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The primary culture of carp (Cyprinus carpio) macrophages and the verification of its phagocytosis activity

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Abstract

This study establishes the primary culture method for red carp (Cyprinus carpio) macrophages in vitro and lays the foundation for further research in the fish immune system. The healthy adult red carp was chosen, and mechanical separation and cell adherent culture methods were used to isolate the primary macrophages. Compared to the traditional method of Percoll discontinuous density gradient isolation, the protocol we reported here makes cell isolation steps more concise and obtains more healthy cells with high macrophage purity. The cells were uniform in size with a clearly visible nucleus. Trypan blue staining and non-radioactive cell proliferation assay were used to detect the cell survival rate. Further, we provide optimum culture conditions which include cell density (1 × 107 cells/mL), culture medium (Leibovitz’s L-15), pH (7.2–7.4), temperature (26°C), and adherent time (24 h). Macrophages have been identified by nonspecific esterase and Wright-Giemsa staining and have shown to grow very well. In addition, the macrophages have a very strong bactericidal activity against three kinds of bacteria, further verifying good growth conditions and proper function.

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References

  • Aderem A, Underhill DM (1999) Mechanisms of phagocytosis in macrophages. Annu Rev Immunol 17:593–623

    Article  PubMed  CAS  Google Scholar 

  • Campbell PA, Canono BP, Drevets DA (2001) Measurement of bacterial ingestion and killing by macrophages. Curr Protoc Immunol Chapter 14, Unit 14 16

  • Chen YH, Fang SW, Jeng SS (2013) Zinc transferrin stimulates red blood cell formation in the head kidney of common carp (Cyprinus carpio). Comp Biochem Physiol A Mol Integr Physiol 166:1–7

    Article  PubMed  CAS  Google Scholar 

  • Cheng A, Wan F, Wang J, Jin Z, Xu X (2008) Macrophage immunomodulatory activity of polysaccharides isolated from Glycyrrhiza uralensis fish. Int Immunopharmacol 8:43–50

    Article  PubMed  CAS  Google Scholar 

  • Chiou P, Khoo J, Bols NC, Douglas S, Chen TT (2006) Effects of linear cationic alpha-helical antimicrobial peptides on immune-relevant genes in trout macrophages. Dev Comp Immunol 30:797–806

    Article  CAS  Google Scholar 

  • Chong L (2001) Molecular cloning—a laboratory manual, 3rd edition. Science 292:446–446

    Article  CAS  Google Scholar 

  • Cossarinidunier M (1987) Effects of the pesticides atrazine and lindane and of manganese ions on cellular-immunity of carp, Cyprinus-Carpio. J Fish Biol 31:67–73

    Article  CAS  Google Scholar 

  • Cossins AR, Crawford DL (2005) Opinion—Fish as models for environmental genomics. Nat Rev Genet 6:324–333

    Article  PubMed  CAS  Google Scholar 

  • Elkamel AA, Hawke JP, Henk WG, Thune RL (2003) Photobacterium damselae subsp piscicida is capable of replicating in hybrid striped bass macrophages. J Aquat Anim Health 15:175–183

    Article  Google Scholar 

  • Fernandez RD, Yoshimizu M, Esura Y, Kimura T (1993) Comparative growth-response of fish cell lines in different media, temperatures, and sodium chloride concentrations. Fish Pathol 28:27–34

    Article  Google Scholar 

  • Fierro-Castro C, Barrioluengo L, Lopez-Fierro P, Razquin BE, Villena AJ (2013) Fish cell cultures as in vitro models of inflammatory responses elicited by immunostimulants. Expression of regulatory genes of the innate immune response. Fish Shellfish Immunol 35:979–987

    Article  PubMed  CAS  Google Scholar 

  • Franke F, Rahn AK, Dittmar J, Erin N, Rieger JK, Haase D, Samonte-Padilla IE, Lange J, Jakobsen PJ, Hermida M, Fernandez C, Kurtz J, Bakker TCM, Reusch TBH, Kalbe M, Scharsack JP (2014) In vitro leukocyte response of three-spined sticklebacks (Gasterosteus aculeatus) to helminth parasite antigens. Fish Shellfish Immunol 36:130–140

    Article  PubMed  CAS  Google Scholar 

  • Fryer JL, Lannan CN (1994) Three decades of fish cell culture: a current listing of cell lines derived from fishes. J Tissue Cult Methods 16:87–94

    Article  Google Scholar 

  • Ganassin RC, Bols NC (1998) Development of a monocyte/macrophage-like cell line, RTS11, from rainbow trout spleen. Fish Shellfish Immunol 8:457–476

    Article  Google Scholar 

  • Gonnou S, Corm ZA (1974) The macrophage. Int Rev Cytol 36:171

    Google Scholar 

  • Haley PJ, Muggenburg BA, Rebar AH, Shopp GM, Bice DE (1989) Bronchoalveolar lavage cytology in cynomolgus monkeys and identification of cytologic alterations following sequential saline lavage. Vet Pathol 26:265–273

    Article  PubMed  CAS  Google Scholar 

  • Hightower LE, Renfro JL (1988) Recent applications of fish cell culture to biomedical research. J Exp Zool 248:290–302

    Article  PubMed  CAS  Google Scholar 

  • Hong S, Li R, Xu Q, Secombes CJ, Wang T (2013) Two types of TNF-alpha exist in teleost fish: phylogeny, expression, and bioactivity analysis of type-II TNF-alpha3 in rainbow trout Oncorhynchus mykiss. J Immunol 191:5959–5972

    Article  PubMed  CAS  Google Scholar 

  • Inaba K, Inaba M, Romani N, Aya H, Deguchi M, Ikehara S, Muramatsu S, Steinman RM (1992) Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J Exp Med 176:1693–1702

    Article  PubMed  CAS  Google Scholar 

  • Ishida Y, Tertinegg I, Heersche JNM (1996) Progesterone and dexamethasone stimulate proliferation and differentiation of osteoprogenitors and progenitors for adipocytes and macrophages in cell populations derived from adult rat vertebrae. J Bone Miner Res 11:921–930

    Article  PubMed  CAS  Google Scholar 

  • Joerink M, Ribeiro CMS, Stet RJM, Hermsen T, Savelkoul HFJ, Wiegertjes GF (2006) Head kidney-derived macrophages of common carp (Cyprinus carpio L.) show plasticity and functional polarization upon differential stimulation. J Immunol 177:61–69

    Article  PubMed  CAS  Google Scholar 

  • Koren HS, Handwerger BS, Wunderlich JR (1975) Identification of macrophage-like characteristics in a cultured murine tumor line. J Immunol 114:894–897

    PubMed  CAS  Google Scholar 

  • Koski IR, Poplack DG, Blaese RM (1976) A nonspecific esterase stain for the identification of monocytes and macrophages. In vitro methods Cell-mediated. Tumor Immuni 2:359–362

    Google Scholar 

  • Li SZ, Sun Y, Liang CP, Thorp EB, Han S, Jehle AW, Saraswathi V, Pridgen B, Kanter JE, Li R, Welch CL, Hasty AH, Bornfeldt KE, Breslow JL, Tabas I, Tall AR (2009) Defective phagocytosis of apoptotic cells by macrophages in atherosclerotic lesions of ob/ob mice and reversal by a fish oil diet. Circ Res 105:1072–U1082

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Luft FC, Dechend R, Muller DN (2012) Immune mechanisms in angiotensin II-induced target-organ damage. Ann Med 44:S49–S54

    Article  PubMed  CAS  Google Scholar 

  • Meseguer J, Lopez-Ruiz A, Angeles Esteban M (1994) Cytochemical characterization of leucocytes from the seawater teleost, gilthead seabream (Sparus aurata L.). Histochemistry 102:37–44

    Article  PubMed  CAS  Google Scholar 

  • Miller NW, Mckinney EC (2012) In vitro culture of fish leukocytes. Anal Tech 341

  • Murray PJ, Wynn TA (2011) Protective and pathogenic functions of macrophage subsets. Nat Rev Immunol 11:723–737

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Ndong D, Chen YY, Lin YH, Vaseeharan B, Chen JC (2007) The immune response of tilapia Oreochromis mossambicus and its susceptibility to Streptococcus iniae under stress in low and high temperatures. Fish Shellfish Immunol 22:686–694

    Article  PubMed  CAS  Google Scholar 

  • Neumann NF, Barreda DR, Belosevic M (2000) Generation and functional analysis of distinct macrophage sub-populations from goldfish (Carassius auratus L.) kidney leukocyte cultures. Fish Shellfish Immunol 10:1–20

    Article  PubMed  CAS  Google Scholar 

  • Ottinger CA, Johnson SC, Ewart KV, Brown LL, Ross NW (1999) Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 123:53–59

    Article  PubMed  CAS  Google Scholar 

  • Ribas JL, da Silva CA, de Andrade L, Galvan GL, Cestari MM, Trindade ES, Zampronio AR, de Assis HC (2014) Effects of anti-inflammatory drugs in primary kidney cell culture of a freshwater fish. Fish Shellfish Immunol 40:296–303

    Article  PubMed  CAS  Google Scholar 

  • Secombes CJ (1990) Isolation of salmonid macrophages and analysis of their killing activity. Technol Fish Immunol 1:137–154

    Google Scholar 

  • Sharp GJE, Secombes CJ (1993) The role of reactive oxygen species in the killing of the bacterial fish pathogen Aeromonas salmonicida by rainbow trout macrophages. Fish Shellfish Immunol 3:119–129

    Article  Google Scholar 

  • Sopinska A (1987) The use of various concentrations of Ficoll for separation of leukocyte population in the carp. Med Weter 43:471–472

    Google Scholar 

  • Sotiropoulou PA, Perez SA, Salagianni M, Baxevanis CN, Papamichail M (2006) Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells 24:462–471

    Article  PubMed  Google Scholar 

  • Stafford JL, McLauchlan PE, Secombes CJ, Ellis AE, Belosevic M (2001) Generation of primary monocyte-like cultures from rainbow trout head kidney leukocytes. Dev Comp Immunol 25(5):447–459

    Article  PubMed  CAS  Google Scholar 

  • Strober W (2001) Wright-Giemsa and nonspecific esterase staining of cells. Curr Protoc Cytom A-3D

  • Tassakka ACMAR, Sakai M (2003) The in vitro effect of CpG-ODNs on the innate immune response of common carp, Cyprinus carpio L. Aquaculture 220:27–36

    Article  CAS  Google Scholar 

  • Underhill DM, Goodridge HS (2012) Information processing during phagocytosis. Nat Rev Immunol 12:492–502

    Article  PubMed  CAS  Google Scholar 

  • Wakefield JS, Gale JS, Berridge MV, Jordan TW, Ford HC (1982) Is Percoll innocuous to cells? Biochem J 202:795–797

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Xia Z, Triffitt JT (2006) A review on macrophage responses to biomaterials. Biomed Mater 1:R1–R9

    Article  PubMed  CAS  Google Scholar 

  • Xu P, Zhang X, Wang X et al (2014) Genome sequence and genetic diversity of the common carp, Cyprinus carpio. Nat Genet 46:1212–1219

    Article  PubMed  CAS  Google Scholar 

  • Yang M, Qiu W, Chen B, Chen J, Liu S, Wu M, Wang KJ (2015) The in vitro immune modulatory effect of bisphenol A on fish macrophages via estrogen receptor alpha and nuclear factor-kappaB signaling. Environ Sci Technol 49:1888–1895

    Article  PubMed  CAS  Google Scholar 

  • Yin Z, Lam TJ, Sin YM (1995) The effects of crowding stress on the non-specific immuneresponse in fancy carp (Cyprinus carpio L.). Fish Shellfish Immunol 5:519–529

    Article  Google Scholar 

  • Yoshida H, Kawane K, Koike M, Mori Y, Uchiyama Y, Nagata S (2005) Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells. Nature 437:754–758

    Article  PubMed  CAS  Google Scholar 

  • Zhou JX, Wang H, Zhu X, Li XW, Lv WL, Zhang DM (2013) The primary culture of mirror carp snout and caudal fin tissues and the isolation of Koi herpesvirus. In Vitro Cell Dev Biol Anim 49:734–742

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Nature Science foundation of China (grants 31470554, 41430644), the Program for Changjiang Scholars and Innovative Research Team in University (IRT13078), the Shanghai Municipal Education Commission (grant 14YZ001). Allan Mock and Dennis Mock of University of California at Los Angeles are thanked for their assistance in polishing the English.

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Authors and Affiliations

  1. School of Environmental and Chemical Engineering, Shanghai University, P.O. Box 144, Shangda Road 99, Shanghai, 200444, China

    Wenhui Qiu, Shuai Liu, Jingsi Chen, Lei Hu, Minghong Wu & Ming Yang

  2. School of Life Sciences, Shanghai University, Shanghai, 200444, China

    Lei Hu

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  1. Wenhui Qiu
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  2. Shuai Liu
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  3. Jingsi Chen
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  4. Lei Hu
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  5. Minghong Wu
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Correspondence to Ming Yang.

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Editor: Tetsuji Okamoto

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Qiu, W., Liu, S., Chen, J. et al. The primary culture of carp (Cyprinus carpio) macrophages and the verification of its phagocytosis activity. In Vitro Cell.Dev.Biol.-Animal 52, 10–19 (2016). https://doi.org/10.1007/s11626-015-9942-7

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  • DOI: https://doi.org/10.1007/s11626-015-9942-7

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