The invention discloses a layered co-culture method for
intramuscular fat cells and
skeletal muscle cells of a
mammal. The layered co-culture method comprises the steps: after washing and
cutting a longissimus tissue on the back of a butchered animal into pieces, digesting the longissimus tissue by using type-I
collagenase, and sieving a digested
mixed solution; collecting filtrate, centrifuging, respectively collecting mature fat
cell sap on the upper layer and
muscle cells on the lower layer, respectively adding a proper amount of serum-
free medium to dilute and wash, and centrifuging; adding the centrifugate on the upper layer and the
muscle cell sap on the lower layer into a culture flask with the capacity of 25cm<2>, and culturing in a culture box with the temperature of 37 DEG C and the CO2 content of 5%. The mature fat cells can float and adhere to a ceiling surface on the upper layer because of small
buoyancy, while
muscle cells sink to the bottom of the culture flask to grow. The
cell culture model can be used for layering and co-culturing the
intramuscular fat cells and the muscle cells of the same
muscle tissue of an animal in the same culture solution, so that a novel in-vitro cell
research model is provided for researching the influence of mature fat cell secreta to muscle cell differentiation or the interaction between the muscle cells and the fat cells.