Patent ID: 11873509
Assignee: OCEAN UNIVERSITY OF CHINA
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
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.