Fibrin Gel In Vitro Angiogenesis Assay Kit

Transcription

1 Fibrin Gel In Vitro Angiogenesis Assay Kit Cat. No. ECM630 FOR RESEARCH USE ONLY Not for use in diagnostic procedures USA & Canada Phone: +1(800) Fax: +1 (909) Europe +44 (0) Australia Germany ISO Registered Worldwide custserv@chemicon.com techserv@chemicon.com

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3 Introduction Angiogenesis is the process of generating new capillary blood vessels. It is a fundamental component of a number of normal (reproduction and wound healing) and pathological processes (diabetic retinopathy, rheumatoid arthritis, tumor growth and metastasis) 1. The CHEMICON Fibrin Gel In Vitro Angiogenesis Assay Kit provides a convenient system for evaluation of tube formation by endothelial cells in 96- well or other formats. When cultured on top or within a fibrin gel, endothelial cells rapidly align and form interconnecting networks that can display patent lumina 2,3. Tube formation is a multi-step process involving cell adhesion, migration, differentiation and growth 4. The formation of intercellular connections and lumina within endothelial cell networks in fibrin gels is dependent upon the actions of VE-cadherin, αvβ3 and α5β1 integrins, the cdc42 and Rac1 GTPases, and membrane-type matrix metalloproteinases (MT- MMPs) 2,3,5,6. Angiogenesis within fibrin gels in vitro is regarded as an accurate model for wound healing and tumor angiogenesis, as tumor cell derived vascular endothelial growth factor/vascular permeability factor promotes leakage of fibrinogen from the tumor vasculature and formation of a fibrin-rich proangiogenic provisional matrix 7. Fibrin gel formation is initiated by enzymatic cleavage of fibrinogen, a heterotrimer, by thrombin 8. The resulting cleaved fibrin molecules form regular, multimolecular arrays that are highly translucent. The concentrations and formulations of the fibrinogen and thrombin in this kit are optimized for maximal tube-formation by HUVEC and easy visualization of these tubes. Application The CHEMICON Fibrin Gel In Vitro Angiogenesis Assay Kit represents a simple model of angiogenesis in which the induction or inhibition of tube formation by exogenous signals can be easily monitored. Fibrin gels are easily and quickly formed in culture dishes by mixing Fibrinogen and Thrombin Solutions. For assaying inhibitors or stimulators of tube formation, simply premix the endothelial cell suspension with different concentrations of the inhibitor or stimulator to be tested, before adding the cells to the top of the fibrin gel. Addition of a second layer of fibrin gel on the day after plating the cells is optional, but will promote optimal survival and a higher degree of network and lumen formation. The assay can be used to monitor the extent of tube assembly in various endothelial cells, e.g. human umbilical vein cells (HUVEC) or bovine capillary endothelial (BCE) cells. 1

4 The CHEMICON In Vitro Angiogenesis Assay Kit is intended for research use only; not for diagnostic or therapeutic applications. Figure 1. HUVEC Cells incubated hours at 37 C in CHEMICON Fibrin Gel (100x magnification). Figure 2. Lumina (arrow) and invasive sprouts of HUVEC sandwiched within CHEMICON Fibrin gel and incubated for 72 h in media supplemented with bfgf, VEGF and PMA (320x magnification). 2

5 Kit Components 1. Fibrinogen Solution: (Part No ) One 10 ml bottle. 2. Thrombin solution: (Part No ) One 7.5 ml bottle x Positive Control Angiogenic Supplement: (Part No ) One 0.5 ml vial. Materials Not Supplied well or other sized Tissue Culture plate 2. Microcentrifuge Tubes, sterile C Tissue Culture Incubator 4. Inverted Light Microscope 5. HUVEC cells or other experimental cell line (any species), and appropriate growth medium and subculturing reagents. Early passage cells are preferred, as they are less prone to cell death in this assay. 6. Pipet capable of delivering µl, preferably a multichannel pipet with 96-well plate 7. Basal medium EBM (Cambrex/BioWhittaker) or MCDB131 (Sigma) supplemented with 1% BSA and antibiotic. 8. PMA (optional) to add to basal media and angiogenic supplement for positive control. Preparation of Reagents 1. Fibrinogen Solution: On the day of the assay, thaw Fibrinogen Solution rapidly and bring to approximately 37ºC by gently agitating in a 37ºC water bath. For short-term storage, store at room temperature for up to 2 weeks. Do not store at 4ºC, as fibrinogen solutions tend to form aggregates when stored at 4ºC. For long-term storage, aliquot into sterile tubes in a sterile laminar flow hood, freeze on dry ice, and store at 20ºC for up to 1 year. 2. Thrombin solution. Thaw thrombin solution at room temperature. Unused portions of thrombin are to be stored at 4ºC for up to 1 month. For longer storage, thrombin solution may be divided into aliquots and stored at 20ºC for up to 1 year. 3

6 3. 100x Positive Control Angiogenic Supplement: Thaw at room temperature. Store unused portion at 4ºC for up to 1 year. Assay Instructions Note: Volumes given in the protocol are for use with a 96-well plate. For use in plates with wells of other sizes, consult the chart below for appropriate volumes. 1. Thaw Thrombin Solution and Fibrinogen Solution as suggested above. 2. Dispense 30 µl Fibrinogen Solution into the desired number of wells in a 96-well plate. Gently shake the plate to ensure that the Fibrinogen Solution covers the bottom of the well. 3. Add 20 µl per well thrombin to the 96-well plate. Shake the plate gently immediately after addition of thrombin to each well. 4. Place plate at 37ºC for min to polymerize. 5. Prepare media to be tested. If desired, prepare positive control media by diluting 100x Positive Control Angiogenic Supplement 1:100 with basal medium, and adding PMA to a final concentration of 50 ng/ml. 6. Trypsinize and count endothelial cells by standard methods. Centrifuge and resuspend at a final concentration of 5 x 10 4 cells/ml in positive control media or test media. Note: Other cell densities may be employed, but higher cell densities result in continuous monolayers that take longer to organize into networks, and lower cell densities have higher numbers of isolated cells and fewer networks. 7. Add 100 µl HUVEC in test medium to wells of 96-well plate. 8. Incubate at 37 C overnight in a tissue-culture incubator. Note: Cellular network structures are highly developed by 12-18h in the presence of PMA, with the first signs apparent after 5-6h. However, cells remain as monolayers when plated on top of fibrin gels in the presence of bfgf or VEGF, and require the addition of another layer of fibrin gel for network formation to occur. The addition of an overlay also reduces the extent of cell death on the fibrin gels. 9. Remove as much of the culture medium as possible without disturbing the gel. For 96-well plate, it is most convenient to invert the plate onto sterile gauze. 10. Add 30 µl per well fibrinogen in each medium to indicated wells of 96 well plate. Shake plate gently to get fibrinogen to evenly cover the gel/cells. 4

7 11. Add 20 µl per well thrombin in each medium to indicated wells. Shake the plate gently immediately after addition of thrombin to each well. 12. Place at 37ºC for 5 min to polymerize. 13. Add 100 µl per well of positive control medium or test medium. 14. Place in 37ºC, 5% CO 2 incubator. 15. In medium containing bfgf and/or VEGF, networks begin to form within 2 h and continue to mature for 48 h. In the presence of PMA, intracellular vacuoles and patent lumina become evident by 24 h after the overlay. Networks remain stable for more than one week. For long-term experiments, replace media every 2-3 days. Suggested Volumes for Different Plates 96-well 48-well 24-well 12-well 6-well Fibrinogen solution Thrombin solution Total gel volume per well Cell suspension/ test media 30 µl 120 µl 200 µl 370 µl 1 ml 20 µl 80 µl 133 µl 245 µl 670 µl 50 µl 200 µl 333 µl 615 µl 1.67 ml 100 µl 580 µl 1 ml 1.8 ml 5 ml 5

8 Quantitation Activated endothelial cells form cellular networks (mesh like structures) from capillary tubes sprouting into the stromal space (see Figure on p.2). The formation of these cellular networks is a dynamic process, starting with cell migration and alignment, followed by the development of capillary tubes, sprouting of new capillaries, and finally the formation of the cellular networks. Although the Fibrin Gel In vitro Angiogenesis Assay Kit is designed as a qualitative assay, it is possible to quantitate to some degree the extent to which cellular networks have formed. A. Pattern recognition Define visual patterns by looking or photographing the cells at 40X 200X magnification at set time at 37 C after seeding on the fibrin gel (end-point assay). Assign a numerical value to each pattern. This way a numerical value is associated with a degree of angiogenesis progression. An example is presented in the table below. Pattern Value Cells isolated or in a sheet-like monolayer 0 Cells begin to migrate and align themselves 1 Capillary tubes visible. No sprouting. 2 Sprouting of new capillary tubes visible. 3 Closed polygons begin to form. 4 Complex mesh like structures develop 5 This pattern/value association criterion should be defined with the type of cells and experimental conditions that will be used in the angiogenesis assay. Several random view-fields (3-10) per well should be examined and the values averaged. This quantitation method will work best in assays involving potent inhibitors or activators of angiogenesis. B. Branch Point Counting A subtler, but more labor-intensive way to quantitate the progression of angiogenesis is to count the capillary tube branch points formed after a set amount of time (end-point assay). The length of the newly formed capillary tubes can also be taken into account when counting (do not count if shorter than an arbitrary predetermined length). Branch points in several random view-fields (3-10) per well should be counted and the values averaged. C. Total Capillary Tube Length Measurement 6

9 An alternative method to branch point counting, suitable particularly for microscopes with imaging capabilities, is to measure the total length of all the capillary tubes in a view-field. The total capillary tube length in several random view-fields (3-10) per well should be examined and the values averaged. D. Quantification of Invasion and Lumen Formation: Under certain conditions, particularly between two layers of fibrin gel in the presence of PMA, HUVEC will invade the gel above or below the original plane at which they were plated. In addition, large vacuoles or lumina are visible within the cells. These indicators of angiogenic invasion and differentiation can be counted in several fields per well, and the mean and standard deviation determined. Storage Store unopened kit materials at -20 C or 70 C for up to their expiration date. Once Fibrinogen Solution has been thawed, store at room temperature for up to two weeks. Thawed Thrombin Solution should be stored at 4ºC for up to one month. Thawed 100x Positive Control Angiogenic Supplement can be stored at 4ºC for up to one year. 7

10 Troubleshooting Problem Cause Remedy Presence of gelled solids in Fibrinogen Solution Aggregation fibrinogen of Thaw Fibrinogen Solution and bring to room temperature rapidly. Store Fibrinogen Solution room temperature, not at 4ºC Centrifuge Fibrinogen Solution at 2000xg in a sterile tube at room temperature. Use supernatant. Grainy or spider-webbed appearance of gel Uneven gelation of fibrinogen Store fibrinogen solution room temperature, not at 4ºC Mix immediately after addition of thrombin solution to fibrinogen solution Presence of bubbles on top of gel Gently pipet medium to dislodge bubbles Cells out of focus at edges Networks form, but many apoptotic cells are evident Presence of a meniscus at the edges Cells are late passage Observe cells at center of the well. Use larger wells to increase observable area. Use early passage cells 8

11 Related References 1. Carmeliet P. and Jain, R.K. (2000) Angiogenesis in cancer and other diseases. Nature 407: Bach, T.L., Barsigian, C., Chalupowicz, D.G., Busler, D., Yaen, C.H., Grant, D.S., and Martinez, J. (1998) VE-Cadherin mediates endothelial capillary tube formation in fibrin and collagen gels. Exp. Cell Res. 238: Bayless, K.J., Salazar, R., and Davis, G.E. (2000) RGD-dependent vacuolation and lumen formation observed during endothelial cell morphogenesis in three-dimensional fibrin matrices involves the αvβ3 and α5β1 integrins. Am. J. Pathol. 156: Carmeliet, P. (2000) Mechanisms of angiogenesis and arteriogenesis. Nat Med. 6: Bayless, K.J. and Davis, G.E. (2002) The Cdc42 and Rac1 GTPases are required for capillary lumen formation in three-dimensional extracellular matrices. J. Cell Sci. 115: Lafleur, M.A., Handsley, M.M., Knauper, V., Murphy, G., and Edwards, D.R. (2002) Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT- MMPs). J. Cell Sci. 115: Dvorak, H.F., Brown, L.F., Detmar, M., and Dvorak, A.M. (1995) Vascular permeability factor/vascular endothelial growth factor, microvascular hyperpermeability, and angiogenesis. Am. J. Pathol. 146: Mann, K.G., and Lorand, L. (1993) Introduction: blood coagulation. Methods Enzymol. 222:

12 Warranty These products are warranted to perform as described in their labeling and in CHEMICON literature when used in accordance with their instructions. THERE ARE NO WARRANTIES, WHICH EXTEND BEYOND THIS EXPRESSED WARRANTY AND CHEMICON DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE. CHEMICON s sole obligation and purchaser s exclusive remedy for breach of this warranty shall be, at the option of CHEMICON, to repair or replace the products. In no event shall CHEMICON be liable for any proximate, incidental or consequential damages in connection with the products. 2003: CHEMICON International, Inc. - By CHEMICON International, Inc. All rights reserved. No part of these works may be reproduced in any form without permissions in writing. Cat. No. ECM630 March 2003 Revision A: 41383