Fabrication of endothelialized tube in collagen gel as starting point for self-developing capillary-like network to construct three-dimensional organs in vitro

2006 ◽  
Vol 95 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Takayuki Takei ◽  
Shinji Sakai ◽  
Tsutomu Ono ◽  
Hiroyuki Ijima ◽  
Koei Kawakami
Keyword(s):  
Three Dimensional ◽  
Collagen Gel ◽  
Starting Point

scholarly journals Targeting hepatocyte growth factor in epithelial–stromal interactions in an in vitro experimental model of human periodontitis

Odontology ◽  
2021 ◽  
Author(s):  
Yoko Yamaguchi ◽  
Akira Saito ◽  
Masafumi Horie ◽  
Akira Aoki ◽  
Patrick Micke ◽  
...  
Keyword(s):  
Growth Factor ◽  
Hepatocyte Growth Factor ◽  
Experimental Studies ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Neutralizing Antibody ◽  
Chronic Inflammatory Disease ◽  
Collagen Degradation ◽  
Hepatocyte Growth

AbstractPeriodontitis is a chronic inflammatory disease leading to progressive connective tissue degradation and loss of the tooth-supporting bone. Clinical and experimental studies suggest that hepatocyte growth factor (HGF) is involved in the dysregulated fibroblast–epithelial cell interactions in periodontitis. The aim of this study was to explore effects of HGF to impact fibroblast-induced collagen degradation. A patient-derived experimental cell culture model of periodontitis was applied. Primary human epithelial cells and fibroblasts isolated from periodontitis-affected gingiva were co-cultured in a three-dimensional collagen gel. The effects of HGF neutralizing antibody on collagen gel degradation were tested and transcriptome analyses were performed. HGF neutralizing antibody attenuated collagen degradation and elicited expression changes of genes related to extracellular matrix (ECM) and cell adhesion, indicating that HGF signaling inhibition leads to extensive impact on cell–cell and cell–ECM interactions. Our study highlights a potential role of HGF in periodontitis. Antagonizing HGF signaling by a neutralizing antibody may represent a novel approach for periodontitis treatment.

Effect of shear stress on microvessel network formation of endothelial cells with in vitro three-dimensional model

2004 ◽  
Vol 287 (3) ◽  
pp. H994-H1002 ◽  
Author(s):  
Akinori Ueda ◽  
Masaki Koga ◽  
Mariko Ikeda ◽  
Susumu Kudo ◽  
Kazuo Tanishita
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Network Formation ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Flow Chamber ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Collagen Gels

Shear stress stimulus is expected to enhance angiogenesis, the formation of microvessels. We determined the effect of shear stress stimulus on three-dimensional microvessel formation in vitro. Bovine pulmonary microvascular endothelial cells were seeded onto collagen gels with basic fibroblast growth factor to make a microvessel formation model. We observed this model in detail using phase-contrast microscopy, confocal laser scanning microscopy, and electron microscopy. The results show that cells invaded the collagen gel and reconstructed the tubular structures, containing a clearly defined lumen consisting of multiple cells. The model was placed in a parallel-plate flow chamber. A laminar shear stress of 0.3 Pa was applied to the surfaces of the cells for 48 h. Promotion of microvessel network formation was detectable after ∼10 h in the flow chamber. After 48 h, the length of networks exposed to shear stress was 6.17 (±0.59) times longer than at the initial state, whereas the length of networks not exposed to shear stress was only 3.30 (±0.41) times longer. The number of bifurcations and endpoints increased for networks exposed to shear stress, whereas the number of bifurcations alone increased for networks not exposed to shear stress. These results demonstrate that shear stress applied to the surfaces of endothelial cells on collagen gel promotes the growth of microvessel network formation in the gel and expands the network because of repeated bifurcation and elongation.

A Continuous-Discrete Finite Element Model of Angiogenesis That Couples Vessel Growth With Matrix Deformation

2013 ◽  
Author(s):  
Lowell T. Edgar ◽  
Steve A. Maas ◽  
James E. Guilkey ◽  
Jeffrey A. Weiss
Keyword(s):  
Type I Collagen ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Element Model ◽  
Fibrillar Structure ◽  
Type I ◽  
Major Pathway ◽  
Vessel Growth ◽  
Discrete Finite Element

Recent developments in tissue engineering have created demand for the ability to create microvascular networks with specific topologies in vitro. During angiogenesis, sprouting endothelial cells apply traction forces and migrate along components of the extracellular matrix (ECM), resulting in neovessel elongation [1]. The fibrillar structure of the ECM serves as the major pathway for mechanotransduction between contact-dependent cells. Using a three-dimensional (3D) organ culture model of microvessel fragments within a type-I collagen gel, we have shown that subjecting the culture to different boundary conditions during angiogenesis can lead to drastically different vascular topologies [2]. Fragments cultured in a rectangular gel that were free to contract grew into a randomly oriented network [3, 4]. When the long-axis of the gel was constrained as to prevent contraction, microvessels and collagen fibers were found aligned along the constrained axis (Fig. 1) [4].

scholarly journals Three-Dimensional In Vitro Staphylococcus aureus Abscess Communities Display Antibiotic Tolerance and Protection from Neutrophil Clearance

2020 ◽  
Vol 88 (11) ◽  
Author(s):  
Marloes I. Hofstee ◽  
Martijn Riool ◽  
Igors Terjajevs ◽  
Keith Thompson ◽  
Martin J. Stoddart ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Immune Cells ◽  
Early Stage ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Maximum Size ◽  
Human Neutrophils ◽  
Content Type

ABSTRACT Staphylococcus aureus is a prominent human pathogen in bone and soft-tissue infections. Pathophysiology involves abscess formation, which consists of central staphylococcal abscess communities (SACs), surrounded by a fibrin pseudocapsule and infiltrating immune cells. Protection against the ingress of immune cells such as neutrophils, or tolerance to antibiotics, remains largely unknown for SACs and is limited by the lack of availability of in vitro models. We describe a three-dimensional in vitro model of SACs grown in a human plasma-supplemented collagen gel. The in vitro SACs reached their maximum size by 24 h and elaborated a fibrin pseudocapsule, as confirmed by electron and immunofluorescence microscopy. The in vitro SACs tolerated 100× the MIC of gentamicin alone and in combination with rifampin, while planktonic controls and mechanically dispersed SACs were efficiently killed. To simulate a host response, SACs were exposed to differentiated PLB-985 neutrophil-like (dPLB) cells and to primary human neutrophils at an early stage of SAC formation or after maturation at 24 h. Both cell types were unable to clear mature in vitro SACs, but dPLB cells prevented SAC growth upon early exposure before pseudocapsule maturation. Neutrophil exposure after plasmin pretreatment of the SACs resulted in a significant decrease in the number of bacteria within the SACs. The in vitro SAC model mimics key in vivo features, offers a new tool to study host-pathogen interactions and drug efficacy assessment, and has revealed the functionality of the S. aureus pseudocapsule in protecting the bacteria from host phagocytic responses and antibiotics.

Expression profiling reveals functionally important genes and coordinately regulated signaling pathway genes during in vitro angiogenesis

2005 ◽  
Vol 22 (1) ◽  
pp. 57-69 ◽  
Author(s):  
C. N. Hahn ◽  
Z. J. Su ◽  
C. J. Drogemuller ◽  
A. Tsykin ◽  
S. R. Waterman ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathways ◽  
Mrna Level ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
Virtual Northern Blot ◽  
In Vitro Angiogenesis

Angiogenesis is a complex multicellular process requiring the orchestration of many events including migration, alignment, proliferation, lumen formation, remodeling, and maturation. Such complexity indicates that not only individual genes but also entire signaling pathways will be crucial in angiogenesis. To define an angiogenic blueprint of regulated genes, we utilized our well-characterized three-dimensional collagen gel model of in vitro angiogenesis, in which the majority of cells synchronously progress through defined morphological stages culminating in the formation of capillary tubes. We developed a comprehensive three-tiered approach using microarray analysis, which allowed us to identify genes known to be involved in angiogenesis and genes hitherto unlinked to angiogenesis as well as novel genes and has proven especially useful for genes where the magnitude of change is small. Of interest is the ability to recognize complete signaling pathways that are regulated and genes clustering into ontological groups implicating the functional importance of particular processes. We have shown that consecutive members of the mitogen-activated protein kinase and leukemia inhibitory factor signaling pathways are altered at the mRNA level during in vitro angiogenesis. Thus, at least for the mitogen-activated protein kinase pathway, mRNA changes as well as the phosphorylation changes of these gene products may be important in the control of blood vessel morphogenesis. Furthermore, in this study, we demonstrated the power of virtual Northern blot analysis, as an alternative to quantitative RT-PCR, for measuring the magnitudes of differential gene expression.

scholarly journals A three-dimensional model of the human blood-brain barrier to analyse the transport of nanoparticles and astrocyte/endothelial interactions

F1000Research ◽  
2016 ◽  
Vol 4 ◽  
pp. 1279 ◽  
Author(s):  
Peddagangannagari Sreekanthreddy ◽  
Radka Gromnicova ◽  
Heather Davies ◽  
James Phillips ◽  
Ignacio A. Romero ◽  
...  
Keyword(s):  
Blood Brain Barrier ◽  
Human Blood ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Cell Types ◽  
Brain Barrier ◽  
Two Dimensional ◽  
Three Dimensional Model ◽  
Blood Brain

The aim of this study was to develop a three-dimensional (3D) model of the human blood-brain barrier in vitro, which mimics the cellular architecture of the CNS and could be used to analyse the delivery of nanoparticles to cells of the CNS. The model includes human astrocytes set in a collagen gel, which is overlaid by a monolayer of human brain endothelium (hCMEC/D3 cell line). The model was characterised by transmission electron microscopy (TEM), immunofluorescence microscopy and flow cytometry. A collagenase digestion method could recover the two cell types separately at 92-96% purity.  Astrocytes grown in the gel matrix do not divide and they have reduced expression of aquaporin-4 and the endothelin receptor, type B compared to two-dimensional cultures, but maintain their expression of glial fibrillary acidic protein. The effects of conditioned media from these astrocytes on the barrier phenotype of the endothelium was compared with media from astrocytes grown conventionally on a two-dimensional (2D) substratum. Both induce the expression of tight junction proteins zonula occludens-1 and claudin-5 in hCMEC/D3 cells, but there was no difference between the induced expression levels by the two media. The model has been used to assess the transport of glucose-coated 4nm gold nanoparticles and for leukocyte migration. TEM was used to trace and quantitate the movement of the nanoparticles across the endothelium and into the astrocytes. This blood-brain barrier model is very suitable for assessing delivery of nanoparticles and larger biomolecules to cells of the CNS, following transport across the endothelium.

scholarly journals Effect of culture methods on cumulus and oocyte morphology and meiotic competence of bovine oocytes from early antral follicles

2005 ◽  
Vol 48 (6) ◽  
pp. 562-571
Author(s):  
L. Kątska-Książkiewicz ◽  
H. Alm
Keyword(s):  
Three Dimensional ◽  
Collagen Gel ◽  
Standard System ◽  
Dimensional Structure ◽  
Culture Methods ◽  
Normal Morphology ◽  
Antral Follicles ◽  
System A ◽  
Meiotic Competence

Abstract. The objective of the present study was to establish a culture system that would maintain the three-dimensional structure of bovine early antral follicles (EAF) or isolated cumulus-oocyte-granulosa complexes (COCGs) and increase the resulting portion of COCs with normal morphology for subsequent IVM. The morphological quality and meiotic competence of oocytes originating from early antral bovine ovarian follicles (0.2 to 0.7 mm and 0.4 to 0.7 mm diameter) were evaluated following culture in vitro for 14 and 7 d, respectively, and subsequent in vitro maturation. Growth culture modifications included culture in the well of the well (WOW) system; a microdroplet of collagen gel (2 x 5 μl vs. 2 x 400 μl; standard system) and culture of EAF in hanging drops of medium (inverted system). Significantly higher (P<0.01) proportions of COCs with normal morphology (60.4%) were obtained from COCGs compared to EAF (4.8%) grown in the WOW system. Embedding of COCGs in microdrops of collagen gel significantly increased proportion of COCs with normal morphology (63.2%) compared to those embedded in standard volume gels (35.3%). Recovery rate of COCs with normal morphology from cultured EAF was improved both by using microdrops of gel (44%) and by culture in the inverted system (39.3%) over that found for the standard system (8.5%).

scholarly journals Epithelia suspended in collagen gels can lose polarity and express characteristics of migrating mesenchymal cells.

1982 ◽  
Vol 95 (1) ◽  
pp. 333-339 ◽  
Author(s):  
G Greenburg ◽  
E D Hay
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Mesenchymal Cells ◽  
Apical Surface ◽  
Collagen Gels ◽  
Mesenchymal Transformation

This study of epithelial-mesenchymal transformation and epithelial cell polarity in vitro reveals that environmental conditions can have a profound effect on the epithelial phenotype, cell shape, and polarity as expressed by the presence of apical and basal surfaces. A number of different adult and embryonic epithelia were suspended within native collagen gels. Under these conditions, cells elongate, detach from the explants, and migrate as individual cells within the three-dimensional lattice, a previously unknown property of well-differentiated epithelia. Epithelial cells from adult and embryonic anterior lens were studied in detail. Elongated cells derived from the apical surface develop pseudopodia and filopodia characteristic of migratory cells and acquire a morphology and ultrastructure virtually indistinguishable from that of mesenchymal cells in vivo. It is concluded from these experiments that the three-dimensional collagen gel can promote dissociation, migration, and acquisition of secretory organelles by differentiated epithelial cells, and can abolish the apical-basal cell polarity characteristic of the original epithelium.

Insulin and IGF-I induce pronounced hypertrophy of skeletal myofibers in tissue culture

1991 ◽  
Vol 260 (3) ◽  
pp. C475-C484 ◽  
Author(s):  
H. H. Vandenburgh ◽  
P. Karlisch ◽  
J. Shansky ◽  
R. Feldstein
Keyword(s):  
Tissue Culture ◽  
Growth Factor ◽  
Three Dimensional ◽  
Collagen Gel ◽  
Insulin Like Growth Factor ◽  
Cell Hyperplasia ◽  
Factor Ii ◽  
Igf I ◽  
Collagen Gel Matrix

Skeletal myofibers differentiated from primary avian myoblasts in tissue culture can be maintained in positive nitrogen balance in a defined serum-free medium for at least 6-7 days when embedded in a three-dimensional collagen gel matrix. Incubation of established myofiber cultures for 3-7 days with insulin (1 microM) or insulin-like growth factor I (IGF-I, 32 nM) stimulates both cell hyperplasia and myofiber hypertrophy. Mean myofiber diameter increases 71-98%. Insulin-like growth factor II stimulates cell hyperplasia but not myofiber hypertrophy. Cell growth results from a 42-62% increase in total protein synthesis and a 28-38% decrease in protein degradation. Myosin heavy-chain content increases 183-258% because of a 55% stimulation of myosin synthesis and 33-61% inhibition of degradation. Associated with myofiber hypertrophy is a 87-148% increase in the number of myofiber nuclei per unit myofiber length. The results indicate that insulin and IGF-I, but not IGF-II, can induce rapid myofiber hypertrophy in vitro, most likely by stimulating myoblast proliferation and/or fusion to established myofibers.

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