• Vedrana Muzic, Ana Katusic Bojanac, Gordana Juric-Lekic, Marta Himelreich, Katarina Tupek, Ljiljana Serman, Nina Marn, Nino Sincic, Maja Vlahovic, and Floriana Bulic-Jakus.
• Ana Katusic Bojanac, Department of Medical Biology, School of Medicine, University of Zagreb, Salata 3, 10000 Zagreb, Croatia, ana.katusic@mef.hr.
• Croat. Med. J. 2013 Oct 28; 54 (5): 489495489-95.

AimTo establish an organotypic in vitro model of limb bud development to verify whether epigenetic drug and teratogen 5-azacytidine (5azaC) has an effect on limb buds independent of its effects on the placenta.MethodsFischer strain rat fore- and hindlimb buds were microsurgically isolated from 13 days old embryos and cultivated in vitro for two weeks at the air-liquid interface in Eagle's minimum essential medium (MEM) with 50% rat serum. 30 μmol of 5azaC was added to the fresh medium. Overall growth was measured by an ocular micrometer. Routine histology, immunohistochemical detection of the proliferating cell nuclear antigen (PCNA), and stereological quantification of PCNA expression were performed.ResultsAt four time points, significantly lower overall growth was detected for fore- and hindlimb bud explants cultivated with 5azaC in comparison to controls. After the culture period, numerical density of the PCNA signal for both types of limb buds was lower than for controls (P<0.001). Limb buds were initially covered by immature epithelium and contained mesenchyme, myotubes, single hemangioblasts, hemangioblast aggregates, blood islands, and capillaries. Regardless of the treatment, cartilage and epidermis differentiated, but cells and structures typical for vasculogenesis disappeared.ConclusionOur findings, obtained outside of the maternal organism, stress the importance of compromised cell proliferation for 5azaC impact on limb buds. This investigation points to the necessity to establish alternatives to in vivo research on animals using teratogenic agents.

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