Immortalization and Characterization of Porcine Macrophages That Had Been Transduced with Lentiviral Vectors Encoding the SV40 Large T Antigen and Porcine Telomerase Reverse Transcriptase

Takenouchi, Takato; Kitani, Hiroshi; Suzuki, Shunichi; Nakai, Michiko; Fuchimoto, Dai-ichiro; Tsukimoto, Mitsutoshi; Shinkai, Hiroki; Sato, Mitsuru; Uenishi, Hirohide

The cells harvested from the mixed primary culture of porcine kidney tissue were passaged twice at a split ratio of 1:10, before being passaged once more at a split ratio of 1:6. No PKM were recovered from the supernatant of the resultant porcine kidney mixed culture. Thus, the macrophage-depleted-porcine kidney mixed culture, which mostly consists of epithelial and fibroblastic cells, was used as a source of feeder cells for the isolation of porcine blood-derived macrophages. Then, based on the findings of a previous study (14) , 1.5 ml of heparinized peripheral blood that had been obtained from a 3-month-old male LDLR-KO pig (15) were directly added to the feeder cell culture in a T-75 flask, and the medium was replaced every 3-4 days. After around 10 days, blood-derived macrophage-like cells appeared among the feeder cells, actively proliferated, and were harvested from the culture supernatant by centrifugation (1,500 rpm for 5 min). The macrophages that became attached to non-tissue culture grade plastic dishes were used for the immortalization experiments.