The global demand for meat is increasing, but traditional methods have environmental drawbacks. Cellular agriculture offers a sustainable alternative by growing meat cells in vitro. While most research focuses on muscle cells, fat is crucial for meat's taste, texture, and tenderness. Currently, vegetable oil is added to cultured meat, but it doesn't replicate the properties of real meat fat. Culturing fat cells in vitro is a better approach. A major challenge is obtaining seed cells that can proliferate stably and maintain high adipogenic potential. Adipocytes are terminally differentiated, so primary cells are used, but their efficiency declines with passages. Immortalized preadipocytes offer a solution, but existing lines often have low adipogenic efficiency or are from non-porcine sources. This study aimed to develop a highly adipogenic immortalized porcine cell line suitable for cell-cultured fat production.

Previous research has explored immortalized preadipocyte cell lines, primarily from rodents (e.g., 3T3-L1) and birds (e.g., IPC1). Several porcine cell lines with adipogenic potential have been reported, capable of maintaining proliferation over 30 passages. However, these lines generally exhibit relatively low adipogenic efficiency and haven’t been validated for cell-cultured fat production. A highly adipogenic, immortalized porcine cell line is needed to advance research and large-scale production of cell-cultured fat.

The researchers established an immortalized porcine preadipocyte cell line, ISP-4, which was created by SV40T immortalization and limiting dilution method. They optimized culture conditions (DMEM vs. DMEM/F12) for growth rate and adipogenic efficiency. They compared two adipogenic protocols: a standard "4+4" protocol (using adipogenic differentiation medium (ADM) for 4 days, then adipogenic maintenance medium (AMM) for 4 days) and a modified "2x5" protocol (using AMM only for 10 days). They also investigated adipogenesis under reduced serum conditions (2% FBS vs. 10% FBS). ISP-4 cells were cultured in 3D alginate hydrogels and textured soy protein (TSP) scaffolds. For large-scale expansion, they utilized a microbead-based upscaled culture system (3D TableTrix microcarriers in a spinner flask). Co-differentiation experiments were conducted by co-culturing ISP-4 with C2C12 myoblasts (mouse) and porcine muscle satellite cells (PMSC), using modified adipogenic and myogenic protocols. Various assays were used to assess cell growth, adipogenic differentiation (Oil Red O staining, BODIPY staining, gene expression analysis of FABP4, ADIPOQ, PLIN1), and myogenic differentiation (immunofluorescence staining, gene expression analysis of Myod1, Myog, Myh1).

ISP-4 maintained its adipogenic potential for over 40 passages. The modified "2x5" protocol, using only AMM, achieved significant lipid accumulation, reducing the need for potentially toxic chemicals in ADM. Adipogenesis occurred effectively with 2% FBS, reducing production costs. ISP-4 successfully differentiated into mature adipocytes in 3D alginate hydrogels, forming cell-cultured fat with a texture similar to real meat fat. The upscaled microcarrier culture system resulted in a 40-fold increase in cell density. In situ differentiation on edible microcarriers was also successful. Co-culturing and co-differentiation of ISP-4 with both C2C12 myoblasts and PMSCs resulted in structures resembling the marbling pattern of real meat, with the successful maturation of both adipocytes and myocytes, confirmed by gene expression analysis and immunostaining.

The findings address the need for a highly efficient and cost-effective cell line for cell-cultured fat production. The successful differentiation of ISP-4 in 3D scaffolds and on microcarriers demonstrates its suitability for large-scale production. The reduced serum requirement and simplified differentiation protocol improve food safety and reduce costs. The co-differentiation with muscle cells is a significant advancement, mimicking the structure of natural meat. The use of ISP-4 may accelerate the development of realistic and sustainable cell-cultured meat products.

This study successfully characterized ISP-4, an immortalized porcine preadipocyte cell strain, with high adipogenic potential and scalability for cell-cultured fat production. The reduced reliance on expensive and potentially hazardous chemicals, reduced serum usage, and efficient upscaling using edible microcarriers represent significant advancements. Future research should focus on creating spontaneously immortalized cell lines and further optimizing culture conditions to eliminate the use of animal-derived products entirely and achieve even higher cell densities. Investigating the co-differentiation potential with porcine muscle cells in larger scale systems is also a key next step.

The immortalization of ISP-4 cells involved the use of a lentiviral vector expressing SV40T, which could raise food safety concerns. The study still utilizes animal-derived products, specifically porcine skin gelatin for the microcarriers. The large-scale production capabilities of ISP-4 were not fully validated due to equipment limitations.