Development and characterization of a continuous macrophage cell line, LRTM, derived from thymus of Labeo rohita (Hamilton 1822) Sanjay C. Rebello & Gaurav Rathore & Peyush Punia & Neeraj Sood & V. Elangovan Received: 8 May 2013 /Accepted: 21 July 2013 / Editor: T. Okamoto # The Society for In Vitro Biology 2013 Abstract A long-term thymic macrophage cell line from the thymus explants of Labeo rohita designated as LRTM (L. rohita thymic macrophages) was established, which has been maintained in culture for more than 1 yr. This cell line desig- nated LRTM cells have been subcultured for 70 passages. The cells shape was initially long and elongated; with subsequent passages, the cells became short and epithelial like. The cells exhibited optimum growth in L-15 containing 10% fetal bo- vine serum and also in Dulbecco’ s modified Eagle’ s medium at 37°C with 5% CO 2 and showed 85+-% viability after 12 mo storage in liquid nitrogen. In addition, cells showed nonspecific esterase and surface expression of Fc receptors for immunoglobulin G and classes I and II major histocompat- ibility complex antigens. These observations confirmed that this cell line had the morphologic and functional features as a macrophage. The cells exhibited phagocytic activity by engulfing yeast cells as well as fluorescent latex beads, which was demonstrated by scanning electron microscopy and Giemsa staining. The long-term cultured cells show rapid production of reactive oxygen and nitrogen intermediates fol- lowing stimulation with lipopolysaccharides and phorbol miristate acetate (PMA). Mostly, all the cells were alpha napthyl esterase acetate positive. After stimulation with PMA and lipopolysaccharide, cultured fish macrophages produced reactive oxygen and nitrogen intermediates. The karyotype analysis showed that these cells have a tetraploid karyotype with 100 chromosomes in each cell, indicating that they are normal L. rohita cells. Amplification, sequencing, and align- ment of fragments of two mitochondrial genes 12S rRNA from rohu confirmed that the cell line originated from L. rohita. This cell line should be useful for studying the role of thymic macrophages in differentiation and maturation of thymocytes and can be source of macrophage-specific enzymes and cyto- kines. The macrophage cell line will be invaluable in studies of pathogen/macrophage interactions, the mechanisms of macro- phage antimicrobial effector functions and the contribution of macrophages to the specific immune responses of teleosts. Keywords Labeo rohita . Cell line . Cytotoxicity . Lysozyme . Macrophage . Nitric oxide . Phagocytosis . Respiratory burst Introduction India is one of the leading countries in freshwater aquaculture. The Indian carps, namely, rohu, Labeo rohita (Hamilton), mrigal, Cirrhinus cirriosus (Bloch), and catla, Catla catla S. C. Rebello : P. Punia (*) : N. Sood National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow 226 002, Uttar Pradesh, India e-mail: ppunia@nbfgr.res.in S. C. Rebello e-mail: sanjayrebello007@gmail.com N. Sood e-mail: sood_neeraj@rediffmail.com G. Rathore Central Institute of Fisheries Education, Versova, Andheri (W), Mumbai 400061, India e-mail: rathore69@rediffmail.com V. Elangovan University Science Instrumentation Centre, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Rai Bareilly Road, Lucknow 226025, India e-mail: elango70@yahoo.com In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-013-9674-5