scholarly journals OP11 Exposure to an inflammatory mix re-induces inflammation in organoids of ulcerative colitis patients, independent of the inflammatory state of the tissue of origin

2020 ◽  
Vol 14 (Supplement_1) ◽  
pp. S011-S012
Author(s):  
K Arnauts ◽  
B Verstockt ◽  
J Sabino ◽  
S Vermeire ◽  
C Verfaillie ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Transcriptional Activation ◽  
Ex Vivo ◽  
Principal Component ◽  
Patient Specific ◽  
Ex Vivo Model ◽  
Mayo Score ◽  
Ex Vivo Culture ◽  
Different Response

Abstract Background Patient-derived intestinal organoids provide a powerful tool to unravel mechanisms underlying inflammatory bowel disease (IBD). Recently, we showed that organoids derived from inflamed regions in ulcerative colitis (UC) patients lose their inflammatory phenotype during ex vivo culture and were indistinguishable from organoids of non-inflamed regions in these patients.1 To study UC in an ex vivo model, we hypothesised that inflammation should be re-induced towards levels corresponding to the in vivo situation. In addition, we aimed to elucidate if organoids derived from inflamed regions are more sensitive towards inflammatory stimulation, compared with organoids from non-inflamed regions of UC patients and non-IBD controls. Methods Biopsies were obtained from 8 patients with active UC (endoscopic Mayo score of ≥2), both in inflamed and non-inflamed regions, and in 8 non-IBD controls. Crypts were isolated and cultured as organoids for at least four weeks. Organoids were subjected to a predefined inflammatory mix (MIX: 100 ng/ml TNF-α, 20 ng/ml IL-1β, 1 µg/ml Flagellin) or medium only (CTRL) for 24 h (Figure 1). RNA was extracted from organoids for RNA sequencing by Lexogen QuantSeq for Illumina. Differential gene expression and pathways were studied through DESeq2 and ingenuity pathway analysis (false discovery rate <0.05). Results Prior to inflammatory stimulation, principal component analysis (PCA) demonstrated separate clustering between organoids derived from non-IBD controls and UC patients. Exposure to the inflammatory mix induced transcriptional activation of inflammatory genes (CXCL1, DUOXA2, IL1β, IL8, IL23α, etc., all p < 0.001) and pathways in all conditions (Figure 2). However, organoids of non-IBD controls clustered separate from organoids of UC patients. Within organoids of UC patients (inflamed vs. non-inflamed origin), we observed no differentially expressed genes after inflammatory stimulation but organoids clustered per patient instead (Figure 3). Inflammatory markers in UC organoids reached transcriptional expression levels (CXCL1, CXCL2, IFNGR1, IL1β, DUOXA2, etc.) and activated pathways (antigen presentation, interferon signalling, granulocyte adhesion and diapedesis) similar to those observed in crypts derived from inflamed biopsies. Conclusion Inflammation can efficiently be (re-)induced in organoids from both UC and non-IBD origin. However, a different response was observed between organoids of non-IBD and UC origin. Of note, in UC organoids, the state of inflammation in the source tissue was irrelevant. In conclusion, we showed that it is essential to re-induce inflammation in patient-specific organoids, but there is no need to obtain biopsies from inflamed regions. Reference

Expression of β-defensin-4 in “an in vivo and ex vivo model” of human osteoarthritic knee meniscus

2011 ◽  
Vol 20 (2) ◽  
pp. 216-222 ◽  
Author(s):  
Giuseppe Musumeci ◽  
Maria Luisa Carnazza ◽  
Rosalia Leonardi ◽  
Carla Loreto
Keyword(s):  
Ex Vivo ◽  
Ex Vivo Model ◽  
Osteoarthritic Knee ◽  
Knee Meniscus

scholarly journals Considerations to Model Heart Disease in Women with Preeclampsia and Cardiovascular Disease

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 899
Author(s):  
Clara Liu Chung Ming ◽  
Kimberly Sesperez ◽  
Eitan Ben-Sefer ◽  
David Arpon ◽  
Kristine McGrath ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiovascular Disease ◽  
Ex Vivo ◽  
Myocardial Damage ◽  
Cardiovascular Disorder ◽  
Model Systems ◽  
Ex Vivo Model ◽  
Disease Manifestation

Preeclampsia is a multifactorial cardiovascular disorder diagnosed after 20 weeks of gestation, and is the leading cause of death for both mothers and babies in pregnancy. The pathophysiology remains poorly understood due to the variability and unpredictability of disease manifestation when studied in animal models. After preeclampsia, both mothers and offspring have a higher risk of cardiovascular disease (CVD), including myocardial infarction or heart attack and heart failure (HF). Myocardial infarction is an acute myocardial damage that can be treated through reperfusion; however, this therapeutic approach leads to ischemic/reperfusion injury (IRI), often leading to HF. In this review, we compared the current in vivo, in vitro and ex vivo model systems used to study preeclampsia, IRI and HF. Future studies aiming at evaluating CVD in preeclampsia patients could benefit from novel models that better mimic the complex scenario described in this article.

scholarly journals Considerations to Model Heart Disease in Women with Preeclampsia and Cardiovascular Disease

2021 ◽  
Author(s):  
Clara Liu Chung Ming ◽  
Kimberly Sesperez ◽  
Eitan Ben-Sefer ◽  
David Arpon ◽  
Kristine McGrath ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Cardiovascular Disease ◽  
Ex Vivo ◽  
Myocardial Damage ◽  
Cardiovascular Disorder ◽  
Model Systems ◽  
Ex Vivo Model ◽  
Disease Manifestation

Preeclampsia is a multifactorial cardiovascular disorder diagnosed after 20 weeks of gestation that is the leading cause of death for both mothers and babies in pregnancy. The pathophysiology remains poorly understood due to variability and unpredictability of disease manifestation when studied in animal models. After preeclampsia, both mothers and offspring have a higher risk of cardiovascular disease (CVD) including myocardial infarction or heart attack and heart failure (HF). Myocardial infarction is an acute myocardial damage that can be treated through reperfusion, however, that therapeutic approach leads to ischemic/reperfusion injury (IRI) often leading to HF. In this review, we compared the current in vivo, in vitro and ex vivo model systems used to study preeclampsia, IRI and HF. Future studies aiming at evaluating CVD in preeclampsia patients could benefit from novel models that better mimic the complex scenario described in this article.

scholarly journals The MEMIC: An ex vivo system to model the complexity of the tumor microenvironment

2021 ◽  
Author(s):  
Libuše Janská ◽  
Libi Anandi ◽  
Nell C. Kirchberger ◽  
Zoran S. Marinkovic ◽  
Logan T. Schachtner ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Cells ◽  
Ex Vivo ◽  
Tumor Stroma ◽  
Stem Cell Differentiation ◽  
Blood Stream ◽  
Direct Access ◽  
Ex Vivo Model

There is an urgent need for accurate, scalable, and cost-efficient experimental systems to model the complexity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) – a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches, and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC provide insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells sense gradual changes in metabolite concentration resulting in multicellular spatial patterns of signal activation and cell proliferation. To illustrate the ease of studying cell-cell interactions in the MEMIC, we show that ischemic macrophages reduce epithelial features in neighboring tumor cells. We propose the MEMIC as a complement to standard in vitro and in vivo experiments, diversifying the tools available to accurately model, perturb, and monitor the tumor microenvironment, as well as to understand how extracellular metabolites affect other processes such as wound healing and stem cell differentiation.

Alterations of nitric oxide synthase expression and activity during rat lung transplantation

2000 ◽  
Vol 278 (5) ◽  
pp. L1071-L1081 ◽  
Author(s):  
Mingyao Liu ◽  
Lorraine Tremblay ◽  
Stephen D. Cassivi ◽  
Xiao-Hui Bai ◽  
Eric Mourgeon ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Lung Transplantation ◽  
Ex Vivo ◽  
Total Activity ◽  
No Production ◽  
Ex Vivo Model ◽  
Gene And Protein Expression ◽  
Lung Transplants ◽  
Inducible Nos

Decreased nitric oxide (NO) production has been reported during lung transplantation in patients. To study the effects of ischemia and reperfusion on endogenous NO synthase (NOS) expression, both an ex vivo and an in vivo lung injury model for transplantation were used. Donor rat lungs were flushed with cold low-potassium dextran solution and subjected to either cold (4°C for 12 h) or warm (21°C for 4 h) ischemic preservation followed by reperfusion with an ex vivo model. A significant increase in inducible NOS and a decrease in endothelial NOS mRNA was found after reperfusion. These results were confirmed in a rat single-lung transplant model after warm preservation. Interestingly, protein contents of both inducible NOS and endothelial NOS increased in the transplanted lung after 2 h of reperfusion. However, the total activity of NOS in the transplanted lungs remained at very low levels. We conclude that ischemic lung preservation and reperfusion result in altered NOS gene and protein expression with inhibited NOS activity, which may contribute to the injury of lung transplants.

Measurement of bone surface strains on the sheep metacarpus in vivo and ex vivo

2003 ◽  
Vol 16 (01) ◽  
pp. 38-43 ◽  
Author(s):  
R. Steck ◽  
C. Gatzka ◽  
E. Schneider ◽  
P. Niederer ◽  
M. L. Tate
Keyword(s):  
Ex Vivo ◽  
Longitudinal Axis ◽  
Peak Strain ◽  
Bone Surface ◽  
Interstitial Fluid Flow ◽  
Ex Vivo Model ◽  
Physiological Strain ◽  
Bending Load ◽  
Walking Speeds

SummaryBone surface strains were measured on the dorsal ovine metacarpus during normal locomotion on a treadmill at different walking speeds to determine physiological strain levels. These measured strains were related to the strains measured in an ex vivo model of the sheep forelimb with two types of load application: loading by two Schanz-screws and loading via the radius. In vivo, the average surface strains were found to be dependent upon body weight as well as the walking speed. The orientation of the peak principal strain corresponded to the longitudinal axis of the bone. Ex vivo, loads applied via Schanz screws in the screw-loading model lead to strains on the dorsal metacarpus that corresponds to strains experienced in vivo during intermittent peak loads. Screw loading imparted primarily a bending load to the metacarpus, with the dorsal aspect in compression and the palmar aspect in tension. Loads, applied via the radius and the hoof in the radius-loading model, resulted in bone surface strains comparable to those measured during slow walking in vivo. In both ex vivo loading situations, peak strain orientation was parallel to the longitudinal axis of the sheep metacarpus. In conclusion, the results show that although the ex vivo loading models do not exactly replicate the load experienced in vivo, the magnitude and orientation of the principal strains on the dorsal metacarpus are within the range of strains occurring during normal physiological loading. These data validate the physiological significance of the ex vivo model and aid in understanding effects of mechanical loading on interstitial fluid flow and mass transport through bone.

scholarly journals Integrin-αvβ3 regulates thrombopoietin-mediated maintenance of hematopoietic stem cells

Blood ◽  
2012 ◽  
Vol 119 (1) ◽  
pp. 83-94 ◽  
Author(s):  
Terumasa Umemoto ◽  
Masayuki Yamato ◽  
Jun Ishihara ◽  
Yoshiko Shiratsuchi ◽  
Mika Utsumi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Matrix Protein ◽  
Ex Vivo ◽  
Extracellular Matrix Protein ◽  
Αvβ3 Integrin ◽  
Hematopoietic Stem ◽  
Β3 Integrin ◽  
Ex Vivo Culture

AbstractThroughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-β3 signaling contributes to HSC maintenance. Specific ligation of β3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvβ3-integrin “inside-out” signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent “outside-in” signaling via phosphorylation of Tyr747 in the β3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between β3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.

Cytokine Treatment of CD34+ Cord Blood Cells with G-CSF, GM-CSF, or SCF During 48 Hours of Ex Vivo Culture Alters CD26/DPPIV Peptidase Activity and Subsequent Engraftment Into NSG Immunodeficient Mice.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1460-1460
Author(s):  
Laura A Paganessi ◽  
Lydia Luy Tan ◽  
Sucheta Jagan ◽  
Robin Frank ◽  
Antonio M. Jimenez ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Gm Csf ◽  
Cytokine Treatment ◽  
Ex Vivo Culture ◽  
Inhibitor Treatment

Abstract Abstract 1460 Many patients with hematologic malignancies choose hematopoietic stem cell transplantation (HSCT) as a treatment option. The most common source of Hematopoietic Stem and Progenitor Cells (HSC/HPC) for adult recipients is mobilized Peripheral Blood (mobPB). Limited quantities of HSC/HPC obtainable from an umbilical cord restricts its use for adult recipients. Ex vivo treatment of umbilical cord blood (CB) with cytokines and growth factors is being used to expand the population of cord blood HSC/HPCs in hopes of obtaining higher numbers of transplantable CB cells. In addition, cytokines and growth factors are often utilized post-transplant in an attempt to improve the rate of immune reconstitution. It has been previously reported that granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-colony-stimulating factor (GM-CSF) up-regulate CD26 (dipeptidyl peptidase IV/DPPIV) activity on freshly isolated CD34+ CB cells within 18 hours of culture [Christopherson, et al. Exp Hematol 2006]. Separate studies have demonstrated that treatment of uncultured CD34+ CB cells with the CD26 inhibitor Diprotin A increases transplant efficiency into immunodeficient mice [Christopherson, et al. Stem Cells Dev. 2007]. We evaluated here the in vitro and in vivo effects of CD26 inhibitor treatment on previously frozen CB CD34+ cells cultured ex vivo with G-CSF, GM-CSF or SCF for 48 hours. We examined CD26 expression by multivariate flow cytometry, CD26 activity using the established chromogenic CD26 substrate, Gly-Pro-p-nitroanilide (Gly-Pro-pNA), and SDF-1α induced migration and adhesion. In vivo, we examined long-term engraftment in NSG (NOD/SCID/IL2Rγnull) immunodeficient mice. After 48 hours of culture with cytokine treatment we observed altered CD26 expression on CD34+ CB cells. There was both an increase in the percentage of CD26+ cells and the mean fluorescence intensity (MFI) of CD26. Additionally, CD26 activity was 1.20, 1.59, 1.58, and 1.65 fold greater after ex vivo culture in untreated, G-CSF, GM-CSF and SCF treated CB CD34+ cells respectively compared to the CD26 activity prior to culture. The increase in CD26 activity as a result of treatment with G-CSF (p≤ 0.01), GM-CSF (p≤ 0.05) or SCF (p≤ 0.01) was significantly higher than the CD26 activity measured in the untreated cells following 48 hours of culture. Post-culture treatment with the CD26 inhibitor, Diprotin A, significantly improved SDF-1α induced migration and adhesion of cultured CD34+ CB cells in vitro, particularly in G-CSF treated cells (p≤ 0.05). Diprotin A treatment of CD34+ CB cells previously treated with G-CSF also significantly increased the long-term in vivo engraftment of stem and progenitor (CD34+CD38-, p=0.032), monocyte (CD14+, p=0.015), and megakaryocyte/platelet (CD61+, p=0.020) cells in the bone marrow of NSG mice. CD26 has been previously shown to cleave SDF-1 (stromal cell-derived factor 1/CXCL12). After cleavage, SDF-1 retains its ability to bind to its receptor (CXCR4) but no longer signals. SDF-1 is a powerful chemoattractant and has been shown to be important in mobilization, homing, and engraftment of HSCs and HPCs. This study demonstrates the influence of ex vivo culture and the effect of cytokine treatment on CD26 activity and subsequent biologic function related to HSCT. All three cytokines studied caused a significant increase in enzymatic activity at 48 hours compared to untreated cells. The up-regulation of CD26 protein expression caused by cytokine treatment for 48 hours, in particular G-CSF, had a significant impact on SDF-1 stimulated migration and adhesion. This was demonstrated in vitro by the improvement in cell function after CD26 inhibitor treatment and in vivo by the improved engraftment seen in the G-CSF treated cells with CD26 inhibitor treatment. These experiments suggest that combining CD26 inhibitor treatment following culture with G-CSF treatment during culture has the greatest overall benefit in engraftment outcome. By increasing our understanding of the effects of exogenous cytokines during culture on trafficking, ex vivo expanded CB has the potential to become a more effective means of not only increasing numbers of CB HSC/HPCs but also engraftment outcomes. This would ultimately allow expanded cord blood to become a more viable option for HSCT. Disclosures: No relevant conflicts of interest to declare.

Detection of allergen-induced airway hyperresponsiveness in isolated mouse lungs

2006 ◽  
Vol 291 (3) ◽  
pp. L466-L472 ◽  
Author(s):  
Martin Witzenrath ◽  
Birgit Ahrens ◽  
Stefanie M. Kube ◽  
Armin Braun ◽  
Heinz G. Hoymann ◽  
...  
Keyword(s):  
Airway Hyperresponsiveness ◽  
Ex Vivo ◽  
Whole Body ◽  
Strongly Correlated ◽  
Ex Vivo Model ◽  
Isolated Lungs ◽  
Spontaneously Breathing

Airway hyperresponsiveness (AHR) is a hallmark of bronchial asthma. Important features of this exaggerated response to bronchoconstrictive stimuli have mostly been investigated in vivo in intact animals or in vitro in isolated tracheal or bronchial tissues. Both approaches have important advantages but also certain limitations. Therefore, the aim of our study was to develop an ex vivo model of isolated lungs from sensitized mice for the investigation of airway responsiveness (AR). BALB/c mice were sensitized by intraperitoneal ovalbumin (Ova) and subsequently challenged by Ova inhalation. In vivo AR was measured in unrestrained animals by whole body plethysmography after stimulation with aerosolized methacholine (MCh) with determination of enhanced pause ( Penh). Twenty-four hours after each Penh measurement, airway resistance was continuously registered in isolated, perfused, and ventilated lungs on stimulation with inhaled or intravascular MCh or nebulized Ova. In a subset of experiments, in vivo AR was additionally measured in orotracheally intubated, spontaneously breathing mice 24 h after Penh measurement, and lungs were isolated further 24 h later. Isolated lungs of allergen-sensitized and -challenged mice showed increased AR after MCh inhalation or infusion as well as after specific provocation with aerosolized allergen. AR was increased on days 2 and 5 after Ova challenge and had returned to baseline on day 9. AHR in isolated lungs after aerosolized or intravascular MCh strongly correlated with in vivo AR. Pretreatment of isolated lungs with the β2-agonist fenoterol diminished AR. In conclusion, this model provides new opportunities to investigate mechanisms of AHR as well as pharmacological interventions on an intact organ level.

Sign in / Sign up

Export Citation Format

Share Document