Establishment of long-term ostracod epidermal culture

Abstract

Primary crustacean cell culture was introduced in the 1960s, but to date limited cell lines have been established. Skogsbergia lerneri is a myodocopid ostracod, which has a body enclosed within a thin, durable, transparent bivalved carapace, through which the eye can see. The epidermal layer lines the inner surface of the carapace and is responsible for carapace synthesis. The purpose of the present study was to develop an in vitro epidermal tissue and cell culture method for S. lerneri. First, an optimal environment for the viability of this epidermal tissue was ascertained, while maintaining its cell proliferative capacity. Next, a microdissection technique to remove the epidermal layer for explant culture was established and finally, a cell dissociation method for epidermal cell culture was determined. Maintenance of sterility, cell viability and proliferation were key throughout these processes. This novel approach for viable S. lerneri epidermal tissue and cell culture augments our understanding of crustacean cell biology and the complex biosynthesis of the ostracod carapace. In addition, these techniques have great potential in the fields of biomaterial manufacture, the military and fisheries, for example, in vitro toxicity testing.

Keywords: Carapace; Crustacean; Culture; Epidermal; Ostracod.

Figure 1.

Ostracod dissection and carapace epidermal removal. (a) A whole ostracod indicating the compound eye, closed carapace and appendages (antennule, antenna and furca). (b) The ostracod is stabilised at the hinge and a single-edged razor blade is inserted into the opening at the furca to cut through the adductor muscle. (c) The carapace is opened and the epithelium is removed using fine microdissection needles.

Figure 2.

X-Ray microtomography reconstruction images of an adult ostracod used to understand ostracod carapace microstructure, prior to microdissection. (a) A whole adult ostracod rotated to reveal the inner body parts and appendages through the furca region of the partially open carapace. (b) Microtomography image slice at a depth of 810 μm from within a 3D image stack of an ostracod. (c) A cross-section of the ostracod bivalve displaying the layering of the carapace. C denotes ostracod carapace and E denotes underlying epidermal cell layer.

Figure 3.

Average percentage cell viability following post-dissection disinfecting steps (n = 10 per disinfectant tested at each time point). Live/dead labelling was used to assess the viability of cells following immersion of carapace halves in 2% povidone–iodine, amphotericin B or a combination of both for 0 to 4 min. Epidermal cell viability decreased with time of exposure to the disinfectants both alone and in combination. Error bars: SEM.

Figure 4.

Percentage cell viability data for epidermal explant and carapace-epithelia culture (n = 15 per culture condition at 0 wk). Cultures in the optimised medium, which comprised a base medium of M199 with supplements, 10% FBS, 0.25 μg/ml amphotericin B, 200 μg/ml penicillin–streptomycin, 10% non-essential amino acids and 2 mM GlutaMAX, showed a high proportion of live cells at up to 4 wk in epidermal explants and up to 3 wk in carapace-epithelia. Error bars: SEM.

Figure 5.

Confirmation of cells in cultured ostracod epidermal explants. Hoechst 33342 nuclear stain was used to confirm location and presence of cells after (a, e) 1, (b, f) 2, (c, g) 3 and (d, h) 4 wk in culture (a–d ×10 magnification, e–h ×40 magnification). The ostracod epidermal layer is not a flat single layer of cells and, therefore, when visualised the nuclei were in different planes and it was not possible to image all nuclei in focus.

Figure 6.

Confirmation of epidermal cell proliferation–EdU incorporation. EdU labelling in the epidermal layer of cultured carapace-epithelia following (a) 24 h and (b) 72 h of incubation. Cell proliferation was 17.0% (n = 5, SEM ± 1.8%) and 9.0% (n = 5, SEM ± 1.9%) of cells at 24 and 72 h, respectively. The ostracod carapace is a curved surface and this surface could not be flattened during sample visualisation. Therefore, when the carapace-epithelia were visualised, the nuclei were in different planes and it was not possible to image all nuclei in focus.

Figure 7.

Confirmation of epidermal cell proliferation–BrdU labelling. (a, b) Fluorescent microscopy images of Rabbit anti-BrdU (ab152095) labelling (red fluorescence) in two of the cultured carapace-epithelia samples visualised following in vitro BrdU incorporation into carapace halves cultured for 48 h. Cellular proliferation was confirmed to be 14.0% (n = 5, SEM ± 2.8%). Cell nuclei = blue following Hoechst 33342 nuclear staining.

Figure 8.

Electron microscopy of cultured epidermal tissue on adult ostracod carapaces at three culture time points (0, 1 and 2 wk, n = 3 at each time point). (a, b) At 0 wk in culture, the tissue is highly organised in structure compared with (c, d) 1 wk and (e, f) 2 wk. The ‘e’ denotes the epidermal cell layer. Images were captured at ×1200 (left-hand column) and ×2500 (right-hand column) magnification. Scale bar: 2 μm.

Figure 9.

Cell dissociation using Dispase I. (a) Cells were in clumps following a 5-min incubation in Dispase I, with tissue remnants (white arrows). At (b) 10 min (white circle) and (c) 40 min (white arrows), cells were dissociated, without clustering. Scale bars: 20 μm Corresponding live (green)/dead (red) cell labelling is shown after (d, g) 5, (e, h) 10 and (f, i) 40-min incubations in Dispase I; (g)–(i) are magnified images of (d)–(f). Scale bars: 100 μm. (j) Percentage epidermal cell viability in culture was affected by incubation time in Dispase I; greatest cell viability in fully dissociated cells was observed after a 10-min incubation. Error bars: SD. Asterisks indicate significant differences in percentage viability between 0 d in culture (*p < 0.05, **p < 0.01) and 1 d in culture (**p < 0.01).

Figure 10.

Phase-contrast images of dissociated cells adhering to substrates (a) laminin, (b) fibronectin, (c) collagen type IV, (d) chitosan, (e) Matrigel and (f) collagen type I. A larger number of cells (arrows) was observed adhered to collagen type I, while cell spreading was most apparent on collagen type IV. Scale bars: 20 μm.

Figure 11.

Ostracod epidermal cell proliferation in dissociated cells at 7 d of culture. (a, b) Proliferating cells (BrdU labelled; red fluorescence) expressed as a percentage of total cells (Hoechst-labelled nuclei; blue fluorescence) on substrates, collagen types I and IV, were quantified. Scale bars: 50 μm. (c) Cell proliferation was not found to be significantly different on these two substrates following 7 d in culture (p = 0.211). Error bars: SD.