Tunable Autocrine Growth Factor Production in Skeletal Muscle Satellite Cells Using Lipid Nanoparticles for Sustainable Cellular Agriculture and Bioproduction

Introduction: The rising world population has induced unsustainable demand for growth on conventional animal protein production, already hampered by the proliferation of climate change. Cellular agriculture (CellAg) offers a method to alleviate this concern by shifting the burden of animal protein production to a cell-based bioreactor production model. However, muscle progenitor cells like bovine satellite cells (BSCs) must be cultured with growth factors such as fibroblast growth factor 2 (FGF2) to maintain proliferation, representing a major cost burden for CellAg scale-up. Recent research has established that genetically modifying BSCs to self-produce FGF2 could help reduce costs by eliminating the need for exogenous FGF2 in culture media.
Here, we propose the use of lipid nanoparticle (LNP)-delivered plasmid DNA (pDNA) to induce transient expression of FGF2, as a method to control growth factor expression and production by satellite cells, and potentially enable tunable engineered autocrine signaling in unmodified primary cells. Specifically, we hypothesize that pDNA-induced FGF2 expression will maintain BSC stemness consistent with exogenous FGF2 supplementation in media.

Materials and

Methods: We produced two LNP formulations containing SM-102 and C12-200 ionizable lipids loaded with green fluorescent protein (GFP)-expressing pDNA. LNPs were analyzed for size, polydispersity index (PDI), zeta potential, encapsulation efficiency (EE). BSCs were cultured with one of the two LNP formulations (untreated cells were used as a control) within a 96-well plate format. Cells were imaged to assess GFP expression at 0, 8, 24, and 48 hours. BSCs in 6-well plates were incubated for 24 hours and evaluated for GFP expression using flow cytometry.

Results, Conclusions, and Discussions: The two generated LNP formulations were produced as described above (N=3 technical replicates). SM-102 LNPs and C12-200 LNPs resulted in uniform average hydrodynamic diameters of 131 nm and 211 nm, respectively. Zeta potential values for SM-102 and C12-200 LNPs were 8.7 mV and 18 mV, respectively. Encapsulation efficiency of pDNA within the LNPs was ~100% for both formulations. We observed high GFP expression using both vehicles (>99% cell expression for both vehicles), but greater fluorescence intensity using SM-102 LNPs (Figure 1), which may suggest greater efficiency in transfection. Therefore, we concluded that pDNA can be effectively delivered into primary bovine satellite cells with LNPs, and that SM-102 may be the preferential vehicle for delivery of pDNA into BSCs.

The present work shows that we are able to produce LNPs loaded with pDNA for use with BSCs, a notoriously difficult cell type to transfect. Flow cytometry evaluation indicates high transfection rates for both vehicles. Current work is targeting delivery of FGF2 pDNA to BSCs, and further refinement of the LNP formulations to decrease production cost and increase efficiency. Further, we are beginning work on a techno-economic analysis to evaluate the potential costs of this approach versus conventional growth factor use in culture media, and whether pDNA-induced autocrine signaling can cut costs for CellAg at scale.

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