Epigenetic drug 5-azacytidine impairs proliferation of rat limb buds in an organotypic model-system in vitro

Abstract

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.

Feasibility of Cell Lines for In Vitro Co-Cultures Models for Bone Metabolism

“…Long bone cultures are supposed to be the best suitable model to investigate linear bone growth and hypertrophic ossification [70][71][72][73][74][75][76][77][78]. Therefore, almost ¾ of ex vivo bone growth studies are performed using ex vivo long bone cultures [79].…”

“…The method was then adapted for long bones of mice [70,78,[80][81][82] or chicken [71]. Preserving the surrounding soft tissue, as done in so called limb bud cultures, promised even better representation of the in vivo situation [72][73][74][75].…”

Influence of hyperthermal regimes on experimental teratoma development in vitro

“…Measures were included in the formula for ellipse area ( A  = π × major diameter × minor diameter/4). All values were normalized by the division to values of the initial measurement and used as the measure of overall growth ( A / A 0 ) as described before 31. Therefore, A / A 0 was 1 for the day 0 when the explants were plated at the air‐liquid surface.…”

Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models

“…Stem cell behavior during linear bone growth and hypertrophic ossification is best observed in the so-called long bone or limb organ cultures. In the literature, different long bone organ cultures are described (Abubakar et al 2019 ; Houston et al 2016 ; Muzic et al 2013 ; Paradis et al 2019 ; Parivar et al 2006 ; Proffit and Ackerman 1964 ; Smith et al 2013 , 2015 ; Uribe and Rosello-Diez 2019 ). Already in the 1960s, ex vivo long bone cultures have been described (Proffit and Ackerman 1964 ): proximal phalanges, metacarpal, and metatarsal bones were dissected from the paws of young rats and kept several days in culture, during which the bones grew and mineralized (Abubakar et al 2019 ; Proffit and Ackerman 1964 ).…”