Primmorphs Cryopreservation: A New Method for Long-Time Storage of Sponge Cells

Abstract

The possibility to cryopreserve cells allows for wide opportunities of flexible handling of cell cultures from different sponge species. Primmorphs model, a multicellular 3D aggregate formed by dissociated sponge cells, is considered one of the best approaches to establish sponge cell culture but, in spite of the available protocols for freezing sponge cells, there is no information regarding the ability of the latter to form primmorphs after thawing. In the present work, we demonstrate that, after a freezing and thawing cycle using dissociated Petrosia ficiformis cells as a model, cells viability was high but it was not possible to obtain primmorphs. The same protocol for cryopreservation was then used to directly freeze primmorphs. In this second case, after thawing, viability and the cellular proliferative level were similar to unfrozen standard primmorphs. Spiculogenesis in thawed primmorphs was evaluated by quantifying the silicatein gene expression level and by assaying the silica amount in the newly formed spicules, then compared with the correspondent values obtained in standard unfrozen primmorphs. Results indicate that the freezing cycle does not affect the spiculogenesis rate. Finally, the expression level of heat shock protein 70, a well-known stress marker, was assayed and the results showed no differences between frozen and unfrozen samples. These findings are likely to promote relevant improvements in sponge cell culture technique, allowing for a worldwide exchange of living biological material, paving the way for cell banking of Porifera.

Development of cultures of the marine sponge Hymeniacidon perleve for genotoxicity assessment using the alkaline comet assay

“…Cell viability for at least 10 d in culture was assessed by their ability to reduce MTT ( Figure 3A,B). After prolonged culture (3 d), structures that appeared morphologically similar ( Figure 2D) to previously reported primmorphs [27], long-lived aggregates of sponge cells that maintain physiological features such as metabolic activity, and an organized inner and outer network of cells were observed. These appeared to be stable for more than 1 wk in culture.…”

“…Levels of Cd, Cr, and Ni were below 1 μg/L as quantified by inductively coupled plasma‐mass spectrometry (ICP‐MS) in both sampled seawater and sponge tissue. Sponges were transported in aerated seawater back to the laboratory, immediately processed into single cells, and cryopreserved in vapor‐phase liquid nitrogen as described and summarized in Figure . Briefly, all solutions were prepared using ultrapure (>12 mΩ · cm) reverse osmosis water, and unless stated otherwise, all chemicals and reagents were purchased from Sigma‐Aldrich UK and were of the highest purity available.…”

Biogeography rather than association with cyanobacteria structures symbiotic microbial communities in the marine sponge Petrosia ficiformis

“…The marine demosponge Petrosia ficiformis (Poiret, 1789) is a sponge species found across the Mediterranean and in the Eastern Atlantic (Guo et al, 1998 ). It has been the focus of diverse studies that investigated: (i) the chemistry of the sponge and its associated microorganisms (Seidel et al, 1986 ; Bringmann et al, 2004 ; Lopez-Gresa et al, 2009 ; Pagliara and Caroppo, 2011 ), (ii) the ability to produce primmorphs (e.g., Mussino et al, 2013 ; Pozzolini et al, 2014 ), (iii) the identity of cyanobacterial symbionts (Usher et al, 2004 ; Steindler et al, 2005 ), and (iv) the molecular mechanisms underlying the interaction between sponge host and cyanobacteria (Arillo et al, 1993 ; Steindler et al, 2007 ). P. ficiformis has usually been described with two different morphs (Sarà et al, 1998 ): (i) a massive, violet-pigmented form, living in illuminated habitats harboring a dense population of intracellular cyanobacteria in the sponge cortex (the superficial layer of the sponge); and (ii) a slender pinkish or white morph, commonly found in shaded habitats (pink) and particularly in dark caves (white), where the sponges are free of phototrophic symbionts (Figure 1 ).…”

Silica-induced fibrosis: an ancient response from the early metazoans

“…Studies focusing on the elucidation of molecular mechanisms in vivo need reproducible and controlled systems, and this is extremely difficult to achieve when dealing with marine sponges, which are very difficult to rear in aquaculture; concomitantly, individuals within the same species show extreme variability in shape, pigmentation, dimensions and developmental stages. Although optimized protocols to obtain in vitro sponge cell cultures have been described (Custodio et al, 1998;Mussino et al, 2013), they overall fail on C. reniformis species as a result of a low cell/extracellular matrix ratio (not shown). However, tissue explants (fragmorphs) can be easily obtained and can be maintained successfully in vitro for several months (Nickel and Brümmer, 2003).…”