A Beefy-R culture medium: Replacing albumin with rapeseed protein isolates

Abstract

The development of cost-effective serum-free media is essential for the economic viability of cultured meat. A key challenge facing this goal is the high-cost of recombinant albumin which is necessary in many serum-free media formulations, including a recently developed serum-free medium for bovine satellite cell (BSC) culture termed Beefy-9. Here we alter Beefy-9 by replacing recombinant albumin with rapeseed protein isolate (RPI), a bulk-protein solution obtained from agricultural waste through alkali extraction (pH 12.5), isoelectric protein precipitation (pH 4.5), dissolution of physiologically soluble proteins (pH 7.2), and concentration of proteins through 3 kDa ultrafiltration. This new medium, termed Beefy-R, was then used to culture BSCs over four passages, during which cells grew with an average doubling time of 26.6 h, showing improved growth compared with Beefy-9. In Beefy-R, BSCs maintained cell phenotype and myogenicity. Together, these results offer an effective, low-cost, and sustainable alternative to albumin for serum-free culture of muscle stem cells, thereby addressing a key hurdle facing cultured meat production.

Keywords: Cellular agriculture; Cultured meat; Muscle satellite cells; Plant protein isolates; Serum-free media.

Figure 1:. Protein isolate generation.

(a) The methods used for generating OPIs involved alkali extraction, isoelectric precipitation, centrifugation, and filtration in order to generate concentrated protein solutions (50 mg/mL). The resulting OPIs were clear or reddish-brown in color, and slightly viscous. (b) Comparisons of global protein meal supply and cost, unoptimized protein yields, and OPI cost based on yields and starting protein meal cost (excluding processing costs from inputs such as chemicals, energy costs, filters, etc.). The results showed substantially reduced cost for SPI, RPI and CPI compared to IPPI. (c) SDS-PAGE of OPIs next to corresponding ladder for each protein. The results revealed heterogeneous mixtures of proteins <75 kDa, with larger average protein fractions for SPI and CPI compared with IPPI and RPI.

Figure 2:. Short-term growth in OPI-supplemented serum-free media.

(a) Four-day cell growth in B8 medium supplemented with various OPIs compared with B8 supplemented with 0.8 mg/mL recombinant albumin (Beefy-9). The results showed that SPI, RPI, and CPI significantly improved cell growth compared to no supplementation, but only RPI was able to recover cell growth comparable to Beefy-9 (1.15 times the growth of Beefy-9, not significant). n = 6 distinct samples; statistical significance was calculated by multiple unpaired t-tests between control (“0”) and OPI concentration with the most growth, as well as between OPI concentration with the most growth and Beefy-9. A Bonferroni correction was applied (α=0.025) to account for multiple comparisons. Statistical significance is indicated for p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). (b) Brightfield images of BSCs on day three of growth in Beef-R or Beefy-9 showed no difference in cell morphology between conditions. Scale bars are 200 μm.

Figure 3:. Multi-passage growth in Beefy-R.

(a) Four-passage cell growth in various media. The results showed that Beefy-R improved growth over Beefy-9, though not to the degree of BSC-GM. n = 3 distinct samples; statistical significance was calculated by two-way ANOVA and is indicated for the final timepoint with p < 0.01 (**). (b) Doubling time calculations for cell growth in (a). While growth slowed for all media types, results showed that Beefy-R maintained doubling times <24 hours for two passages, compared with one for Beefy-9 and three for BSC-GM. n = 3 distinct samples; statistical significance was calculated by two-way ANOVA and is indicated for p < 0.05 (*), p < 0.01 (**).

Figure 4:. Phenotypic analysis of cells cultured in various media.

(a) qPCR of proliferative cells (Pro.) cultured in various media, and cells following two days of differentiation (Diff.). In proliferating cells, results showed increased MyoD expression for BSC-GM and increased Myogenin expression for Beefy-R and Beefy-9. In differentiated cells, results showed increased MyoD, Myogenin, and MHC expression in Beefy-R and Beefy-9 compared with BSC-GM. n = 3 distinct samples; statistical significance was calculated by two-way ANOVA (or one-way ANOVA for Myogenin Pro. insert) and is indicated for p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****). (b) Immunostaining of proliferative cells in Beefy-R for nuclei (DAPI, blue), Pax7 (magenta), and MyoD (green). The results showed ubiquitous staining for Pax7 and heterogeneous staining for MyoD. Similar staining for Beefy-9 and BSC-GM is given in Fig. S3. Scale bars are 200 μm. (c) Pax7 quantification revealed >99% Pax7+ for all three media types (Beefy-R = 99.98%; Beefy-9 = 99.70%; BSC-GM = 99.34%. n = 12 (4 images each for 3 distinct samples). (d) Immunostaining of differentiated cells from Beefy-R for nuclei (DAPI), MHC (magenta), and actin (green). Results showed robust formation of MHC-positive multinucleated myotubes. Similar staining for Beefy-9 and BSC-GM is given in Fig. S5. Scale bars are 200 μm. (e) Fusion index analysis of differentiated myotubes revealed significantly enhanced differentiation for Beefy-R and Beefy-9 compared with BSC-GM. n = 13–15 images for one distinct sample; statistical significance was calculated by two-way ANOVA and is indicated for p < 0.01 (**).

Figure 5:. Growth with immortalized BSCs in Beefy-R prepared with multiple lots of rapeseed protein cakes.

(a) Short-term growth screen. Both lots of RPI showed improved growth compared with B8 (0 mg/mL of RPI supplementation). Lot #2 showed optimal growth at 0.2 mg/mL, and both lots showed a significant improvement over B8 at this concentration, though Lot #2 showed significantly reduced growth compared with Lot #1. n = 3 distinct samples; statistical significance was calculated by multiple unpaired t-tests between RPIs at 0.2 mg/mL, as well between RPIs of each lot at 0.2 mg/mL and B8 (0 mg/mL) controls. A Bonferroni correction was applied (α=0.025) to account for multiple comparisons. Statistical significance is indicated for p < 0.025 (*) and p < 0.001 (***). (b) Month-long immortalized BSC growth in serum-free media. The results showed that Beefy-R produced with 0.2 mg/mL of RPI from Lot #1 of rapeseed protein cakes (sourced in 2022) demonstrated improved growth compared with Beefy-R produced with Lot #2 rapeseed cakes (sourced in 2023). All serum-free media showed continuous growth over one month for iBSCs. n = 3 distinct samples.

Figure 6:. Gene ontology (GO) classification of proteins in OPIs, weighted by Normalized Spectral Abundance Factor (NSAF).

(a) GO terms of proteins classified under “Biological Process”. The results showed increased prevalence of “biological regulation,” “cellular process,” and “metabolic process” proteins in SPI and RPI compared with CPI, and increased concentrations of “developmental process,” “multicellular organismal process,” “reproduction,” and “reproductive process” proteins in CPI. (b) GO terms of proteins classified under “Cellular Component”. The results showed increased prevalence of “cytoplasm” proteins for SPI and RPI compared with CPI, increased concentrations of “endoplasmic reticulum” proteins for SPI, and increased concentrations of “membrane” proteins for CPI. (c) GO terms of proteins classified under “Molecular Function”. The results showed increased concentrations of “binding” and “catalytic activity” proteins for SPI and RPI compared with CPI, and increased concentrations of “nutrient reservoir activity” proteins for CPI.