| Marine bivalves with high commercial value are mainstay of mariculture in China.Recently,massive mortality of maricultured bivalves frequently occurs, there is an urgent need to establish cell culture system of marine bivalves to explain pathogenesis at the cellular and molecular levels for establishing methods for detection and prevention of such diseases.Cell culture facilitates cell engineering applied to breeding to breed new species with increased stress resistance,which is also one way to resolve the disease.Marine bivalves possess with rich germplasm resources.Cell lines of marine bivalves will benefit germplasm preservation for long time.The role of marine bivalves in monitoring environmental pollution also increases as environment deteriorates.The scallop C. farreri is an important mariculture bivalve along the northern coast of China. Long term culture of the scallop C.farreri in vitro can set up base for the study of immunology,pathogenesis,species conservation,genetic breeding,monitoring environment,also for the establishment of cell lines from the scallop C.farreri.Optimization of single factor of the scallop C.farreri heart primary culture was studied.L-15 medium gave the best result for cell migration from the explant and maintained heart cells for a long time in vitro,followed by L-15+M199 medium. M199 medium was the worst.5%FBS gave the best result for cell migration and maintained cells for a long time,followed by 10%FBS.15%FBS and 20%FBS were the worst.In L-15 medium combined with 5-10%FBS most cells were maintained for two weeks,some cells could be maintained for up to 50 days.Cardiomyocytes in vitro beat at regular intervals.At cell confluency,myotubes and cardiomyocytes bunches could be observed.Ultrastructure of cardiomyocytes in vitro showed myofilament in local region of the cell when cultured for morethan 10 days.Sarcoplasmic reticulum enlarged to become some vacuoles when cultured for longer time.Activity of Succinatedehydrogenase of cardiomyocytes in vitro weaken along with cell culture, while activity of alkaline phosphatase showed relatively stable than succinatedehydrogenase.Orthogonal design was designed to investigate the optimum combination of FBS, taurine,Ca2+ in L-15 medum.5%FBS benefited cells migration from the explant.As the concentration of FBS increased,the number of cells migrating from the explants decreased.Cells could survived for 1-2 weeks longer than before under 5%FBS in combined with taurine.The promoted effect of high concentration of taurine(50mM) was more noticeable,in which cell survived for 10-30 days longer than low concentration of taurine(5,20 mM),and a few cells could survived up for 60 days. Most cells maintained two weeks in vitro under 20%FBS in combined with taurine, and some cardiomyocytes with increased volume and many granules in cytoplasmic could survived for one month.In 20%FBS with high concentration taurine(50mM),a few cells showed high activity of succinatedehydrogenase,some cells lying in mitosis telophase were observed.Under the same culture condition,a few cells at early stage of development experienced proliferation.These cells gradually differentiated along culture,and degenerated with vacuoles in cytoplasmic.These cells could maintained for 41 days in vitro.In this paper,we tried to transfer SV40LT to the primary heart cells of the scallop C.farreri with the lentiviral vectors.This is the first time for using SV40LT to transform the primary cells of marine bivalves.Lentiviral vectors recombined with SV40LT infected primary heart cells cultured for 3 days,and there was no apparent cell proliferation for up to 10 days after infection.The experimental conditions of transduction of the scallop C.farreri primary heart cells with SV40LT will needed to be optimized. |
