Questions regarding the spheroid based sprouting assay:

• How do you prepare the collagen stock solution needed for the angiogenesis assay?
  To prepare a collagen stock solution you need 250ml steril filtered acetic acid diluted 1:1000 in aqua dest. (cell culture quality), 2 rat tails (stored at -20°C), 2x500ml 70% Ethanol. Place the rat tails for 20min in 70% ethanol, remove the skin. Wash the naked tails in ethanol. Break each second (depends on the length of the tails) vertebral and extract the tendons. Be sure to get clean tendons without attached connective tissue! When you finished that preparation, take all the tendons and place them in the second 500ml clean ethanol for 20min. Dry the tendons under the lamina air flow for 20-30min. Put the tendons in 250ml acetic acid (see above) and place them in the refrigerator for 48 hours shaking the solution at least two times each day. Aliquot the final solution in autoclaved tubes and centrifuge them at 4°C at 17.000g for 1h. Collect the clear supernatant. Be sure not to collect the debris!!! The clear supernatant/collagen stock solution can be stored in the refrigerator for at least 6 month. For equilibration, carefully mix 4ml of the stock solution with 0,5ml 10xHBSS and keep the mixed solution on ice for at least 15min. In most cases the collagen concentration of the stock solution is too high and the mixed solution will become a gel. In that case dilute the collagen stock solution (use 1:1000 sterile filtered acetic acid) and mix it with 10xHBSS as described above. Repeat diluting the stock solution till the mixed solution stays liquid. Now, the collagen stock solution is ready to use. To keep the EC baseline sprouting level low, it is advisable to prepare the stock solution at least 4 weeks before use (freshly prepared collagen by itself has the capability to induce sprouting of EC).
  
  
• How do you prepare the collagen gels for the spheroid based angiogenesis assay?
  For 8 spheroid containing collagen gels (1ml/well in a 24-well-plate) put 4,5ml mixed collagen solution (0,5ml 10xHBSS + 4ml collagen stock solution) and 0,2N NaOH on ice. Harvest 400 spheroids in one 50ml tube and centrifuge (3min, 300-500g). Remove the supernatant and shortly scratch the tube over a rough underground to loosen the pellet (don´t let the pellet stay for more than 15-30min, otherwise the spheroids will stick together). Overlay (do NOT mix!) the pellet with 4-4,5ml FCS containing methocel stock solution (10-40%FCS, 90-60% methocel stock solution; FCS content depends on the cell type; note that the final FCS content in the gel is only the half of the concentration in this methocel solution). Right before use, neutralize the HBSS/collagen solution using ice cold NaOH (the phenol red pH indicator in HBSS should switch the color from yellow to flesh colored; mix fast and very carefully by inverting the tube to avoid polymerization. After neutralization keep the HBSS/collagen solution on ice. The neutralized collagen solution should be clear). Mix 4ml neutralized collagen with the 4ml spheroid containing methocel solution (again: do it fast and carefully). Divide the collagen/methocel solution on a PREWARMED 24-well-plate (8x1ml). Immediately place the plate into an incubator (37°C, 100% humidity). Let the collagen polymerize for at least 30min. Thereafter you can easily overlay the collagen gels with 100-200µl medium/supernatant. If you prepare the sprouting assay in the way described above, you must keep in mind that the supernatants to be tested first have to penetrate the collagen before they can affect the sprouting of the cells in the spheroids. To bring the spheroids in direct contact with agents to be tested use the following protocol: For each 1ml standard collagen gel, harvest 48 spheroids (half of a 96 well plate) and transfer them into a 15ml Falcon tube (48spheroids/tube). After centrifugation, remove the supernatant and shortly scratch the tube over a rough underground to loosen the pellet (don´t let the pellet stay for more than 15-30min, otherwise the spheroids will stick together). Overlay each pellet with 0,5ml methocel solution containing an appropriate amount of FCS (i.e. 20% FCS if the final concentration in the gel should be 10%; you also can use a medium containing only 40% methocel but the more methocel you use the slower the spheroids will sink to the bottom before the collagen becomes polymerized). Do NOT suspend the spheroids in the methocel solution! Add the agents to be tested to the methocel solution (again: do NOT mix!). Finally, each 0,5ml spheroids/agents-containing methocel solution is carefully mixed several times with 0,5ml neutralized collagen solution (see above) till the solution becomes homogenous and rapidly transferred into a PREWARMED 24-well-plate. Immediately after preparation of all collagen gels place the plate into an incubator (37°C, 100% humidity). Notes: For rapid collagen polymerization it is important that the plate is prewarmed to 37°C before the spheroid containin collagen/methocel-solution is filled in. Adequate mixing and rapid distribution of the final spheroid containing collagen/methocel-solution in a prewarmed 24-well plate is VERY important for homogenous dispersion of the spheroids in collagen gels.
  
  
• How can I decrease baseline sprouting in spheroid based angiogenesis assays?
  To reduce the baseline sprouting, we culture the HUVECs as well as the HUVEC spheroids in medium containing all supplements (ECGS, FCS ect.). To keep the baseline sprouting low, the FCS content in collagen gels should be the same as in the culture medium. If your culture medium contains 10%FCS the collagen gel also should contain 10% FCS. You should never add any supplements in the collagen gel. This will dramatically increase the baseline sprouting. If the baseline sprouting is too high, just culture the spheroids for two days (instead of 24h) before use or decrease the FCS concentration (not below 5%). Note that some cell types endogenously produce FGF-2 (i.e. BAEC) and show a high baseline sprouting which can be decreased by adding FGF-2-neutralizing antibody.
  
  
• How long let you run the spheroid based sprouting assay?
  Usually, up to seven days with BAEC or PAEC spheroids; up to three days with HUVEC spheroids.