Drone pupae extract enhances Hanwoo myosatellite cell function for cultivated meat production

Abstract

In this study, we analyzed effects of drone pupae aqueous extract powder (DEP) on proliferation and differentiation of Hanwoo myosatellite cells (HSC). Results of amino acid, vitamin, and mineral analysis of drone pupae revealed the presence of branched-chain amino acids, Glu, essential amino acids, vitamins B6, C and Mg, K, and so on. Additionally, drone pupae were shown to have an antioxidant ability. HSC were cultured for proliferation by adding 0, 10, 100, 200, and 400 μg/mL DEP to the medium. As a result of MTS analysis, DEP increased the proliferation capacity of HSC, with cell viability being significantly higher after treatment with DEP, especially when DEP was used at 100 μg/mL (p < 0.05). To measure the differentiation ability of HSC, 0 and 100 μg/mL DEP (CON, D100) were added to the medium, and cells were cultured. Myotube formation was confirmed through images using immunofluorescence staining. Fusion index and myotube area in the D100 were higher than those in the CON (p < 0.01). DEP promoted differentiation ability and myotube formation by increasing the expression of MYH2, MYOG, and DES genes and MYH2 and DES proteins in HSC. Additionally, in HSC differentiation culture, proteome expression intensity was higher in D100 than in CON. Proteins upregulated in the D100 group included Myosin, IL18, MYO1D, and so on. In conclusion, characteristics of various components present in DEP could improve the proliferation and differentiation ability of HSC. This suggests that drone pupae can be used as a functional substance to enhance muscle growth.

Keywords: Cultivated meat; Differentiation; Drone pupae extract; Functional substance; Hanwoo myosatellite cell; Proliferation.

Fig. 1.. Drone pupae extract powder manufacturing process.

Fig. 2.. Cytotoxic effects of drone pupae extract powder against H2O2-induced oxidative stress in Hanwoo myosatellite cells.

HSC were treated with 700 μM H₂O₂ for 1 h after treatment with DEP (0, 10, 100, 200, and 400 μg/mL) for 24 h. Cell viability was determined by MTS assay. ^a,bDifferent superscripts indicate statistically significant differences (p < 0.05). CON, no addition; DEP, drone pupae extract powder.

Fig. 3.. Experimental results of Hanwoo myosatellite cell proliferation include (A) images of control and treatment groups after 5 days (×40) and (B) MTS assay results showing cell viability after 1, 3, and 5 days of culture.

CON, no addition; D10, DEP 10 μg/GM 1 mL; D100, DEP 100 μg/GM 1; D200, DEP 200 μg/GM 1 mL; D400, DEP 400 μg/GM 1 mL. ^a,bDifferent superscripts indicate statistically significant differences (p < 0.05). DEP, drone pupae extract powder.

Fig. 4.. (A) Representative images of nuclei and myosin of each treatment group after 4 days of differentiation of Hanwoo myosatellite cells according to the concentration of drone pupae extract powder in DM (x10, scale bar 100 μm), (B) images of control and treatment groups after 4 days of differentiation (×40), and (C) the fusion index and myotube area of immunofluorescence-stained Hanwoo myosatellite cells.

CON, no addition; D100, DEP 100 μg/DM 1 mL. **p < 0.01. DEP, drone pupae extract powder.

Fig. 5.. Expression levels of (A) MYH2, MYOG, and DES genes assessed using RT-q-PCR, with ACTB acting as the housekeeping gene for normalization.

Following this, (B) expression levels of MYH2 and DES proteins in Hanwoo myosatellite cells were determined by Western blot analysis. Subsequently, (C) western blot results of Hanwoo myosatellite cells were validated based on band intensities, with ACTB serving as the housekeeping protein for normalization. CON, no addition; D100, DEP 100 μg /DM 1 mL. **p < 0.01, ***p < 0.001. RT-qPCR, reverse transcription and quantitative polymerase chain reaction; DEP, drone pupae extract powder.

Fig. 6.. Heat maps showing clustering of differentially expressed proteins in terms of intensity based on comparative proteomic analysis.

CON, no addition; D100, DEP 100 μg /DM 1 mL.