In vitro formation by reverse dialysis of collagen gels containing highly oriented arrays of fibrils

We previously showed that so-called de-differentiated fat (DFAT) cells, which are derived from mature white adipocytes, spontaneously differentiate into beating cardiomyocytes. Our aim in this study was to investigate if DFAT cells also differentiate into endothelial cells (ECs) in vitro, and to further examine the cellular origin of DFAT cells as well as adipose stromal cells (ASCs) using lineage tracing. First, we examined DFAT and ASCs prepared from aP2-Cre+/+;LacZ ROSA(R26R)+/+ double transgenic mice, which express LacZ under the aP2 promoter. The results revealed that 99.9% of DFAT cells and 45% of the ASCs stained positive for LacZ, supporting that the DFAT cells and part of the ASCs are of adipocytic origin. Second, we allowed newly isolated DFAT cells to spontaneously undergo EC differentiation, which was monitored by expression of EC lineage markers as determined by real-time PCR, immunofluorescence, and FACS. Expression of the EC markers CD31 and VE-cadherin increased progressively during 2 weeks in culture, the percentage of CD31(+) cells increased from 0.0% to 8.3%, and the cells formed multi-cellular tube structures when placed in Matrigel™/Collagen gels. The data supported that a fraction of the DFAT cells differentiate into ECs. Furthermore, the EC differentiation could be enhanced in DFAT cells by treatment with bone morphogenetic protein (BMP)-4 and BMP-9. In addition to EC differentiation, the DFAT cells also expressed markers of other cardiovascular lineages including smooth muscle cells and pericytes. The multipotency of DFAT cells suggests that cellular de-differentiation might be a way for differentiated cells to regain stem cell-like properties. Thus, white mature adipocytes maybe a new stem cell source for cardiovascular regeneration.

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Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.418 ◽

2017 ◽

Vol 37 (suppl_1) ◽

Author(s):

Martin Liu ◽

Angelos Karagiannis ◽

Matthew Sis ◽

Srivatsan Kidambi ◽

Yiannis Chatzizisis

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Type I Collagen ◽

Smooth Muscle Actin ◽

Muscle Cells ◽

Type I ◽

Collagen Gels ◽

Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

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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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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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Fibroblasts from cancer patients display a mixture of both foetal and adult-like phenotypic characteristics

Journal of Cell Science ◽

10.1242/jcs.90.3.401 ◽

1988 ◽

Vol 90 (3) ◽

pp. 401-407 ◽

Cited By ~ 3

Author(s):

S.L. Schor ◽

A.M. Schor ◽

G. Rushton

Keyword(s):

Breast Cancer ◽

Cancer Patient ◽

Cancer Patients ◽

Normal Adult ◽

Population Doubling ◽

Collagen Gels ◽

Phenotypic Characteristics ◽

Biochemical Basis ◽

Adult Cells

We have previously reported that foetal and adult fibroblasts display distinctive migratory phenotypes when cultured on three-dimensional collagen gels. Both skin and tumour-derived fibroblasts from a significant proportion of patients with breast cancer were subsequently observed to display foetal-like migratory behaviour. In the accompanying paper concerned with the biochemical basis of these observations, we presented evidence that foetal fibroblasts and the foetal-like fibroblasts of cancer patients produce a soluble migration-stimulating factor (MSF) not made by normal adult cells. Data are presented here indicating that: (1) the spontaneous foetal-to-adult transition in migratory phenotype that foetal fibroblasts undergo after approximately 50–55 population doublings in vitro is correlated with a cessation of MSF production; (2) breast cancer patient fibroblasts do not undergo such a phenotypic transition and continue to produce MSF for their entire in vitro lifespan. These foetal-like cancer patient fibroblasts do, however, resemble normal adult cells by a number of other criteria, including population doubling potential, enhanced migration in the presence of serum compared to platelet-poor plasma, saturation cell density and morphology in confluent culture. These data indicate that the fibroblasts of breast cancer patients express a mixture of both foetal and adult phenotypic characteristics. Such a finding is consistent with published information indicating that foetal-to-adult transitions in various fibroblast phenotypic characteristics occur in a temporally disparate fashion during normal development, and further imply that cancer patient fibroblasts have undergone only certain of these transitions.

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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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Abstract 061: Interstitial Matrix Flow-Regulation:Myocardial Fibroblasts Respond to Colloidosmotic Pressure

Circulation Research ◽

10.1161/res.113.suppl_1.a061 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Maria P McGee ◽

Michael Morykwas ◽

Rui Wang ◽

James Jordan ◽

Louis Argenta

Keyword(s):

At Risk ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Linear Regression Analysis ◽

P Value ◽

Control Mechanisms ◽

Collagen Gels ◽

Fluid Transfer

Ischemia-reperfusion injury induces large differences in hydration potential between ischemic and nonischemic areas of the myocardium. They influence the rate and volume of fluid transfer in myocardial explants; temperature-dependent hydration-potential differences of approximately 100 mmHg in at-risk areas, as opposed to contiguous areas that are not at risk, suggest prompt interstitial fluid-transfer control mechanisms (Circ. Res . 2012;11:A235) . Aim: We adapted osmotic-stress techniques to determine whether myocardial fibroblasts, which are known to respond to mechanical and flow signals, also respond to hydration-potential changes. Methods: Fibroblasts were isolated from midwall regions of the left ventricles of healthy pigs using standard procedures. They were incorporated into 3-dimensional collagen gels of 500 mm 3 volume at 150 cells/mm 3 and equilibrated overnight in cultures using the nontoxic, inert polymer polyethylene glycol 8000 (molecular radius ~26.5 Å; concentration range 0-10% w/w) to adjust colloidosmotic pressure from approximately 5 to 205 mmHg . After the gels were detached from the dish, fluid flux was derived from any time-dependent changes in their dimensions. Results: The volume of gels without fibroblasts did not change significantly at any coloidosmotic pressure. In those with fibroblasts, the volume decreased, more slowly in those subjected to higher-than-plasma pressure levels. Progression curves conformed well to a two-exponential term model (R 2 >0.9) suggesting that two parallel processes contribute to matrix efflux, one relatively fast, with decay constant 0.274 ± 0.014 (n = 3) , and another ~50-fold slower. Initial rates were calculated from the fitted curves, and linear regression analysis used to examine their dependence on colloidosmotic pressure. Initial efflux rates decreased with pressure, mean slope, - 0.29 ± 0.08 µl/h/mmHg (R 2 = 0.7; P-value = 0.006). Conclusion: In vitro , fibroblasts in collagen matrices regulate fluid efflux in response to colloidosmotic stresses within the range of hydration-potential differences measured in myocardial explants after ischemia-reperfusion injury.

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