Martin Humphries
LAB

Protocols - Produce Cell-derived Matrices (CDM) using human foreskin fibroblast (HFF)

Written by Guillaume Jacquemet


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- Clean coverslips:


(If you are growing the matrix in MATTEK glass bottomed dishes, or plastic tissue culture dishes you do not need to do this.)

  1. Boil coverslips 3 times in MilliQ water to clean them
  2. Autoclave coverslips in MilliQ water to sterilise
  3. Transfer coverslips to 24-well plate to grow matrix


- Prepare coverslips:


  1. Wash with PBS
  2. Incubate with 0.2% sterile gelatin for 60 min at 37°C (2% gelatin, Sigma G1393 diluted in PBS)(250μl per well for 24-well plate)
  3. Wash 3 times with PBS
  4. Crosslink with 1% sterile glutaraldehyde in PBS for 30 min at RT (250μl per well for 24-well plate)
  5. Wash 3 times with PBS
  6. Quench crosslinker with 1M sterile glycine in PBS for 20 min at RT
  7. Wash 3 times with PBS
  8. Incubate in growth media for 30 min at 37°C
  9. Wash 3 times with PBS
  10. Use immediately or store at 4°C. If you store them, re-incubate with media before seeding cells (If you are growing the matrix on plastic, crosslinking and quenching (steps 4-7) not necessary)


- Plating HFF:


  1. Detach cells with trypsin as normal. Resuspend T75cm flask in 10 ml growth media (including serum) and count on hemocytometer
  2. Dilute cells and plate as appropriate: 6 well plate, 105 / ml, plate 2 ml per well 9-cm plate, 105 / ml, plate 12.5 ml 24 well plate, 5x104 / ml, plate 1 ml per well
  3. Culture overnight 37°C, 5% CO2 (Cells should be confluent throughout generation of the matrix. If they appear subconfluent at this stage, grow for another day.)
  4. Change media to complete media supplemented with 50 ug/ml Ascorbic acid (Sigma).
  5. Change ascorbic acid media every day until denudation. The ascorbic acid increases collagen production and stabilises the matrix. Without it the matrix will not stick to the plate properly. We wanted a fibronectin-rich matrix so we only changed the ascorbic acid media every other day.
  6. Culture for: HFFs 11-14 days (we used 8-12 days) 3T3 5-9 days

We used HFFs and did not see improvement in matrix after 8 days for the first batch of cells, but needed longer for a second batch. You might want to try different culture times for your batch of cells and staining the fibronectin to see what they look like. Growing for too long will not do any harm.


- Denuding cells:


  1. Aspirate medium and wash cells with PBS
  2. Gently add pre-warmed extraction buffer (20 mM NH4OH, 0.5% Triton X-100 in PBS). 1.5 ml for 6-well plate, 6 ml for 9-cm plate
  3. Cell lysis is virtually instantaneous. Lyse for about 2 min until no intact cells visible by phase. The matrix is delicate, so must be treated very gently
  4. Aspirate extraction buffer partially (remove half of buffer gently) and wash with PBS containing calcium and magnesium (BioWhittaker UK, Ltd)
  5. Repeat wash gently with PBS containing calcium and magnesium x 2
  6. Remove all PBS and digest residual DNA with 10 ug/ml DNase I (Roche) in PBS containing calcium and magnesium. Incubate 30 min, 37°C, 5% CO2
  7. Aspirate DNase and wash twice with PBS containing calcium and magnesium
  8. Store at 4°C in PBS containing calcium and magnesium with pen/strep and fungizone for up to a month. You should be able to see the matrix by phase so will be able to check that it is intact before use


- Preparation of matrix for plating cells:


  1. Wash twice with PBS containing calcium and magnesium
  2. Block with heat-denatured BSA (10 mg/ml >99% pure BSA in PBS, heated to 85°C for 13 min, and then cooled and filter through 0.45 um acrodisc). NB: Can block with FCS-containing medium if using this in the assay, in which case omit step 3 below and wash 1x with FCS-containing medium instead
  3. Wash three times with PBS containing calcium and magnesium
  4. Plate cells at appropriate density: 9-cm plate, 105 / ml, plate 8 ml for biochemical assay 6-well plate, 105 / ml, plate 2 ml for migration assay 24 well plate, 5x104 / ml, plate 1 ml per well Cells take about 4 hours to spread and start migrating on cell-derived matrix

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