Co-culture of carp ( Cyprinus carpio) kidney haematopoietic cells with feeder cells resulting in long-term proliferation of T-cell lineages

To characterise fish haematopoietic stem/progenitor cells, it is necessary to develop a culture system that supports proliferation and differentiation of these cells. In the present study, we established cell lines from various tissues of carp ( Cyprinus carpio) and ginbuna ( Carassius auratus langsdorfii). By using these cell lines, we developed a culture system in which carp haematopoietic cells proliferated and were successively passaged. Cell lines from carp thymus (KoT), carp fin (KoF1) and ginbuna thymus (GTS6 and GTS9) were newly established. In addition to these cell lines, ginbuna fin (CFS) cell lines were also used as feeder layers. Kidney haematopoietic cells co-cultured with these feeder layers proliferated rapidly and were passaged over 20 times for more than 60 days. To characterise the proliferating cells, expression of marker genes for blood cell development were analysed. In the primary culture, marker genes for myeloid/erythroid progenitors ( gata1), haematopoietic stem cells ( gata2), neutrophils ( mpx/ mpo), B-cells ( IgH) and T-cells ( lck, TCRβ and gata3) were detected by reverse transcriptase polymerase chain reaction (RT-PCR). Expression of most of the genes disappeared after the third passage, only T-cell marker genes were highly expressed after passages. These results indicate that multiple blood cells developed in the primary culture and then T-cell lineages dominantly proliferated after several passages.