栉孔扇贝心脏细胞的体外培养

体外培养细胞对研究生物的资源保护、功能基因及病害发生机理与防治等均具有重要意义。然而,目前海洋双壳贝类中可以长期存活的组织细胞是有限的。本研究采用植块法启动栉孔扇贝(Chlamys farreri)心脏细胞的原代培养,通过优化培养基的方法,建立了可使其长期存活的原代培养体系。根据组织块迁出细胞的数量和细胞存活时间,确定L-15是3个培养基(L-15,M199,L-15+M199)中最适宜栉孔扇贝心脏细胞的基础培养基。通过三因子三水平正交实验,得出栉孔扇贝心脏细胞适宜的添加物组合:L-15基础培养基中添加5%胎牛血清、50 mmol/L牛磺酸和6 mmol/L Ca~(2+)。原代培养产物中以心肌细胞为主要的细胞类型,其中部分心肌细胞在原代培养14 d内可进行节奏性搏动;部分细胞可局部形成心肌束和肌管样结构;心肌细胞在体外存活时间达2个月。本研究将为栉孔扇贝基础生物学和功能基因的研究提供细胞平台。

Cell cultures from Chlamys farreri heart cells

Cells cultured in vitro play an important role in organism-resource protection and functional identification of genes, as well as mechanism exploration and prevention of animal disease. However, tissue cells that can survive for a long time in vitro are limited at present in marine shellfish. In this study, primary cultures of heart cells from the scallop Chlamys farreri were started using an explant method, and a primary culture system-which can keep the cells alive for a long time in vitro-was established using an optimizing-medium method. Effects of three basic media (L-15, M199, and L-15+M199) on the number of migrated cells from the explants and cell survival time were compared; the L-15 medium was verified to be the optimal basic medium for C. farreri heart cells. Furthermore, the optimal supplemental-factor combination for C. farreri heart cells was the L-15 medium supplemented with 5% FBS, 50 mmol/L taurine and 6 mmol/L Ca²⁺ (orthogonal experiment of three factors and three levels), in which cardiomyocytes could survive for 2 months in vitro. In this study, the results show that low concentration serum (5% FBS) was most favorable for the migration of cells from the tissue mass. With an increase in FBS concentration, the cell-migration ability was weaker, and the survival time of primary cells became shorter. After the addition of taurine, the survival time of primary cells was significantly prolonged. However, the Ca²⁺ (2 mmol/L, 4 mmol/L, and 6 mmol/L) had no significant effect on C. farreri heart-cell cultures. Perhaps the additive taurine regulated intracellular Ca²⁺ to a suitable level, resulting in the addition of Ca²⁺ without significant improvement in the primary culture. In this primary culture, most cells were cardiomyocytes; part of the cardiomyocytes beat at regular intervals within a short time, and cardiomyocytes and myotubes were formed in part of the region. This phenomenon was also reported in the primary culture of heart cells of Crassostrea gigas. This study provides a useful foundation for further studies on C. farreri basic biology and functional genes.