained the extracellular composition better and may therefore have improved recellularization and other downstream bioengineering applications. These two novel types of mouse ovarian scaffolds serve as promising scaffold-candidates for future ovarian bioengineering experiments.

Introduction: Uterus transplantation has recently proved that infertility in women with uterine factor infertility can be cured. It is still an experimental procedure with numerous critical details remaining to be established, including tolerance to warm and cold ischemic insults. In preparation for human uterus transplantation trials, most teams use the sheep as a model system for research and team training, since the vasculature and the uterus is of similar size as in the human. We, therefore, aimed to develop an ex vivo sheep uterus reperfusion platform that mimics the reperfusion situation so that initial assessments and comparisons can be performed without the need for costly and labor-intensive in vivo transplantation experiments. Material and methods: Isolated sheep uteri were perfused with the preservation solution IGL-1 and were then exposed to cold ischemia for either 4 (n = 6) or 48 hours (n = 7). Uteri were then reperfused for 48 hours under normothermic conditions with an oxygenated recirculating perfusate containing growth factors and synthetic oxygen carriers. Histological and biochemical analysis of the perfusate was conducted to assess reperfusion injury. Results: Quantification of cell density indicated no significant edema in the myometrium or in the endometrium of uteri exposed to 4 hours cold ischemia and then a normothermic ex vivo reperfusion for 48 hours. Only the outer serosa layer and the inner columnar luminal epithelial cells were affected by the reperfusion. However, a much faster and severe reperfusion damage of all uterine layers were evident during the reperfusion experiment following 48 hours of cold ischemia. This was indicated by major accumulation of extracellular fluid, presence of apoptotic-labeled glandular epithelial layer and vascular endothelium. A significant accumulation of lactate was measured in the perfusate with a subsequent decrease in pH. Conclusions: We developed a novel ex vivo sheep uterus model for prolonged perfusion. This model proved to be able to distinguish reperfusion injury-related differences associated to organ preservation. The experimental setup is a platform that

Here we report the fabrication of a novel composite gel from decellularized gal-gal-knockout porcine skin and human peripheral blood mononuclear cells (hPBMCs) for full-thickness skin wound healing. Decellularized skin extracellular matrix (ECM) powder was prepared via chemical treatment, freeze drying, and homogenization. The powder was mixed with culture medium containing hyaluronic acid to generate a pig skin gel (PSG). The effect of the gel in regeneration of full-thickness wounds was studied in nude mice. We found significantly accelerated wound closure already on day 15 in animals treated with PSG only or PSG + hPBMCs compared to untreated and hyaluronic acid-treated controls (p < 0.05). Addition of the hPBMCs to the gel resulted in marked increase of host blood vessels as well as the presence of human blood vessels. At day 25, histologically, the wounds in animals treated with PSG only or PSG + hPBMCs were completely closed compared to those of controls. Thus, the gel facilitated generation of new skin with well-arranged epidermal cells and restored bilayer structure of the epidermis and dermis. These results suggest that porcine skin ECM gel together with human cells may be a novel and promising biomaterial for medical applications especially for patients with acute and chronic skin wounds.

Scaffolds derived from decellularized tissue possess many advantages for bioengineering applications, including for novel infertility treatments. However, the decellularization process results in allogenic‐independent damage‐associated molecular patterns (DAMPs). This field is poorly studied, in particular for uterus bioengineering applications. An increased knowledge concerning the immune system activation after transplantation of decellularized tissue will enable safer construct development and thereby accelerate translation from research to clinic. We therefore transplanted rat uterus scaffolds produced by three different decellularization protocols based on Triton X‐100 (P1 and P2) or sodium deoxycholate (P3) in a syngeneic animal model and assessed the immune response towards DAMPs exposed by the decellularization process. Biopsies were retrieved on day 5, 15, and 30 post transplantation and immunohistochemistry‐stained CD45+ (leucocytes), CD4+ (T‐cells), CD8a+ (cytotoxic T‐cells), CD22+ (B‐cells), NCR1+ (NK‐cells), CD68+ (pan‐macrophages), and CD163+ (M2 macrophages) cells within the grafts were quantified. The gene expression for interferon γ, interleukin (IL)‐1β, IL‐2, IL‐6, and tumor necrosis factor (TNF) eotaxin‐2, RANTES, MCP‐1, MIP‐1α, MIP‐3α, IL‐8 were also measured. Scaffolds from P1 induced a rapid cell infiltration after transplantation, presumably induced by DNA‐based DAMPs. However, this response was only transient. Protocol 3 derived scaffolds induced an early pro‐inflammatory cytokine response at the transcript level which remained high throughout the study. This response may be caused by the stronger decellularization detergent that could expose more extracellular matrix‐related DAMPs. However, earlier proteomics analysis also identified significantly more abundant heat shook proteins‐related DAMPs in this scaffold type. Protocol 2 caused the least immunogenic scaffolds and should thus be the future focus for in vivo uterus bioengineering applications.

Background Fertility preservation is particularly challenging in young women diagnosed with hematopoietic cancers, as transplantation of cryopreserved ovarian cortex in these women carries the risk for re-introducing cancer cells. Therefore, the construction of a bioengineered ovary that can accommodate isolated small follicles was proposed as an alternative to minimize the risk of malignancy transmission. Various options for viable bioengineered scaffolds have been reported in the literature. Previously, we reported three protocols for producing mouse ovarian scaffolds with the decellularization technique. The present study examined these scaffolds further, specifically with regards to their extracellular composition, biocompatibility and ability to support recellularization with mesenchymal stem cells. Material and methods Three decellularization protocols based on 0.5% sodium dodecyl sulfate (Protocol 1; P1), or 2% sodium deoxycholate (P2), or a combination of the two detergents (P3) were applied to produce three types of scaffolds. The levels of collagen, elastin and sulfated glycosaminoglycans (sGAGs) were quantified in the remaining extracellular matrix. Detailed immunofluorescence and scanning electron microscopy imaging were conducted to assess the morphology and recellularization efficiency of the constructs after 14 days in vitro utilizing red fluorescent protein-labelled mesenchymal stem cells. Results All protocols efficiently removed the DNA while the elastin content was not significantly reduced during the procedures. The SDS-protocol (P1) reduced the sGAG and the collagen content more than the SDC-protocol (P2). All scaffolds were biocompatible and recellularization was successful, particularly in several P2-derived scaffolds. The cells were extensively distributed throughout the constructs, with a denser distribution observed towards the ovarian cortex. The cell density was not significantly different (400 to 550 cells/mm 2 ) between scaffold types. However, there was a tendency towards a higher cell density in the SDC-derived constructs. Scanning electron microscope images showed fibrous scaffolds with a dense repopulated surface structure. Conclusions While there were differences in the key structural macromolecules between protocols, all scaffolds were biocompatible and showed effective recellularization. The results indicate that our SDC-protocol might be better than our SDS-protocol. However, additional studies are necessary to determine their suitability for attachment of small follicles and folliculogenesis.

Genetically modified pigs have been considered favorable resources in xenotransplantation. Microinjection of randomly integrating transgenes into zygotes, somatic cell nuclear transfer, homologous recombination, zinc finger nucleases, transcription activator-like effector nucleases, and most recently, clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9) are the techniques that have been used to generate these animals. Here, we provide an overview of the CRISPR approaches that have been used to modify genes which are vital in improving xenograft survival rate, including cytidine monophosphate-N-acetylneuraminic acid hydroxylase, B1,4N-acetylgalactosaminyltransferase, isoglobotrihexosylceramide synthase, class I MHC, von Willebrand factor, C3, and porcine endogenous retroviruses. In addition, we will mention the importance of potential candidate genes which could be targeted using CRISPR/Cas9.