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

In our previous study, the cells harvested from a mixed primary culture of porcine kidney tissue (from a 6-day-old crossbred female pig) were cryopreserved (12) . The frozen porcine kidney cells were rapidly thawed at 37°C, split into three T-75 tissue culture flasks (Sumitomo Bakelite Co., Ltd., Tokyo, Japan), and cultured in growth medium composed of Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO, USA) containing 10% heatinactivated fetal bovine serum (Sanko Junyaku Co., Ltd., Tokyo, Japan), supplemented with 25 µM monothioglycerol (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 10 µg/ml insulin (Sigma), 100 µg/ml streptomycin (Life Technologies, Carlsbad, CA, USA), 100 U/ml penicillin (Life Technologies), and 5 µg/ml Fungin (InvivoGen, San Diego, CA, USA). The culture medium was replaced every 3-4 days. After around 14 days, a sheet-like mixed cell monolayer formed, and macrophage-like cells began to actively proliferate on the cell sheet. The proliferating cells were loosely attached to the cell sheet and so were harvested from the culture supernatant by centrifugation (1,500 rpm for 5 min). Since the macrophages readily attached to non-tissue culture grade plastic dishes, they were selectively isolated from the other types of cells on the basis of this feature (13) .