Animal-free edible scaffolds from soy protein isolate for the scalable production of cultured meat

Abstract

Large-scale production of cultured meat requires muscle cell culture in bioreactors, where microcarriers (MCs) support cell attachment, growth, and differentiation. However, most MCs are composed of inedible materials, requiring a cell detachment step, and/or contain animal-derived components, which are undesirable for cultured meat production. Therefore, we developed animal-free edible microcarriers based on soy protein isolate (SPI) that support muscle cell growth. SPI MCs supported cell attachment and growth similar to commercial collagen-coated dextran MCs, as bovine myoblasts expanded 24-fold over 8 days in a bioreactor. Moreover, myoblasts could differentiate into myotubes on the SPI-MCs. Importantly, SPI supported cell attachment in serum-free medium, as opposed to methacrylated gelatin (GelMA). Proteomics analysis revealed that, during SPI processing, cell adhesion peptides become available on the biomaterial, which also partially leach into the cell culture medium and replace serum components. To conclude, our study demonstrates the feasibility of growing and differentiating bovine muscle cells on edible, fully plant-based MCs, providing a scalable system for the production of cultured meat.

Keywords: Bovine myoblasts; Cultured meat; Microcarriers; Plant-based film; Proteomics; Soy protein isolate.

Graphical abstract

Fig. 1

SPI film production, sterilization and characterization A. Images of 5 wt% SPI with no glycerol after enzymatic crosslinking with 5 wt% or 10 wt% transglutaminase, and 10 wt% SPI film with 10 wt% glycerol crosslinked with 5 wt% transglutaminase, and 20 wt% SPI, without enzymatic crosslinking or glycerol. B. Cryo-SEM images of 10 wt% SPI in water heated to 90 °C (left) and 10 wt% SPI heated to 90 °C in water with 10 wt% glycerol and crosslinked through 5 wt% transglutaminase (right). C. Static contact angle (°) of fresh 10 wt% SPI 10 wt% glycerol 5 wt% transglutaminase film (SPI) and after ethanol sterilization (SPI EtOH). Significant differences are indicated on the graph (∗∗∗∗, p < 0.0001). D. Amplitude sweep of 10 wt% SPI 10 wt% glycerol 5 wt% transglutaminase film (N = 3, n = 3). E. Results of texture analysis of 10 wt% SPI 10 wt% glycerol 5 wt% transglutaminase film (N = 4, n > 3).

Fig. 2

SPI supports C2C12 cell adhesion in both serum and serum-free conditions. A. CTG assay showing relative cell viability of C2C12 cells seeded on GelMA and 10 wt% SPI 10 wt% glycerol 5 wt% transglutaminase films (SPI films) in serum-free medium (SFM) and growth medium (GM), normalized to cells (C) on NTC plastic in GM (set to 100 %) (N > 4, n > 4). B. Calcein-AM staining of live C2C12 cells on GelMA and SPI films in GM and SFM. Scale bar = 100 μm. C. CTG luminescence assay comparing relative C2C12 viability on TCP in SFM (N = 3, n = 3) and enriched medium (N = 3, n > 5) containing leached SPI components, normalized to cells on TCP in GM (C GM = 100 %) (N = 3, n > 3). Significant differences are indicated on the graphs (∗, p < 0.05; ∗∗p < 0.01; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001).

Fig. 3

SPI contains and leaches several proteins/peptides that possess one or more cell adhesion motifs. A. Amount of detected proteins per sample (SPI powder, 10 wt% SPI 10 wt% glycerol 5 wt% transglutaminase film, SPI leach) determined through proteomic and/or peptidomic analysis. B. Venn-diagram displaying the mutual detected proteins between the samples. C. Number of cell adhesion motifs (DGEA, RGD, REDV, KRSR, LDV or GRKRK) present in each sample. D. Venn-diagram displaying the mutual detected proteins that contain an RGD sequence between samples. E. Venn-diagram displaying the mutual detected proteins that contain an LDV sequence.

Fig. 4

SPI films and MCs support attachment, expansion and differentiation of bovine myoblasts. A. Metabolic activity of bovine myoblasts expanded on 10 wt% SPI 10 wt% glycerol 5 wt% transglutaminase films (SPI films) determined with resazurin assay and normalized to the metabolic activity of 3 × 10⁵ cells grown in a culture well. B. Representative images of bovine myoblast growth on SPI films over the course of 14 days. Live cells (green) are stained with Calcein AM. C. Bovine myoblast differentiation on SPI films. Myotubes (green) are detected with anti-tropomyosin antibody, nuclei (blue) are stained with Hoechst. White arrows indicate multinucleated myotubes. D. Phase contrast images of hydrated Cytodex 3 and SPI MCs. E. Representative images of bovine myoblast grown on Cytodex 3 and SPI MCs. Nuclei (blue) are stained with Hoechst solution. Live cells (green) are stained with Calcein AM solution. F. Bovine myoblast differentiation on a SPI MC. Myotubes (green) are detected with anti-tropomyosin antibody, nuclei (blue) are stained with Hoechst. G. Cell count after 8 days expansion on SPI and Cytodex 3 MCs in a spinner flask. H. Expression of CD56 in myoblasts expanded on MCs in comparison to myoblasts expanded in a standard 2D culture (on gelatin coated plastic). I. Fusion index of myoblasts expanded on MC in comparison to myoblasts expanded in a standard 2D culture (on gelatin coated plastic). For that, myoblasts were detached from MCs, seeded in culture wells and differentiated following the fusion assay protocol. Paired two-tailed test was used to compare the means. Bars represent mean ± SD. Significant differences are indicated on the graphs (ns, non-significant; ∗, p < 0.05; ∗∗p < 0.01).