Effects of hempseeds on cell viability and proliferation in 2D and 3D culture systems: Implications for cellular agriculture

The growing interest in cultured meat (CM) highlights the urgent need for sustainable and ethically acceptable culture media, particularly alternatives to animal-derived ingredients. In this study, we evaluated hempseed protein isolate (HSPI) as a functional supplement in both 2D and 3D cell culture systems using murine C2C12 muscle cells. Proteomic and peptidomic analyses identified 231 proteins and more than 500 endogenous peptides, including several bioactive sequences with antioxidant, ACE-inhibitory, and immunomodulatory properties. In 2D cultures, HSPI at 0.01 mg/mL supported cell viability at 24 h (110.10 ± 3.33%) and 48 h (118.93 ± 2.66%), with values comparable to the FBS control (117.83 ± 3.64% and 121.62 ± 0.81%, respectively). Morphological observations, together with the expression of Cyclin D1 and Cyclin-Dependent Kinase 4, confirmed that HSPI was able to sustain cell proliferation. Long-term supplementation in 2.5% FBS did not significantly affect cell proliferation before or after cryopreservation over a one-month period; however, it significantly increased the total antioxidant activity in cell lysates. In 3D-bioprinted constructs, HSPI maintained high cell viability over seven days when either added to the culture medium or incorporated directly into the bioink. Notably, HSPI incorporated into the bioink preserved cell viability at levels comparable to FBS even at day 7. Overall, these findings suggest that HSPI represents a promising plant-based ingredient for CM culture media, while simultaneously enhancing the antioxidant profile of cultured cells, highlighting its potential application in the development of functionalized cell-based products.