Mimicking osteocytes in vivo using 3D collagen gels: development of a novel tool to study osteocyte biology

Melanoma phenotype plasticity underlies tumour dissemination and resistance to therapy, yet its regulation is incompletely understood. In vivo switching between a more differentiated, proliferative phenotype and a dedifferentiated, invasive phenotype is directed by the tumour microenvironment. We found that treatment of partially dedifferentiated, invasive A375M2 cells with two structurally unrelated p38 MAPK inhibitors, SB2021920 and BIRB796, induces a phenotype switch in 3D collagen, as documented by increased expression of melanocyte differentiation markers and a loss of invasive phenotype markers. The phenotype is accompanied by morphological change corresponding to amoeboid–mesenchymal transition. We performed RNA sequencing with an Illumina HiSeq platform to fully characterise transcriptome changes underlying the switch. Gene expression results obtained with RNA-seq were validated by comparing them with RT-qPCR. Transcriptomic data generated in the study will extend the present understanding of phenotype plasticity in melanoma and its contribution to invasion and metastasis.

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The selective binding and transmigration of monocytes through the junctional complexes of human endothelium.

Journal of Experimental Medicine ◽

10.1084/jem.168.5.1865 ◽

1988 ◽

Vol 168 (5) ◽

pp. 1865-1882 ◽

Cited By ~ 43

Author(s):

N A Pawlowski ◽

G Kaplan ◽

E Abraham ◽

Z A Cohn

Keyword(s):

Silver Staining ◽

Umbilical Vein ◽

Collagen Gel ◽

Collagen Gels ◽

Silver Stain ◽

Term Survival ◽

Topographical Distribution ◽

Junctional Complexes

Human monocytes show a high affinity for vascular endothelium both in vitro and in vivo. To explore monocyte-endothelial interaction in greater detail, we have developed a new in vitro model for growth of human endothelial cells (EC). Human umbilical vein EC (HUVEC) cultured upon collagen gels form confluent monolayers of EC that bind silver at their intercellular border similar to cells in situ. Intercellular junctional structures, both adherens and tight junctions, were identified. In contrast, HUVEC grown on plastic surfaces did not stain with silver. The silver-staining characteristic of EC-collagen monolayers was reversible and related to their in vitro maturation and senescence. Silver staining of EC borders provided a grid by which the location of monocyte binding to the luminal surface of individual EC could be assessed. Using this technique, we found that monocytes preferentially bound to the margins of EC, in approximation to the silver-staining junctions. These results suggest that EC determinants recognized by monocytes occur in a unique topographical distribution on the apical face of EC. After binding, monocytes migrated through the EC monolayers at high basal rates. The lack of penetration of collagen gels in the absence of an EC monolayer suggested the generation of EC-specific chemotactic signal(s). Monocytes were observed to pass between EC without evidence of disruption of the monolayer. Silver stain remained present during all phases of migration, and under transmission electron microscopy, junctional complexes were found proximal to monocytes that had just completed their passage through the monolayer. After orientation to the basal surface of the EC monolayer, monocytes migrated randomly into the underlying collagen gel. Monocyte adherence, penetration, migration, and long term survival can be studied under these conditions.

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Matrix metalloproteinase-9 activates TGF-β and stimulates fibroblast contraction of collagen gels

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00015.2014 ◽

2014 ◽

Vol 306 (11) ◽

pp. L1006-L1015 ◽

Cited By ~ 73

Author(s):

Tetsu Kobayashi ◽

HuiJung Kim ◽

Xiangde Liu ◽

Hisatoshi Sugiura ◽

Tadashi Kohyama ◽

...

Keyword(s):

Matrix Metalloproteinase ◽

Human Fibroblasts ◽

Matrix Metalloproteinase 9 ◽

Lung Fibroblasts ◽

Type I ◽

Image Analysis System ◽

Collagen Gels ◽

3D Collagen ◽

Metalloproteinase 9 ◽

Tgf Β1

Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 small-interfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-β1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-β1 and reduced several TGF-β1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-β1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-β1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts.

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The Effects of Geometry and Static Boundary Conditions on Microvessel Outgrowth in a 3D Model of Angiogenesis

ASME 2010 Summer Bioengineering Conference, Parts A and B ◽

10.1115/sbc2010-19587 ◽

2010 ◽

Cited By ~ 1

Author(s):

Clayton J. Underwood ◽

Laxminarayanan Krishnan ◽

Lowell T. Edgar ◽

Steve Maas ◽

James B. Hoying ◽

...

Keyword(s):

Boundary Condition ◽

Boundary Conditions ◽

Direct Relationship ◽

3D Model ◽

Mechanical Strain ◽

Matrix Stiffness ◽

Collagen Gels ◽

3D Collagen ◽

In Vitro Angiogenesis

We reported previously that, in addition to mechanical strain, a constrained boundary condition alone can alter the organization of microvessel outgrowth during in vitro angiogenesis [1]. After 6 days of culture in vitro, microvessels aligned parallel to the long axis of rectangular 3D collagen gels that had constrained edges on the ends. However, unconstrained cultures did not show any alignment of microvessels. The ability to direct microvessel outgrowth during angiogenesis has significant implications for engineering prevascularized grafts and tissues in vitro, therefore an understanding of this process is important. Since there is direct relationship between the ability of endothelial cells to contract 3D gels and matrix stiffness [2], we hypothesize that some constrained boundary conditions will increase the apparent matrix stiffness and in turn will limit gel contraction, prevent microvessel alignment, and reduce microvessel outgrowth. The objective of this study was to compare microvessel growth and alignment under several different static boundary conditions.

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Association between tension and orientation of periodontal ligament fibroblasts and exogenous collagen fibres in collagen gels in vitro

Journal of Cell Science ◽

10.1242/jcs.58.1.125 ◽

1982 ◽

Vol 58 (1) ◽

pp. 125-138

Author(s):

C.G. Bellows ◽

A.H. Melcher ◽

J.E. Aubin

Keyword(s):

Three Dimensional ◽

Lower Surface ◽

Cytochalasin D ◽

Collagen Gels ◽

Collagen Fibres ◽

Periodontal Ligament Fibroblasts ◽

The Relationship ◽

Develop Tension

The relationship between the development of tension in sheets of fibroblasts and the orientation of these cells and collagen fibres in collagen gels was examined. Cell-containing, three-dimensional collagen gels were established in agarose-coated Epon dies measuring 10 mm X 4 mm X 4 mm, to which pieces of demineralized tooth and bone had been attached at opposite ends. Contraction of the gel into an opaque structure suspended between the two particles occurred over 24 h and resulted in concave upper and lateral surfaces and a flat to slightly concave lower surface. Initial orientation of the fibres along the tooth-bone axis was followed by similar orientation of the cells. Gels cast without cells exhibited no change in dimensions. Release of the tooth particle after 12 or 24 h of incubation led to shortening of the contracted gels 5 min following release. This shortening was significantly greater (P less than 0.001) than that of uncontracted or slightly contracted gels (1 h and 3 h incubation). Gels attached at one end only compacted around the site of attachment but did not show orientation of cells or fibres. Gels containing colcemid or cytochalasin D were only slightly compacted and did not develop tension. Collagen fibres, but not cell in colcemid-containing gels, showed some alignment; neither were aligned in the presence of cytochalasin D. These data suggest that both microtubules and microfilaments are necessary for alignment of cells and the establishment of tension between two points of attachment in collagen gels. Furthermore, they lend support to our previously advanced hypothesis that the development of tension between two points can result in the orientation of the cells along an axis connecting the points of attachment. This could provide a mechanism for the development of oriented fibre systems in vivo.

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From local to global matrix organization by fibroblasts: a 4D laser-assisted bioprinting approach

Biofabrication ◽

10.1088/1758-5090/ac40ed ◽

2021 ◽

Author(s):

Camille Douillet ◽

Marc Nicodeme ◽

Loïc Hermant ◽

Vanessa Bergeron ◽

Fabien Guillemot ◽

...

Keyword(s):

High Resolution ◽

Soft Tissue ◽

Dynamic Properties ◽

Unmet Need ◽

Specific Pattern ◽

Matrix Remodeling ◽

Collagen Gels ◽

Lattice Contraction

Abstract Fibroblasts and myofibroblasts play a central role in skin homeostasis through dermal organization and maintenance. Nonetheless, the dynamic interactions between (myo)fibroblasts and the extracellular matrix (ECM) remain poorly exploited in skin repair strategies. Indeed, there is still an unmet need for soft tissue models allowing to study the spatial-temporal remodeling properties of (myo)fibroblasts. In vivo, wound healing studies in animals are limited by species specificity. In vitro, most models rely on collagen gels reorganized by randomly distributed fibroblasts. But biofabrication technologies have significantly evolved over the past ten years. High-resolution bioprinting now allows to investigate various cellular micropatterns and the emergent tissue organizations over time. In order to harness the full dynamic properties of cells and active biomaterials, it is essential to consider “time” as the 4th dimension in soft tissue design. Following this 4D bioprinting approach, we aimed to develop a novel model that could replicate fibroblast dynamic remodeling in vitro. For this purpose, (myo)fibroblasts were patterned on collagen gels with laser-assisted bioprinting (LAB) to study the generated matrix deformations and reorganizations. First, distinct populations, mainly composed of fibroblasts or myofibroblasts, were established in vitro to account for the variety of fibroblastic remodeling properties. Then, LAB was used to organize both populations on collagen gels in even isotropic patterns with high resolution, high density and high viability. With maturation, bioprinted patterns of fibroblasts and myofibroblasts reorganized into dispersed or aggregated cells, respectively. Stress-release contraction assays revealed that these phenotype-specific pattern maturations were associated with distinct lattice tension states. The two populations were then patterned in anisotropic rows in order to direct the cell-generated deformations and to orient global matrix remodeling. Only maturation of anisotropic fibroblast patterns, but not myofibroblasts, resulted in collagen anisotropic reorganizations both at tissue-scale, with lattice contraction, and at microscale, with embedded microbead displacements. Following a 4D bioprinting approach, LAB patterning enabled to elicit and orient the dynamic matrix remodeling mechanisms of distinct fibroblastic populations and organizations on collagen. For future studies, this method provides a new versatile tool to investigate in vitro dermal organizations and properties, processes of remodeling in healing, and new treatment opportunities.

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Displacement fields using correlation methods as a tool to investigate cell migration in 3D collagen gels

Journal of Microscopy ◽

10.1111/jmi.12825 ◽

2019 ◽

Vol 275 (3) ◽

pp. 172-182

Author(s):

ARNOLD FERTIN ◽

LAURE LAFORGUE ◽

ALAIN DUPERRAY ◽

VALÉRIE M. LAURENT ◽

YVES USSON ◽

...

Keyword(s):

Cell Migration ◽

Collagen Gels ◽

Displacement Fields ◽

Correlation Methods ◽

3D Collagen

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Environmental Restrictions: A New Concept Governing HIV-1 Spread Emerging from Integrated Experimental-Computational Analysis of Tissue-Like 3D Cultures

Cells ◽

10.3390/cells9051112 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1112 ◽

Cited By ~ 1

Author(s):

Samy Sid Ahmed ◽

Nils Bundgaard ◽

Frederik Graw ◽

Oliver Fackler

Keyword(s):

In Silico ◽

Computational Analysis ◽

Virus Transmission ◽

Target Cells ◽

Virus Spread ◽

Transmission Modes ◽

3D Collagen ◽

Cell Densities ◽

Hiv 1

HIV-1 can use cell-free and cell-associated transmission modes to infect new target cells, but how the virus spreads in the infected host remains to be determined. We recently established 3D collagen cultures to study HIV-1 spread in tissue-like environments and applied iterative cycles of experimentation and computation to develop a first in silico model to describe the dynamics of HIV-1 spread in complex tissue. These analyses (i) revealed that 3D collagen environments restrict cell-free HIV-1 infection but promote cell-associated virus transmission and (ii) defined that cell densities in tissue dictate the efficacy of these transmission modes for virus spread. In this review, we discuss, in the context of the current literature, the implications of this study for our understanding of HIV-1 spread in vivo, which aspects of in vivo physiology this integrated experimental–computational analysis takes into account, and how it can be further improved experimentally and in silico.

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Prolactin effects on ion transport across cultured mouse mammary epithelium

AJP Cell Physiology ◽

10.1152/ajpcell.1981.240.3.c110 ◽

1981 ◽

Vol 240 (3) ◽

pp. C110-C115 ◽

Cited By ~ 21

Author(s):

C. A. Bisbee

Keyword(s):

Cell Cultures ◽

Short Circuit ◽

Collagen Gel ◽

Mammary Epithelium ◽

Short Circuit Current ◽

Sodium Absorption ◽

Collagen Gels ◽

Mouse Mammary Epithelium

Prolactin is a known osmoregulatory hormone in lower vertebrates, and recent evidence indicates that this hormone modulates ionic concentrations in milk. In an ultrastructurally and biochemically differentiated primary cell culture system in which mouse mammary epithelium is maintained on floating collagen gels, prolactin causes an increase in short-circuit current (Isc) of monolayers of cells derived from midpregnant (24.6 to 48.0 microA . cm-2) and lactating (10.4 to 16.1 microA . cm-2) glands. Transepithelial potential differences (basal side ground) average about -12 mV and are similar to those seen in vivo. Prelactating mammary epithelial cell cultures have transepithelial resistances ranging from 374 omega . cm2 (prolactin present) to 507 omega . cm2 (prolactin absent), and lactating cell cultures have resistances averaging almost 1,000 omega . cm2. Prolactin effects require at most one day of culture maintenance in prolactin-containing medium, and the effects are not due to known contamination of prolactin preparations with arginine vasopressin or growth hormone. Medium concentrations of prolactin as low as 1 ng/ml can elicit these effects. In prelactating cell cultures not treated with prolactin, the Isc is equal to the rate of sodium absorption. Prolactin increases sodium absorption fourfold but increases Isc only twofold. Clearly, prolactin induces other active transport; neither potassium nor chloride movements can account for this additional transport. Resistance values, current-voltage plots, and permeability coefficients indicate that in vitro mammary epithelium is a moderately “tight” tissue. Comparisons with intact glands indicate that in vitro mammary epithelium closely resembles its in vivo counterpart. Floating collagen gel cultures appear suitable for elucidating transport properties in cellularly heterogeneous and structurally complex mammalian tissues.

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