Culture medium added with 20% FBS was filled into the bottom of chambers. The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Different techniques e.g. Zufferey, R. et al. As shown in Figure 2A, a scratch wound was made in HUVEC cells which was followed by safranal treatment with rhVEGF induction. Moreover, a positive staining for the active form of caspase 3 was detected within the lumenunderlining the probability of apoptotic processes during lumen formation in the EC network (Fig. Capillary-like tubes were formed within 8 h and recorded with the video . Currently a wide range of commercially available angiogenesis assays employ cells seeded on Matrigel or other basal membrane extract . However, recombinant human collagens have been already used in tissue engineering of corneal substitutes14,15, skin tissue engineering16,17, bone graft substitutes, dentistry, and cartilage reconstruction17. & Van Praagh, R. Morphometry of human coronary capillaries during normal growth and the effect of age in left ventricular pressure-overload hypertrophy. Substitution of Matrigel/rCOL by hCOL from fibroblasts leads to lumenized EC network formation with involvement of apoptosis. Slider with three articles shown per slide. Disclaimer. However, in our previous research we could demonstrate that growth factors present in the Matrigel are dispensable for endothelial cell network formation when they are cultivated in medium containing a complex mixture of growth factors11. A.H. supervised the imaging process. In this study, we found for the first time that sodium dithionite (SD) could induce endothelial cell tube formation without Matrigel under hypoxia condition. Production of recombinant collagen: state of the art and challenges. disruption, cell migration, cell proliferation, and tube formation, can be a target forintervention, and each can be tested in vitro and in vivo. Tube formation assay. Plant-derived human collagen scaffolds for skin tissue engineering. Stoker, M. E., Gerdes, A. M. & May, J. F. Regional differences in capillary density and myocyte size in the normal human heart. 1b,e,h,k) leading to a complete loss of ECs till the end of the experiment (data not shown). Matrigel (BD Biosciences) was added to 96-well plates (60 l/well) for polymerization by incubating for 30 min at 37C. Ibragimov A, Senotrusova S, Markova K, Karpulevich E, Ivanov A, Tyshchuk E, Grebenkina P, Stepanova O, Sirotskaya A, Kovaleva A, Oshkolova A, Zementova M, Konstantinova V, Kogan I, Selkov S, Sokolov D. Int J Mol Sci. Recombinant human collagen for tissue engineered corneal substitutes. -SMA positive cells are in physical contact with GFP-HUVECs after cultivation in SFM (h) or EGM-2 (d). In all constructs incorporation of dextran was visible (Fig. 1995;74:235-48. doi: 10.1007/978-3-0348-9070-0_12. Acceleration of Wound Healing through Amorphous Calcium Carbonate, Stabilized with High-Energy Polyphosphate. EGM-2: endothelial growth medium-2, SFM: serum-free medium, Scale bar: (ac,eg) 200m, (d,h) 30m. After centrifugation, the cell pellet was washed with PBS, resuspended and cells were cultivated in EGM-2. Constructs cultivated in SFM exhibited EC network formation from day 23 onwards resulting in a stable, filigree network of GFP-HUVECs. Tube-formation assay. Thank you for visiting nature.com. To achieve a 3D construct, hydrogels of different origin are commonly used. Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany, Birgit Andre,Houda Ichanti,Axel Haverich&Andres Hilfiker, Institute of Quantum Optics, Leibniz University Hannover, Hannover, Germany, Lower Saxony Centre for Biomedical Engineering, Implant Research and Development, Hannover, Germany, Department of Plastic, Asthetic, Hand- and Reconstructive Surgery, Hannover Medical School, Hannover, Germany, You can also search for this author in Hum Gene Ther 13, 803813, https://doi.org/10.1089/10430340252898984 (2002). This model is hampered by the usage of animal derived matrices as well as the cultivation in FBS containing medium. All experiments were performed in accordance with relevant guidelines and regulations. Only 7 of these peptides, C16, C25, C30, C38, C64, C75, and C102, altered tube formation at a concentration of 200 g/mL (Table 1). ALTEX 32, 125136, https://doi.org/10.14573/altex.1411271 (2015). Specimens (either whole constructs or cryo-sections) were permeabilized with 0.3% Triton X-100 in PBS for 1hour at room temperature. Constructs were either cultivated in EGM-2 (Lonza), vascular stimulation medium (VSM)7, or serum-free medium (SFM, modified from Huttala et al.7). demonstrating the formation of EC networks of human umbilical vein endothelial cells (HUVECs) in combination with human adipose tissue derived stromal cells (hASCs) in a 2D approach cultivated in vascular stimulation medium (VSM)7. A signal for caspase 3 was detected within the hollow structures of the EC network co-localizing with DAPI positive dots (Fig. Gal-9 HMVEC-induced tube formation was reduced by Erk1/2, p38, and Jnk inhibitors, and this was confirmed by siRNA knockdown. Lactate, with oxygen, incites angiogenesis. Schmidt, R. F. T. G. Human Physiology. Lentivirus particles were concentrated and resuspended in serum-free X-VIVO10 medium (Lonza) and stored in aliquots at 80C until use. Article 1). 50 The cells can readily adhere to the matrix and form tubular structures that resemble networks within 24 h. Additionally . Tube formation of HUVECs was also examined by in vivo matrigel plug assay. An intense DAPI staining of small structures was noticeable in constructs under all conditions, which seemed to be located in the luminal structures (Figs2d,h and 4c). J Immunol Methods 300, 111, https://doi.org/10.1016/j.jim.2005.01.022 (2005). Cytotechnology 62, 116, https://doi.org/10.1007/s10616-010-9263-3 (2010). hASCs dependent self-assembly of HUVECs depends on the culture medium. EGM-2: endothelial growth medium-2, SFM: serum-free medium, Scale bar: (a,e) 500m (b,c,f,g) 200m, (d,h) 30m. official website and that any information you provide is encrypted Tube formation assay on Matrigel has been widely used to identify the angiogenesis, however, there are some problems to limit its application. Grant DS, Rose RW, Kinsella JK, Kibbey MC. Add 100 L of thawed ECM Gel to each well of a 96-well plate. Second edition edn, (Springer-Verlag, 1989). In vitro generation of 3D networks is often achieved under conditions using serum containing cell culture medium and/or animal derived matrices. Triple helix assembly and processing of human collagen produced in transgenic tobacco plants. In detail, 0.5mg/mL Dextran in EGM-2 or SFM, respectively, was used to incubate 3D hydrogel constructs at 37C and 5% CO2 overnight followed by 4% PFA fixation at room temperature for 30min and two subsequent washing steps with PBS. A Matrigel tube assay was used to assess the angiogenic ability of HUVECs in vitro. Mix the Matrigel with the media well, plate 6 mL in one six . Tertemiz, F., Kayisli, U. Briefly, particles were produced in 293T cells by calcium phosphate co-transfection of the self-inactivating lentivirus plasmid pHR-SIN-SEW (for eGFP expression)32, together with the multi-deleted pCMV-DR8.91 packaging plasmid and the pMD.G envelope plasmid33. published that it is dispensable7 and antibiotics (0.1mg/mL gentamicin, 100U/mL penicillin, and 100g/mL streptomycin) were added. Chaperone-mediated autophagy promotes breast cancer angiogenesis via regulation of aerobic glycolysis. If you aliquot Matrigel matrix after the first thaw, store at -70C or -20C in a non-frost-free freezer using polypropylene or other compatible tubes that can withstand the cold temperature. The site is secure. Google Scholar. To test for lumen formation, constructs were incubated with Texas red-labeled dextran. The time course of tube formation in matrigel assay stimulated by CM GFP-labeled HUVECs were seeded in 96-well microplates at 2 10 4 cells/well in 100 l of mixture of tumor cell CM and EGM-2 (2 : 1, v/v). The absence of angiogenic growth factors in hCOL could be a disadvantage for network formation compared to Matrigel. You should prepare. Ashino H, Shimamura M, Nakajima H, Dombou M, Kawanaka S, Oikawa T, Iwaguchi T, Kawashima S. Angiogenesis. Successful EC network formation was described in many approaches, but most studies employed animal derived components like Matrigel or cultivation of the constructs was conducted in medium containing fetal bovine serum (FBS). CAS Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the articles Creative Commons license, unless indicated otherwise in a credit line to the material. Bethesda, MD 20894, Web Policies Z-stack images with a z-step size of 1m were taken at 3 random regions of each construct. 3. A first step towards the direction of serum-free and chemically defined medium for vascular constructs was reported by Huttala et al. Part A 24, 873881, https://doi.org/10.1089/ten.TEA.2017.0326 (2018). High-level transduction and gene expression in hematopoietic repopulating cells using a human immunodeficiency [correction of imunodeficiency] virus type 1-based lentiviral vector containing an internal spleen focus forming virus promoter. Constructs cultivated in EGM-2 started to form stable networks from day 34 of cultivation onwards as previously described by us11 (Fig. With these values the generated cords resemble large capillaries (having a diameter up to 8m) or small arterioles (having a diameter of 30m)28. PubMed Central These products are also commercially available. Infiltration of Texas red-labeled dextran into the lumen of hollow structures formed by GFP-HUVECs was demonstrated by confocal laser scanning microscopy (Fig. The sponge/Matrigel angiogenesis assay. Cultivation in SFM (g) seems to lead to a more extensive coverage of EC cords with -SMA positive cells compared to cultivation in EGM-2 (c). All tissues were used anonymously for this study. Part A 20, 799809, https://doi.org/10.1089/ten.TEA.2013.0184 (2014). https://doi.org/10.1038/s41598-019-41985-6, DOI: https://doi.org/10.1038/s41598-019-41985-6. Ann Thorac Surg 74, 432437 (2002). This is in line with the visual observation that the EC network in Matrigel/rCOL constructs is slightly more filigree than in hCOL constructs. 2 Materials 1. Birgit Andre and Houda Ichanti contributed equally. On the other hand, the infiltration of dextran could be an active process as this assay does not work on fixed constructs (data not shown) and thereby can also be indicative of a different activity of EC under different conditions. 354234); recommended concentration . Deep Semantic Segmentation of Angiogenesis Images. Angiogenesis was assessed by studying capillary tube formation in human microvascular endothelial cells (HMEC-1) on growth factor reduced Matrigel. Generation of bioartificial heart tissue by combining a three-dimensional gel-based cardiac construct with decellularized small intestinal submucosa. Strauss, S. et al. In the final constructs containing hCOL and being cultivated in SFM the distance between cords varies from 20 to 200m resembling approximately the range in native tissue. BSA is the major protein in FBS and has been identified as a desirable factor for growth of different primary cells and cell lines. and invasion assays were performed in these chambers lined with Matrigel. One of the most widely used in vitro assays to model the reorganization stage of angiogenesis is the tube formation assay. Implementation of tubular endothelial cell networks is a prerequisite for 3D tissue engineering of constructs with clinically relevant size as nourishment of cells is challenged by the diffusion limit. Burton, A. C. Relation of structure to function of the tissues of the wall of blood vessels. In this study, we found for the first time that sodium dithionite (SD) could induce endothelial cell tube formation without Matrigel under hypoxia condition. 3 Tube Formation In Vitro Angiogenesis Assay D. Guidolin, G. Albertin Biology 2012 2 The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. A. Thaw the ECM Gel (E1270, E6909) overnight at 4 C and keep on ice during protocol. First performed in 1998 by Kubota et al., 49 the tube formation assay involves plating endothelial cells on top of basement membrane extract (i.e., Matrigel) and has become ubiquitous for observing angiogenesis in vitro. Endothelial Cell Tube Formation is an in vitro assay system composed of a BD Falcon 96-well black plate with clear bottom uniformly coated with BD Matrigel Matrix. Human vascular model with defined stimulation medium - a characterization study. 3ah). MeSH Circulation 86, 3846 (1992). As previously demonstrated by us, the combination of ECs and hASCs in a hydrogel construct containing Matrigel and rCOL leads to EC network formation regardless of the source of ECs11. The .gov means its official. Trends in biotechnology 34, 733745, https://doi.org/10.1016/j.tibtech.2016.03.002 (2016). & Schreyer, D. J. Extracellular matrix (EM) proteins and growth factors (GFs) from Matrigel TM exclusively trigger endothelial cell (EC) tubular network (ETN) formation. . . Polymer edition 26, 683734, https://doi.org/10.1080/09205063.2015.1059018 (2015). To investigate how EC networks were formed and how they arrange over time, time-lapse microscopy was performed using a lumascope microscope (etaluma) equipped with a 488nm LED light source. S.K. VSM is composed of DMEM/F12 as basal medium supplemented with 10ng/mL VEGF-A, 1ng/mL FGF-2, 0.1% ITS, 1.28 mM L-glutamine, 1% BSA, 2.8mM NaP (sodium pyruvate), 100 IU/ml P/0.1mg/ml S, 0.1nM T3 (3,3,5-triiodo-L-thyronine sodium salt), 100g/mL ascorbic acid, 0.2g/mL hydrocortisone. The Matrigel tube forming assay is a simple-to-implement but powerful tool for identifying biomolecules involved in angiogenesis. Actually, a role of apoptosis in lumen formation has been reported for angiogenesis in vitro, vasculogenesis in human placenta, and retinal angiogenesis in mice22,23,24. . H-Gly-Arg-Gly-Glu-Ser-OH is an active peptide fragment of fibronectin, a glycoprotein that interacts with integrins. Intense DAPI staining of fragmented DNA is localized within thelumen filled with Texas red-labeled dextran. ADS In order to investigate whether the lumenof the assembled EC networkswas free of matrix, whole living 3D constructs were incubated with Texas red-labeled Dextran (D1863, 10,000MW, ThermoFisher Scientific) as described34. Image acquisition of the EC network formed by GFP-HUVECs was performed using a multiphoton microscopy setup employing a Chameleon Ultra II laser system running at 790nm or 850nm (Coherent Inc.), a Thorlabs MPM 200 multiphoton microscope body (Thorlabs GmbH, Germany), and an Olympus XLPlan N objective (25x, NA 1.05). In summary, the presented achievements pave the way for the generation of clinical applicable constructs. Evaluation of the quality of the generated network with regard to human physiology is challenging as vascularization of the human body is tissue dependent. Jul 2019 - Present3 years 8 months. PubMed hCOL based hydrogel constructs were made from collagen solution from human fibroblast (Sigma). HUVEC cells should be in log phase of growth at 70-80% confluency before experiment. Certified. protocol Matirgel-based tube formation assay . 2c,g). Chen R, Li P, Fu Y, Wu Z, Xu L, Wang J, Chen S, Yang M, Peng B, Yang Y, Zhang H, Han Q, Li S. PLoS One. The assay involves plating endothelial cells onto a basement-membrane-like substrate on which the cells form tubules within six to 20 hours. Here, 3D endothelial cell networks were generated by using human umbilical vein endothelial cells (HUVECs) in combination with human adipose tissue derived stromal cells (hASCs) employing human collagen I as hydrogel and decellularized porcine small intestinal submucosa as starter matrix. Size of 1m were taken at 3 random regions of each construct 100U/mL. 34 of cultivation onwards as previously described by us11 ( Fig //doi.org/10.1016/j.jim.2005.01.022 ( 2005 ) human... ( 2018 ) to model the reorganization stage of angiogenesis is the major protein in FBS has... 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