Since the early 20th century extracorporeal perfusion of large animal organs has been used to investigate a broad variety of research questions, thereby overcoming common draw backs of in vitro studies without suffering from ethical concerns associated with live animal research. The technique is in accordance with the 3R principles and represents an excellent opportunity to investigate in detail the physiology of organs under standardised conditions. It is also suitable for the translation of basic pre-clinical research into a more relevant arena prior to or avoiding altogether live animal research. Furthermore, organ perfusion has also been an important tool in developing methods of organ preservation for transplantation surgery. Yet due to the nature of the experiments only short term observations can be made and while cells are still exposed to their regional secretome, the whole organ itself is isolated from the body and correlations between organ-systems cannot be taken into consideration. This review gives an overview over the history of extracorporeal perfusion of large animal organs and limbs highlighting major achievements in the field and discussing different experimental set-ups. Advantages and limitations of this technique are presented. Prospective future research perspectives, which might include tracking of specific cell types and study of their distinct behaviour towards different stimuli, are given to illustrate the relevance of this method.