Questions regarding the spheroid culture technique:

• Which cell culture plastic do you use to generate spheroids?
  Non adhesive cell culture plastic for suspension culture. We recommend 96-well-plates for suspension culture, U-form, cat#: 650185 from Greiner.
  
  
• Which cell types have been tested for their spheroid forming capacity?
  So far, we tested HUVEC, HUAEC, HUSMC, HMVEC, human cytotrophoblast cells, BAEC, PAEC 10T1/2, C6-glioma and different epithelial cell types. All of the tested cell types performed well in spheroid based assays.
  
  
• How do you prepare the methocel stock solution which is needed for spheroid culture?
  The preparation of methocel stock solution is very critical. If the concentration of methocel is too low or the solution is containing methylcellulose debris, single cells will stick to the wall and several small spheroids are formed in each well. We use methylcellulose from sigma (cat#: m-0512, 4000 centipoises). The methocel stock solution should have an extremly high viscosity. We autoclave the pure powder (6g) in a 500ml flask containing a magnetic stirrer (the methylcellulose powder is resistant to this procedure). The autoclaved methylcellulose is dissolved in preheated 250ml basal medium (60°C) for 20min (using the magnetic stirrer). Thereafter, 250ml basal medium (room temperature) is added to a final volume of 500ml and the whole solution is mixed for 1-2h (4°C). The final stock solution is aliqoted and cleared by centrifugation (5000g, 2h, room temperature). Only the clear highly viscous supernatant should be used for the spheroid assay (about 90-95% of the stock solution). For spheroid generation we use 20% of the stock solution and 80% culture medium.
  
  
• How do you generate the shperoids containig a precise number of cells?
  The number of cells in a spheroid depends on the experimental procedure. If you want to analyze spheroids morphologically or biochemically (paraffin embedding, western blot, RNA analysis ect.), spheroids should contain at least 2250 cells. Spheroids which shall be embedded into collagen gels for functional anaysis (sprouting assays) should contain not more than 750 cells: For morphological/biochemical analysis trypsinize one petridish (55-75cm2) of a confluent EC monolayer. Suspend these cells in 10ml medium. Count the cells and seed a defined number of cells in methocel containing medium (20% methocel stock solution (see above), 80% culture medium [supplements/FCS: cell type dependent]). Distribute the medium to 96-well-plates (after trypsinization you count 3.000.000 cell in 10 ml, you need 4x96 spheroids (150µl/well) and one spheroid should contain ca. 2250 cells. Based on that, you need 2x1,5ml from the 10ml cell suspension. Dilute each 1,5ml in 30ml methocel containing medium and distribute it over 4x96 wells [1,5ml contains 450.000 cells; divided by 200 (exactly 2x96 wells) you get ca.2250 cells/well]). For morphological/biochemical analysis trypsinize one petridish (55-75cm2) of a confluent EC monolayer. Suspend these cells in 10ml medium. Count the cells and seed a defined number of cells in methocel containing medium (20% methocel stock solution (see above), 80% culture medium [supplements/FCS: cell type dependent]). Distribute the medium to 96-well-plates (after trypsinization you count 3.000.000 cell in 10 ml, you need 4x96 spheroids (150µl/well) and one spheroid should contain ca. 750 cells. Based on that, you need 2x0,5ml from the 10ml cell suspension. Dilute each 0,5ml in 30ml methocel containing medium and distribute it over 4x96 wells [1,5ml contain 150.000 cells; divided by 200 (exactly 2x96 wells) you get ca.750 cells/well]).
  
  
• How long can you culture the spheroids?
  It depends on the cell type and culture conditions. HUVEC spheroids can be cultured for up to one week in media containing supplements like ECGS and FCS (10%). However, the cells in the center of the spheroids will undergo apoptosis. BAEC spheroids can be cultured for prolonged times (up to four weeks) in media containing FCS (10%). Because of their endogenous FGF-2 expression, the amount of apoptosis in the spheroid center is reduced.
  
• How do you harvest the spheroids?
  We harvest the spheroids from 96 well plates using standard pipette tips (1ml, blue) transferring them into a 15/50ml tube. To enlarge the tiphole, 1-2mm (not more!) of the front of the tip is cut away.
  
  
• How fragile are the spheroids?
  Whether the spheroids are fragile or not depends on the cell type you use and the culture conditions. For example, HUVEC spheroids are more fragile than BAEC spheroids. Generally, spheroids show a stable cellular organization. It is unlikely to disrupt spheroids by pipetting. However, cutting the pipette tip (1-2mm) before harvesting the spheroids should prevent their disruption. Note that EGTA-treatment induces disruption of HUVEC and BAEC spheroids.