Name: an ex vivo choroid sprouting assay of ocular microvascular angiogenesis
Uploaded: 2020-08-06T16:30:00
Description: Watch this Scientific Journal Video about An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis at JoVE.com

The work was supported by Grants from the Manpei Suzuki Diabetic Foundation (YT), Boston Children&#39;s Hospital OFD/BTREC/CTREC Faculty Career Development Grant, Boston Children&#39;s Hospital Ophthalmology Foundation, BCH Pilot Award, BCH Manton Center Fellowship, and Little Giraffe Foundation (ZF), The German Research Foundation (DFG; to BC [CA1940/1-1]), NIH R24EY024868, EY017017, R01EY01717-13S1, EY030904-01, BCH IDDRC (1U54HD090255), Massachusetts Lions Eye Foundation (LEHS).

All animal experiments described were approved by the Institutional Animal Care and Use Committee at Boston Children&#8217;s Hospital (ARCH protocol number 19-04-3913R).
1. Preparation
<ol>
	<li>Add 5 mL of Penicillin/Streptomycin (10000 U/mL) and 5 mL and 10 mL of commercially available supplements to 500 mL of complete classic medium with serum. Aliquot 50 mL of the medium initially. 
	NOTE: Do not return any medium back to the stock to avoid contamination.</li>
	<li>Put an aliquot of...

<ol>
	<li>Zarbin, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+in+the+pathogenesis+of+age-related+macular+degeneration.">Current concepts in the pathogenesis of age-related macular degeneration.</a> Arch Ophthalmol. 122 (4), 598-614 (2004).</li><li>Pemp, B., Schmetterer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+blood+flow+in+diabetes+and+age-related+macular+degeneration.">Ocular blood flow in diabetes and age-related macular degeneration.</a> Canadian Journal of Ophthalmology. 43 (3), 295-301 (2008).</li><li>Murakami, Y., Ishikawa, K., Nakao, S., Sonoda, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+response+in+retinal+homeostasis+and+inflammatory+disorders.">Innate immune response in retinal homeostasis and inflammatory disorders.</a> Progress in Retinal and Eye Research. 74, 100778 (2020).</li><li>Fu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dyslipidemia+in+retinal+metabolic+disorders.">Dyslipidemia in retinal metabolic disorders.</a> EMBO Molecular Medicine. 11 (10), 10473 (2019).</li><li>Daruich, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+macular+edema:+Beyond+the+surface.">Mechanisms of macular edema: Beyond the surface.</a> Progress in Retinal and Eye Research. 63, 20-68 (2018).</li><li>Tomita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Acting+FGF21+Inhibits+Retinal+Vascular+Leakage+in+In+Vivo+and+In+Vitro+Models.">Long-Acting FGF21 Inhibits Retinal Vascular Leakage in In Vivo and In Vitro Models.</a> International Journal of Molecular Sciences. 21 (4), 21041188 (2020).</li><li>Maisto, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ARPE-19-derived+VEGF-containing+exosomes+promote+neovascularization+in+HUVEC:+the+role+of+the+melanocortin+receptor+5.">ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5.</a> Cell Cycle. 18 (4), 413-424 (2019).</li><li>Mazzoni, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+Inhibitor+Apcdd1+Coordinates+Vascular+Remodeling+and+Barrier+Maturation+of+Retinal+Blood+Vessels.">The Wnt Inhibitor Apcdd1 Coordinates Vascular Remodeling and Barrier Maturation of Retinal Blood Vessels.</a> Neuron. 96 (5), 1055-1069 (2017).</li><li>Fu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adiponectin+Mediates+Dietary+Omega-3+Long-Chain+Polyunsaturated+Fatty+Acid+Protection+Against+Choroidal+Neovascularization+in+Mice.">Adiponectin Mediates Dietary Omega-3 Long-Chain Polyunsaturated Fatty Acid Protection Against Choroidal Neovascularization in Mice.</a> Investigative Ophthalmology and Visual Sciences. 58 (10), 3862-3870 (2017).</li><li>Gong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+P450+Oxidase+2C+Inhibition+Adds+to+omega-3+Long-Chain+Polyunsaturated+Fatty+Acids+Protection+Against+Retinal+and+Choroidal+Neovascularization.">Cytochrome P450 Oxidase 2C Inhibition Adds to omega-3 Long-Chain Polyunsaturated Fatty Acids Protection Against Retinal and Choroidal Neovascularization.</a> Arteriosclerosis, Thrombosis and Vascular Biology. 36 (9), 1919-1927 (2016).</li><li>Nicosia, R. F., Zorzi, P., Ligresti, G., Morishita, A., Aplin, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+regulation+of+angiogenesis+by+different+cell+types+in+the+aorta+ring+model.">Paracrine regulation of angiogenesis by different cell types in the aorta ring model.</a> International Journal of Developmental Biology. 55 (4-5), 447-453 (2011).</li><li>Bellacen, K., Lewis, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+ring+assay.">Aortic ring assay.</a> Journal of Visulaized Experiments. (33), e1564 (2009).</li><li>Masson, V. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Aortic+Ring+Assay:+A+New+Approach+of+the+Molecular+Genetics+of+Angiogenesis.">Mouse Aortic Ring Assay: A New Approach of the Molecular Genetics of Angiogenesis.</a> Biological Procedures Online. 4, 24-31 (2002).</li><li>Katakia, Y. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+model+for+studying+endothelial+tip+cells:+Revisiting+the+classical+aortic-ring+assay.">Ex vivo model for studying endothelial tip cells: Revisiting the classical aortic-ring assay.</a> Microvascular Research. 128, 103939 (2020).</li><li>Rezzola, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+and+ex+vivo+retina+angiogenesis+assays.">In vitro and ex vivo retina angiogenesis assays.</a> Angiogenesis. 17 (3), 429-442 (2014).</li><li>Rezzola, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+ex+vivo+murine+retina+angiogenesis+(EMRA)+assay.">A novel ex vivo murine retina angiogenesis (EMRA) assay.</a> Experimental Eye Research. 112, 51-56 (2013).</li><li>Shao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroid+sprouting+assay:+an+ex+vivo+model+of+microvascular+angiogenesis.">Choroid sprouting assay: an ex vivo model of microvascular angiogenesis.</a> PLoS One. 8 (7), 69552 (2013).</li><li>Tomita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+fatty+acid+receptor+4+activation+protects+against+choroidal+neovascularization+in+mice.">Free fatty acid receptor 4 activation protects against choroidal neovascularization in mice.</a> Angiogenesis. 23, 385-394 (2020).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+TWIST1+promotes+pathological+ocular+angiogenesis.">Endothelial TWIST1 promotes pathological ocular angiogenesis.</a> Investigative Ophthalmology and Vision Science. 55 (12), 8267-8277 (2014).</li><li>Liu, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+microRNA-150+is+an+intrinsic+suppressor+of+pathologic+ocular+neovascularization.">Endothelial microRNA-150 is an intrinsic suppressor of pathologic ocular neovascularization.</a> Proceedings of the National Academy of Science U. S. A. 112 (39), 12163-12168 (2015).</li><li>Zhou, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LncEGFL7OS+regulates+human+angiogenesis+by+interacting+with+MAX+at+the+EGFL7/miR-126+locus.">LncEGFL7OS regulates human angiogenesis by interacting with MAX at the EGFL7/miR-126 locus.</a> Elife. 8, 40470 (2019).</li><li>Kobayashi, S., Fukuta, M., Kontani, H., Yanagita, S., Kimura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+assay+for+angiogenesis+of+cultured+choroidal+tissues+in+streptozotocin-diabetic+Wistar+and+spontaneously+diabetic+GK+rats.">A quantitative assay for angiogenesis of cultured choroidal tissues in streptozotocin-diabetic Wistar and spontaneously diabetic GK rats.</a> Japanese Journal of Pharmacology. 78 (4), 471-478 (1998).</li><li>Kobayashi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+effects+of+tetrandrine+and+related+synthetic+compounds+on+angiogenesis+in+streptozotocin-diabetic+rodents.">Inhibitory effects of tetrandrine and related synthetic compounds on angiogenesis in streptozotocin-diabetic rodents.</a> Biological and Pharmaceutical Bulletin. 22 (4), 360-365 (1999).</li><li>Kobayashi, S., Shinohara, H., Tsuneki, H., Nagai, R., Horiuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N(epsilon)-(carboxymethyl)lysine+proliferated+CD34(+)+cells+from+rat+choroidal+explant+in+culture.">N(epsilon)-(carboxymethyl)lysine proliferated CD34(+) cells from rat choroidal explant in culture.</a> Biological and Pharmaceutical Bulletin. 27 (9), 1382-1387 (2004).</li><li>Kobayashi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overproduction+of+N(epsilon)-(carboxymethyl)lysine-induced+neovascularization+in+cultured+choroidal+explant+of+streptozotocin-diabetic+rat.">Overproduction of N(epsilon)-(carboxymethyl)lysine-induced neovascularization in cultured choroidal explant of streptozotocin-diabetic rat.</a> Biological and Pharmaceutical Bulletin. 27 (10), 1565-1571 (2004).</li><li>Bergers, G., Song, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+pericytes+in+blood-vessel+formation+and+maintenance.">The role of pericytes in blood-vessel formation and maintenance.</a> Neuro-Oncology. 7 (4), 452-464 (2005).</li><li>Browning, A. 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The choroidal sprouting assay aids research in neovascular AMD9,10,18,19,20. Choroid explants can be isolated from mice as well as rats and humans17,21. The choroid explant includes ECs, macrophages, and pericytes17. In this assay the interaction between choroidal ECs and adjace...

Choroidal angiogenesis dysregulation is associated with neovascular age-related macular degeneration (AMD)1. The choroid is a microvascular bed present underneath the retinal pigment epithelium (RPE). It has been shown that reduced blood flow in the choroid is associated with progression of AMD2. The intricate relationship between vascular endothelium, RPE, macrophages, pericytes and other cells is responsible for the homeostasis of the tissue3,4,5. Therefore, a reproducible assay modeling choroidal microenvironment is critical for the study of neovascular AMD.
Ex vivo angiogenesis assays and in vitro endothelial cell cultures can complement studies of microvascular behavior in vivo, for testing new drugs and for studies of pathogenesis. Endothelial cells such as human retinal microvascular endothelial cells (HRMECs), Human Umbilical Vein Endothelial Cells (HUVEC), isolated primary animal brain or retinal ECs are often used in in vitro studies for ocular angiogenesis research6,7,8. HRMECs in particular have been widely used as a model of in vitro choroidal neovascularization (CNV)9 by assessing endothelial proliferation, migration, tubular formation, and vascular leakage to evaluate interventions6,10. However, ECs in culture are limited as a model of CNV because of the lack of interactions with other cell types found in the choroid and because most EC used in these assays do not originate from choroid. Mouse choroidal ECs are difficult to isolate and maintain in culture.
The aortic ring assay is widely used as a model of macro vascular proliferation. Vascular sprouts from aortic explants include ECs, pericytes and macrophages11. The aortic ring assay models large vessel angiogenesis well12,13,14. However, it has limitations as a model of choroidal neovascularization as aortic rings are a macrovascular tissue lacking the characteristic choroidal microvascular environment, and sprouts from large vessels may differ from sprouts from capillary networks involved in microvascular pathology. Recently a group published an ex-vivo retinal assay15,16. Although, it is suitable for retinal neovascular disease, it is not as appropriate for choroidal neovascularization as seen in AMD.
The choroidal sprouting assay using mouse RPE, choroid, and scleral explanted tissue was developed to better model CNV. The tissue can easily be isolated from mouse (or other species) eyes17. This assay allows reproducible evaluation of pro- and anti-angiogenic potential of pharmacologic compounds and evaluation of the role of specific pathways in choroidal neovascularization using tissue from genetically modified mice and controls18. This choroidal sprouting assay has been referenced in many subsequent publications9,10,18,19,20. Here, the method involved in the use of this assay are demonstrated.

<table>
	<tbody>
		<tr>
			<td>AnaSed (Xylazine)</td>
			<td>AKORN</td>
			<td>59339-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Basal membrane extract (BME) Matrigel</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td>10cm</td>
		</tr>
		<tr>
			<td>Complete classic medium with serum and CultureBoost</td>
			<td>Cell systems</td>
			<td>4Z0-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol 200 Proof</td>
			<td>Pharmco</td>
			<td>111000200</td>
			<td>use for 70%</td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEISS</td>
			<td>Axio Observer Z1</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>GIBCO</td>
			<td>15140</td>
			<td>10000 U/mL</td>
		</tr>
		<tr>
			<td>Tissue culture plate (24-well)</td>
			<td>Olympus</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr>
			<td>VetaKet CIII (Ketamine)</td>
			<td>AKORN</td>
			<td>59399-114-10</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ex vivo choroid sprouting assay of ocular microvascular angiogenesis

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell (EC) proliferation assays using human retinal microvascular endothelial cells (HRMECs) or isolated primary retinal ECs are widely used in vitro models to study retinal angiogenesis. However, isolating pure murine retinal endothelial cells is technically challenging and retinal ECs may have different proliferation responses than choroidal endothelial cells and different cell/cell interactions. A highly reproducible ex vivo choroidal sprouting assay as a model of choroidal microvascular proliferation was developed. This model includes the interaction between choroid vasculature (EC, macrophages, pericytes) and retinal pigment epithelium (RPE). Mouse RPE/choroid/scleral explants are isolated and incubated in growth-factor-reduced basal membrane extract (BME) (day 0). Medium is changed every other day and choroid sprouting is quantified at day 6. The images of individual choroid explant are taken with an inverted phase microscope and the sprouting area is quantified using a semi-automated macro plug-in to the ImageJ software developed in this lab. This reproducible ex vivo choroidal sprouting assay can be used to assess compounds for potential treatment and for microvascular disease research to assess pathways involved in choroidal micro vessel proliferation using wild type and genetically modified mouse tissue.

Comparison of choroid sprouting growth per day
We dissected the choroid with sclera, embedded in BME and cultured them for 6 days (Figure 1). The choroid sprouting in C57BL/6J mice from day 3 to day 6 were examined with a microscope and quantified with SWIFT-Choroid a semi-automated quantification method in ImageJ. In a representative case, the choroidal sprouting area (the vessels extending from the explant, excluding the explant itself) was 0.38...

Watch this Scientific Journal Video about An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis at JoVE.com

An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis

This protocol presents choroid sprouting assay, an ex vivo model of microvascular proliferation. This assay can be used to assess pathways involved in proliferating choroidal micro vessels and assess drug treatments using wild type and genetically modified mouse tissue.

Pathological choroidal angiogenesis, a salient feature of age-related macular degeneration, leads to vision impairment and blindness. Endothelial cell ...

This Choroid assay is highly reproducible and pertinent to Choroidal Angiogenesis research and Age related Macular Degeneration, AMD It can compliment in vivo studies of microvascular behavior. This assay can be used to screen compounds as potential treatments for neovascular AMD or to assess pathways involved in Choroidal Neovascularization using wild type and genetically modified mouse tissue. To begin, add five milliliters of penicillin, streptomycin and 10 and five milliliters of commercially available supplements to 500 milliliters of complete classic medium with serum then aliquot 50 milliliters of the medium thaw the basal membrane extract or BME overnight in a refrigerator at two to eight degrees Celsius.Put an aliquot of complete classic medium on ice. Clean the dissecting microscope, forceps and scissors with 70%ethanol. Prepare to cell culture dishes and to place one on the dissection microscope and the other on ice.Then add 10 milliliters of complete classic medium to each dish. Keep the eyes in complete classic medium on ice before dissection. Remove the connective tissue and optic nerve then use a micro scissor to circumferentially cut 0.5 millimeters posterior to the corneal Limbus.Remove the cornea Iris complex, vitreous and to the lens. Make a one millimeter incision perpendicular to the cut edge towards the optic nerve and to cut a circumferential one millimeter wide band then separate the central and peripheral regions of the complex. Use forceps to peel the retina from the RPE choroid sclera complex.Keep the peripheral choroid band in complete classic medium on ice. Isolate the other eye and repeat the process to cut a second band. Cut the circular band into five to six approximately equal square pieces.Add 30 microliters of the thawed BME into the center of each well of a 24 well tissue culture plate. Make sure that the droplet of BME forms a convex dome at the bottom of the plate without touching the edges. Place the tissue in the middle of the BME.Do not flatten the choroid X plant. Let the tissue expand within the BME. Incubate the plate at 37 degrees Celsius for 10 minutes to let the gel solidify then add 500 microliters of complete classic medium into each well.Change the classic medium every other day. Choroid sprouting can be observed after three days with a microscope. Open the choroid sprouting image with image J and check image, type and eight bit with gray scale.Then optimize the contrast by selecting image, adjust, brightness contrast and adjusting it. Use the magic wand function to outline and remove the choroid tissue which are present in the center of the sprouts. Remove the background of the image with the free selection tools.Next, go to image, adjust threshold, and use the threshold function to define the microvascular sprouts against the background in periphery. Click F2 and a summary will appear. Click save to save an image of the selected area in the same folder as the original image for future reference.After a group of samples is measured, copy the recorded data for analysis. This protocol was used to examine and quantify choroid sprouting in C 57, black six J mice from day three to day six. In a representative case, the choroidal sprouting area was 0.38, 1.47, 5.62 and 10.09 millimeters squared at days three, four, five, and six, respectively.The effects of loss of FFAR4 on choroidal vascular sprouting were evaluated using the choroid sprouting assay in FFAR4 four knockout and FFAR4 wild type mice. The sprouting area in FFAR4 knockout mice increased choroidal vascular growth compared to FFAR4 wild type at day six. Furthermore, treatment with one micromolar FFAR4 agonist reduced the choroidal sprouting area compared to untreated mice at day six.This assay allows reproducible evaluation of anti angiogenic potential of pharmacological compounds and the variation of the role of a specific pathways in Choroidal Neovascularization using genetically modified mouses tissue.

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Biology

Christopher Hughes:  An in vitro model for the Study of Angiogenesis (Interview)

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher Hughes:  An in vitro model for the Study of Angiogenesis Interview

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro. He explains the importance of fibroblasts that secrete a critical, yet unidentified, soluble factor that allow endothelial cells to form vessels in culture that branch, form proper lumens, and undergo anastamosis.

Christopher C.W. Hughes describes the utility of his culture system for studying angiogenesis in vitro.  He explains the importance of fibroblasts that ...

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1&#160;hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

The tube formation assay is a fast, quantifiable method for measuring in vitro angiogenesis. Endothelial cells are combined with conditioned media and plated on basement membrane extract. Tube formation occurs within hours and newly formed tubules easily quantified.

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique tissue important for proper function of the synovial joint and is constituted by a dense extracellular matrix (ECM), rich in proteoglycan and type II collagen. Chondrocytes are the only cell type present within cartilage and are widespread and relatively low in number. Altered external stimuli and cellular signalling can lead to changes in ECM composition and deterioration, which are important pathological hallmarks in diseases such as osteoarthritis (OA) and rheumatoid arthritis.
Ex vivo cartilage models allow 1) profiling of chondrocyte mediated alterations of cartilage tissue turnover, 2) visualizing the cartilage ECM composition, and 3) chondrocyte rearrangement directly in the tissue. Profiling these alterations in response to stimuli or treatments are of high importance in various aspects of cartilage biology, and complement in vitro experiments in isolated chondrocytes, or more complex models in live animals where experimental conditions are more difficult to control.
Cartilage explants present a translational and easily accessible method for assessing tissue remodeling in the cartilage ECM in controllable settings. Here, we describe a protocol for isolating and culturing live bovine cartilage explants. The method uses tissue from the bovine knee, which is easily accessible from the local butchery. Both explants and conditioned culture medium can be analyzed to investigate tissue turnover, ECM composition, and chondrocyte function, thus profiling ECM modulation.

Here, we present a protocol describing isolation and culturing of cartilage explants from bovine knees. This method provides an easy and accessible tool to describe tissue changes in response to biological stimuli or novel therapeutics targeting the joint.

Ex vivo culture systems cover a broad range of experiments dedicated to studying tissue and cellular function in a native setting. Cartilage is a unique ...

Glomerular Outgrowth as an Ex Vivo Assay to Analyze Pathways Involved in Parietal Epithelial Cell Activation

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of pathways involved in parietal epithelial cell activation could therefore be a tool to attenuate the progression of glomerular diseases. This article describes a method to culture and analyze parietal epithelial cell outgrowth of encapsulated glomeruli isolated from mouse kidney. After dissecting isolated mouse kidneys, the tissue is minced, and glomeruli are isolated by sieving. Encapsulated glomeruli are collected, and single glomeruli are cultured for 6 days to obtain glomerular outgrowth of parietal epithelial cells. During this period, parietal epithelial cell proliferation and migration can be analyzed by determining the cell number or the surface area of outgrowing cells. This assay can therefore be used as a tool to study the effects of an altered gene expression in transgenic- or knockout-mice or the effects of culture conditions on parietal epithelial cell growth characteristics and signaling. Using this method, important pathways involved in the process of parietal epithelial cell activation and consequently in glomerulosclerosis can be studied.

This article describes a method for culturing and analyzing glomerular parietal epithelial cell outgrowths of encapsulated glomeruli isolated from mouse kidney. This method can be used to study pathways involved in parietal epithelial cell proliferation and migration.

Parietal epithelial cell (PEC) activation is one of the key factors involved in the development and progression of glomerulosclerosis. Inhibition of ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...