On large-scale culturing Ctenopharyngodon idellus cells on 

a microcarrier to proliferate grass carp reovirus genotype Ⅱ 

Abstract: Grass carp (Ctenopharyngodon idella) is one of the most important cultured fish species in China, it is appreciated by the majority of breeders and consumers for its fast growth rate, diverse feed sources and delicious meat. Grass carp hemorrhagic disease caused by grass carp reovirus genotype Ⅱ (GCRV Ⅱ) has seriously hindered the development of the grass carp farming industry with a long onset season, wide epidemic scope, rapid transmission and high mortality rate. Vaccination is the most effective method for the control of viral diseases. However, viral vaccine production requires a largescale culture of the host cells. In the current race for virus vaccine production, microcarrier and suspension culture systems are amongst the most promising techniques available. As the microcarrier bead has a large surface area for cell attachment, it can produce a larger number of cells than the conventional monolayer culture. The current challenge in GCRV Ⅱonly induces a few fish cells proliferating and does not produce cytopathic effects (CPE), making it difficult to obtain high titers of the virus. Grass carp swim bladder cells (CiSB) are sensitive to GCRV Ⅱ and can be used in the proliferation of GCRV Ⅱ.

To optimize the largescale cell culture of grass carp swim bladder cells (CiSB) for improving the antigenic content of GCRV Ⅱ, the Cytodex 1 microcarrier suspension culture system was chosen to cultivate CiSB cells and propagate GCRV Ⅱ. At the same time, the related indicators of proliferation dynamics were determined. The initial distribution of attached cells was recognized as the most critical stage for evaluating attachment efficiency. Suitable intermittent stirring could improve the contact time and also the attachment rate. During the apposition period, a speed of 30 r·min-1 and stirring for 2 min every 30 min were the best parameters for CiSB cell growth, and the cell apposition rate could reach more than 96% after 3 h. Next, in the cell proliferation stage, the conditions such as the initial inoculation density, stirring speed, microcarrier concentration and loading way of CiSB cells were explored and optimized. Results showed that when the cell density was 2×105 cells·mL-1, the cells showed good linear growth, and the highest harvest concentration was reached after 5 days. The cells proliferated quickly and the medium consumption was low when the microcarrier dosage of 2 g·L-1 was used, which provided the coverage rate of cells on the microcarrier of more than 95% and the ratio was about 20 cells per microcarrier. When the concentration of microcarrier increased to 3 g·L-1 or 5 g·L-1 , the cell density increased but more cell metabolites were also produced, leading to faster consumption of medium and harmful growth environment. In addition, a large number of damaged cells appeared later due to the shear force generated by the increased probability of collision between microcarriers. When the stirring speed was set at 30 r·min-1, the cell could obtain a stable flow culture environment, the proliferation rate was fast, and the attachment rate in the later stage of culture was good. When the agitation rate increased, the initial cell rate increased, but the increase of shear force resulted in a large number of cell damage. Adding 5% FBS to the medium and replacing the medium in half on the third day of culture could achieve the maximum yield of cells while maintaining the monolayer growth state of cells, and reduce the production cost while adding nutrients and diluting harmful metabolites. Under the optimal culture conditions, CiSB cells displayed a typical fibroblast morphology with good growth and uniform distribution, and could fully cover the microcarriers after 5 days. The cells could be dissociated from the microcarriers by treatment of preheated 0.5% trypsin containing 0.02% EDTA for 3 min in a 125 mL stirring bottle. The released and welldispersed cells were then used for scaleup culture in a 1 L bioreactor. High cell density (1.71×106 cells·mL-1) could be reached in the bioreactor with the efficient use of microcarriers, which confirmed the data obtained from the 125 mL stirring bottle. Moreover, CiSB cells were cultured under optimized parameters and infected with GCRV Ⅱ. The proliferation dynamics of GCRV were observed by measuring the copy number: the virus infection entered the logarithmic growth phase on the second day and reached a peak value (6.2×105 copy·μL-1) on the 5th day, which was 3 times higher than that in the adherent cell flask, indicating that this process was suitable for amplification of GCRV Ⅱ.

Herein, a suspension culture system of grass carp swim bladder cells （CiSB） was developed using Cytodex 1 as microcarrier in a stirred bioreactor. This can could serve as a reference for future studies on the development of largescale production of vaccines against grass carp hemorrhagic disease.

Keywords: grass carp swim bladder cells (CiSB); genotype Ⅱ grass carp reovirus; microcarrier culture; technology optimization