Methods in Cell Science

, Volume 25, Issue 3–4, pp 137–148 | Cite as

Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges



This study focuses on recent improvement in epithelial monolayer cultures originating from whole extirpated Botryllus schlosseri (Urochordata) buds. Buds (n = 2,000) were taken at different (‘A’ to ‘D’) blastogenic stages. We tested the suitability of 35 combinations of various substrates and media on attachment, cell spread, epithelial growth frequencies and on monolayer lifespans. Under favorable conditions, cultured buds at blastogenic stages ‘B’ to ‘D’ (but not stage ‘A’) started to attach to the substrates following a 3-day transient period that leads to formation of spheres and attached monolayers. Substrate type is important for the attachment and the development of monolayers. Under various culture conditions, some of stages ‘B’ and ‘C’ buds develop (3–20 days) one or more large (1 mm diameter) spheres. Stage ‘D’ buds develop monolayers (up to 20% of buds) without going through a sphere phase. Neither spheres nor attached monolayers of epithelium were observed in stage ‘A’ bud cultures. Spheres grew at a rate of 60 μm in diameter per day using specific medium types and did not attach unless the appropriate substrate was present. When attached, epithelial monolayers expanded at a rate of 200 μm in diameter per day, for 3–15 days, and subsequently detached and died. Sixteen types of media were tested. Medium and substrate combinations were found to determine epithelial lifespan. These results revealed significant improvements in the culture of epithelial monolayers from Botryllus palleal buds. However, an early senescence of the developed epithelial sheets (up to two weeks from onset of appearance) may indicate an internal ageing clock that should be taken into consideration in future approaches.


Botryllus Cellular senescence Epithelial cells Invertebrates 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Berrill NJ (1950). The Tunicata. London: Ray SocietyGoogle Scholar
  2. 2.
    Burighel P, Brunetti R (1971). The circulatory system in the blastozooid of the colonial ascidian Botryllus schlosseri (Pallas). Boll Zool 38: 273–289Google Scholar
  3. 3.
    Burighel P, Schiavinato A (1984). Degenerative regression of the digestive tract in the colonial ascidian Botryllus schlosseri (Pallas). Cell Tissue Res 235: 309–318Google Scholar
  4. 4.
    Corbo JC, Di Gregorio A, Levine M (2001). The ascidian as a model organism in developmental and evolutionary biology. Cell 106: 535–538Google Scholar
  5. 5.
    Dehal P, Satou Y, Campbell RK et al. (2002). The draft genome of Ciona intestinalis: Insights into chordate and vertebrate origins. Science 298: 2157–2167CrossRefPubMedGoogle Scholar
  6. 6.
    De Tomaso AW, Saito Y, Ishizuka KJ, Palmeri KJ, Weissman IL (1998). Mapping the genome of a model protochordate. I. A low-resolution genetic map encompassing the fusion/histocompatibility (Fu/HC) locus of Botryllus schlosseri. Genetics 149: 277–287Google Scholar
  7. 7.
    Freeman G (1970). The reticuloendothelial system of tunicates. J Reticuloendothelial Soc 7: 183–194Google Scholar
  8. 8.
    Georges D (1985). Presence of cells resembling serotonergic elements in four species of tunicates. Cell Tissue Res 242: 341–348Google Scholar
  9. 9.
    Grosberg RK, Quinn JF (1986). The genetic control and consequences of kin recognition by the larvae of a colonial marine invertebrate. Nature 322: 456–459Google Scholar
  10. 10.
    Ilan M, Contini H, Carmeli S, Rinkevich B (1996). Progress towards cell cultures from a marine sponge that produces bioactive compounds. J Mar Biotechnol 4: 145–149Google Scholar
  11. 11.
    Kawamura K, Fujiwara S (1995). Establishment of cell lines from multipotent epithelial sheet in the budding tunicate, Polyandrocarpa misakiensis. Cell Struct Funct 20: 97–106PubMedGoogle Scholar
  12. 12.
    Khalturin K, Becker M, Rinkevich B, Bosch TCG (2003). Urochordates and the origin of natural killer cells: Identification of a CD94/NKR-P1-related receptor in blood cells of Botryllus. Proc Natl Acad Sci USA 100: 622–627Google Scholar
  13. 13.
    Lauzon RJ, Change W, Dewing S (1996). Evidence for transcriptional modulation but not acid phosphatase expression during programmed cell death in the colonial tunicate Botryllus schlosseri. Micros Res Tech 34: 218–227Google Scholar
  14. 14.
    Lauzon RJ, Ishizuka KJ, Weissman IL (1992). A cyclical, developmentally-regulated death phenom-enon in a colonial urochordate. Dev Dyn 194: 71–83Google Scholar
  15. 15.
    Lauzon RJ, Ishizuka KJ, Weissman IL (2002). Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regneration. Dev Biol 249: 333–348Google Scholar
  16. 16.
    Lauzon RJ, Patton CW, Weissman IL (1993). A morphological and immunohistochemical study of pro-grammed cell death in Botryllus schlosseri (Tunicata, Ascidiacea). Cell Tissue Res 272: 115–127Google Scholar
  17. 17.
    Lauzon RJ, Rinkevich B, Patton CW, Weissman IL (2000). A morphological study of nonrandom senescence in a colonial urochordate. Biol Bull 198: 367–378Google Scholar
  18. 18.
    Magor BG, De Tomaso A, Rinkevich B, Weissman IL (1999). Allorecognition in colonial tunicates: Protection against predatory cell lineages? Immunol Rev 167: 69–79Google Scholar
  19. 19.
    Marshall DJ, Styan CA, Keough MJ (2002). Sperm environment affects offspring quality in broadcast spawning marine invertebrates. Ecol Letters 5: 173–176Google Scholar
  20. 20.
    Mukai H (1974). A histological study on the degeneration of zooids in a compound ascidian, Botryllus primigenus. Zool Magn 83: 18–23Google Scholar
  21. 21.
    Mukai H, Watanabe H (1976). Studies on the formation of germ cells in a compound ascidian Botryllus schlosseri Oka. J Morphol 148: 337–362Google Scholar
  22. 22.
    Peddie CM, Richest AC, Smith VJ (1995). Proliferation of undifferentiated blood cells from the solitary ascidian, Ciona intestinalis in vitro. Dev Comp Immunol 19: 377–387Google Scholar
  23. 23.
    Raftos DA, Cooper EL (1991). Proliferation of lymphocyte-like cells from the solitary tunicate, Styela clava, in response to allogeneic stimuli. J Exp Zool 260: 391–400Google Scholar
  24. 24.
    Raftos DA, Cooper EL, Habicht GS, Beck G (1991). Invertebrate cytokines: tunicate cell proliferation stim-ulated by an interlukin 1 – like molecule. Proc Natl Acad Sci USA 88: 9518–9522Google Scholar
  25. 25.
    Raftos DA, Stillman DL, Cooper EL (1990). In-vitro culture of tissue from the tunicate Styela clava. In vitro Cell Dev Biol 26A: 962–970Google Scholar
  26. 26.
    Rinkevich B (1999). Cell cultures from marine invertebrates: obstacles, new approaches and recent improvements. J Biotech 70: 133–153CrossRefGoogle Scholar
  27. 27.
    Rinkevich B (2002). The colonial urochordate Botryllus schlosseri: from stem cells and natural tissue transplantation to issues in evolutionary ecology. BioEssays 24: 730–740Google Scholar
  28. 28.
    Rinkevich B, Lauzon RJ, Brown BWM, Weissman IL (1992). Evidence for a programmed life span in a colonial protochordate. Proc Natl Acad Sci USA 89: 3546–3550Google Scholar
  29. 29.
    Rinkevich B, Rabinowitz C (1993). In vitro culture of blood cells from the colonial protochordate Botryllus schlosseri. In vitro Cell Dev Biol 29A: 79–85Google Scholar
  30. 30.
    Rinkevich B, Rabinowitz C (1994). Acquiring embryo-derived cell culture and aseptic metamor-phosis of larvae from the colonial protochordate Botryllus schlosseri. Invertebrate Reprod Dev 25: 59–72. MICS ART NO. 03008 PIPS NO. 5152087 147Google Scholar
  31. 31.
    Rinkevich B, Rabinowitz C (1997). Initiation of epithelial cell cultures from palleal buds of Botryllus schlosseri, a colonial tunicate. In vitro Cell Dev Biol 33A: 422–424Google Scholar
  32. 32.
    Rinkevich B, Shlemberg Z, Fishelson L (1995). Whole-body protochordate regeneration from totipotent blood cells. Proc Natl Acad Sci USA 92: 7695–7699Google Scholar
  33. 33.
    Rinkevich B, Shlemberg Z, Fishelson L (1996). Survival budding processes in the colonial tunicate Botrylloides from the Mediterranean sea: The role of totipotent blood cells. In: Maramorosch K and Loeb MJ (eds), Invertebrate cell culture: looking towards the 21st century, pp 1–9Google Scholar
  34. 34.
    Sawada T, Zhang J, Cooper EL (1994). Sustained viability and proliferation of hemocytes from the cultured pharynx of Styela clava. Mar Biol 119: 597–603Google Scholar
  35. 35.
    Scofield VL, Schlumpberger JM, West LA, Weissman IL (1982). Protochordate allorecognition is controlled by an MHC-like gene system. Nature 295: 499–502CrossRefPubMedGoogle Scholar
  36. 36.
    Stoner D, Rinkevich B, Weissman IL (1999). Heritable germ and somatic cell lineage competitions in chimeric protochordates. Proc Natl Acad Sci USA 96: 9148–9153Google Scholar
  37. 37.
    Stoner DS, Ben-Shlomo R, Rinkevich B, Weissman IL (2002). Genetic variability of Botryllus schlosseri invasions to the east and west coasts of the U.S.A. Mar Ecol Prog Ser 243: 93–100Google Scholar
  38. 38.
    Voskoboynic A, Reznick AZ, Rinkevich B (2002). Rejuvenescence and extension of a urochordate life span following a single, acute administration of an anti-oxidant, butylated hydroxytoluene. Mech Aging Dev 123: 1203–1210Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  1. 1.Israel Oceanographic and Limnological ResearchNational Institute of OceanographyTel ShikmonaHaifaIsrael
  2. 2.Israel Oceanographic and Limnological ResearchNational Institute of OceanographyHaifaIsrael

Personalised recommendations