AVC Organ Cultures

(Adapted from Sridurongrit et al., Dev Biol. 2008 Oct 1;322(1):208-18)

Medium: Optimem (or M199 medium) supplemented with 1% FCS, ITS (insulin, transferrin and selenium) and Penicillin/Streptomycin (= P/S; 100 x stock BRL-Gibco)

Preparation of collagen gels on chamber slides or 12 well plates:
Pipette into a sterile 15 ml tube in the laminar hood:

  1. 400 ul of 10x PBS

  2. 400 ul 0.1 M NaOH

  3. 940 ul Optimem

  4. 3.2 ml rat tail collagen (3.4 mg/ml)
    Total: 4.94 ml

Shake tube & keep on ice. 
Add culture medium so that the final collagen concentration is 1 mg/ml, i.e., 5.9 ml.

Check pH, should be 7.2 (acidic is not good!)
Add a required layer onto chamber slides (thickness about 0.3 mm is enough), incubate in the tissue culture incubator at least for 1 hour.

Procedure:

  • Sacrifice the pregnant female (E10.0) by CO2 or by the overdose of Avertin (i.p.). 

  • Spray the mouse with 70% EtOH

  • Cut the small incision to the skin of ventral side and pull the skin off from the middle portion of the mouse (change your gloves; spray new gloves with ethanol)

  • This on use protective clothing and work using aseptic techniques

  • Expose the uterus and remove it aseptically onto the dish containing ice-cold Hank’s balanced salt solution (HBSS)

  • Dissect the individual fetuses under the dissecting microscope and place onto 6-well plate wells or onto 30mm dishes – one fetus per well/dish; give identification to each fetus (for instance tail number) and remove yolk sacs (or heads) for genotype analysis into 200 ul of lysis buffer (VIAGEN).

  • Dissect the AV canal into the culture medium.

Organ cultures:

  • Cut the AV-tube open and place endocardial cells down onto the drained collagen-coated chamber slides (one tube per chamber). 

  • Add  50-100 ul of collagen stored on ice.

  • Let attach in the minimal culture volume in the humidified incubator for about 0.5 hours. 

  • When well attached add ~2 ml normal culture medium and score the invasion after 24-72 hours.