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Procedures for the preparation and culture of 'reconstructed' rainbow trout branchial epithelia

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Methods in Cell Science

Abstract

Techniques for the in vitro 'reconstruction' of freshwater rainbow trout branchial epithelia using the primary culture of gill cells on permeable polyethylene terephthalate cell culture filter supports are described. Representing models of the freshwater fish gill, epithelia grown by two separate techniques are composed of branchial pavement cells with or without the inclusion of mitochondria-rich (MR) cells. The generation of epithelia consisting of pavement cells only (via a method called single seeded inserts = SSI) involves an initial period of flask culture during which time MR cells, that appear unable to attach to the culture flask base, are excluded from the general cell populace. Alternately, the generation of a heterogeneous epithelia consisting of both pavement cells and MR cells (via a method called double seeded inserts = DSI) is facilitated by the direct seeding of cells into cell culture filter inserts. Critical to this second procedure is the repeat seeding of filter inserts over a two day period. Repeat seeding appears to allow MR cells to nest amongst the attached cell layer generated by the first day's seeding. The use of cell culture filter supports allows free access to both the apical and basolateral compartment of the epithelium and is ideal for experimental manipulation. Cells are grown under symmetrical conditions (apical media/basolateral media) and epithelium growth is measured as a function of transepithelial resistance (TER). When the epithelia exhibit a plateau in growth they can be subjected to asymmetrical conditions (freshwater apical/media basolateral) in order to assess gill cell function as in vivo.

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References

  1. Avella M, Ehrenfeld J (1997). Fish gill respiratory cells in culture: A model for Cl-secreting epithelia. J Membrane Biol 156: 87-97.

    Google Scholar 

  2. Bastian SEP, Walton PE, Ballard FJ, et al. (1999). Transport of IGF-1 across epithelial cell monolayers. J Endocrinol 162: 361-369.

    Google Scholar 

  3. Bath RN, Eddy FB (1979). Salt and water balance in rainbow trout (Salmo gairdneri) rapidly transferred from freshwater to seawater. J Exp Biol 83: 193-202.

    Google Scholar 

  4. Burgess DW, Marshall WS, Wood CM (1998). Ionic transport by the opercular epithelia of freshwater acclimated tilapia (Oreochromis niloticus) and killifish (Fundulus heteroclitus). Comp Biochem Physiol 121A: 155-164.

    Google Scholar 

  5. Candia O, Mia AJ, Yorio T (1993). Influence of filter supports on transport characteristics of cultured A6 kidney cells. Am J Physiol 265: C1479-C1488.

    Google Scholar 

  6. Carlsson C, Pärt P (Submitted). EROD induction in rainbow trout (Oncorhynchus mykiss) gill epithelium cultured on permeable supports: Asymmetrical distribution of substrate metabolites. Aquat Toxicol.

  7. Carlsson, C, Pärt, P, Brunström, B (1999). Induction of EROD-activity in gill epithelial cells. Aquat Toxicol 47: 117-128.

    Google Scholar 

  8. Cereijido M, Meza I, Martinez-Palomo A (1981). Occluding junctions in cultured epithelial monolayers. Am J Physiol 240: C96-C102.

    Google Scholar 

  9. Cheek JM, Kim K-J, Crandall ED (1989). Tight monolayers of rat alveolar epithelial cells: Bioelectric properties and active sodium transport. Am J Physiol 256: C688-C693.

    Google Scholar 

  10. Claiborne JB (1997). Acid-base regulation. In: Evans DH (ed) The Physiology of Fishes, 2nd edition, pp 179-200. New York: CRC Press.

    Google Scholar 

  11. Dickman KG, Renfro JL (1986). Primary culture of flounder renal tubule cells: Transepithelial transport. Am J Physiol 251: F424-F432.

    Google Scholar 

  12. Fernandes MN, Eddy FB, Penrice WS (1995). Primary cell culture from gill explants of rainbow trout. J Fish Biol 47: 641-651.

    Google Scholar 

  13. Fletcher M, Kelly SP, Pärt P, et al. (2000, in press). Transport properties of cultured branchial epithelia from freshwater rainbow trout: a novel preparation with mitochondria-rich cells. J Exp Biol 203: 1523-1537.

    Google Scholar 

  14. Foskett JK, Logsdon DC, Turner T, et al. (1981). Differentiation of the chloride extrusion mechanism during seawater adaptation of a teleost fish, the cichild Sarotherodon mossambicus. J Exp Biol 93: 209-224.

    Google Scholar 

  15. Gilmour KM, Fletcher M, Pärt P (1998a). Transepithelial potential of cultured branchial epithelia from rainbow trout under symmetrical conditions. In Vitro Cell Dev Biol 34: 436-438.

    Google Scholar 

  16. Gilmour KM, Pärt P, Prunet P, et al. (1998b). Permeability and morphology of a cultured branchial epithelium from the rainbow trout during prolonged apical exposure to freshwater. J Exp Zool 281: 531-545.

    Google Scholar 

  17. Goss GG, Perry SF, Laurent (1995). Ultrastructural and morphometric studies on ion and acid-base transport processes in freshwater fish. In: Wood CM, Shuttleworth TJ (eds) Fish Physiology, vol 14, pp 257-284. Cellular and molecular approaches to fish ionic regulation. San Diego: Academic Press.

    Google Scholar 

  18. Hughes GM, Morgan M (1973). The structure of fish gills in relation to their respiratory function. Biol Rev 48: 419-475.

    Google Scholar 

  19. Jönsson M, Carlsson C, Smith R, et al. (Submitted) Effect of waterborne copper on barrier properties and EROD activity of cultured rainbow trout gill epithelia. Comp Biochem Physiol.

  20. Karnaky, KJ, Degnan, KJ and Zadunaisky, JA (1977). Chloride transport across isolated opercular epithelium of killifish: A membrane rich in chloride cells. Science 195: 203-205.

    Google Scholar 

  21. Kirschner LB (1997). Extrarenal mechanisms in hydromineral metabolism and acid-base regulation in aquatic vertebrates. In: Dantzler WH (ed) The Handbook of Physiology, pp 577-622. Bethesda MD: American Physiological Society.

    Google Scholar 

  22. Laurent P, Hebibi N (1989). Gill morphometry and fish osmoregulation. Can J Zool 67: 3055-3063.

    Google Scholar 

  23. Leguen I, Pisam M, Bidet M, et al. (1998). pHi regulation and ultrastructural analysis in cultured gill cells from freshwater and seawater-adapted trout. Fish Physiol Biochem 18: 297-309.

    Google Scholar 

  24. Leguen I, Carlsson C, Perdu-Durand E, et al. (2000, In press). Xenobiotic and steroid biotransformation activities in rainbow trout gill epithelial cells in culture. Aquat Toxicol 48: 165-176.

    Google Scholar 

  25. Marshall, WS (1977). Transepithelial potential and short-circuit current across the isolated skin of Gillichthys mirabilis (Teleostei: Gobiidae), acclimated to 5% and 100% seawater. J Comp Physiol 114B: 157-165.

    Google Scholar 

  26. Marshall, WS, Bryson SE, Wood CM (1992). Calcium transport by isolated skin of rainbow trout. J Exp Biol 166: 297-316.

    Google Scholar 

  27. Marshall, WS (1995). Transport processes in isolated epithelia: Opercular epithelium and urinary bladder. In: Wood CM, Shuttleworth TJ (eds) Fish Physiology, vol 14, Cellular and molecular approaches to fish ionic regulation, pp 1-23. San Diego: Academic Press.

    Google Scholar 

  28. Marshall, WS, Bryson SE, Burghardt JS, et al. (1995). Ca2+ transport by opercular epithelium of the freshwater adapted euryhaline teleost, Fundulus heteroclitus. J Comp Physiol 165B: 297-316.

    Google Scholar 

  29. Marshall WS, Bryson SE, Darling P, et al. (1997). NaCl transport and ultrastructure of opercular epithelium from a freshwater-adapted euryhaline teleost Fundulus heteroclitus. J Exp Zool 277: 23-37.

    Google Scholar 

  30. Marshall WS, Emberley TR, Singer TD, et al. (1999). Time course of salinity adaptation in a strongly euryhaline estuarine teleost, Fundulus heteroclitus: A multivariable approach. J Exp Biol 202: 1535-1544.

    Google Scholar 

  31. McCormick SD, Hasegawa S, Hirano T (1992). Calcium uptake in the skin of a freshwater teleost. Proc Natn Acad Sci USA 89: 3635-3638.

    Google Scholar 

  32. Pärt P, Norrgren L, Bergström E, et al. (1993). Primary cultures of epithelial cells from rainbow trout gills. J Exp Biol 175: 219-232.

    Google Scholar 

  33. Perry SF, Flik G (1988). Characterization of branchial transepithelial calcium fluxes in freshwater trout, Salmo gairdneri. Am J Physiol 1988; 254: R491-R498.

  34. Perry SF (1997). The chloride cell: Structure and 162 function in the gills of freshwater fishes. Ann Rev Physiol 59: 325-347.

    Google Scholar 

  35. Rutten MJ (1992). Use of commercially available cell culture inserts for primary culture and electrophysiological studies of guinea pig gastric mucous epithelial cells. J Tissue Cult Meth 14: 235-246.

    Google Scholar 

  36. Sandbacka M, Lilius H, Enkvist MOK, et al. (1998). Rainbow trout gill epithelial cells in primary culture communicate through gap junctions as demonstrated by dye-coupling. Fish Physiol Biochem 19: 287-292.

    Google Scholar 

  37. Sandbacka M, Pärt P, Isomaa B (1999). Gill epithelial cells as tools for toxicity screening-comparison between primary cultures, cells in suspension and epithelia on filters. Aquat Toxicol 46: 23-32.

    Google Scholar 

  38. Steele RE, Handler JS, Preston A, et al. (1992). A device for sterile measurement of transepithelial electrical parameters of cultured cells. J Tissue Cult Meth 14: 259-264.

    Google Scholar 

  39. Witters H, Berckmans P, Vangenechten C (1996). Immunolocalization of Na+-K+-ATPase in gill epithelium of rainbow trout, Oncorhynchus mykiss. Cell Tiss Res 283: 461-468.

    Google Scholar 

  40. Wood CM (1988). Acid-base and ionic exchanges at the gills and kidney after exhaustive exercise in the rainbow trout. J Exp Biol 136: 461-481.

    Google Scholar 

  41. Wood CM, Marshall WS (1994). Ion balance, acidbase regulation and chloride cell function in the common killifish, Fundulus heteroclitus-a euryhaline estuarine teleost. Estuaries 17: 34-52.

    Google Scholar 

  42. Wood, CM, Pärt, P (1997). Cultured branchial epithelia from freshwater fish gills. J Exp Biol 200: 1047-1059.

    Google Scholar 

  43. Wood CM, Gilmour KM, Pärt P (1998). Passive and active transport properties of a gill model, the cultured branchial epithelium of the freshwater rainbow trout (Oncorhynchus mykiss). Comp Biochem Physiol 119A: 87-96.

    Google Scholar 

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Authors
  1. Scott P. Kelly
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  2. Mary Fletcher
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  3. Peter Pärt
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  4. Chris M. Wood
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Kelly, S.P., Fletcher, M., Pärt, P. et al. Procedures for the preparation and culture of 'reconstructed' rainbow trout branchial epithelia. Methods Cell Sci 22, 153–163 (2000). https://doi.org/10.1023/A:1009816923262

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