US 11,873,509 B2
Method for continuous culture of shrimp cells
Huarong Guo, Qingdao (CN); Xin Song, Qingdao (CN); and Yang Zhou, Qingdao (CN)
Assigned to Ocean University of China, Qingdao (CN)
Filed by Ocean University of China, Qingdao (CN)
Filed on Apr. 28, 2020, as Appl. No. 16/860,315.
Claims priority of application No. 201910622127.X (CN), filed on Jul. 10, 2019.
Prior Publication US 2021/0009943 A1, Jan. 14, 2021
Int. Cl. C12N 5/07 (2010.01)
CPC C12N 5/0601 (2013.01) [C12N 2500/30 (2013.01); C12N 2500/32 (2013.01); C12N 2500/34 (2013.01); C12N 2500/60 (2013.01); C12N 2500/82 (2013.01); C12N 2501/11 (2013.01); C12N 2501/115 (2013.01); C12N 2513/00 (2013.01)] 12 Claims
 
1. A method for passage of three-dimensional (3D) cultured shrimp cells, wherein the method for passage comprises steps of: 3D culturing shrimp cells, washing the 3D cultured shrimp cells with phosphate-buffered saline (PBS) pre-cooled at 4° C., adding a cell recovery solution with an osmotic pressure that is the same as a complete medium for shrimp cells, and subsequently dissolving an Engelbreth-Holm-Swarm mouse sarcoma matrix at 0 to 4° C.; centrifuging the dissolved Engelbreth-Holm-Swarm mouse sarcoma matrix cell suspension for 5 to 10 min at 3,000×g at VC, and discarding supernatant; re-suspending pellets with the complete medium for shrimp cells, and seeding on the Engelbreth-Holm-Swarm mouse sarcoma matrix of a new culture plate for passage;
wherein the step of 3D culturing shrimp cells is performed by adding a shrimp cell suspension to culture wells with Engelbreth-Holm-Swarm mouse sarcoma matrix, replenishing the complete medium for shrimp cells, culturing in a CO2 incubator, and changing the complete medium for shrimp cells in a culture period;
wherein the culture wells with Engelbreth-Holm-Swarm mouse sarcoma matrix are prepared by adding the Engelbreth-Holm-Swarm mouse sarcoma matrix pre-cooled at 4° C. into culture wells at a proportion of 55 to 80 μL/cm2, spreading out and solidifying the Engelbreth-Holm-Swarm mouse sarcoma matrix;
the complete medium for shrimp cells is a shrimp cell basal medium supplemented with 15% fetal bovine serum, 20% shrimp ovarian extract, 20 μg/L basic fibroblastic growth factor (bFGF) and 20 μg/L epidermal growth factor (EGF) before use; a formulation of the shrimp cell basal medium comprises: 20.55 g/L Leibovitz's L-15 medium powder, 5 g/L NaCl, 2 g/L glucose, 1 g/L NaHCO3, 166.7 mg/L histidine, 50 mg/L lysine, 50 mg/L methionine, 33.3 mg/L tryptophan, 30 mg/L proline, 30 mg/L taurine, 0.25 mg/L amphotericin B, 100 IU/L penicillin, and 100 mg/L streptomycin, at a pH of 7.2 to 7.4.