270 Ex Vivo Treatment of Infection in Human Donor Lungs

AbstractAllogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

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Ex vivo Treatment of Cytomegalovirus in Human Donor Lungs Using a Novel Chemokine-Based Immunotoxin

The Journal of Heart and Lung Transplantation ◽

10.1016/j.healun.2021.10.010 ◽

2021 ◽

Author(s):

Rafaela V.P. Ribeiro ◽

Terrance Ku ◽

Aizhou Wang ◽

Layla Pires ◽

Victor H. Ferreira ◽

...

Keyword(s):

Ex Vivo ◽

Human Donor

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Evaluation of Cytotoxicity of Perfluorocarbons for Intraocular Use by Cytotoxicity Test In Vitro in Cell Lines and Human Donor Retina Ex Vivo

Translational Vision Science & Technology ◽

10.1167/tvst.8.5.24 ◽

2019 ◽

Vol 8 (5) ◽

pp. 24 ◽

Cited By ~ 3

Author(s):

Mario R Romano ◽

Mariantonia Ferrara ◽

Claudio Gatto ◽

Barbara Ferrari ◽

Laura Giurgola ◽

...

Keyword(s):

Cell Lines ◽

Ex Vivo ◽

Cytotoxicity Test ◽

Human Donor

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Insights Into Thrombopoiesis From Infused Human Megakaryocytes Into Mice

Blood ◽

10.1182/blood.v122.21.1165.1165 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1165-1165

Author(s):

Yuhuan Wang ◽

Vincent M. Hayes ◽

Prasuna Paluru ◽

Stella T. Chou ◽

Deborah L. French ◽

...

Keyword(s):

Half Life ◽

Size Range ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Human Donor ◽

Nsg Mice ◽

Infusion Study ◽

Recipient Animal ◽

Post Infusion

Abstract Thrombopoiesis is the process by which megakaryocytes (Megs) release platelets (Plts), but issues remain as to the detailed in vivo mechanisms underlying this process. We now report new insights into this process by studying infused human Megs into immunocompromized NOD/SCID, gamma-interferon deleted (NSG) mice. Prior in situ microscopy has suggested that Megs release varied-size cytoplasmic fragments up to whole Megs in size into the medullary vascular space. Other studies have suggested that at least a portion of thrombopoiesis occurs by Megs lodged in the lungs. We previously infused ex vivo-generated murine Megs into mice and found that these Megs become entrapped in the animals’ lungs, and in <1.5 hrs, release functional Plts (termed here “Meg-Plts”) that have a similar half-life as infused mouse donor-derived Plts (termed here “Donor-Plts”). To better understand the biology of thrombopoiesis, we have infused ex vivo-generated human Megs into NSG mice. These studies replicated many of the observations seen with infused murine Megs: Human Megs were entrapped in the lungs with delayed release of human Meg-Plts, and these Meg-Plts had the same half-life as infused human Donor-Plts. Human Plts differ from murine Plts in size so this parameter was analyzed following infusion of human Megs using forward cell scatter analysis. We noted that 10 mins post-infusion, the Meg-Plt size range was wide and displayed a non-bell-shaped distribution. This distribution was in contrast to the tight bell-shaped curves seen for the endogenous murine Plts and for infused human Donor-Plts. However, by 3 hrs post-human Meg infusion - at the time of peak Meg-Plt counts - the human Meg-Plts now displayed an identical bell-shaped distribution curve as infused human Donor-Plt. The smaller, human Meg-Plts had disappeared. The size and distribution of these Meg-Plts then remained near identical to Donor-Plts for the remaining portion of the 48 hr post-infusion study. However, after impairing macrophage clearance in NSG recipient mice with clodronate-ladened liposome infusion, the small Meg-Plts did not disappear and were present at 48 hrs. Using thiazole orange (TO) to stain platelets for RNA content, we noted that ∼70% of all Meg-Plts were initially TO+ compared to the steady-state of ∼10% for mouse endogenous platelets. This high TO+ state decreased to near 10% by 24 hrs post-infusion. Up to ∼6 hrs, all of the large Meg-Plts were TO+, while the smaller-sized Meg-Plts were predominantly TO-. Unless the mice were treated with clodronate-ladened liposomes, these TO-, small Meg-Plts disappeared before 6 hrs. In conclusion, these data support that ex vivo-generated human Megs release physiologic platelets in the pulmonary vascular bed of NSG mice with the same size range/distribution and survival as infused human Donor-Plts. Mean Meg-Plt size depends on the species of origin of the infused Megs rather than on the species of the recipient animal. We did not detect large Meg cytoplasmic fragments that underwent further size reduction although our technique may not be capable of detecting small numbers of such fragments or the small size changes that would accompany platelet maturation from preplatelets. Our data also suggest that Megs generated in culture release a wide size range of non-physiologic Plt-like particles that when infused are cleared rapidly by macrophages. Disclosures: No relevant conflicts of interest to declare.

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High CXCL6 drives matrix expression and correlate with markers of poor outcome in IPF

10.1101/2021.06.22.449424 ◽

2021 ◽

Author(s):

Harinath Bahudhanapati ◽

Jiangning Tan ◽

Rosa-Marie Apel ◽

Benjamin Seeliger ◽

Xiaoyun Li ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Ex Vivo ◽

Cyclic Adenosine Monophosphate ◽

Adenosine Monophosphate ◽

Collagen I ◽

Lung Fibroblasts ◽

Dependent Manner ◽

Human Donor ◽

Poor Outcomes ◽

Matrix Expression

Signaling via G protein-coupled receptors (GPCRs) can modulate levels of cyclic adenosine monophosphate (cAMP) and shape the functions of fibroblasts in idiopathic pulmonary fibrosis (IPF). We have identified Chemokine (C-X-C) Motif Ligand 6 (CXCL6) as a potential pro-fibrotic GPCR ligand. We tested the function of CXCL6 in ex vivo human donor and fibrotic lung fibroblasts and in an animal model of pulmonary fibrosis. We also measured levels of CXCL6 in the blood and bronchoalveolar lavage (BAL) of patients with IPF. CXCL6 decreased cAMP levels in a dose-dependent manner in Donor and IPF Fibroblasts. CXCL6 mRNA and protein were localized to epithelial cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased Collagen I and α-SMA levels in Donor and IPF Fibroblasts. Silencing of CXCR1/2 as well as Reparixin, a CXCR1/2 inhibitor, blocked effects of CXCL6. Treprostinil blocked effects of CXCL6 only on levels of α-SMA but not on Collagen I. CXCL6 levels in the BAL of two separate cohorts of patients with IPF was associated with poor survival. We conclude that high CXCL6 drives fibroblast function and correlates with poor outcomes in IPF.

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Electrocardiographic Imaging of Repolarization Abnormalities

Journal of the American Heart Association ◽

10.1161/jaha.120.020153 ◽

2021 ◽

Vol 10 (9) ◽

Author(s):

Laura R. Bear ◽

Matthijs Cluitmans ◽

Emma Abell ◽

Julien Rogier ◽

Louis Labrousse ◽

...

Keyword(s):

Sinus Rhythm ◽

Correlation Coefficient ◽

Ex Vivo ◽

Data Sets ◽

Human Donor ◽

Electrocardiographic Imaging ◽

Significant Difference ◽

Highly Correlated ◽

Repolarization Abnormalities

Background Dispersion and gradients in repolarization have been associated with life‐threatening arrhythmias, but are difficult to quantify precisely from surface electrocardiography. The objective of this study was to evaluate electrocardiographic imaging (ECGI) to noninvasively detect repolarization‐based abnormalities. Methods and Results Ex vivo data were obtained from Langendorff‐perfused pig hearts (n=8) and a human donor heart. Unipolar electrograms were recorded simultaneously during sinus rhythm from an epicardial sock and the torso‐shaped tank within which the heart was suspended. Regional repolarization heterogeneities were introduced through perfusion of dofetilide and pinacidil into separate perfusion beds. In vivo data included torso and epicardial potentials recorded simultaneously in anesthetized, closed‐chest pigs (n=5), during sinus rhythm, and ventricular pacing. For both data sets, ECGI accurately reconstructed T‐wave electrogram morphologies when compared with those recorded by the sock (ex vivo: correlation coefficient, 0.85 [0.52–0.96], in vivo: correlation coefficient, 0.86 [0.52–0.96]) and repolarization time maps (ex‐vivo: correlation coefficient, 0.73 [0.63–0.83], in vivo: correlation coefficient, 0.76 [0.67–0.82]). ECGI‐reconstructed repolarization time distributions were strongly correlated to those measured by the sock (both data sets, R 2 ≥0.92). Although the position of the gradient was slightly shifted by 8.3 (0–13.9) mm, the mean, max, and SD between ECGI and recorded gradient values were highly correlated ( R 2 =0.87, 0.75, and 0.86 respectively). There was no significant difference in ECGI accuracy between ex vivo and in vivo data. Conclusions ECGI reliably and accurately maps potentially critical repolarization abnormalities. This noninvasive approach allows imaging and quantifying individual parameters of abnormal repolarization‐based substrates in patients with arrhythmogenesis, to improve diagnosis and risk stratification.

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A Decellularized Human Corneal Scaffold for Anterior Corneal Surface Reconstruction

10.21203/rs.3.rs-109219/v1 ◽

2020 ◽

Author(s):

Naresh Polisetti ◽

Anke Schmid ◽

Ursula Schlötzer-Schrehardt ◽

Philip Maier ◽

Stefan Lang ◽

...

Keyword(s):

Extracellular Matrix ◽

Ex Vivo ◽

Sodium Deoxycholate ◽

Nuclear Material ◽

Biological Properties ◽

Host Cells ◽

Human Cornea ◽

Human Donor ◽

Short Period

Abstract Allogenic transplants of the cornea are prone to rejection, especially in repetitive transplantation and in scarred or highly vascularized recipient sites. Patients with these ailments would particularly benefit from the possibility to use non-immunogenic decellularized tissue scaffolds for transplantation, which may be repopulated by host cells in situ or in vitro. So, the aim of this study was to develop a fast and efficient decellularization method for creating a human corneal extracellular matrix scaffold suitable for repopulation with human cells from the corneal limbus. To decellularize human donor corneas, sodium deoxycholate, deoxyribonuclease I, and dextran were assessed to remove cells and nuclei and to control tissue swelling, respectively. We evaluated the decellularization effects on the ultrastructure, optical, mechanical, and biological properties of the human cornea. Scaffold recellularization was studied using primary human limbal epithelial cells, stromal cells, and melanocytes in vitro and a lamellar transplantation approach ex vivo. Our data strongly suggest that this approach allowed the effective removal of cellular and nuclear material in a very short period of time while preserving extracellular matrix proteins, glycosaminoglycans, tissue structure, and optical transmission properties. In vitro recellularization demonstrated good biocompatibility of the decellularized human cornea and ex vivo transplantation revealed complete epithelialization and stromal repopulation from the host tissue. Thus, the generated decellularized human corneal scaffold could be a promising biological material for anterior corneal reconstruction in the treatment of corneal defects.

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325 PRODUCTION OF GAL KNOCKOUT/hA20 TRANSGENIC PIGS WITH IMPROVED XENOPROTECTIVE PROPERTIES

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab325 ◽

2013 ◽

Vol 25 (1) ◽

pp. 310

Author(s):

H. E. Ahrens ◽

B. Petersen ◽

S. Petkov ◽

J. Hauschild-Quintern ◽

H. Niemann

Keyword(s):

Mrna Expression ◽

Fas Ligand ◽

Zinc Finger Protein ◽

Ex Vivo ◽

Sleeping Beauty ◽

Coagulation System ◽

Transgenic Pigs ◽

Human Donor ◽

Fetal Fibroblasts ◽

Human Xenotransplantation

Pig-to-human xenotransplantation is promising for overcoming the shortage of suitable human donor organs, but is hampered by immunological barriers. The next immunological hurdle is the acute vascular rejection (AVR), which is associated with activation of the endothelium and the coagulation system. Recently, we demonstrated that transgenic expression of the zinc finger protein A20 protects porcine cells against apoptotic and inflammatory stimuli (Oropeza et al. 2009 Xenotransplantation 16, 522–534). Compared with other anti-apoptotic proteins, A20 also has immune-modulatory potential as shown in an CD95(Fas)Ligand assay. However, in that study, hA20 was only expressed in skeletal muscle, heart and porcine aortic endothelial cells of transgenic pigs. For use in xenotransplantation, it is critical to produce pigs with ubiquitous expression of hA20. Here, we constructed a new vector based on the Sleeping Beauty transposon plasmid pT2/HB containing the hA20 cDNA driven by the ubiquitously and strongly expressing CAGGS-promoter and co-transfected gal–/– porcine fibroblasts (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010) together with the SB transposase 100X plasmid (pT2/HB and SB transposase both kindly provided by Dr. Zoltan Ivics). Cells were selected with 400 µg of G418/mL of medium for 14 days. Subsequently, cells were screened by PCR. Transfected cell clones were pooled and used as donor cells in somatic cell nuclear transfer. Reconstructed embryos were transferred to 2 synchronized sows. Both remained pregnant on Day 25 of gestation. One recipient is expected to deliver in September 2012. The second sow, which received 104 embryos, was sacrificed on Day 26 of pregnancy and 2 fetuses could be obtained (cloning efficiency 1.92%). The fetuses had integrated the transgene into their genome as shown by PCR. Real-time PCR results from fetal fibroblasts indicated similar hA20 mRNA expression levels in both fetuses, whereas wild type controls were negative. The hA20 expression level was 2.5 and 3.1 times lower than in the original pooled of transfected cells. Fetus #1, which showed a slightly higher hA20 mRNA expression, was used for recloning. In total, 3 more recipients received an average of 104 hA20 transgenic embryos each. Currently, the hA20 protein level in fetal fibroblasts is determined by fluorescence activated cell sorting analysis. Once pigs are born, the tissue distribution of hA20 will be analysed. In parallel, the function of the transgene will be studied in the CD95(Fas)Ligand assay. Hearts and kidneys of the hA20-transgenic pigs will be further tested in ex vivo perfusion assays and in a pig-to-baboon xenotransplantation. This approach is promising for advancing pig-to-human xenotransplantation to preclinical application.

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