The authors thank the technical staff at Nordic Bioscience for laboratory support, as well as the Danish Research Foundation for general support of our research.

1. Tissue isolation
<ol>
	<li>Tissue sourcing
	<ol>
		<li>Perform the entire tissue sourcing section outside a laminar flow hood in an aseptic environment.</li>
		<li>From the local slaughterhouse, obtain an entire fresh bovine tibiofemoral knee joint from calves between 1.5 and 2 years of age.</li>
		<li>Gently dissect the calf knee by first removing the excess flesh, uncovering the condyles, meniscus, tendons, and synovial membrane. Cut the tendons and synovial membrane, allowing the joint to dismember. Remove the meniscus to expose the femoral condyles.</li>
		<li>Isolate explants from the load-bearing area of the femoral condyles using a 3 mm biopsy puncher and release them from the articular surface by cutting with a scalpel parallel to and as close to the subchondral bone as possible. The hard structure of the subchondral bone should ensure that explants do not contain calcified matrix. Strive for explants with uniform height.</li>
		<li>Immediately store and mix the explants in DMEM/F12- GlutaMAX + 1% P/S culture medium in a 50 mL tube or Petri dish. Do not mix explants from different cow knees but keep separate for each study.</li>
	</ol>
	</li>
	<li>Tissue culturing
	<ol>
		<li>Transfer the explants to a sterile 96-well plate in a laminar flow hood.</li>
		<li>Wash the explants 3 times in culture medium or PBS and culture them in 200 &#956;L of culture medium per well until the start of the experiment. Use a washout period of 1 day to synchronize biopsy cellular activity and passive biomarker release.</li>
		<li>Culture the explants up to 10 weeks in a 37 &#176;C incubator with 5% CO2. Place all replicates within each group diagonally in the culture plate to minimize the variation induced by evaporation. To further avoid evaporation of the supernatant, add PBS to the outer wells of the culture plate.</li>
	</ol>
	</li>
</ol>
2. Bovine cartilage explant treatment and assessment of metabolic activity
<ol>
	<li>Culture medium change and treatment
	<ol>
		<li>Change the culture medium every 2-3 days in a laminar flow hood.</li>
		<li>If applying any treatments, prepare these prior to changing the medium. Prepare the treatments to the wanted concentration in the explant wells by dilution in the culture medium.</li>
		<li>Gently remove the supernatant from each well and transfer it to a new 96 well plate. Store the supernatant with sealing tape at &#8722;20 &#176;C for biomarker analysis of tissue turnover and protein expression.</li>
		<li>Immediately add 200 &#181;L of fresh culture medium or treatment per well. Do not let the explants dry out during the medium change and ensure that all the explants are completely submerged in the new medium.</li>
	</ol>
	</li>
	<li>Resazurin staining
	<ol>
		<li>Measure metabolic activity once weekly as an indirect measurement of cell viability. The resazurin test is an easy way to assess if the metabolic activity of the explants deteriorates for an individual explant due to cell death or cellular changes. Explants in culture medium alone have a relatively stable resazurin reading throughout the experiment period.</li>
		<li>Make a solution of culture medium with 10% resazurin.</li>
		<li>Harvest the supernatant as described in step 2.1.3.</li>
		<li>Immerse the explants in 10% resazurin solution for 3 h at 37 &#176;C or until the supernatants turn purple. Include 4 wells without explants as background controls.</li>
		<li>Transfer the conditioned and background control resazurin solution to a black microtiter plate and measure fluorescence at 540 nm excitation/590 nm emission.</li>
		<li>Wash thoroughly 3 times in culture medium or PBS and submerge the explants in wash medium for 5-10 min to allow the resazurin to completely diffuse out. Add new culture medium or treatments if used.</li>
	</ol>
	</li>
</ol>
3. Termination, fixation, and sample storage
<ol>
	<li>Termination of culturing period
	<ol>
		<li>Measure the metabolic activity as described in step 2.2. Add 200 &#181;L of PBS per well.</li>
	</ol>
	</li>
	<li>Fixation and storage
	<ol>
		<li>Remove the PBS, add 200 &#181;L of formaldehyde per well, and leave for 2 h at room temperature.</li>
		<li>Dispose of the formaldehyde and add 200 &#181;L of PBS per well. Cover the plate with sealing tape, and store at 4 &#176;C for histochemical analysis. We recommend performing histochemical analysis within 3 months.</li>
	</ol>
	</li>
</ol>
4. Tissue turnover biomarkers (ELISA)
<ol>
	<li>Indirect competitive ELISAs
	<ol>
		<li>Coat a streptavidin-plate with the specific biotinylated assay target-peptide diluted 1:100 in assay buffer (100 &#181;L per well) for 30 min at 20 &#176;C.</li>
		<li>Wash 5 times with standard washing buffer and add sample-supernatant (20 &#181;L per well) together with primary monoclonal antibody against the assay target-peptide diluted 1:93.3 for ProC2 and 1:100 in assay buffer for AGNx1 (100 &#181;L per well) and incubate for 2 h at 20 &#176;C with shaking for ProC2 and 3 h at 20 &#176;C for AGNx1. 
		NOTE: The sample volume is directly taken from the stored supernatant plates. If the measured concentration is out of the assay measuring range, dilute the supernatant in a v-bottomed dilution plate in PBS or assay buffer.</li>
		<li>Wash 5 times with standard washing buffer and incubate with peroxidase-labeled secondary antibody diluted 1:100 in assay buffer (100 &#181;L per well) for 1 h at 20 &#176;C.</li>
		<li>Wash 5 times with standard washing buffer and incubate with shaking for 15 min in the dark at 20 &#176;C with tetramethylbenzidine (TMB) as a peroxidase substrate (100 &#181;L per well).</li>
		<li>End the reaction with standard stop solution, 0.1 M H2SO4 (100 &#181;L per well).</li>
		<li>Read the colorimetric reaction at 450 nm absorbance using a reference absorbance at 650 nm on a standard laboratory plate reader.</li>
	</ol>
	</li>
	<li>Direct Competitive ELISAs for measurement of the cartilage tissue turnover in the supernatant 
	NOTE: This quantifies C2M.
	<ol>
		<li>Coat a streptavidin-plate with specific biotinylated assay target-peptide diluted 1:100 in assay buffer (100 &#181;L per well) for 30 min at 20 &#176;C.</li>
		<li>Wash 5 times with washing buffer and add sample-supernatant together with 100 &#181;L of peroxidase-labeled monoclonal antibody against the assay target-peptide diluted 1:100 in assay buffer (20 &#181;L per well). Incubate for 20 h at 2&#8211;8 &#176;C with shaking. 
		NOTE: The sample volume is directly taken from the stored supernatant plates. If the measured concentration is out of the assay measuring range, dilute the supernatant in a v-bottomed dilution plate in PBS or assay buffer.</li>
		<li>Wash 5 times with standard washing buffer and incubate with shaking for 15 min in the dark at 20 &#176;C with TMB as a peroxidase substrate (100 &#181;L per well).</li>
		<li>End the reaction with standard stop solution, 0.1 M H2SO4 (100 &#181;L per well).</li>
		<li>Read the colorimetric reaction at 450 nm absorbance with a reference absorbance at 650 nm on a standard laboratory plate reader.</li>
	</ol>
	</li>
	<li>AGNx1
	<ol>
		<li>Quantify aggrecan degradation by measuring the release of the AGNx1 neo-epitope. This indirect competitive ELISA assay targets the aggrecan C-terminal peptide (NITEGE373) generated by ADAMTS-4 and 5 cleavage. The monoclonal antibody recognizes all fragments with an exposed NITEGE epitope. The experimental details of the assay have been published elsewhere19.</li>
	</ol>
	</li>
	<li>ProC2
	<ol>
		<li>Quantify type II collagen formation by measuring the release of the profragment of type II collagen. This indirect competitive ELISA assay targets the epitope of the PIIBNP propeptide (QDVRQPG) generated by N-propeptidases during trimming of newly synthesized type II collagen. The experimental details of the assay have been published elsewhere16.</li>
	</ol>
	</li>
	<li>C2M
	<ol>
		<li>Quantify type II collagen degradation by measuring the release of the C2M neo-epitope fragment. This direct competitive ELISA recognizes the MMP-cleaved C-terminal peptide (KPPGRDGAAG1053). This assay differs from AGNx1 and ProC2 as it is the primary antibody that is peroxidase-labeled and thus, used as detector. The experimental details of the assay have been published elsewhere20.</li>
	</ol>
	</li>
</ol>
5. Histological analysis
<ol>
	<li>Infiltration, embedding, and cutting
	<ol>
		<li>Place the fixated explants (see step 3.2) into individually labeled cassettes. Include both a label within the cassette and label cassettes to ensure identification.</li>
		<li>Transfer the cassettes containing explants to a tissue processor machine. Then infiltrate the explants with paraffin in a series of dehydration and paraffin infiltration steps.
		<ol>
			<li>Dehydrate with 96% ethanol for 90 min with no temperature adjustment. Repeat this step 3 times.</li>
			<li>Clear the ethanol with toluene for 90 min with no temperature adjustment. Repeat this step 2 times.</li>
			<li>Clear the ethanol with toluene for 90 min at 60 &#176;C.</li>
			<li>Infiltrate with paraffin wax for 30 min at 60 &#176;C.</li>
			<li>Infiltrate with paraffin wax for 60 min at 60 &#176;C.</li>
			<li>Infiltrate with paraffin wax for 90 min at 60 &#176;C.</li>
			<li>For each step, add the solutions into the sample chamber with slow pump-out and pump-in flows under 33&#8211;34 kPa. Run the infiltration process in a pressure/vacuum cycle with a maximum vacuum of &#8722;65 to &#8722;70 kPa.</li>
		</ol>
		</li>
		<li>Following infiltration, place the cassettes on a heating block to allow careful removal of the explants from the cassette. Gently embed the infiltrated explants into individual paraffin blocks. With heated forceps, place the explants with the superficial articular cartilage and subchondral bone sides perpendicular to the cutting surface, ensuring visualization of the different cartilage layers within each specimen section.</li>
		<li>Cut 5 &#181;m sections of cooled paraffin-blocks with embedded explants on a microtome. Transfer the cut sections to a cold-water bath. If necessary, sections can be separated using either a scalpel or a cover glass.</li>
		<li>Using an uncoated glass slide carefully, transfer the sections to a warm water bath (50 &#176;C), where the sections unfold. Lift each section onto a labeled cover slide and place on a hot plate for 30 min.</li>
		<li>Place the slides in a basket and incubate at 60 &#176;C for 1 h and then keep them overnight at 37 &#176;C. Hereafter, store slides in closed containers at 4 &#176;C until staining.</li>
	</ol>
	</li>
	<li>Safranin O/Fast Green staining and visualization
	<ol>
		<li>Place the slides to be stained in a basket and incubate the slides at 60 &#176;C for 1 h.</li>
		<li>Prepare and filter all reagents with a 0.45 mm filter.</li>
		<li>In preparation for staining, pour the filtered reagents in beakers to a volume that allows the solutions to completely cover the slides when submerging the basket. The beakers used required a volume of 250 mL to cover the slides.</li>
		<li>Deparaffinize the melted slides by submerging the basket in toluene for 10 min twice, 99% ethanol for 2 min twice, 96% ethanol for 2 min twice, and 70% ethanol for 2 min twice. Then, hydrate the slides in water for 2 min.</li>
		<li>Stain the deparaffinized and hydrated slides by submerging the basket in Weigert&#8217;s Iron Hematoxylin solution (pH 1.5) for 10 min, dip in 1% HCl once, and rinse with running tap water for approximately 5 min or until excess color has washed away.</li>
		<li>Next, stain in 0.05% Fast Green solution (pH: 5.75) for 5 min, dip in 1% CH3COOH once, and stain in 0.1% Safranin O (pH: 6.5) for 20 min.</li>
		<li>Dehydrate and clear the slides by dipping twice in 70% ethanol, 96% ethanol for 2 min twice, 99% ethanol for 2 min twice, and toluene 2 min twice.</li>
		<li>Mount the uncoated glass slides with resinous medium covering the histology slides.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cope, P. J., Ourradi, K., Li, Y., Sharif, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+osteoarthritis:+the+good,+the+bad+and+the+promising.">Models of osteoarthritis: the good, the bad and the promising.</a> Osteoarthritis and Cartilage. 27 (2), 230-239 (2018).</li><li>Thysen, S., Luyten, F. P., Lories, R. J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targets,+models+and+challenges+in+osteoarthritis+research.">Targets, models and challenges in osteoarthritis research.</a> Disease Models &amp; Mechanisms. 8 (1), 17-30 (2015).</li><li>Reker, D., 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=Articular+cartilage+from+osteoarthritis+patients+shows+extracellular+matrix+remodeling+over+the+course+of+treatment+with+sprifermin+(recombinant+human+fibroblast+growth+factor+18).">Articular cartilage from osteoarthritis patients shows extracellular matrix remodeling over the course of treatment with sprifermin (recombinant human fibroblast growth factor 18).</a> Osteoarthritis and Cartilage. 26, S43 (2018).</li><li>Kjelgaard-Petersen, C., 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=Synovitis+biomarkers:+ex+vivo+characterization+of+three+biomarkers+for+identification+of+inflammatory+osteoarthritis.">Synovitis biomarkers: ex vivo characterization of three biomarkers for identification of inflammatory osteoarthritis.</a> Biomarkers. 20 (8), 547-556 (2015).</li><li>Henriksen, K., 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+specific+subtype+of+osteoclasts+secretes+factors+inducing+nodule+formation+by+osteoblasts.">A specific subtype of osteoclasts secretes factors inducing nodule formation by osteoblasts.</a> Bone. 51 (3), 353-361 (2012).</li><li>Gigout, 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=Sprifermin+(rhFGF18)+enables+proliferation+of+chondrocytes+producing+a+hyaline+cartilage+matrix.">Sprifermin (rhFGF18) enables proliferation of chondrocytes producing a hyaline cartilage matrix.</a> Osteoarthritis and Cartilage. 25 (11), 1858-1867 (2017).</li><li>Reker, D., 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=Sprifermin+(rhFGF18)+modulates+extracellular+matrix+turnover+in+cartilage+explants+ex+vivo.">Sprifermin (rhFGF18) modulates extracellular matrix turnover in cartilage explants ex vivo.</a> Journal of Translational Medicine. 15 (1), 3560 (2017).</li><li>Karsdal, 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=Introduction.">Introduction.</a> Biochemistry of Collagens, Laminins and Elastin. , (2016).</li><li>Heineg&#229;rd, D., Saxne, T. <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+the+cartilage+matrix+in+osteoarthritis.">The role of the cartilage matrix in osteoarthritis.</a> Nature Reviews Rheumatology. 7 (1), 50-56 (2011).</li><li>Karsdal, M. 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=Osteoarthritis&#8211;+a+case+for+personalized+health+care?.">Osteoarthritis&#8211; a case for personalized health care?.</a> Osteoarthritis and Cartilage. 22 (1), 7-16 (2014).</li><li>Karsdal, M. A., Bay-Jensen, A. C., Henriksen, K., Christiansen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathogenesis+of+osteoarthritis+involves+bone,+cartilage+and+synovial+inflammation:+may+estrogen+be+a+magic+bullet?.">The pathogenesis of osteoarthritis involves bone, cartilage and synovial inflammation: may estrogen be a magic bullet?.</a> Menopause International. 18 (4), 139-146 (2012).</li><li>Loeser, R. F., Goldring, S. R., Scanzello, C. R., Goldring, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis:+a+disease+of+the+joint+as+an+organ.">Osteoarthritis: a disease of the joint as an organ.</a> Arthritis and Rheumatism. 64 (6), 1697-1707 (2012).</li><li>Goldring, M. B., Goldring, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritis.">Osteoarthritis.</a> Journal of Cellular Physiology. 213 (3), 626-634 (2007).</li><li>Karsdal, M. 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=The+coupling+of+bone+and+cartilage+turnover+in+osteoarthritis:+opportunities+for+bone+antiresorptives+and+anabolics+as+potential+treatments?.">The coupling of bone and cartilage turnover in osteoarthritis: opportunities for bone antiresorptives and anabolics as potential treatments?.</a> Annals of the Rheumatic Diseases. 73 (2), 336-348 (2014).</li><li>Genovese, F., Karsdal, 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=Protein+degradation+fragments+as+diagnostic+and+prognostic+biomarkers+of+connective+tissue+diseases:+understanding+the+extracellular+matrix+message+and+implication+for+current+and+future+serological+biomarkers.">Protein degradation fragments as diagnostic and prognostic biomarkers of connective tissue diseases: understanding the extracellular matrix message and implication for current and future serological biomarkers.</a> Expert Review of Proteomics. 13 (2), 213-225 (2016).</li><li>Gudmann, N. 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=Cartilage+turnover+reflected+by+metabolic+processing+of+type+II+collagen:+a+novel+marker+of+anabolic+function+in+chondrocytes.">Cartilage turnover reflected by metabolic processing of type II collagen: a novel marker of anabolic function in chondrocytes.</a> International Journal of Molecular Sciences. 15 (10), 18789-18803 (2014).</li><li>Madej, W., van Caam, A., Davidson, E. B., Buma, P., van der Kraan, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unloading+results+in+rapid+loss+of+TGF&#946;+signaling+in+articular+cartilage:+role+of+loading-induced+TGF&#946;+signaling+in+maintenance+of+articular+chondrocyte+phenotype?.">Unloading results in rapid loss of TGF&#946; signaling in articular cartilage: role of loading-induced TGF&#946; signaling in maintenance of articular chondrocyte phenotype?.</a> Osteoarthritis and Cartilage. 24 (10), 1807-1815 (2016).</li><li>Kjelgaard-Petersen, C. F., 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=Translational+biomarkers+and+ex+vivo+models+of+joint+tissues+as+a+tool+for+drug+development+in+rheumatoid+arthritis.">Translational biomarkers and ex vivo models of joint tissues as a tool for drug development in rheumatoid arthritis.</a> Arthritis &amp; Rheumatology. 70 (9), 1419-1428 (2018).</li><li>Wang, B., 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=Suppression+of+MMP+activity+in+bovine+cartilage+explants+cultures+has+little+if+any+effect+on+the+release+of+aggrecanase-derived+aggrecan+fragments.">Suppression of MMP activity in bovine cartilage explants cultures has little if any effect on the release of aggrecanase-derived aggrecan fragments.</a> BMC Research Notes. 2 (4), 259 (2009).</li><li>Bay-Jensen, A. C., 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=Enzyme-linked+immunosorbent+assay+(ELISAs)+for+metalloproteinase+derived+type+II+collagen+neoepitope,+CIIM&#8212;Increased+serum+CIIM+in+subjects+with+severe+radiographic+osteoarthritis.">Enzyme-linked immunosorbent assay (ELISAs) for metalloproteinase derived type II collagen neoepitope, CIIM&#8212;Increased serum CIIM in subjects with severe radiographic osteoarthritis.</a> Clinical Biochemistry. 44 (5-6), 423-429 (2011).</li><li>Lories, R. J., Luyten, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bone-cartilage+unit+in+osteoarthritis.">The bone-cartilage unit in osteoarthritis.</a> Nature Reviews Rheumatology. 7 (1), 43-49 (2011).</li></ol>

CST, ACBJ and MK are employees of Nordic Bioscience. ACBJ and MK holds shares in Nordic Bioscience. The remaining authors have nothing to disclose.

The protocol presented here for the profiling of cartilage tissue turnover in bovine cartilage explants can be used for characterizing treatment effects of many types of drugs, including inhibitors of inflammatory intracellular pathways, inhibitors of proteolytic enzymes, or anabolic growth factors.
Two different setups were described in this protocol: an anabolic setup where explants were stimulated with insulin-like growth factor 1 (IGF-1), and a catabolic setup comprising stimulation with T...

Chondrocytes produce and maintain the cartilage matrix. In order to study the biology of chondrocytes and how they and the surrounding ECM react to external stimuli, it is crucial to interrogate them in their native environment1,2. Studying cartilage tissue turnover is important to augment the understanding of the underlying mechanisms in joint diseases such as OA, a disease for which there is currently no disease modifying treatment. Consequently, there is a significant need for better translation models2.
Ex vivo characterization of cell and tissue effects is essential to complement other preclinical models, both in vitro, such as chondrocyte monolayer cultures, and in vivo, such as surgery-induced OA models or the autoimmune collagen-induced arthritis model (CIA). Numerous studies have highlighted the differences between how cells behave in 2D monolayer cultures and 3D structures or in their native tissue3,4. Many cells in 2D layers adopt unnatural morphologies, including differences in cell polarity and tissue attachment, resulting in both visual and functional differences in cells within native tissues5. The differences are also apparent in the functionality of the cells, which may shift protein expression, leading to profoundly altered differentiation patterns, regulatory machinery, and cell functionality5,6,7,8.
The cartilage ECM consists mainly of type II collagen providing a matrix framework, and aggrecan, a proteoglycan that helps retain fluid within the tissue. Other matrix molecules such as collagen type IV, VI, IX, X, XI, XII, fibronectin, cartilage oligomeric protein (COMP), biglycan, decorin, and perlecan are also present9.
While the aetiology of OA remains unclear10,11, the onset of the disease is believed to be caused by imbalances in tissue turnover and repair processes12,13. The degradation of the articular cartilage is a hallmark of OA. Cartilage-resident chondrocytes or cells in the surrounding tissues increase their release of cytokines, stimulating elevated production of proteinases such as matrix metalloproteinases (MMPs) and aggrecanases, which increase degradation of cartilage ECM14. This degradation results in the release of small unique protein fragments called neo-epitopes, which can be quantified in serum, urine, or culture medium15. Upon formation and maturation of collagen, so-called profragments are also released; these can be quantified as a measure of matrix production16.
The aim of this protocol is to establish an ex vivo cartilage model to compare the effect of stimulation and/or drug treatment on ECM tissue turnover. Cartilage turnover is profiled by measuring matrix-derived neo-epitope biomarkers directly in the conditioned culture medium using ELISA: AGNx1 (reflecting aggrecanase activity), C2M (reflecting matrix MMP activity), and ProC2 (reflecting type II collagen formation). The findings can be verified by histological staining of the ECM, which also visualizes the organization of chondrocytes in the individual explants. The described protocol can be used to test the effect of novel treatments on chondrocyte function and cartilage ECM turnover. A number of studies have used cartilage explants to describe biological processes or the effect of intervention on cytokine-challenged explants using quantitative histological or immunohistochemical approaches, mRNA, protein expression, or proteomics2,17,18. However, these protocols are outside the scope of the current manuscript.

<table>
	<tbody>
		<tr>
			<td>45% Iron(III) chloride solution</td>
			<td>Sigma-Aldrich</td>
			<td>12322</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>1.00056.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Alamar Blue</td>
			<td>Life tech Invitrogen</td>
			<td>DAL1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy processing cassettes &#8211; green</td>
			<td>IHCWORLD</td>
			<td>BC-0109G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch W/Plunger (3 mm)</td>
			<td>Scandidat</td>
			<td>MTP-33-32</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine cartilage (Bovine knees)</td>
			<td>Local slaughterhouse</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C2M</td>
			<td>Nordic Bioscience</td>
			<td></td>
			<td>Fee for service</td>
		</tr>
		<tr>
			<td>Corning 96-well plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS7007</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass &#216; 13 mm</td>
			<td>VWR</td>
			<td>631-0150P</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12-GlutaMAX Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12) without HEPES</td>
			<td>Gibco</td>
			<td>31331-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol &#8805;96%</td>
			<td>VWR</td>
			<td>83804.36</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute &#8805;99.5%</td>
			<td>VWR</td>
			<td>83813.36</td>
			<td></td>
		</tr>
		<tr>
			<td>exAGNx1</td>
			<td>Nordic Bioscience</td>
			<td></td>
			<td>Fee for service</td>
		</tr>
		<tr>
			<td>exPRO-C2</td>
			<td>Nordic Bioscience</td>
			<td></td>
			<td>Fee for service</td>
		</tr>
		<tr>
			<td>Fast green</td>
			<td>Sigma-Aldrich</td>
			<td>F7252</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution 4%</td>
			<td>Merck</td>
			<td>1004965000</td>
			<td></td>
		</tr>
		<tr>
			<td>GM6001</td>
			<td>Sigma-Aldrich</td>
			<td>M5939-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>H3136</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Merck</td>
			<td>30721-M</td>
			<td></td>
		</tr>
		<tr>
			<td>IGF-1</td>
			<td>Sigma-Aldrich</td>
			<td>I3769-50UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Oncostatin M</td>
			<td>Sigma-Aldrich</td>
			<td>O9635-10UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin (P/S)</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Pertex (mounting medium for light microscopy)</td>
			<td>HistoLab</td>
			<td>811</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>Safranin O</td>
			<td>Sigma-Aldrich</td>
			<td>S2255</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Standard Scalpels</td>
			<td>Integra Miltex</td>
			<td>12-460-451</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfuric acid</td>
			<td>Sigma-Aldrich</td>
			<td>30743</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERFROST PLUS Adhesion Microscope Slides</td>
			<td>Thermo scientific</td>
			<td>J1800AMNT</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-alpha</td>
			<td>R&#38;D Systems</td>
			<td>210-TA-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>Merck</td>
			<td>1.08327.2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Filtration &#34;rapid&#34;-Filtermax</td>
			<td>TPP</td>
			<td>99955</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Bovine full-depth explants were isolated, cultured, and treated for 3 weeks (Figure 1). The culture medium was changed with the addition of treatment 3 times per week. Once weekly, metabolic activity was measured by the resazurin assay. Biomarkers of ECM turnover were measured in the supernatant harvested from the culture plate 3 times per week. Explants were divided into 4 groups for treatment: 1) Oncostatin M and TNF&#945; (O+T); 2) O+T + GM6001 (GM6001); 3) Insulin like Growth Factor-1 (I...

Watch this Scientific Journal Video about An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants at JoVE.com

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

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 ...

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Research

JoVE Journal

Biology

9.2K Views.  Nordic Bioscience. 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.

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

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

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, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...

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.

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 ...