Assaying Growth and Differentiation Factors

The effect of growth and differentiation factors on explanted tissue can be assayed in different ways. Factors that are available as purified proteins can be assayed simply by adding them to the culture medium (17), or by deriving them to appropriate beads and then implanting the beads within the collagen. Alternatively, if purified protein is not available, but full-length clones encoding a protein are available, attempts can be made to produce protein in COS cell aggregates transiently, which are then implanted in the collagen. The technique is as follows:

1. Seed COS-1 cells at approx 2.5 x 105 cells/35-mm dish on d 1.

2. On d 2, transfect expression vector containing clone under examination into COS-1 cells, using standard techniques (e.g., lipofection, electroporation, DEAE trans-fection).

3. On d 3, clump transfected cells. Treat cell layers with either trypsin or enzymefree dissociation buffer. Wash cells twice with DMEM with 10% HIFCS, and resuspend in DMEM with 1% HIFCS (0.25 mL/35-mm dish [18]).

4. Place 20-| L drops of the cell suspension onto the lids of 60-mm culture dishes, and invert over dishes containing 5 mL of DMEM. Incubate the hanging drop cultures for 14-16 h.

5. Harvest cell aggregates into L15-air medium, and trim as required with tungsten needles for use in explant culture.

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