scholarly journals In-vitro and In-vivo Feasibility Study for a Mobile VV-ECMO and ECCO2R System

2021 ◽  
Author(s):  
Lasse Johannes Strudthoff ◽  
Hanna Lüken ◽  
Sebastian Victor Jansen ◽  
Jan Petran ◽  
Peter Schlanstein ◽  
...  
Keyword(s):  
Physical Exercise ◽  
Feasibility Study ◽  
Rescue Therapy ◽  
Integrated System ◽  
Large Animal Model ◽  
Large Animal ◽  
In Vitro Experiments ◽  
Blood Damage

Extracorporeal membrane oxygenation (ECMO) is an established rescue therapy for patients with chronic respiratory failure waiting for lung transplantation (LTx). The therapy inherent immobilization may result in fatigue, consecutive deteriorated prognosis and even lost eligibility for transplantation. We conducted a feasibility study on a novel system designed for the deployment of a mobile ECMO device, enabling physical exercise of awake patients prior to LTx. The system comprises a novel mobile oxygenator with a directly connected blood pump, a double lumen cannula, gas blender and supply, as well as control, and energy management. In-vitro experiments included tests regarding performance, efficiency, and blood damage. A reduced system was tested in vivo for feasibility using a novel large animal model. Six anesthetized pigs were first positioned in supine position, followed by a 45° angle, simulating an upright position of the patients. We monitored performance and vital parameters. All in-vitro experiments showed good performance for the respective subsystems and the integrated system. The acute invivo trials of 8h duration confirmed the results. The novel mobile ECMO-system enables adequate oxygenation and decarboxylation sufficient for, e.g., physical exercise of designated LTx-recipients. These results are promising and suggest further preclinical studies on safety and efficacy to facilitate translation into clinical application.

scholarly journals Light-degradable hydrogels as dynamic triggers for gastrointestinal applications

2020 ◽  
Vol 6 (3) ◽  
pp. eaay0065 ◽  
Author(s):  
Ritu Raman ◽  
Tiffany Hua ◽  
Declan Gwynne ◽  
Joy Collins ◽  
Siddartha Tamang ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Implantable Devices ◽  
Large Animal Model ◽  
Large Animal ◽  
Gi Tract ◽  
Biomedical Device ◽  
Precise Tool ◽  
And Performance

Triggerable materials capable of being degraded by selective stimuli stand to transform our capacity to precisely control biomedical device activity and performance while reducing the need for invasive interventions. Here, we describe the development of a modular and tunable light-triggerable hydrogel system capable of interfacing with implantable devices. We apply these materials to two applications in the gastrointestinal (GI) tract: a bariatric balloon and an esophageal stent. We demonstrate biocompatibility and on-demand triggering of the material in vitro, ex vivo, and in vivo. Moreover, we characterize performance of the system in a porcine large animal model with an accompanying ingestible LED. Light-triggerable hydrogels have the potential to be applied broadly throughout the GI tract and other anatomic areas. By demonstrating the first use of light-degradable hydrogels in vivo, we provide biomedical engineers and clinicians with a previously unavailable, safe, dynamically deliverable, and precise tool to design dynamically actuated implantable devices.

ZFN-Mediated Gene Targeting at the Albumin Locus in Liver Results in Therapeutic Levels of Human FIX in Mice and Non-Human Primates

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 200-200 ◽  
Author(s):  
Thomas Wechsler ◽  
Kathleen E. Meyer ◽  
S. Kaye Spratt ◽  
Judith Greengard ◽  
Yolanda Santiago ◽  
...  
Keyword(s):  
Hemophilia B ◽  
Large Animal Model ◽  
Zinc Finger Nucleases ◽  
Clotting Factor ◽  
Large Animal ◽  
Spontaneous Bleeding ◽  
Virus Serotype ◽  
Donor Template

Abstract Hemophilia is an attractive target for gene therapy, since activity levels as low as 1% to 2% of normal are beneficial and levels of ~5% prevent spontaneous bleeding. Our goal was to provide a single treatment that permanently enables hepatic production of therapeutic levels of hFIX activity to decrease or potentially eliminate the need for prophylactic treatment in hemophilia B patients. We performed targeted in vivo genome editing using 1) two zinc finger nucleases (ZFNs) targeting intron 1 of the albumin locus, and 2) a human F9 donor template construct. The ZFNs and donor template are encoded on separate hepatotropic adeno-associated virus serotype 2/6 (AAV2/6) vectors injected intravenously, resulting in targeted insertion of a corrected copy of the hF9 gene into the albumin locus in a proportion of liver hepatocytes. The albumin locus was selected as a "safe harbor" as production of this most abundant plasma protein exceeds 10 g/day, and moderate reductions in those levels are well-tolerated. These genome edited hepatocytes produce normal hFIX in therapeutic quantities, rather than albumin, driven by the highly active albumin enhancer/promoter, to treat hemophilia B; the genetic modification is expected to be sustained even in the face of hepatocyte turnover, making this approach attractive for treating young children with hemophilia before the appearance of significant organ damage. Transformed and primary human hepatocytes transduced in vitro with AAV2/6 encoding human albumin ZFNs and a promoterless hF9 transgene were shown to secrete hFIX. Extensive molecular analyses demonstrated that this was due to targeted integration of the hF9 transgene at the albumin locus and splicing of this gene into the albumin transcript. By employing AAV2/6 delivery of murine-specific ZFNs in vivo, stable levels of hFIX were observed in blood of mice injected with the albumin ZFNs and hF9 transgene donor. C57BL/6 mice were administered vehicle (n=20) or AAV2/6 vectors (n=25) encoding mouse surrogate reagents at 1.0 x1013 vector genome (vg)/kg via tail vein injection. ELISA analysis of plasma hFIX in the treated mice showed peak levels of 50-1053 ng/mL that were sustained for the duration of the 6-month study. Analysis of FIX activity from mouse plasma confirmed bioactivity commensurate with expression levels. Next, we report the feasibility of this approach in non-human primates (NHPs), showing that a single intravenous co-infusion of AAV2/6 vectors encoding the NHP targeted albumin-specific ZFNs and a human F9 donor at 1.2x1013 vg/kg (n=5/group) resulted in >50 ng/mL (>1% of normal) in this large animal model. The use of higher AAV2/6 doses (up to 1.5x1014 vg/kg) yielded plasma hFIX levels up to 1000 ng/ml (or 20% of normal) in several animals and up to 2000 ng/ml (or 50% of normal) in a single animal, for the duration of the study (3 months). The treatment was well tolerated in mice and NHPs, with no significant toxicological findings related to AAV2/6 ZFN + donor treatment in either species at therapeutic doses. Together, these data support a clinical trial to determine if a single co-administration of ZFN and donor AAV vectors is sufficient to enable therapeutic and potentially lifelong production of the clotting factor for the treatment of Hemophilia B. Disclosures Wechsler: Sangamo BioSciences: Employment. Meyer:Sangamo Biosciences Inc: Employment. Spratt:Sangamo Biosciences Inc: Employment. Greengard:Sangamo Biosciences Inc: Employment. Santiago:Sangamo Biosciences Inc: Employment. Sproul:Sangamo Biosciences Inc: Employment. Surosky:Sangamo Biosciences Inc: Employment. Paschon:Sangamo Biosciences Inc: Employment. Dubois-Stringfellow:Sangamo Biosciences Inc: Employment. Ando:Sangamo Biosciences Inc: Employment. Nichol:Sangamo Biosciences Inc: Employment. Rebar:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment.

scholarly journals Effect of cariporide on ram sperm pH regulation and motility: possible role of NHE1

Reproduction ◽  
2018 ◽  
Vol 155 (5) ◽  
pp. 433-445 ◽  
Author(s):  
Stefania Muzzachi ◽  
Lorenzo Guerra ◽  
Nicola Antonio Martino ◽  
Maria Favia ◽  
Giuseppe Punzi ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Prolonged Exposure ◽  
Ph Regulation ◽  
Equatorial Region ◽  
Large Animal Model ◽  
Large Animal ◽  
Significant Drop

Sperm motility, a feature essential forin vivofertilization, is influenced by intracellular pH (pHi) homeostasis. Several mechanisms are involved in pHiregulation, among which sodium–hydrogen exchangers (NHEs), a family of integral transmembrane proteins that catalyze the exchange of Na+for H+across lipid bilayers. A preliminary characterization of NHE activity and kinetic parameters, followed by analysis of the expression and localization of the protein in ram spermatozoa was performed. NHE activity showed an apparentKmfor external Na+of 17.61 mM. Immunoblotting revealed a molecular mass of 85 kDa. Immunolocalization pattern showed some species-specific aspects, such as positive labeling at the equatorial region of the sperm head. Cariporide, a selective NHE1 inhibitor, significantly reduced pHirecovery (85%). Similarly, exposure to cariporide significantly inhibited different motility parameters, including those related to sperm capacitation.In vitrofertilization (IVF) was not affected by cariporide, possibly due to the non-dramatic, although significant, drop in motility and velocity parameters or due to prolonged exposure during IVF, which may have caused progressive loss of its inhibitory effect. In conclusion, this is the first study documenting, in a large animal model (sheep) of well-known translational relevance, a direct functional role of NHE on sperm pHiand motility. The postulated specificity of cariporide toward isoform 1 of the Na+/H+exchanger seems to suggest that NHE1 may contribute to the observed effects on sperm cell functionality.

scholarly journals KRASG12D and TP53R167H Cooperate to Induce Pancreatic Ductal Adenocarcinoma in Sus Scrofa Pigs

2018 ◽  
Author(s):  
Daniel R. Principe ◽  
Nana Haahr Overgaard ◽  
Alex J. Park ◽  
Andrew M. Diaz ◽  
Carolina Torres ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Sus Scrofa ◽  
Cancer Biology ◽  
Ductal Adenocarcinoma ◽  
Large Animal Model ◽  
Large Animal ◽  
Desmoplastic Stroma ◽  
Immunocompromised Mice

AbstractAlthough survival has improved in recent years, the prognosis of patients with advanced pancreatic ductal adenocarcinoma (PDAC) remains poor. Despite substantial differences in anatomy, physiology, genetics, and metabolism, the overwhelming majority of preclinical testing relies on transgenic mice. Hence, while mice have allowed for tremendous advances in cancer biology, they have been a poor predictor of drug performance/toxicity in the clinic. Given the greater similarity of sus scrofa pigs to humans, we engineered transgenic sus scrofa expressing a LSL-KRASG12D-TP53R167H cassette. By applying Adeno-Cre to pancreatic duct cells in vitro, cells self-immortalized and established tumors in immunocompromised mice. When Adeno-Cre was administered to the main pancreaticduct in vivo, pigs developed extensive PDAC at the injection site hallmarked by excessive proliferation and desmoplastic stroma. This serves as the first large animal model of pancreatic carcinogenesis, and may allow for insight into new avenues of translational research not before possible in rodents.

scholarly journals Generation of tumorigenic porcine pancreatic ductal epithelial cells: toward a large animal model of pancreatic cancer

2018 ◽  
Author(s):  
Neeley Remmers ◽  
Jesse L. Cox ◽  
James A. Grunkemeyer ◽  
Shruthi Aravind ◽  
Christopher K. Arkfeld ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Animal Model ◽  
Cell Lines ◽  
Large Animal Model ◽  
Large Animal ◽  
Murine Models ◽  
Transformed Cell ◽  
Transformed Cell Lines

AbstractBackground. A large animal model of pancreatic cancer would permit development of diagnostic and interventional technologies not possible in murine models, and also would provide a more biologically-relevant platform for penultimate testing of novel therapies, prior to human testing. Here, we describe our initial studies in the development of an autochthonous, genetically-defined, large animal model of pancreatic cancer, using immunocompetent pigs.Methods. Primary pancreatic epithelial cells were isolated from pancreatic duct of domestic pigs; epithelial origin was confirmed with immunohistochemistry. Three transformed cell lines subsequently were generated from these primary cells using expression of oncogenic KRAS and dominant negative p53, with/without knockdown of p16 and SMAD4. We tested these cell lines using in vitro and in vivo assays of transformation and tumorigenesis.Results. The transformed cell lines outperformed the primary cells in terms proliferation, population doubling time, soft agar growth, 2D migration, and Matrigel invasion, with the greatest differences observed when all four genes (KRAS, p53, p16, and SMAD4) were targeted. All three transformed cell lines grew tumors when injected subcutaneously in nude mice, demonstrating undifferentiated morphology, mild desmoplasia, and staining for both epithelial and mesenchymal markers. Injection into the pancreas of nude mice resulted in distant metastases, particularly when all four genes were targeted.Conclusions. Tumorigenic porcine pancreatic cell lines were generated. Inclusion of four genetic “hits” (KRAS, p53, p16, and SMAD4) appeared to produce the best results in our in vitro and in vivo assays. The next step will be to perform autologous or syngeneic implantation of these cell lines into the pancreas of immunocompetent pigs. We believe that the resultant large animal model of pancreatic cancer could supplement existing murine models, thus improving preclinical research on diagnostic, interventional, and therapeutic technologies.

Immune-Modulatory Effects of Mesenchymal Stromal Cell Infusions for the Treatment of Factor VIII Inhibitor in Hemophilia A.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1299-1299
Author(s):  
Hideto Matsui ◽  
Lori Harpell ◽  
Sandra Powell ◽  
Moutih Rafei ◽  
Jacques Galipeau ◽  
...  
Keyword(s):  
Cellular Therapy ◽  
Hemophilia A ◽  
Conflicts Of Interest ◽  
Large Animal Model ◽  
Large Animal ◽  
Factor Viii Inhibitor ◽  
Immunoglobulin Production ◽  
Wide Range

Abstract Abstract 1299 Poster Board I-321 The current most serious treatment-related complication in hemophilia A is the development of antibodies to FVIII following exposure to FVIII replacement therapy. We have previously demonstrated that the injection of syngeneic mesenchymal stromal cells (MSCs) into hemophilia A mice with FVIII antibodies lead to a robust decrease in FVIII specific IgG levels because the secretome of MSCs suppresses plasma cell immunoglobulin production, induces plasmablast proliferation, and leads to IL-10-mediated blockade both in vitro and in vivo. Therefore, we wanted to pursue this study in a large animal model. In the current study, we harvested MSCs from bone marrow aspirates obtained from 2 hemophilia A dogs with both human and canine FVIII inhibitors. The isolated MSCs were expanded ex vivo for the infusions. Prior to the infusions, we performed in vitro characterization of the canine MSCs and found that the cells secrete a wide range of cytokines and chemokines including CC chemokine ligand 2 (CCL2) and CCL2 that has been cleaved to its truncated inhibitory form by proteolysis with matrix metalloproteinases (MMPs). These results are identical to those seen in the previous mouse experiments. Before the MSC infusions, the hemophilia A dogs with inhibitors were immunized by multiple intravenous infusions of both human FVIII and canine cryoprecipitate to further boost the development of FVIII antibodies. Expanded autologus MSCs (4.5-6.0×106 cells/Kg) were infused intravenously into the first dog twice, with a month in between each injection. After the second infusion, the titer of anti-human FVIII inhibitory activity was significantly decreased from 6.2 to 1.9 BUs and a transient but significant decrease from 3.5 to 2.2 BUs against canine FVIII. Second MSC infusion trial is currently ongoing in a second dog. In parallel with in vivo study, the inhibitory response mediated by MSCs on the FVIII antibody producing B cells was assessed by ELISPOT assay. In conclusion, MSCs may play an immunosuppressive role in modulating Immunoglobulin production by plasma cells via MMP processing of paracrine CCL2 secretion. This may represent a novel cellular therapy for attenuation of pathological humoral responses such as anti-FVIII antibody development. Disclosures No relevant conflicts of interest to declare.

scholarly journals Two novel direct SPIO labels and in vivo MRI detection of labeled cells after acute myocardial infarct

2017 ◽  
Vol 6 (8) ◽  
pp. 205846011771840 ◽  
Author(s):  
Riikka M Korpi ◽  
Kirsi Alestalo ◽  
Timo Ruuska ◽  
Eveliina Lammentausta ◽  
Ronald Borra ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Myocardial Infarct ◽  
Superparamagnetic Iron Oxide ◽  
Large Animal Model ◽  
Large Animal ◽  
Differentiation Potential ◽  
Acute Myocardial Infarct ◽  
Incubation Method

Background Acute myocardial infarction (AMI) is a leading cause of morbidity and mortality worldwide. Cellular decay due hypoxia requires rapid and validated methods for possible therapeutic cell transplantation. Purpose To develop direct and rapid superparamagnetic iron oxide (SPIO) cell label for a large-animal model and to assess in vivo cell targeting by magnetic resonance imaging (MRI) in an experimental AMI model. Material and Methods Bone marrow mononuclear cells (BMMNCs) were labeled with SPIO particles using two novel direct labeling methods (rotating incubation method and electroporation). Labeling, iron incorporation in cells and label distribution, cellular viability, and proliferation were validated in vitro. An AMI porcine model was used to evaluate the direct labeling method (rotating incubation method) by examining targeting of labeled BMMNCs using MRI and histology. Results Labeling (1 h) did not alter either cellular differentiation potential or viability of cells in vitro. Cellular relaxation values at 9.4 T correlated with label concentration and MRI at 1.5 T showing 89 ± 4% signal reduction compared with non-labeled cells in vitro. In vivo, a high spatial correlation between MRI and histology was observed. The extent of macroscopic pathological myocardial changes (hemorrhage) correlated with altered function detected on MRI. Conclusion We demonstrated two novel direct SPIO labeling methods and demonstrated the feasibility of clinical MRI for monitoring targeting of the labeled cells in animal models of AMI.

scholarly journals In VivoCaprine Model for Osteomyelitis and Evaluation of Biofilm-Resistant Intramedullary Nails

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Nhiem Tran ◽  
Phong A. Tran ◽  
John D. Jarrell ◽  
Julie B. Engiles ◽  
Nathan P. Thomas ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Rapid Screening ◽  
Large Animal Model ◽  
Large Animal ◽  
Intramedullary Nails ◽  
Formidable Challenge ◽  
Intramedullary Implant ◽  
Metal Organic

Bone infection remains a formidable challenge to the medical field. The goal of the current study is to evaluate antibacterial coatingsin vitroand to develop a large animal model to assess coated bone implants. A novel coating consisting of titanium oxide and siloxane polymer doped with silver was created by metal-organic methods. The coating was testedin vitrousing rapid screening techniques to determine compositions which inhibitedStaphylococcus aureusgrowth, while not affecting osteoblast viability. The coating was then applied to intramedullary nails and evaluatedin vivoin a caprine model. In this pilot study, a fracture was created in the tibia of the goat, andStaphylococcus aureuswas inoculated directly into the bone canal. The fractures were fixed by either coated (treated) or non-coated intramedullary nails (control) for 5 weeks. Clinical observations as well as microbiology, mechanical, radiology, and histology testing were used to compare the animals. The treated goat was able to walk using all four limbs after 5 weeks, while the control was unwilling to bear weight on the fixed leg. These results suggest the antimicrobial potential of the hybrid coating and the feasibility of the goat model for antimicrobial coated intramedullary implant evaluation.

Transplantation of Hepatocytes: An In-Vitro and In-Vivo Study in Canines

1994 ◽  
Vol 3 (2) ◽  
pp. 193-201 ◽  
Author(s):  
Pedro A. Rivas ◽  
Alfredo J. Fabrega ◽  
Daniel Schwartz ◽  
William Dagiantis ◽  
Michael G. Ward ◽  
...  
Keyword(s):  
Large Animal Model ◽  
Large Animal ◽  
Uw Solution ◽  
University Of Wisconsin ◽  
Native Liver ◽  
Yield And Purity ◽  
Intrasplenic Transplantation ◽  
Optimal Site

We isolated and transplanted hepatocytes in the canine, large animal model to evaluate hepatocyte yield and purity as well as the optimal site for hepatocyte engraftment (i.e., the spleen or the portal bed). We obtained viable, pure, single hepatocyte suspensions that were readily preserved at 4°C in University of Wisconsin (UW) solution for up to 3 days. Both intrasplenic and portal vein injection were well tolerated, with minimal recipient morbidity and mortality when 1-2 × 109 hepatocytes were injected into immunosuppressed allogeneic hosts. We noted the embolization of hepatocytes into the parenchyma of the native liver within 7 days of intrasplenic transplantation that produced a mild reversible derangement of liver function and histology. These results warrant consideration prior to clinical trials of hepatocyte transplantation in man.

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