The authors are most grateful to the medical and surgical retina colleagues, nurses and whole staff of the Diabetic Unit and Vitreoretinal Surgery Unit at the Department of Ophthalmology, Helsinki University Hospital for actively participating in the recruitment of patients. We thank Biomedicum Molecular Imaging Unit for imaging facilities. We thank Anastasiya Chernenko for excellent technical assistance. This study was supported by grants from the Academy of Finland (KL), University of Helsinki (KL), Sigrid Juselius Foundation (KL), K. Albin Johansson Foundation (KL), Finnish Cancer Institute (KL), Karolinska Institutet (KL), Finnish Eye Foundation (SL), Eye and Tissue Bank Foundation (SL), Mary and Georg C. Ehrnrooth Foundation (SL), and HUCH Clinical Research Grants (TYH2018127 after TYH2016230, SL), Diabetes Research Foundation (SL, KL, AK, EG) as well as the Doctoral Programme in Biomedicine (EG).

This research was approved by the Institutional Review Board and Ethical committee of Helsinki University Hospital. Signed informed consent was obtained from each patient.
1. Preparation of Solutions, Media and Equipment
<ol>
	<li>Prepare the following equipment prior to collection of the fibrovascular tissue (FT) to ensure rapid processing.
	<ol>
		<li>Sterile-autoclave two microdissection tweezers.</li>
		<li>Prepare 1x phosphate-buffered saline (PBS) by dissolving 1 pre-weighed PBS tablet...

<ol>
	<li>Cho, N. 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=IDF+Diabetes+Atlas:+Global+estimates+of+diabetes+prevalence+for+2017+and+projections+for+2045.">IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045.</a> Diabetes Research and Clinical Practice. 138, 271-281 (2018).</li><li>Liew, G., Wong, V. W., Ho, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mini+Review:+Changes+in+the+Incidence+of+and+Progression+to+Proliferative+and+Sight-Threatening+Diabetic+Retinopathy+Over+the+Last+30+Years.">Mini Review: Changes in the Incidence of and Progression to Proliferative and Sight-Threatening Diabetic Retinopathy Over the Last 30 Years.</a> Ophthalmic Epidemiology. 24 (2), 73-80 (2017).</li><li>Loukovaara, 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=Indications+of+lymphatic+endothelial+differentiation+and+endothelial+progenitor+cell+activation+in+the+pathology+of+proliferative+diabetic+retinopathy.">Indications of lymphatic endothelial differentiation and endothelial progenitor cell activation in the pathology of proliferative diabetic retinopathy.</a> Acta Ophthalmologica. 93 (6), 512-523 (2015).</li><li>Stitt, A. W., 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+progress+in+understanding+and+treatment+of+diabetic+retinopathy.">The progress in understanding and treatment of diabetic retinopathy.</a> Progress in Retinal and Eye Research. 51, 156-186 (2016).</li><li>Duh, E. J., Sun, J. K., Stitt, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+retinopathy:+current+understanding,+mechanisms,+and+treatment+strategies.">Diabetic retinopathy: current understanding, mechanisms, and treatment strategies.</a> JCI Insight. 2 (14), (2017).</li><li>Gross, J. G., 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=Five-Year+Outcomes+of+Panretinal+Photocoagulation+vs+Intravitreous+Ranibizumab+for+Proliferative+Diabetic+Retinopathy:+A+Randomized+Clinical+Trial.">Five-Year Outcomes of Panretinal Photocoagulation vs Intravitreous Ranibizumab for Proliferative Diabetic Retinopathy: A Randomized Clinical Trial.</a> JAMA Ophthalmology. 136 (10), 1138-1148 (2018).</li><li>Robinson, R., Barathi, V. A., Chaurasia, S. S., Wong, T. Y., Kern, T. 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S., Montesano, R., Mandriota, S. J., Orci, L., Vassalli, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis:+a+paradigm+for+balanced+extracellular+proteolysis+during+cell+migration+and+morphogenesis.">Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis.</a> Enzyme, Protein. 49 (1-3), 138-162 (1996).</li><li>Lafleur, M. A., Handsley, M. M., Knauper, V., Murphy, G., Edwards, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+tubulogenesis+within+fibrin+gels+specifically+requires+the+activity+of+membrane-type-matrix+metalloproteinases+(MT-MMPs).">Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT-MMPs).</a> Journal of Cell Science. 115 (Pt 17), 3427-3438 (2002).</li><li>Loukovaara, 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=Quantitative+Proteomics+Analysis+of+Vitreous+Humor+from+Diabetic+Retinopathy+Patients.">Quantitative Proteomics Analysis of Vitreous Humor from Diabetic Retinopathy Patients.</a> Journal of Proteome Research. 14 (12), 5131-5143 (2015).</li><li>Abu El-Asrar, A. M., Struyf, S., Opdenakker, G., Van Damme, J., Geboes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+stem+cell+factor/c-kit+signaling+pathway+components+in+diabetic+fibrovascular+epiretinal+membranes.">Expression of stem cell factor/c-kit signaling pathway components in diabetic fibrovascular epiretinal membranes.</a> Molecular Vision. 16, 1098-1107 (2010).</li><li>Loukovaara, 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=Ang-2+upregulation+correlates+with+increased+levels+of+MMP-9,+VEGF,+EPO+and+TGFbeta1+in+diabetic+eyes+undergoing+vitrectomy.">Ang-2 upregulation correlates with increased levels of MMP-9, VEGF, EPO and TGFbeta1 in diabetic eyes undergoing vitrectomy.</a> Acta Ophthalmologica. 91 (6), 531-539 (2013).</li><li>Sugiyama, N., 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=EphA2+cleavage+by+MT1-MMP+triggers+single+cancer+cell+invasion+via+homotypic+cell+repulsion.">EphA2 cleavage by MT1-MMP triggers single cancer cell invasion via homotypic cell repulsion.</a> Journal of Cell Biology. 201 (3), 467-484 (2013).</li><li>Tatti, O., 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=MMP16+Mediates+a+Proteolytic+Switch+to+Promote+Cell-Cell+Adhesion,+Collagen+Alignment,+and+Lymphatic+Invasion+in+Melanoma.">MMP16 Mediates a Proteolytic Switch to Promote Cell-Cell Adhesion, Collagen Alignment, and Lymphatic Invasion in Melanoma.</a> Cancer Research. 75 (10), 2083-2094 (2015).</li><li>Zudaire, E., Gambardella, L., Kurcz, C., Vermeren, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computational+tool+for+quantitative+analysis+of+vascular+networks.">A computational tool for quantitative analysis of vascular networks.</a> PLoS One. 6 (11), e27385 (2011).</li><li>Senger, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+framework+for+angiogenesis:+a+complex+web+of+interactions+between+extravasated+plasma+proteins+and+endothelial+cell+proteins+induced+by+angiogenic+cytokines.">Molecular framework for angiogenesis: a complex web of interactions between extravasated plasma proteins and endothelial cell proteins induced by angiogenic cytokines.</a> American Journal of Pathology. 149 (1), 1-7 (1996).</li><li>Senger, D. R., Davis, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis.">Angiogenesis.</a> Cold Spring Harbor Perspectives in Biology. 3 (8), a005090 (2011).</li><li>Bishop, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+macromolecules+and+supramolecular+organisation+of+the+vitreous+gel.">Structural macromolecules and supramolecular organisation of the vitreous gel.</a> Progress in Retinal and Eye Research. 19 (3), 323-344 (2000).</li><li>Marmorstein, A. D., Marmorstein, L. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+modeling+macular+degeneration+in+mice.">The challenge of modeling macular degeneration in mice.</a> Trends in Genetics. 23 (5), 225-231 (2007).</li><li>Kim, L. 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=Characterization+of+cells+from+patient-derived+fibrovascular+membranes+in+proliferative+diabetic+retinopathy.">Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy.</a> Molecular Vision. 21, 673-687 (2015).</li><li>Avery, R. L., 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=Intravitreal+bevacizumab+(Avastin)+in+the+treatment+of+proliferative+diabetic+retinopathy.">Intravitreal bevacizumab (Avastin) in the treatment of proliferative diabetic retinopathy.</a> Ophthalmology. 113 (10), e1691-e1615 (2006).</li><li>Zhao, L. Q., Zhu, H., Zhao, P. Q., Hu, Y. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systematic+review+and+meta-analysis+of+clinical+outcomes+of+vitrectomy+with+or+without+intravitreal+bevacizumab+pretreatment+for+severe+diabetic+retinopathy.">A systematic review and meta-analysis of clinical outcomes of vitrectomy with or without intravitreal bevacizumab pretreatment for severe diabetic retinopathy.</a> The British Journal of Ophthalmology. 95 (9), 1216-1222 (2011).</li><li>Carrion, B., Janson, I. A., Kong, Y. P., Putnam, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+safe+and+efficient+method+to+retrieve+mesenchymal+stem+cells+from+three-dimensional+fibrin+gels.">A safe and efficient method to retrieve mesenchymal stem cells from three-dimensional fibrin gels.</a> Tissue Engineering. 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The authors have nothing to disclose.

Considering the importance of relevant tissue microenvironment for reliable functional cell and molecular mechanistic results, it is imperative to find appropriate experimental models that provide this tissue environment. The herein described ex vivo PDR culture model for the fibrin-embedded FTs allows the investigation of the mechanisms of PDR pathophysiology in the native, complex and multicellular context of the PDR clinical samples.
Critical steps within the protocol are the prope...

DR is a serious ocular complication of diabetes, a disease that has reached enormous proportions in the last three decades1. Twenty years after diagnosis, virtually every patient with type 1 diabetes and 60% of patients with type 2 diabetes present signs of retinopathy, making diabetes per se one of the leading causes of blindness in working age adults2. According to the level of microvascular degeneration and ischemic damage, DR is classified into non-proliferative DR (non-PDR) and proliferative DR (PDR). The end-stage disease, PDR, is characterized by ischemia- and inflammation-induced neovascularization and fibrotic responses at the vitreoretinal interface. In untreated conditions, these processes will lead to blindness due to vitreous hemorrhage, retinal fibrosis, tractional retinal detachment, and neovascular glaucoma3,4. Despite recent advances, current treatment options target only DR stages, including diabetic macular edema and PDR, when retinal damage has already ensued. Moreover, a great proportion of DR patients does not benefit from current treatment armamentarium, indicating an urgent need for improved therapies4,5,6.
Multiple other in vivo disease/developmental models and diabetic animal models have been developed to date, but none of them recapitulates the full range of pathologic features observed in human PDR7,8. Moreover, increasing evidence indicates that treatment responses are tightly connected to the ECM composition as well as the spatial arrangement and interaction between the cellular and acellular microenvironment9. We, therefore, set out to develop a clinically relevant model of human PDR by utilizing the FT pathological material that is commonly excised from eyes undergoing vitrectomy as part of the surgical management of PDR10.
This manuscript describes the protocol for the 3D ex vivo culture and characterization of the surgically-excised, PDR patient-derived pathological FT. The method described here has been used in a recent publication that demonstrated successful deconstruction of the native 3D PDR tissue landscape, and recapitulation of features of PDR pathophysiology including angiogenic and fibrotic responses of the abnormal vascular structures11. This model also revealed novel features that cannot be easily appreciated from thin histological sections, such as spatially confined apoptosis and proliferation as well as vascular islet formation11. Vitreous fluid has been successfully used by others on 3D endothelial spheroid cultures to evaluate its angiogenic potential and the efficacy of angiostatic molecules12. When combined with an in vitro 3D lymphatic endothelial cell (LEC) spheroid sprouting assay using PDR vitreous as stimulant, our model revealed the contribution of both soluble vitreal factors as well as local cues within the neovascular tissue to the as yet poorly understood LEC involvement in PDR pathophysiology3,11. In the management of PDR, vitreoretinal surgery is a routinely performed yet challenging procedure. As surgical instrumentations and techniques are seeing continuous advancement and sophistication, timely and conservative removal of fibrovascular proliferative specimen not only improves vision outcome but also provides invaluable tissue material for the investigation of PDR pathophysiology and treatment responses in the complex translational aspects of the live human tissue microenvironment.

<table>
	<tbody>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microforceps</td>
			<td>Medicon</td>
			<td>07.60.03</td>
			<td>Used for handling the FTs</td>
		</tr>
		<tr>
			<td>Disposable Scalpels - Sterile</td>
			<td>Swann-Morton</td>
			<td>0513</td>
			<td>Used for FT dissection</td>
		</tr>
		<tr>
			<td>Culture dish, vented, 28 ml (60mm)</td>
			<td>Greiner Bio-One</td>
			<td>391-3210</td>
			<td>Used for dissection and for testing fibrin gel formation</td>
		</tr>
		<tr>
			<td>Cell culture plates, 12-well</td>
			<td>Greiner Bio-One</td>
			<td>392-0049</td>
			<td>Used for FT dissection and whole-mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Reagent/centrifuge tube with screw cap, 15 mL</td>
			<td>Greiner Bio-One</td>
			<td>391-3477</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent/centrifuge tube with screw cap, 50 mL</td>
			<td>Greiner Bio-One</td>
			<td>525-0384</td>
			<td></td>
		</tr>
		<tr>
			<td>Millex-GV Syringe Filter Unit, 0.22&#160;&#181;m, PVDF</td>
			<td>Millipore</td>
			<td>SLGV033RS</td>
			<td>Used to sterile-filter the fibrinogen solution</td>
		</tr>
		<tr>
			<td>Syringe, 10 mL</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td>Used to sterile-filter the fibrinogen solution</td>
		</tr>
		<tr>
			<td>Polypropylene Microcentrifuge Tubes, 1.5 mL</td>
			<td>Fisher</td>
			<td>FB74031</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Culture Treated Multidishes, 24-well</td>
			<td>Nunc</td>
			<td>142475</td>
			<td>Used for casting the FT/fibrin gels for native FT characterization and ex vivo culture</td>
		</tr>
		<tr>
			<td>Cell culture plates, 96-well, U-bottom</td>
			<td>Greiner Bio-One</td>
			<td>392-0019</td>
			<td>Used for whole-mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Round/Flat Spatulas, Stainless Steel</td>
			<td>VWR</td>
			<td>82027-528</td>
			<td>Used for whole-mount immunofluorescence</td>
		</tr>
		<tr>
			<td>Coverslips 22x22mm #1</td>
			<td>Menzel/Fisher</td>
			<td>15727582</td>
			<td>Used for mounting</td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher</td>
			<td>Kindler K102</td>
			<td>Used for mounting</td>
		</tr>
		<tr>
			<td>Absorbent paper</td>
			<td>VWR</td>
			<td>115-0202</td>
			<td>Used for mounting</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS tablets</td>
			<td>Medicago</td>
			<td>09-9400-100</td>
			<td>Used for preparing 1x PBS</td>
		</tr>
		<tr>
			<td>Fibrinogen, Plasminogen-Depleted, Human Plasma</td>
			<td>Calbiochem</td>
			<td>341578</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution</td>
			<td>Sigma-Aldrich</td>
			<td>H9394-500ML</td>
			<td>Used for preparing the fibrinogen and TA solution</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10270106</td>
			<td>Used for preparing the blocking solution</td>
		</tr>
		<tr>
			<td>Human Serum</td>
			<td>Sigma-Aldrich</td>
			<td>H4522</td>
			<td>Aliquoted in -20 &#176;C, thaw before preparing the ex vivo culture media</td>
		</tr>
		<tr>
			<td>Gentamicin Sulfate 10mg/ml</td>
			<td>Biowest</td>
			<td>L0011-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial cell media MV Kit</td>
			<td>Promocell</td>
			<td>C-22120</td>
			<td>Contains 500 ml of Endothelial Cell Growth Medium MV, 25 mL of fetal calf serum, 2 mL of endothelial cell growth supplement,&#160; 500 &#956;L of recombinant human epidermal growth factor (10 &#956;g/ mL) and 500 &#956;L of hydrocortisone (1 g/ mL)</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td>Used for storage of the native and ex vivo cultured FTs. TOXIC: wear protective gloves and/or clothing, and eye and/or face protection. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>32201-2.5L-M</td>
			<td>Used to prepare the post-fixation solution. HARMFUL: wear protective gloves and/or clothing. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>32213</td>
			<td>Used to prepare the post-fixation solution. TOXIC: wear protective gloves and/or clothing. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Triton X-100 (octyl phenol ethoxylate)</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td>Used for whole-mount immunofluorescence. HARMFUL: wear protective gloves and/or clothing.</td>
		</tr>
		<tr>
			<td>Hoechst 33342, 20mM</td>
			<td>Life Technologies</td>
			<td>62249</td>
			<td>For nuclei counterstaining. HARMFUL: wear protective gloves and/or clothing, and eye and/or face protection.</td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Wear protective gloves and/or clothing, and eye protection. Use in fume hood.</td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Mounting medium with nuclei counterstaining. Wear protective gloves and/or clothing, and eye protection. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Eukitt Quick-hardening mounting medium</td>
			<td>Sigma-Aldrich</td>
			<td>03989-100ml</td>
			<td>TOXIC: Wear protective gloves and/or clothing, and eye protection. Use in fume hood.</td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma, lyophilized powder</td>
			<td>Sigma-Aldrich</td>
			<td>T9549-500UN&#160;</td>
			<td>Dissolve at 100 units/ mL, aliquote and store at -20 &#176;C, avoid repeated freeze/ thaw</td>
		</tr>
		<tr>
			<td>Aprotinin from bovine lung, lyophilized powder</td>
			<td>Sigma</td>
			<td>A3428</td>
			<td>Dissolve at 50 mg/ mL, aliquote and store at -20 &#176;C, avoid repeated freeze/ thaw</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Growth factors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human VEGFA</td>
			<td>R&#38;D Systems</td>
			<td>293-VE-010</td>
			<td>50 ng/ mL final concentration</td>
		</tr>
		<tr>
			<td>Recombinant human VEGFC</td>
			<td>R&#38;D Systems</td>
			<td>752-VC-025</td>
			<td>200 ng/ mL final concentration</td>
		</tr>
		<tr>
			<td>Recombinant human TGF&#946;</td>
			<td>Millipore</td>
			<td>GF346</td>
			<td>1 ng/ mL final concentration</td>
		</tr>
		<tr>
			<td>Recombinant human bFGF</td>
			<td>Millipore</td>
			<td>01-106</td>
			<td>50 ng/ mL final concentration</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 (JC70A)</td>
			<td>Dako</td>
			<td>M0823</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>CD34 (QBEND10)</td>
			<td>Dako</td>
			<td>M716501-2</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>CD45 (2B11+PD7/26)</td>
			<td>Dako</td>
			<td>M070129-2</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>CD68</td>
			<td>ImmunoWay</td>
			<td>RLM3161</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>Cleaved caspase-3 (5A1E)</td>
			<td>Cell Signalling</td>
			<td>9664</td>
			<td>Used at 1:200 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>ERG (EP111)</td>
			<td>Dako</td>
			<td>M731429-2</td>
			<td>Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Dako</td>
			<td>Z0334</td>
			<td>Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>Ki67</td>
			<td>Leica Microsystems</td>
			<td>NCL-Ki67p</td>
			<td>Used at 1:1500 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>Lyve1</td>
			<td>R&#38;D Systems</td>
			<td>AF2089</td>
			<td>Used at 1:100 dilution, Donkey anti Goat Alexa 568 Secondary Ab</td>
		</tr>
		<tr>
			<td>NG2</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td>Used at 1:100 dilution, Goat anti Rabbit Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>Prox1</td>
			<td>ReliaTech</td>
			<td>102-PA32</td>
			<td>Used at 1:200 dilution, Goat anti Rabbit Alexa 568 Secondary Ab</td>
		</tr>
		<tr>
			<td>Prox1</td>
			<td>R&#38;D Systems</td>
			<td>AF2727</td>
			<td>Used at 1:40 dilution, Chicken anti Goat Alexa 594 Secondary Ab</td>
		</tr>
		<tr>
			<td>VEGFR3 (9D9F9)</td>
			<td>Millipore</td>
			<td>MAB3757</td>
			<td>Used at 1:100 dilution, Donkey anti Mouse Alexa 488 Secondary Ab</td>
		</tr>
		<tr>
			<td>&#945;-SMA (1A4)</td>
			<td>Sigma</td>
			<td>C6198</td>
			<td>Used at 1:400 dilution, Cy3 conjugated</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor488 Donkey Anti-Mouse IgG</td>
			<td>Life Technologies</td>
			<td>A-21202</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor594 Goat Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-11012</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor568 Donkey anti-Goat IgG</td>
			<td>Thermo Scientific</td>
			<td>A-11057</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor568 Goat anti-Rabbit IgG</td>
			<td>Thermo Scientific</td>
			<td>A-11036</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor594 Chicken Anti-Goat IgG</td>
			<td>Molecular Probes</td>
			<td>A-21468</td>
			<td>Used at 1:500 dilution</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Microscopes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Axiovert 200 inverted epifluorescence microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>For imaging of the fresh and fibrin-embedded FT</td>
		</tr>
		<tr>
			<td>SZX9 upright dissection stereomicroscope</td>
			<td>Olympus</td>
			<td></td>
			<td>For FT dissection</td>
		</tr>
		<tr>
			<td>LSM 780 confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>For imaging of whole-mount immunostained FT</td>
		</tr>
		<tr>
			<td>AxioImager.Z1 upright epifluorescence microscope with Apotome</td>
			<td>Zeiss</td>
			<td></td>
			<td>For imaging of whole-mount immunostained FT</td>
		</tr>
	</tbody>
</table>

an ex vivo tissue culture model for fibrovascular complications in proliferative diabetic retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Deeper understanding of the PDR fibrovascular tissue properties and protein expression has relied mainly on vitreous samples and thin histological FT sections3,15,16,17. To develop a method for thorough investigation of the 3D tissue organization and multicellular physiopathological processes of PDR, we set out to utilize the surgically excised, patient-derived ...

Watch this Scientific Journal Video about An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy at JoVE.com

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...

Diabetic retinopathy is the most common microvascular complication of diabetes.This method can help answer key questions on the pathophysiology of the end stage disease proliferative diabetic retinopathy.This technique allows the deconstruction of the native three dimensional tissue landscape and the investigation of tissue pathophysiology in live patient material with direct clinical relevance.Refer to the text protocol for details on the surgery and the instrumentation utilized in the dissection.To begin, place fibrovascular tissue that has been excised from human subjects undergoing transconjunctival microincision vitreoretinal surgery in a cell culture dish containing sterile PBS.Using an upright dissection stereo microscope, cut the fibrovascular tissue into approximately one square millimeter pieces.Submerge the tissue in PBS and hold the tissue in place with micro dissection tweezers.Make clear cuts in the tissue with a sterile scalpel taking care to avoid tearing the fibrovascular tissue.Place each individual piece of tissue into a well of a 12 well cell culture plate containing one milliliter of sterile PBS.First, prepare 25 microliter aliquots of fibrinogen solution at room temperature.Place one piece of fibrovascular tissue at the center of the well of a 24 well plate and remove any excess PBS.Next, add 25 microliters of TA solution to one of the prepared fibrinogen aliquots.And using another pipette set to 50 microliters mix by pipetting.Dispense the mixture onto the piece of fibrovascular tissue and pipette up and down to insure the tissue piece is contained within the droplet.The time of fibrinogen formation critical for the three dimensionality of the ex vivo culture, is tested in an empty droplet prior tissue embedding as described in the protocol.Incubate the plate in a cell culture incubator.After 30 to 60 minutes tilt the plate to check that the fibrin gel is completely formed.Next, overlay the fibrovascular tissue fibrin gels with ex vivo culture medium supplemented with aprotinin.After this, return the culture plates to the cell culture incubator for the desired time period.First, replace the ex vivo culture medium with one milliliter of paraformaldehyde solution per well to fix the cultures.After one hour, rinse the gels with three five-minute PBS washes.Next, replace the PBS wash with two milliliters of sodium azide solution per well.Store the fixed gels at four degrees Celsius.Use a stainless steel squared edge spatula to lift the droplets from the plate carefully, starting at the edges before lifting the center of the droplet.Using a round edged spatula transfer the droplets to individual wells of a 12 well plate containing one milliliter of PBS.After this, tilt the plate on a support and postfix the droplets with one milliliter of ice-cold acetone methanol solution for one minute.Then, rinse with three to five two milliliter washes of PBS.Centrifuge the blocking solution for 15 minutes to remove any debris.Then, tilt the plate on a support and incubate the droplets in 500 microliters of blocking solution for two hours at room temperature.Prepare the primary antibody mixture according to the text protocol.Then use a round edged spatula to transfer the droplets to a round bottom 96 well plate.Incubate the droplets in 30 microliters of primary antibody mixture per droplet overnight at four degrees celsius.The next day, transfer the droplets to a 12 well plate containing two milliliters of washing solution per well.Rinse the droplets with three five-minute washes.Then, wash the droplets with nine 30-minute washes.The following day, rinse the droplets three times with PBS.Prepare the appropriate fluorophore-conjugated secondary antibody mixture according to the text protocol.Transfer the droplets to a round bottom 96 well plate, as previously demonstrated, and incubate the plate with 30 microliters of secondary antibody mixture for four hours at room temperature, protected from light.After four hours, transfer the droplets to a 12 well plate containing two milliliters of washing solution per well.First, rinse the droplets with three five-minute washes.Then, wash the droplets with four 30-minute washes.The next day, rinse the droplets five times with washing solution for 30 minutes, and three times with PBS for 30 minutes.The next day, transfer the droplets to a round bottom 96 well plate, as previously demonstrated.Counterstain the droplets with Hoechst nuclear stain for 30 minutes at room temperature.Transfer the droplets to a 12 well plate containing two milliliters of PBS per well and rinse three times with PBS.Next, apply a narrow layer of quick hardening mounting medium along the edges of a square cover glass and allow it to dry for one minute.Then, rinse the droplet by dipping it into a well of a 12 well plate containing deionized water.And transfer the droplet to a microscope slide.Next, dispense 15 microliters of non-hardening anti-fade mounting medium onto the droplet.Gently position the cover glass over the droplet with the mounting medium facing the slide and let it settle.Let the slides dry for two hours at room temperature and store them at four degrees Celsius overnight.Finally, image the slides with an upright epifluorescence microscope equipped with an optical sectioning function at 20x or 40x objective.In this protocol fibrovascular tissue was prepared and imaged.The representative data showed that PDR ex vivo cultures induce the sprouting of CD-31 positive endothelium and vasculature preservation in response to VEGFA.Conversely, TGF

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7.6K Aufrufe.  University of Helsinki. Diabetische Retinopathie ist die häufigste mikrovaskuläre Komplikation von Diabetes. Diese Methode kann helfen, schlüsselweisen Fragen zur Pathophysiologie der proliferativen diabetischen Retinopathie im Endstadium zu beantworten. Diese Technik ermöglicht die Dekonstruktion der einheimischen dreidimensionalen Gewebelandschaft und die Untersuchung der Gewebepathophysiologie in lebendem Patientenmaterial mit direkter klinischer Relevanz. Im Textprotokoll finden Sie Details zur Operation und...