.png){kind=logo}
Printing Muscle-Scale Fibers from Polysaccharide-Free Soy and Pea Protein
A team at McMaster University's School of Biomedical Engineering, led by Pegah Saraf and Ravi Selvaganapathy, developed a coagulation-assisted extrusion method that keeps the final scaffold polysaccharide-free — composed of SPI or PPI.
Based on: Saraf, P., & Selvaganapathy, P. R. (2026). Coagulation-assisted extrusion 3D-printing of soy and pea protein isolate scaffolds for cultivated meat applications. Food Hydrocolloids, 183, 113193.
Protein First, Polysaccharide-Free
Cultivated meat production requires scaffolds that replicate the fibrous, muscle-scale architecture of native tissue. Fibrous structure is not cosmetic: it provides the alignment that guides myotube formation, the structural integrity to withstand contraction, and the surface area that supports nutrient exchange and cell adhesion. Native muscle fibers in species such as chicken and beef measure roughly 20–100 µm in diameter, and this microscale organization, which can contribute to tissue organization and eventually meat-like texture.
Soy protein isolate (SPI, 88–90% protein content) and pea protein isolate (PPI, 80–85%) are among the most concentrated plant-based protein sources available, and both are inherently cell-adhesive — soy carries the RGD sequence within its lunasin peptide, permitting cell attachment without additional coating. The limitation is rheological: native SPI and PPI are globular proteins that do not form continuous filaments under extrusion. Printed without modification, the ink remains with poor shape retention, and is disrupted by handling or washing.
Prior work addressing this limitation has consistently required polysaccharide reinforcement. Formulations incorporating alginate, pectin, or others improve printability but reduce the effective protein fraction of the final construct.
Formulation constraint
A team at McMaster University's School of Biomedical Engineering, led by Pegah Saraf and Ravi Selvaganapathy,,developed a coagulation-assisted extrusion method that keeps the final scaffold polysaccharide-free — composed of SPI or PPI. With no permanent additive available to reinforce the ink, printability instead had to be engineered directly into the protein itself — unfolding its native structure with urea and sodium sulfite, tuning its flow behavior through concentration and aging, and triggering near-instant aggregation the moment it left the nozzle via the coagulation bath's chemistry.
Every non-protein reagent used in the process is transient: present only while the ink is being formulated and extruded, and removed before the scaffold is exposed to cells.
Transient unfolding of the native protein
Native SPI and PPI are globular proteins, folded into compact structures rather than existing as extended chains. A folded, globular protein cannot align into a continuous fiber under shear.
To address this, the protein powder is dissolved in 8 M urea containing 1.1% (w/w) sodium sulfite. The two reagents act on different bonds. The combined effect unfolds the compact structure into an extended chain capable of aligning under the shear imposed during extrusion.
The dissolved protein is aged for three to seven days at room temperature before printing, in the same urea–sulfite solution. This step was not optional: without aging, the protein still precipitated in the coagulation bath, but only as irregular aggregates rather than continuous filaments. Aging allows the material to develop the viscosity and network uniformity required for stable filament formation.
Concentration screening and printability mapping
Establishing a printable formulation required systematic screening of protein concentration. SPI inks below 11% w/w and PPI inks below 8% w/w failed to form continuous filaments, as insufficient intermolecular interaction prevented coherent precipitation in the coagulation bath.
At 12% (SPI) and 9% (PPI), continuous fibers formed but lacked mechanical integrity and fractured during handling. Concentrations of 18% (SPI) and 15% (PPI) produced fibers that retained structural integrity after drying, and were used for all subsequent experiments.
Beyond concentration, printability was mapped directly against process parameters — flow rate and print speed — at 12, 15, and 18% w/w for each protein, with outcomes classified into four regimes: no extrusion, intermittent extrusion, stable filament formation, and wavy or buckled deposition. Among PPI concentrations, 15% produced the broadest and most robust stable-printing window; SPI required the higher 18% concentration to reach comparable robustness, consistent with its lower storage modulus (G′) at matched concentration.
Quantifying removal of urea and sulfite
After printing, the scaffold is dried and then washed repeatedly in water before any crosslinking begins — this is the step that clears out the urea used earlier to dissolve the protein. The wash worked: residual urea dropped by roughly 90%, from about 64 mg/dL down to just 4–6 mg/dL. That's already below the natural urea level found in milk (18–40 mg/dL), and far under the levels shown elsewhere to affect cells. Residual sulfite was checked two ways: quick test strips (which showed a reading below their detection limit after washing) and a more precise lab kit. That second test told the more interesting story — washed and unwashed scaffolds had almost the same low sulfite level, around 5–10 mg/L either way. In other words, washing wasn't really what removed the sulfite. Most of it was already gone by the time washing happened, cleared out earlier during centrifugation.
Fixing the fiber shape: two distinct mechanisms
The coagulation bath — a mix of citric acid, sodium sulfate, and water — grabs the extruded strand the instant it leaves the nozzle. Dropping the ink into that bath is what makes it hold a fiber shape right away — without this step, the printed strand just stays liquid and falls apart when you try to handle it. Almost immediately, the strand holds its fiber shape. But that's only a physical hold, not a chemical one. Left as-is, even scaffolds printed at the highest protein concentration tested fell apart into fragments after just one day in a cell-culture-like liquid (PBS). Coagulation gives the fiber its shape — it doesn't make it stable in water. That stability came from two separate reinforcement steps, done in sequence. First, genipin — a natural plant-derived crosslinker — forms real chemical bonds between protein chains. At high doses or long exposure, genipin is well known for turning things deep blue, which isn't ideal for something meant to look like meat. So the authors used a low dose, kept at body temperature for one to two days — enough to reinforce the structure without the strong color. Before drying, scaffolds also got a quick soak in a glycerol solution, which keeps them from turning brittle during the next step: a heat-and-vacuum treatment (65°C, 17 hours) that adds more reinforcement without any added chemicals. Neither step alone was enough — heat-and-vacuum alone made a stiff scaffold that snapped rather than bent, and low-dose genipin alone wasn't strong enough for regular handling. Combined, the two held up to repeated bending without breaking, and stayed pink instead of turning blue, unlike scaffolds treated with a higher genipin dose. A separate spectroscopy test confirmed the crosslinking was really happening at the molecular level, with the strongest signal in scaffolds that got both treatments. Soy showed a stronger crosslinking signal than pea under the same conditions — likely because soy has more of the reactive amino-acid sites that genipin needs to latch onto.
Digestibility was checked, not assumed
Because the scaffold is intended as an edible construct, the authors assessed whether crosslinking compromised gastric digestibility using a pepsin–OPA hydrolysis assay. Uncrosslinked pea and soy scaffolds already retained less hydrolysis signal than the corresponding loose protein powders (72.2% and 49.1%, respectively) — attributed to the fibrous architecture limiting enzyme access relative to a dispersed powder. Crosslinking reduced this further: DHT-only and genipin + DHT scaffolds retained 40.0% and 43.1% of the pea powder signal, and 34.3% and 35.4% of the soy powder signal. Crosslinked scaffolds retained measurable, quantifiable hydrolysis in all cases — indicating that crosslinking increased structural stability while only partially reducing apparent digestibility, rather than blocking it outright.
Biological validation
Cell studies used two lines: C2C12 murine myoblasts, for early-stage optimization, and primary bovine satellite cells (Opo-Moo-M17), as the more directly relevant model. Metabolic activity was tracked with an Alamar Blue (resazurin) assay at days 1, 3, and 7 (n = 6 biological replicates, blank-subtracted, unpaired two-tailed t-tests). Both scaffold types supported a time-dependent increase in activity for both cell types. C2C12 activity was significantly higher on PPI at every timepoint measured, with the largest difference at day 7; bovine satellite cells showed a comparable increase over time on both materials, with only minor differences between SPI and PPI, at overall lower absolute activity than C2C12.
Cell-associated biomass was estimated independently with a BCA protein assay: scaffolds were extracted in 0.1 M NaOH (60°C, 2 hours), and absorbance was converted to protein concentration using a BSA standard curve, then to an estimated cell-equivalent count using a separate calibration curve built from known cell numbers (1, 3, 6, 10, and 15 million cells, extracted under identical conditions). Background-corrected signal was higher for PPI (24.82 mg/mL extract) than SPI (23.84 mg/mL), consistent with the Alamar Blue trend. Against the cell-only calibration curve, this corresponded to estimated cell-equivalent biomass of roughly 60.6 million (PPI) and 58.2 million (SPI) per scaffold; because both values exceeded the upper range of the calibration curve, the authors report them as biomass estimates rather than direct cell counts.
Depth-resolved infiltration was quantified by dividing the ≈1000 µm-thick scaffold into top, middle, and bottom zones, cryosectioning a 100 µm sub-region from the center of each zone, and acquiring confocal Z-stacks at 10 µm intervals across five fields of view per zone, in three independent scaffold replicates. DAPI-positive nuclei were segmented and counted per stack volume. Cell density was highest in the middle zone for both materials, consistent with a two-sided seeding protocol in which cells migrate inward from both surfaces; DAPI-positive nuclei were also detected in the bottom zone, at lower density, confirming that infiltration extended cellular distribution remained depth-dependent and heterogeneous. Myogenic differentiation was confirmed by immunostaining for myosin heavy chain (MF20 antibody), which showed elongated, aligned, multinucleated structures on both SPI and PPI, for both cell types.
Soy vs. pea, side by side
The two protein sources reached broadly comparable performance, through somewhat different routes. Soy required a higher concentration to achieve comparable mechanical robustness and formed a more tightly crosslinked network under identical treatment (per FTIR). Pea supported a broader stable-printing window at lower concentration and showed higher metabolic activity in C2C12 cells at every measured timepoint (Alamar Blue assay, days 1, 3, and 7; largest difference at day 7). Bovine satellite cells showed lower overall metabolic activity than C2C12 and comparable, less scaffold-composition-sensitive activity between SPI and PPI.
What this changes?
This work opens the way for whole-cut cultivated meat constructs built without polysaccharide scaffolding — closing a specific bottleneck that had confined plant-protein bioprinting to two unsatisfying options: coarse, millimeter-scale strands with limited architectural control, or fine resolution achieved only by diluting the protein with alginate, pectin, or gum at the cost of nutritional authenticity. By showing that a fully food-grade, polysaccharide-free protein ink can be aligned and fixed into continuous, muscle-scale fibers using transient processing aids and a standard extrusion setup, the method removes that trade-off rather than managing it.
The TissueLabs Team is delighted to share projects like this reflect the dedication, effort, and passion that define quality scientific work — and seeing this research advance and produce such positive results is a source of great joy for all of us. We're cheering on the continuation of this journey and all the achievements yet to come. Congratulations to the whole team — you've earned it!
Reference
Saraf, P., & Selvaganapathy, P. R. (2026). Coagulation-assisted extrusion 3D-printing of soy and pea protein isolate scaffolds for cultivated meat applications. Food Hydrocolloids, 183, 113193. doi.org/10.1016/j.foodhyd.2026.113193
