Caspase inhibition supports proper gene expression in ex vivo mouse limb cultures

We standardized conditions for ex vivo mouse limb culture to study cartilag e maturation and joint formation. We compared 12.5 d.p.c. mouse forelimbs t hat were cultured either mounted or freely rotating for up to 72 h. Limb ou tgrowth progressed ex vivo at a variable rate as compared to its developmen t in vivo, spanning approximately 48 h. Although cartilage maturation and j oint formation developed grossly normal, aberrant expression of skeletal ma rker genes was seen. Interestingly, no regression of the interdigital webs took place in mounted cultures, in contrast to limited webbing under freely rotating conditions. Caspase inhibition, by addition of zVAD-fmk to the cu lture medium of freely rotating limbs, supported proper gene expression ass ociated with skeletal development, and prevented interdigital web regressio n. Taken together, a freely rotating ex vivo culture for mouse limb outgrow th that is combined with caspase inhibition provides a good model to study cartilage maturation and joint formation.