METHOD FOR CULTIVATING BIOMASS OF MYOBLASTS OBTAINED FROM STERLET MUSCLES Russian patent published in 2023 - IPC C12N5/71 

FIELD: biotechnology; cell biology.

SUBSTANCE: invention relates to a method for cultivating the biomass of myoblasts obtained from sterlet muscles in order to obtain a cellular meat product. The implementation of this method is carried out in three stages: at the first stage, myoblasts are isolated from the sterlet fry, at the second stage, the cultivation of myoblast cells is performed, at the third stage, the proliferation index of sterlet myoblasts is determined as the ratio of the number of grown cells to the number of seeded cells. To carry out the first stage, first a sterlet carcass is taken at the age of up to 1.5 months, sterilized, placed in sterile conditions, the skin is removed from the dorsal side, then the white muscle tissue is cut off. Next, the nutrient medium DMEM is taken and 100 u/ml penicillin and 100 µg/ml streptomycin are added to obtain culture medium. After that, the cut muscle tissue is crushed, placed in the resulting cultural nutrient medium in the ratio of muscle tissue: cultural nutrient medium = 1 : 10, and centrifuged until a precipitate is formed. The supernatant is poured out, 0.25% crab collagenase solution and 0.1% solution of trypsin-versene are added to the sediment at the rate of 1 ml of solutions per 1 g of sediment and subjected to enzymatic cleavage for 50–80 min at room temperature at a constant rocking. Next, the split muscle tissue is centrifuged 2 times, adding a pure DMEM nutrient medium, the supernatant is drained and not used further, and the precipitate is resuspended in a 0.1% trypsin-versene solution at a temperature of 37 °C and incubated for 20–22 minutes at room temperature, then centrifuged, the isolated cells of myoblasts are obtained by sedimentation. The myoblast cells are then washed away from trypsin-versene by centrifugation with DMEM nutrient medium supplemented with penicillin and streptomycin to obtain target myoblast cells. The second stage is carried out as follows. First, the growth medium L-15 with the addition of 5% FBS and 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine is added to the target myoblast cells obtained at the first stage, in a weight ratio of target cells : growth medium = 1 : 20. Next, the resulting suspension of myoblasts is sown on a culture container pre-treated with a 0.1% gelatin solution with an L-15 growth medium, the seeded cells are kept on the culture container for 30 minutes, after which the non-attached cells are drained together with the medium. Then, the attached cells are poured into new clean L-15 growth medium and incubated at 20°C. After that, when the myoblasts reach a density of 70%, sieving is carried out on cultural plastic, removing the myoblasts from the bottom of the culture container with 0.25% trypsin-versene, culturing is carried out for at least 96 hours, and the target product is obtained, consisting of the biomass of sterlet myoblasts. And the third stage is carried out as follows. First, myoblasts are removed from the bottom of the culture container with 0.25% trypsin-versene, and the number of cells in 1 ml of suspension is counted using a Goryaev camera; the myoblasts are then plated in a 24-well gelatin-coated plate and cultured at 20°C. To establish the growth rate, the grown myoblasts are removed from the surface of the culture vessel using 0.5% trypsin-versene and the number of cells in suspension is counted and the proliferation index of sterlet myoblasts is determined in order to check the quality of the target product.

EFFECT: invention makes it possible to obtain a biomass of sterlet myoblasts with a high proliferation index exceeding the proliferation index of most sturgeon myoblasts suitable for further use in the food industry.

1 cl, 2 ex