scholarly journals Ex vivo engineering of blood and lymphatic microvascular networks

2019 ◽  
Vol 1 (1) ◽  
pp. H17-H22 ◽  
Author(s):  
Jaana Schneider ◽  
Marianne Pultar ◽  
Wolfgang Holnthoner
Keyword(s):  
Endothelial Cells ◽  
3D Printing ◽  
Laser Processing ◽  
Interstitial Fluid ◽  
Ex Vivo ◽  
Cell Types ◽  
Supporting Cell ◽  
Microvascular Networks ◽  
Culture Systems ◽  
Engineered Tissues

Upon implantation, engineered tissues rely on the supply with oxygen and nutrients as well as the drainage of interstitial fluid. This prerequisite still represents one of the current challenges in the engineering and regeneration of tissues. Recently, different vascularization strategies have been developed. Besides technical approaches like 3D printing or laser processing and de-/recelluarization of natural scaffolds, mainly co-cultures of endothelial cells (ECs) with supporting cell types are being used. This mini-review provides a brief overview of different co-culture systems for the engineering of blood and lymphatic microvascular networks.

scholarly journals Collagen scaffolds functionalized with triple-helical peptides support 3D HUVEC culture

2020 ◽  
Vol 7 (5) ◽  
pp. 471-482
Author(s):  
Jean-Daniel Malcor ◽  
Emma J Hunter ◽  
Natalia Davidenko ◽  
Daniel V Bax ◽  
Ruth Cameron ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Three Dimensional ◽  
Cell Types ◽  
Cross Linking ◽  
Collagen Scaffolds ◽  
Mechanical Stiffness ◽  
Helical Peptides ◽  
Surface Receptors ◽  
Engineered Tissues

Abstract Porous biomaterials which provide a structural and biological support for cells have immense potential in tissue engineering and cell-based therapies for tissue repair. Collagen biomaterials that can host endothelial cells represent promising tools for the vascularization of engineered tissues. Three-dimensional collagen scaffolds possessing controlled architecture and mechanical stiffness are obtained through freeze–drying of collagen suspensions, followed by chemical cross-linking which maintains their stability. However, cross-linking scaffolds renders their biological activity suboptimal for many cell types, including human umbilical vein endothelial cells (HUVECs), by inhibiting cell–collagen interactions. Here, we have improved crucial HUVEC interactions with such cross-linked collagen biomaterials by covalently coupling combinations of triple-helical peptides (THPs). These are ligands for collagen-binding cell-surface receptors (integrins or discoidin domain receptors) or secreted proteins (SPARC and von Willebrand factor). THPs enhanced HUVEC adhesion, spreading and proliferation on 2D collagen films. THPs grafted to 3D-cross-linked collagen scaffolds promoted cell survival over seven days. This study demonstrates that THP-functionalized collagen scaffolds are promising candidates for hosting endothelial cells with potential for the production of vascularized engineered tissues in regenerative medicine applications.

scholarly journals Dynamic Interplay between Pericytes and Endothelial Cells during Sprouting Angiogenesis

Cells ◽  
2019 ◽  
Vol 8 (9) ◽  
pp. 1109 ◽  
Author(s):  
Giulia Chiaverina ◽  
Laura di Blasio ◽  
Valentina Monica ◽  
Massimo Accardo ◽  
Miriam Palmiero ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ex Vivo ◽  
Cell Types ◽  
Blood Capillary ◽  
Sprouting Angiogenesis ◽  
Mural Cells ◽  
Cellular Recruitment ◽  
Vascular Physiology ◽  
Experimental Approaches ◽  
Dynamic Interplay

Vascular physiology relies on the concerted dynamics of several cell types, including pericytes, endothelial, and vascular smooth muscle cells. The interactions between such cell types are inherently dynamic and are not easily described with static, fixed, experimental approaches. Pericytes are mural cells that support vascular development, remodeling, and homeostasis, and are involved in a number of pathological situations including cancer. The dynamic interplay between pericytes and endothelial cells is at the basis of vascular physiology and few experimental tools exist to properly describe and study it. Here we employ a previously developed ex vivo murine aortic explant to study the formation of new blood capillary-like structures close to physiological situation. We develop several mouse models to culture, identify, characterize, and follow simultaneously single endothelial cells and pericytes during angiogenesis. We employ microscopy and image analysis to dissect the interactions between cell types and the process of cellular recruitment on the newly forming vessel. We find that pericytes are recruited on the developing sprout by proliferation, migrate independently from endothelial cells, and can proliferate on the growing capillary. Our results help elucidating several relevant mechanisms of interactions between endothelial cells and pericytes.

MO005GENERATION OF NOVEL 3D CO-CULTURE SYSTEMS TO STUDY PODOCYTOPATHIES EX VIVO IN A PERSONALIZED MANNER*

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Victoria Rose ◽  
Nina Sopel ◽  
Alexandra Ohs ◽  
Christina Warnecke ◽  
Mario Schiffer ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Cell Lines ◽  
Ex Vivo ◽  
Time Lapse ◽  
Cell Types ◽  
Glomerular Diseases ◽  
Glomerular Cell ◽  
Glomerular Endothelial Cells ◽  
Culture Model

Abstract Background and Aims The smallest filtration unit of the kidney, the glomerulus, is composed of capillaries formed by glomerular endothelial cells (GEC), glomerular basement membrane and podocytes. Furthermore, glomerular mesangial cells (GMC) between the capillary loops give structural support. It is known that loss of podocyte foot processes is leading to a dysfunctional glomerular filtration barrier and is seen in glomerular diseases like focal segmental glomerulosclerosis and other podocytopathies. Indeed, it is hard to investigate podocytes in culture because podocytes are terminally end-differentiated cells that do not proliferate, lack foot processes and cell type-specific markers. Although conditionally immortalized human podocytes regained the capacity of proliferation, marker expression and behaviour differ between cell lines. The overarching aims of this study are to generate a 3D glomerular co-culture model that better reflects the in vivo phenotype of glomerular cell types. We want to investigate cell-cell contact, interaction and communication and extracellular matrix production in 3D glomerular co-cultures. Furthermore, patient-derived hiPSC-podocytes will be used in the glomerular co-cultures to investigate podocyte disease in a personalized manner and to identify potential therapeutic targets. Method The hanging droplet method was used to produce 3D glomerular spheroids. Therefore, human differentiated immortalized podocytes, human GECs and human GMCs were inserted in a medium-droplet hanging from the lid of a petri dish and harvested at different time points. Fluorescent cell lines of the different glomerular cell types were tracked in a time-lapse experiment to study if cell attachment and spheroid formation undergoes a specific order and structure. Glomerular spheroids were further characterized regarding the expression of podocyte-specific markers and extracellular matrix synthesis by immunohistochemistry, electron microscopy and qPCR and were compared to human cells isolated from glomeruli. Furthermore, scRNA-sequencing analysis was performed in 2D mono-cultures of human GECs, GMCs and immortalized podocytes and on 3D co-cultures to see if this change in culture conditions leads to transcriptomic alterations. For the generation of patient-derived podocytes, skin fibroblast of patients with podocyte mutations (INF2 mutation and WT1 mutation) and from healthy controls were reprogramed in iPSCs and differentiated into podocytes that keep the patient’s mutation. Results First time-lapse experiments of glomerular co-cultures showed that human podocytes and human glomerular endothelial cells attach to each other (Fig. 1a) and histological sections revealed that the glomerular spheroids are encapsulated by a monolayer of cells (Fig. 1b). SEM allowed ultrastructural characterization of the 3D spheroid-like structures (Fig. 1c). TEM revealed cell protrusions of podocytes that were not seen in monocultures (Fig. 1d, e). We could also demonstrate production of extracellular matrix by the cells (Fig. 1f). Immunohistochemistry and qPCR showed expression of collagen-IV and laminin. During the reprogramming of patient-derived fibroblasts, size of the generated hiPSC decreased and the nuclei to cell body ratio increased. HiPSCs formed colonies with distinct boarders and the proliferation rate increased. Furthermore, generated hiPSC showed similar gene expression of pluripotency markers compared to a commercial hiPSC control cell line and podocytes derived from these hiPSC expressed synaptopodin (Fig. 2). Conclusion We generated a 3D co-culture model that better represents the complexity of the glomerulus ex vivo. It is indicated that this model provides better physiological conditions. By an insertion of patient-specific hiPSC-derived podocytes in the 3D co-culture we will investigate glomerular diseases in a personalized manner in the future.

scholarly journals Distinct Roles of Adenovirus Vector-Transduced Dendritic Cells, Myoblasts, and Endothelial Cells in Mediating an Immune Response against a Transgene Product

2002 ◽  
Vol 76 (6) ◽  
pp. 2899-2911 ◽  
Author(s):  
Stéphanie Mercier ◽  
Hanne Gahéry-Segard ◽  
Martine Monteil ◽  
Renée Lengagne ◽  
Jean-Gérard Guillet ◽  
...  
Keyword(s):  
Immune Response ◽  
Endothelial Cells ◽  
Dendritic Cells ◽  
Recombinant Adenovirus ◽  
Ex Vivo ◽  
Adenovirus Vector ◽  
Cell Types ◽  
T Cell Response ◽  
Specific Response ◽  
Melanoma Tumor

ABSTRACT Adenovirus-mediated gene delivery via the intramuscular route efficiently promotes an immune response against the transgene product. In this study, a recombinant adenovirus vector encoding β-galactosidase (AdβGal) was used to transduce dendritic cells (DC), which are antigen-presenting cells, as well as myoblasts and endothelial cells (EC), neither of which present antigens. C57BL/6 mice received a single intramuscular injection of AdβGal-transduced DC, EC, or myoblasts and were then monitored for anti-β-galactosidase (anti-β-Gal) antibody production, induction of gamma interferon-secreting CD8+ T cells, and protection against melanoma tumor cells expressing β-Gal. While all transduced cell types were able to elicit an antibody response against the transgene product, the specific isotypes were distinct, with exclusive production of immunoglobulin G2a (IgG2a) antibodies following injection of transduced DC and EC versus equivalent IgG1 and IgG2a responses in mice inoculated with transduced myoblasts. Transduced DC induced a strong ex vivo CD8+ T-cell response at a level of 50% of the specific response obtained with the AdβGal control. In contrast, this response was 6- to 10-fold-lower in animals injected with transduced myoblasts and EC. Accordingly, only animals injected with transduced DC were protected against a β-Gal tumor challenge. Thus, in order to induce a strong and protective immune response to an adenovirus-encoded transgene product, it is necessary to transduce cells of dendritic lineage. Importantly, it will be advantageous to block the transduction of DC for adenovirus-based gene therapy strategies.

scholarly journals Expression of α2-macroglobulin by the interaction between hepatocytes and endothelial cells in coculture

1998 ◽  
Vol 275 (1) ◽  
pp. R203-R211
Author(s):  
Mark A. Talamini ◽  
Michael P. McCluskey ◽  
Timothy G. Buchman ◽  
Antonio De Maio
Keyword(s):  
Endothelial Cells ◽  
Ex Vivo ◽  
Apparent Molecular Weight ◽  
Rat Hepatocytes ◽  
Cell Types ◽  
Hepatic Function ◽  
Cellular Interactions ◽  
Ex Vivo Model ◽  
Extracellular Medium ◽  
Isolated Rat Hepatocytes

The interaction between distinct cell types within the liver seems to be important in regulating hepatic function. However, these interactions have not been well characterized because of difficulty in reproducing the hepatic environment in an ex vivo model. In the present study a coculture system of hepatocytes and endothelial cells was established to investigate the communication between parenchymal and nonparenchymal cells. Freshly isolated rat hepatocytes were placed onto a monolayer of primary aortic rat endothelial cells. Analysis of the proteins secreted into the extracellular medium after pulse labeling with radioactive amino acids revealed the presence of a 180,000-apparent molecular weight glycoprotein, BBB-180, which was not detected in the extracellular medium of hepatocytes or endothelial cells when they were cultured separately. This glycoprotein was identified as α2-macroglobulin after sequencing of the proteolytic peptides derived from the purified protein. This finding was confirmed by Northern and Western blotting, immunoprecipitation, and RT-PCR. The expression of α2-macroglobulin required direct contact between hepatocytes and viable endothelial cells. These findings suggest that endothelial cells modulate hepatocyte gene expression by direct cellular interactions.

scholarly journals Culture of human cells in experimental units for spaceflight impacts on their behavior

2016 ◽  
Vol 242 (10) ◽  
pp. 1072-1078
Author(s):  
Alessandra Cazzaniga ◽  
Claudia Moscheni ◽  
Jeanette AM Maier ◽  
Sara Castiglioni
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Biomedical Research ◽  
Normal Growth ◽  
Cell Types ◽  
Growth Conditions ◽  
Potential Hazard ◽  
Culture Dish ◽  
Bone Mesenchymal Stem Cells ◽  
Culture Systems

Because space missions produce pathophysiological alterations such as cardiovascular disorders and bone demineralization which are very common on Earth, biomedical research in space is a frontier that holds important promises not only to counterbalance space-associated disorders in astronauts but also to ameliorate the health of Earth-bound population. Experiments in space are complex to design. Cells must be cultured in closed cell culture systems (from now defined experimental units (EUs)), which are biocompatible, functional, safe to minimize any potential hazard to the crew, and with a high degree of automation. Therefore, to perform experiments in orbit, it is relevant to know how closely culture in the EUs reflects cellular behavior under normal growth conditions. We compared the performances in these units of three different human cell types, which were recently space flown, i.e. bone mesenchymal stem cells, micro- and macrovascular endothelial cells. Endothelial cells are only slightly and transiently affected by culture in the EUs, whereas these devices accelerate mesenchymal stem cell reprogramming toward osteogenic differentiation, in part by increasing the amounts of reactive oxygen species. We conclude that cell culture conditions in the EUs do not exactly mimic what happens in a culture dish and that more efforts are necessary to optimize these devices for biomedical experiments in space. Impact statement Cell cultures represent valuable preclinical models to decipher pathogenic circuitries. This is true also for biomedical research in space. A lot has been learnt about cell adaptation and reaction from the experiments performed on many different cell types flown to space. Obviously, cell culture in space has to meet specific requirements for the safety of the crew and to comply with the unique environmental challenges. For these reasons, specific devices for cell culture in space have been developed. It is important to clarify whether these alternative culture systems impact on cell performances to allow a correct interpretation of the data.

Paracrine induction of angiogenesis in vitro by Swiss 3T3 fibroblasts

1993 ◽  
Vol 105 (4) ◽  
pp. 1013-1024
Author(s):  
R. Montesano ◽  
M.S. Pepper ◽  
L. Orci
Keyword(s):  
Endothelial Cells ◽  
Conditioned Medium ◽  
Collagen Gel ◽  
Cell Types ◽  
Microvascular Endothelial Cells ◽  
3T3 Cells ◽  
Culture Systems ◽  
3T3 Fibroblasts ◽  
Swiss 3T3 ◽  
Swiss 3T3 Fibroblasts

During angiogenesis, normally quiescent endothelial cells that line existing microvessels are induced to invade the surrounding extracellular matrix and to form capillary sprouts in response to paracrine factors released by neighboring cell types. In an attempt to mimic the physiological conditions under which angiogenesis is triggered in vivo, we have designed two co-culture systems that are suitable for the study of paracrine interactions between microvascular endothelial cells and cell types that might produce angiogenic factors. In the first model, cells to be co-cultured with endothelial cells were suspended within a collagen gel and overlaid with an additional collagen gel devoid of cells, onto which bovine microvascular endothelial cells were subsequently seeded and grown to confluence. In the second model, a small collagen gel disc containing a suspension of endothelial cells was embedded into a larger, cell-free collagen gel disc, which in turn was surrounded by an annular collagen gel containing other cell types. We show that Swiss 3T3 fibroblasts and their 3T3-L1 substrain induce the formation of capillary-like tubes by endothelial cells in these co-culture systems, whereas Balb 3T3 cells, as well as a number of other fibroblasts and epithelial cells, lack this ability. Thus, endothelial cells grown on a collagen gel containing Swiss 3T3 or 3T3-L1 cells invaded the underlying matrix to form a network of interconnecting tubules. In addition, Swiss 3T3 and 3T3-L1 cells stimulated extensive radial outgrowth of tubular sprouts from the periphery of endothelial-cell-containing collagen discs. Conditioned medium from Swiss 3T3 cells mimicked the effect of co-culture by inducing formation of capillary-like tubes, and also increased plasminogen activator activity in endothelial cells. Conditioned medium from Balb 3T3 cells, in contrast, lacked these activities. Preliminary evidence suggests that the factor(s) in Swiss 3T3 conditioned medium that induces tubule formation by endothelial cells may be different from a number of well-characterized angiogenic cytokines. The co-culture systems described here should prove to be useful for the identification of physiological regulators of angiogenesis produced by various cell types.

scholarly journals Matrix Metalloproteinase Activity in Infections by an Encephalitic Virus, Mouse Adenovirus Type 1

2017 ◽  
Vol 91 (6) ◽  
Author(s):  
Shanna L. Ashley ◽  
Carla D. Pretto ◽  
Matthew T. Stier ◽  
Padma Kadiyala ◽  
Luiza Castro-Jorge ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Ex Vivo ◽  
Adenovirus Type ◽  
Cell Types ◽  
Tight Junction Proteins ◽  
Mock Infection ◽  
Junction Proteins

ABSTRACT Mouse adenovirus type 1 (MAV-1) infection causes encephalitis in susceptible strains of mice and alters the permeability of infected brains to small molecules, which indicates disruption of the blood-brain barrier (BBB). Under pathological conditions, matrix metalloproteinases (MMPs) can disrupt the BBB through their proteolytic activity on basement membrane and tight junction proteins. We examined whether MAV-1 infection alters MMP activity in vivo and in vitro. Infected MAV-1-susceptible SJL mice had higher MMP2 and MMP9 activity in brains, measured by gelatin zymography, than mock-infected mice. Infected MAV-1-resistant BALB/c mice had MMP activity levels equivalent to those in mock infection. Primary SJL mouse brain endothelial cells (a target of MAV-1 in vivo) infected ex vivo with MAV-1 had no difference in activities of secreted MMP2 and MMP9 from mock cells. We show for the first time that astrocytes and microglia are also infected in vivo by MAV-1. Infected mixed primary cultures of astrocytes and microglia had higher levels of MMP2 and MMP9 activity than mock-infected cells. These results indicate that increased MMP activity in the brains of MAV-1-infected susceptible mice may be due to MMP activity produced by endothelial cells, astrocytes, and microglia, which in turn may contribute to BBB disruption and encephalitis in susceptible mice. IMPORTANCE RNA and DNA viruses can cause encephalitis; in some cases, this is accompanied by MMP-mediated disruption of the BBB. Activated MMPs degrade extracellular matrix and cleave tight-junction proteins and cytokines, modulating their functions. MAV-1 infection of susceptible mice is a tractable small-animal model for encephalitis, and the virus causes disruption of the BBB. We showed that MAV-1 infection increases enzymatic activity of two key MMPs known to be secreted and activated in neuroinflammation, MMP2 and MMP9, in brains of susceptible mice. MAV-1 infects endothelial cells, astrocytes, and microglia, cell types in the neurovascular unit that can secrete MMPs. Ex vivo MAV-1 infection of these cell types caused higher MMP activity than mock infection, suggesting that they may contribute to the higher MMP activity seen in vivo. To our knowledge, this provides the first evidence of an encephalitic DNA virus in its natural host causing increased MMP activity in brains.

scholarly journals From Clones to Buds and Branches: The Use of Lung Organoids to Model Branching Morphogenesis Ex Vivo

2021 ◽  
Vol 9 ◽  
Author(s):  
Ana Ivonne Vazquez-Armendariz ◽  
Susanne Herold
Keyword(s):  
Cell Fate ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
3D Culture ◽  
Cell Types ◽  
Branching Morphogenesis ◽  
Cell Fate Decisions ◽  
Culture Systems

Three-dimensional (3D) organoid culture systems have rapidly emerged as powerful tools to study organ development and disease. The lung is a complex and highly specialized organ that comprises more than 40 cell types that offer several region-specific roles. During organogenesis, the lung goes through sequential and morphologically distinctive stages to assume its mature form, both structurally and functionally. As branching takes place, multipotent epithelial progenitors at the distal tips of the growing/bifurcating epithelial tubes progressively become lineage-restricted, giving rise to more differentiated and specialized cell types. Although many cellular and molecular mechanisms leading to branching morphogenesis have been explored, deeper understanding of biological processes governing cell-fate decisions and lung patterning is still needed. Given that these distinct processes cannot be easily analyzedin vivo, 3D culture systems have become a valuable platform to study organogenesisin vitro. This minireview focuses on the current lung organoid systems that recapitulate developmental events occurring before and during branching morphogenesis. In addition, we also discuss their limitations and future directions.

Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies

1979 ◽  
Author(s):  
S. Korach ◽  
D. Ngo
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Vascular Endothelial Cells ◽  
Intercellular Junctions ◽  
Cell Types ◽  
Endothelial Damage ◽  
Antibody Concentration ◽  
High Yields ◽  
Scanning Em

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.

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