This is a cached page for the URL (http://homepage.uab.edu/wzhang/3dCellCultureOC.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
3D growth of cells on Collagen

Three Dimensional growth of cells on Collagen: Cell Invasion Assay

Apply appropriate aseptic techniques at each step!

Materials

  1. 6-well transwell plate
  2. BD collagen sollution (cat# 354236) (If use dried collagen, refer to method for the preparation of collagen solution) (4oC)
  3. 10X DMEM, sterile (4oC)
  4. 1N sterile NaOH
  5. Autoclaved MilliQ H2O
  6. Growth media: DMEM (Mediatech MT10-013-CV) + 10% FBS + 1% PS
  7. 10% buffered formalin
  8. 50ml Falcon centrifuge tubes
  9. Sereological pipets (sterile)
  10. Micropipetters and tips
  11. Laminar flow hood
  12. Centrifugate with 50 ml tube adapters
  13. Ice
  14. Surgical blade
  15. Staining apparatus and solutions

Procedure

  1. In Laminar flow hood, place collagen, 10X DMEM, 1N NaOH and sterile H2O tubes on ice.
  2. To a 50ml centrifuge tube, add H2O, 10X DMEM, 1N NaOH and collagen sollution as in table 1 and mix carefully by pipetting up and down and avoid producing bubbles.
    3. (If bubbles produced, centrifuge briefly at 4oC)

table 1. Calculation of collagen gel components

Total Volume Original collagen concentration Final collagen concentration H2O 10X DMEM 1N NaOH Collagen
ml mg/ml 2.2 mg/ml ml ml µl ml
  1. Pour onto Transwell at 1.5ml/well for 6-transwell plate. Spread evenly by gently shaking. Incubate at 37oC for at least 10 min.
  2. Trypsinize cells by standard precedure. Count cells and allocate certain amount of cells (fibroblasts: 500K/well; Tumor cells: 200K/well in our case) into fresh sterile tubes, pellet to remove trypsin and EDTA.
  3. Resuspend cells in 2 ml regular growth media and put onto collagen gel.
  4. Fill the lower camb with 3 ml media after incubation for 1-3 hours.
  5. Incubate at regular condition and change media periodically as needed (usually every 2-3 days).
  6. Terminate culture at the 6th day by replacing media with 10% formalin solution and fix gel overnight at 4oC. (Longer culture time could be needed if cell invasion is weak).
  7. Cut gel along Transwell inner edge with surgical blade and transfer into tissue cassettes. Put in plastic container submerged in 10% formalin and send to Procurement Center for embedding.
  8. Cut two sections per gel at 5 um using microtome and stain (see H&E stain).
  9. Observe cells' migration under microscope and take pictures.