Abstract
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.
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References
Berrill NJ (1950). The Tunicata. London: Ray Society
Burighel P, Brunetti R (1971). The circulatory system in the blastozooid of the colonial ascidian Botryllus schlosseri (Pallas). Boll Zool 38: 273–289
Burighel P, Schiavinato A (1984). Degenerative regression of the digestive tract in the colonial ascidian Botryllus schlosseri (Pallas). Cell Tissue Res 235: 309–318
Corbo JC, Di Gregorio A, Levine M (2001). The ascidian as a model organism in developmental and evolutionary biology. Cell 106: 535–538
Dehal P, Satou Y, Campbell RK et al. (2002). The draft genome of Ciona intestinalis: Insights into chordate and vertebrate origins. Science 298: 2157–2167
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: 277–287
Freeman G (1970). The reticuloendothelial system of tunicates. J Reticuloendothelial Soc 7: 183–194
Georges D (1985). Presence of cells resembling serotonergic elements in four species of tunicates. Cell Tissue Res 242: 341–348
Grosberg RK, Quinn JF (1986). The genetic control and consequences of kin recognition by the larvae of a colonial marine invertebrate. Nature 322: 456–459
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: 145–149
Kawamura K, Fujiwara S (1995). Establishment of cell lines from multipotent epithelial sheet in the budding tunicate, Polyandrocarpa misakiensis. Cell Struct Funct 20: 97–106
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: 622–627
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: 218–227
Lauzon RJ, Ishizuka KJ, Weissman IL (1992). A cyclical, developmentally-regulated death phenom-enon in a colonial urochordate. Dev Dyn 194: 71–83
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: 333–348
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: 115–127
Lauzon RJ, Rinkevich B, Patton CW, Weissman IL (2000). A morphological study of nonrandom senescence in a colonial urochordate. Biol Bull 198: 367–378
Magor BG, De Tomaso A, Rinkevich B, Weissman IL (1999). Allorecognition in colonial tunicates: Protection against predatory cell lineages? Immunol Rev 167: 69–79
Marshall DJ, Styan CA, Keough MJ (2002). Sperm environment affects offspring quality in broadcast spawning marine invertebrates. Ecol Letters 5: 173–176
Mukai H (1974). A histological study on the degeneration of zooids in a compound ascidian, Botryllus primigenus. Zool Magn 83: 18–23
Mukai H, Watanabe H (1976). Studies on the formation of germ cells in a compound ascidian Botryllus schlosseri Oka. J Morphol 148: 337–362
Peddie CM, Richest AC, Smith VJ (1995). Proliferation of undifferentiated blood cells from the solitary ascidian, Ciona intestinalis in vitro. Dev Comp Immunol 19: 377–387
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: 391–400
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: 9518–9522
Raftos DA, Stillman DL, Cooper EL (1990). In-vitro culture of tissue from the tunicate Styela clava. In vitro Cell Dev Biol 26A: 962–970
Rinkevich B (1999). Cell cultures from marine invertebrates: obstacles, new approaches and recent improvements. J Biotech 70: 133–153
Rinkevich B (2002). The colonial urochordate Botryllus schlosseri: from stem cells and natural tissue transplantation to issues in evolutionary ecology. BioEssays 24: 730–740
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: 3546–3550
Rinkevich B, Rabinowitz C (1993). In vitro culture of blood cells from the colonial protochordate Botryllus schlosseri. In vitro Cell Dev Biol 29A: 79–85
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 147
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: 422–424
Rinkevich B, Shlemberg Z, Fishelson L (1995). Whole-body protochordate regeneration from totipotent blood cells. Proc Natl Acad Sci USA 92: 7695–7699
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 1–9
Sawada T, Zhang J, Cooper EL (1994). Sustained viability and proliferation of hemocytes from the cultured pharynx of Styela clava. Mar Biol 119: 597–603
Scofield VL, Schlumpberger JM, West LA, Weissman IL (1982). Protochordate allorecognition is controlled by an MHC-like gene system. Nature 295: 499–502
Stoner D, Rinkevich B, Weissman IL (1999). Heritable germ and somatic cell lineage competitions in chimeric protochordates. Proc Natl Acad Sci USA 96: 9148–9153
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: 93–100
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: 1203–1210
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Rabinowitz, C., Rinkevich, B. Epithelial cell cultures from Botryllus schlosseri palleal buds: accomplishments and challenges. Methods Cell Sci 25, 137–148 (2004). https://doi.org/10.1007/s11022-004-2087-9
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DOI: https://doi.org/10.1007/s11022-004-2087-9