Long-term Cultivation of Bovine Hoof Cells in a Novel Perfusion Chamber System

The aim of this study was to establish a co-culture of bovine hoof keratinocytes and fibroblasts in different types of perfusion chambers under defined conditions. The perfusion chamber PCS3c (Oligene, Berlin) was used to culture dermal fibroblasts and epidermal keratinocytes separated by a Millicell® Insert (Millipore, Schwalbach). In addition novel perfusion chambers developed by Dirk Hoffmann were used in combination with S & S membrane filters (Schleicher & Schuell, Dassel). First, fibroblasts were seeded onto one side of the insert/membrane and allowed to grow for 4 days. Subsequently keratinocytes were seeded onto the other side and allowed to adhere for one day. Then the chambers were connected to the tubing system with an attached 8-channel pump and a gas permeable media bag. We worked with different media and flow rates ranging from 0.035 ml/min to 0.35 ml/min. The system was run for up to 20 days. After a few days in culture the cells had grown to confluence. Then keratinocytes began to differentiate and built up stratified colonies. Within these colonies the cells showed the characteristic morphology of a stratified squamous epithelium. The use of perfusion chambers allows three-dimensional cultures to grow and survive for weeks, because of an unlimited medium supply and gas exchange. Additional, perfusion chamber systems enable the co-cultivation of fibroblasts and keratinocytes separated by membranes, which permit the exchange of molecules like growth factors. Furthermore, it is possible to perfuse the two different parts of the perfusion chamber with various and even different types of media. This provides the opportunity to add a growth factor to the medium for only one cell type and to study the effect of this factor to the other cell type. This work was supported by the European Communities under the Lamecow project QLK5-CT-2002-00969.The authors are solely responsible and the work does not necessarily represent the opinion of the European Communities.