In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract

Arnaoutova, Irina; Kleinman, Hynda K.

doi:10.1038/nprot.2010.6

Cited by 625 publications

(527 citation statements)

References 28 publications

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“…Our analysis is also consistent with studies that identified a period in which the vascular plexus underwent a slight deformation after formation (14). On the other hand, the second observed phase of cell spreading (where the cells spread while exhibiting minimal movement, presumably forming filopodia and lamellipodia, before elongating to form cell-cell contacts) and the fourth observed phase of plexus stabilization (a distinct period where the plexus stabilizes before it rearranges) have not been identified as discrete events to our knowledge (14,15,34,35).…”

Section: Discussionsupporting

confidence: 72%

Uncovering the behaviors of individual cells within a multicellular microvascular community

Parsa

Upadhyay²,

Sia³

2011

Proc. Natl. Acad. Sci. U.S.A.

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Although individual cells vary in behavior during the formation of tissues, the nature of such variations are largely uncharacterized. Here, we tracked the morphologies and motilities of ∼300 human endothelial cells from an initial dispersed state to the formation of capillary-like structures, distilling the dynamics of tissue morphogenesis into an array of ∼36,000 numerical phenotypes. Quantitative analysis of population averages revealed two previously unidentified phases in which the cells spread before forming connections with neighboring cells and where the microvascular plexus stabilized before spatially reorganizing. Analysis at the single-cell level showed that in contrast to the population-averaged behavior, most cells followed distinct temporal patterns that were not reflected in the bulk average. Interestingly, some of these behavioral patterns correlated to the cells' final structural role within the plexus. Knowledge of how individual cells or groups of cells behave enhances our understanding of how native tissues self-organize and could ultimately enable more precise approaches for engineering tissues and synthesizing multicellular communities.vasculogenesis | systems biology | single-cell tracking | multispectral fluorescence microscopy | variability V ariations in the behaviors of individual cells during the morphogenesis of human tissues are likely to be important in shaping the evolution of multicellular structures (1). Knowledge of how individual cells behave and self-organize in native systems are also important in the design of synthetic systems, such as engineered tissues (1) and complex multicellular communities (2, 3). By contrast, population-averaged measurements, although widely used, are the end results of a large number of possible underlying statistical distributions and mask critical cell-to-cell variations (4). For example, the same population-averaged measurement could reflect either all cells behaving close to the average or the sum of many unique cellular behaviors. For the morphogenesis of a human tissue, the extent of cell-to-cell variations has thus far not been systematically studied and is currently largely unknown.Here, we directly tracked the behavior of every individual primary endothelial cell during the early stages of formation of human microvascular structures. Formation of microvessels is central to the etiology of many diseases (5) and critical for vascularizing newly engineered tissues for regenerative medicine (6). Formation of new capillaries, the smallest of the microvessels, can take place via vasculogenesis-the de novo formation of vascular networks from dispersed endothelial cells-both during prenatal development and in adults. For nearly three decades, microvasculogenesis has been studied by using well-established in vitro models (7,8). In particular, beginning with Folkman and up to recent studies (7, 9, 10), soft gels such as Matrigel have remained the most well-established in vitro system for controllably studying the initial steps of microvasculogenesis...

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Section: Discussionsupporting

confidence: 72%

Uncovering the behaviors of individual cells within a multicellular microvascular community

Parsa

Upadhyay²,

Sia³

2011

Proc. Natl. Acad. Sci. U.S.A.

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“…We are aware that any in vitro assay is not more than an indication of what happens in vivo . In general, the tube formation assay is considered to be a quick and quantifiable way of measuring angiogenesis in vitro 48, 49. It is a useful method to determine growth factors that potentially play a role in vivo vascularization.…”

Section: Discussionmentioning

confidence: 99%

Stimulation of vascularization of a subcutaneous scaffold applicable for pancreatic islet‐transplantation enhances immediate post‐transplant islet graft function but not long‐term normoglycemia

Smink

Swart

et al. 2017

J Biomedical Materials Res

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The liver as transplantation site for pancreatic islets is associated with significant loss of islets, which can be prevented by grafting in a prevascularized, subcutaneous scaffold. Supporting vascularization of a scaffold to limit the period of ischemia is challenging and was developed here by applying liposomes for controlled release of angiogenic factors. The angiogenic capacity of platelet‐derived growth factor, vascular endothelial growth factor, acidic fibroblast growth factor (aFGF), and basic FGF were compared in a tube formation assay. Furthermore, the release kinetics of different liposome compositions were tested. aFGF and L‐α‐phosphatidylcholine/cholesterol liposomes were selected to support vascularization. Two dosages of aFGF‐liposomes (0.5 and 1.0 μg aFGF per injection) were administered weekly for a month after which islets were transplanted. We observed enhanced efficacy in the immediate post‐transplant period compared to the untreated scaffolds. However, on the long‐term, glucose levels of the aFGF treated animals started to increase to diabetic levels. These results suggest that injections with aFGF liposomes do improve vascularization and the immediate restoration of blood glucose levels but does not facilitate the long‐term survival of islets. Our data emphasize the need for long‐term studies to evaluate potential beneficial and adverse effects of vascularization protocols of scaffolds. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2533–2542, 2017.

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“…26,27 The cultured LN LECs readily formed tube structures at early P4 (n = 6 independent cell isolates) as well as late P22 passages (n = 2 independent cell isolates), with examples shown in Figure 3. Early and late LEC cultures therefore both maintain the same ability to migrate and form vessel-like structures.…”

Section: Jordan-williams and Ruddellmentioning

confidence: 99%

Culturing Purifies Murine Lymph Node Lymphatic Endothelium

Jordan-Williams

Ruddell

2014

Lymphatic Research and Biology

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Background: Lymph node (LN) lymphatic sinuses transport lymph, cells, and antigens from the periphery through the LN. The lymphatic endothelium lining these sinuses appears to be an important contributor to the lymph node immune response. It has been challenging to obtain sufficient LN lymphatic endothelial cells for investigation of their functions, as they are minor constituents of LNs. Methods and Results: A procedure was developed to purify lymphatic endothelial cells (LEC) from murine LNs, which yields large numbers of primary LN LEC. Two-dimensional in vitro cultures of dissociated LN stromal cells initially consist of multiple cell types, and then rapidly evolve to produce pure cultures of lymphatic endothelium within a few passages. One million LEC can be harvested after 4 weeks of culture, and much larger cell numbers can be obtained by continued culturing over long periods. The LEC cultures maintain endothelial morphology and expression of LEC markers, and preserve the same slow growth characteristics over at least 20 passages. The LEC cultures readily form tubes in Matrigel at early and at late passages, resembling those formed by LEC lines. Conclusions: A simple and economical approach to obtain purified primary murine LN LEC was developed for in vitro studies of their function. The morphology, growth characteristics, and functional behavior of these cells in tube formation assays did not change between initial and long-term passages. Large numbers of these cells can be harvested after long-term passage, so that they can be studied in biochemical and biological assays.

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In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract

Cited by 625 publications

References 28 publications

Uncovering the behaviors of individual cells within a multicellular microvascular community

Uncovering the behaviors of individual cells within a multicellular microvascular community

Stimulation of vascularization of a subcutaneous scaffold applicable for pancreatic islet‐transplantation enhances immediate post‐transplant islet graft function but not long‐term normoglycemia

Culturing Purifies Murine Lymph Node Lymphatic Endothelium

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