Novel methods to establish whole-body primary cell cultures for the cnidarians Nematostella vectensis and Pocillopora damicornis

Abstract

Cnidarians are emerging model organisms for cell and molecular biology research. However, successful cell culture development has been challenging due to incomplete tissue dissociation and contamination. In this report, we developed and tested several different methodologies to culture primary cells from all tissues of two species of Cnidaria: Nematostella vectensis and Pocillopora damicornis. In over 170 replicated cell cultures, we demonstrate that physical dissociation was the most successful method for viable and diverse N. vectensis cells while antibiotic-assisted dissociation was most successful for viable and diverse P. damicornis cells. We also demonstrate that a rigorous antibiotic pretreatment results in less initial contamination in cell cultures. Primary cultures of both species averaged 12-13 days of viability, showed proliferation, and maintained high cell diversity including cnidocytes, nematosomes, putative gastrodermal, and epidermal cells. Overall, this work will contribute a needed tool for furthering functional cell biology experiments in Cnidaria.

Figure 1

Dissociation process of N. vectensis and P. damicornis tissue. (A) MgCl₂ treated N. vectensis with an initial scalpel cut for mechanical dissociation. (B) N. vectensis tissue clump being spontaneously dissociated into cells in culture 24 h post-dissociation (pd). (C) Diverse N. vectensis cell suspension after full dissociation 48 h pd. (D) Green autofluorescent P. damicornis tissue being sloughed off of the skeleton 8 h into antibiotic-facilitated dissociation. (E) P. damicornis cells immediately after dissociation with intact bailed-out polyps (black arrowhead) and abundant zooxanthellae (white arrowhead). (F) Autofluorescent P. damicornis cells 12 h post-dissociation with various cell types. Red fluorescence indicates algal symbionts, Symbiodiniaceae.

Figure 2

Cnidarian cell culture growth and viability. (A) Distribution of N. vectensis cell culture longevity over 123 cell cultures (n = 123, binwidth = 2 days). The dashed vertical lines indicate the 95% confidence interval after a one sample t-test (11.41879, 13.39421). (B) Histogram of distributions of P. damicornis cell culture longevity over 51 cell cultures (n = 51, binwidth = 2 days). The dashed vertical lines indicate the 95% confidence interval after a one sample t-test (10.79539, 14.53794). (C,D) Comparisons of viability between culture media in N. vectensis and P. damicornis. Each grey rectangle represents the 95% confidence interval for viable days in culture for all recorded cell cultures using the listed media. Two-tailed t-test between each medium were performed, a and b represent significantly different intervals (*** = P < 0.001) (ACCM anemone cell culture medium, CCCM coral cell culture medium, FSM full strength medium, AGM anemone gentamicin medium, CGM coral gentamicin medium, FDL fish disease lab media, see supplementary figures for media formulae). (E,F) Comparison of cells counts for N. vectensis and P .damicornis maintained in seawater (11ppt seawater for N. vectensis) or cell culture media over a 14-day period. Cell counts were plotted as log10 in cells/well of culture against time (in days). Statistical significance was determined using a non-parametric Kruskal–Wallis between counts on days 0 and 7, 7 and 14, and 0 and 14 for both species. (***P < 0.001, **P < 0.01).

Figure 3

Cnidarian cell culture cell type diversity. (A) N. vectensis cells 48 h pd with several intact nematosomes (white arrowhead). (B) N. vectensis cells isolated only from the mesenteries 6 days pd. This cell suspension had mostly granulated round cells with dark vacuoles (black arrowhead) and large, occasionally mobile round clusters (white arrow). (C) Cells isolated only from the oral region 6 days pd. A diverse suspension of unidentified smaller round ectodermal cells (black arrowhead) along with cnidocytes (white arrowhead). (D) Proliferative N. vectensis cell culture 7 days pd with diverse cells of various shapes and sizes, including “pointed” round cells (white arrowhead) and globular cells, mostly unidentifiable based on morphology. (E) P. damicornis cell culture 21 days pd with low zooxanthellae counts, giving an observable variety of mostly unidentified host cnidarian cells in culture such as abundant rounded cells (arrowhead) (F) Diverse P. damicornis culture 25 days pd with smaller putative digestive round cells (arrowhead). (G) P. damicornis culture 28 days pd with 3 types of viable cnidocytes (arrows) (along with a piece of coral aragonite in frame) (H) P. damicornis culture 6 days pd with a large discharged cnidocyte (arrow).