Nacre formation by epithelial cell cultures from mantle of the black-lip pearl oyster, Pinctada margaritifera

Abstract

Mantle tissue from the black-lip pearl oyster, Pinctada margaritifera, was cultured in vitro using sterilized seawater supplemented with 0.1% yeast extract as the culture medium. Granular and agranular epithelial cells, hyalinocytes, and fibroblast-like cells were observed in the initial stages of culture. Epithelial cells later formed pseudopodial cell networks containing clusters of granulated cells, which upon maturation released their colored granules. These granules induced formation of nacre crystal deposits on the bottom of the culture plate. Cultures comprised of only granulated epithelial cells were established through periodic sub-culturing of mantle cells and maintained for over 18 mo in a viable condition. Reverse transcriptase PCR of cultured cells demonstrated gene expression of the shell matrix protein, nacrein. To further evaluate the functional ability of cultured granulated epithelial cells, nuclear shell beads were incubated in culture medium containing these cells to induce nacre formation on the beads. Observation of the bead surface under a stereomicroscope at periodic intervals showed the gradual formation of blackish yellow colored nacre deposits. Examination of the bead surface by scanning electron microscopy and energy dispersive X-ray analysis at periodic intervals revealed a distinct brick and mortar formation characteristic of nacre, comprised of aragonite platelets and matrix proteins. Calcium, carbon, and oxygen were the major elements in all stages examined. Our study shows that mantle epithelial cells in culture retain the ability to secrete nacre and can therefore form the basis for future studies on the biomineralization process and its application in development of sustainable pearl culture.

Cell Culture and Gene Expression Studies in Relation to Biomineralization in the Black-lip Pearl Oyster, Pinctada margaritifera

“…Results of gene expression studies of the mantle cell cultures were in agreement with that in the mantle tissue, which demonstrated that in vitro cultured mantle epithelial cells could synthesize shell matrix proteins similar to the mantle epithelia in vivo. The amplification results of nacrein are in accordance with previous reports in P. fucata by Gong et al (2008) and in P. margaritifera by Jayasankar et al (2018). Gong et al (2008) reported the expression of nacrein in mantle cell cultures of P. fucata.…”

“…Cultures with a mixture of epithelial cells, hyalinocytes, hemocytes and other cell types as previously reported for P. fucata (Dharmaraj and Suja, 2002, 2006Suja and Lakshmana Senthil, 2019), abalone mantle cells (Auzoux-Bordenave et al 2007;O'Neill et al, 2013;Suja 2003;Suja and Dharmaraj, 2005) and P. margaritifera (Jayasankar et al, 2018) were obtained from mantle explants. Morphology and cellular behaviour similar to that described in the previous cell cultures were confirmed during this study.…”

“…However, in this study a relatively simple and cost effective culture medium composed of sterilized seawater supplemented with 0.1% yeast, developed by Jayasankar et al (2018) was used successfully for culture of P. margaritifera mantle explants. Sea water, with or without added factors has been used in earlier studies too as a culture medium for invertebrate cells (Cecil, 1969;Qiao et al, 2003;Suja et al, 2017;van der Merwe et al 2010).The close resemblance of these media to natural seawater, the habitat of marine molluscs, in terms of pH and osmolarity is what makes it an attractive _________________________________________________________________________________________________ Journal of Coastal Research, Special Issue No.…”

“…The decontaminated mantle tissues were given a final rinse in SSW and minced into 2-3 mm 2 pieces. The culture procedure was performed as previously described by Jayasankar et al (2018). Briefly, tissue explants were transferred to 12-well culture plates and left undisturbed for ~10 min until the explants adhered to the bottom of the wells, following which 2 ml of SSW medium containing 0.1% kanamycin antibiotic solution (Sigma-Aldrich, St. Louis, USA), with 75µl/ml of 0.1% yeast extract (Sigma-Aldrich, St. Louis, USA) as supplement was added to each well.…”

“…Therefore, development of an in vitro culture system of viable mantle cells forms an attractive alternative for producing pearls under controlled conditions. Trials for in-vitro pearl production through tissue culture methods have been employed for the last two decades from different pearl producing molluscs (Dharmaraj and Suja, 1998, 2001, 2002, 2006Jayasankar et al, 2018;Suja, 2003Suja, , 2009Dharmaraj, 2005, 2019). However, keeping mantle epithelial cells in a continuously proliferating condition, and ensuring that they retain their shell formation functions for a long period of time are issues that need to be resolved before this application can become fully successful.…”