Materials

We obtained porcine gelatin powder produced from mild acid treatment, with bloom value ~300, from Sigma-Aldrich, St. Louis, MO, USA (Sigma G2500, CAS 9005-70-8, Type 300A). Chemical crosslinking agents obtained from Sigma were EDC (Sigma product #03450) and NHS (Sigma product #130672). Calcium-independent mTG was obtained from Modernist Pantry, Eliot, ME, USA (ActivaT1 mTG) and was used without further purification: this enzyme is supplied as a proprietary formulation with a maltodextrin support (Ajinomoto ActivaTI: 1% enzyme and 99% maltodextrin) and is reported to have a specific activity of 100 U/g.65 Unless stated otherwise, gelatin solutions were prepared by dissolving gelatin in deionized water at 50 °C. Prior to fiber spinning, solutions were maintained at 50 °C by storage in a heated water bath.

Cells

Primary RbSkMCs (Rb150-05, Lot #2430) and BAOSMCs (B354-05, Lot #1190) were obtained from a commercial vendor (Cell Applications, San Diego, CA, USA) and cultured according to the manufacturer’s recommendations.

Food products

We obtained fresh uncooked rabbit hind limbs, beef tenderloin, and ground beef from a local supplier of fresh meat products. We obtained bacon, prosciutto, cured turkey breast, and processed “fish ball” meat products from local suppliers. All meats were cut into cylindrical samples by circular biopsy punch with 1 cm diameter and ~1.5 mm thickness. To obtain fresh rabbit muscle, we isolated the gracilis muscle from the hind limb and subsequently cut samples as outlined above. These sample geometries were used for rheometry, TPA, and histological sectioning.

Shear rheology of gelatin solutions

A TA Instruments Discovery Hybrid 3 Rheometer (TA Instruments, New Castle, DE, USA) with a cone plate geometry was used to study 20% (w/w) gelatin solutions and 1:10 ratio solutions of 20% (w/w) gelatin crosslinked with 50% (w/w) mTG solutions. The cone geometry had a 60 mm diameter, 2° angle, and a 400 µm truncation gap. Both the solutions and the rheometer plate were heated to 50 °C before loading and were maintained at 50 °C during testing. Evaporation plates were used to prevent solution loss during experiments. Crosslinked samples were mixed immediately before loading onto the rheometer with minimal conditioning time before starting experiments. Non-crosslinked samples were given 60 s to equilibrate followed by 60 s of pre-shear stress at a rate of 100 s−1 prior to collecting data. Solutions were then sampled for 1 h at a 10% strain rate with points recorded every 6 s. Storage and loss moduli, and other rheological parameters, were derived from the data using manufacturer-supplied software (Trios software v4.5.0.42498, TA Instruments, New Castle, DE, USA).

Immersion rotary jet spinning

The iRJS extrudes precursor solutions into a precipitation bath through perforations in the wall of a rotating reservoir. For gelatin fiber spinning, we built custom-machined stainless steel open-top reservoirs with volume = 5 mL and dual 0.5 mm extrusion orifices in the reservoir walls. For fiber production, reservoirs were fitted to high-speed motors and gelatin was extruded from the rotating reservoir walls at a fixed rotation rate of 15 kRPM. Gelatin solutions were all prepared in deionized water and maintained at 50 °C in a water bath prior to spinning. They were then transferred to 50 mL syringe tubes and fed by controlled air pressure (10 kPa applied pressure) to the spinning iRJS reservoir at a rate of 10 mL/min for a total of 5 min per production run. In preliminary experiments, we verified gelatin fiber production using three different concentrations (4%, 10%, and 20% w/w in deionized water) in pure ethanol baths. We then varied the bath composition (ethanol:water = 100:0, 80:20, 70:30, 60:40, and 30:70) and observations by optical microscopy revealed that bath water concentrations higher than 30% led to fiber fusion and partial scaffold dissolution in the precipitation bath. We therefore spun replicate samples for three fiber-producing bath conditions (ethanol:water = 100:0, 80:20, 70:30), using 20% gelatin and a constant iRJS reservoir rotation rate of 15kRPM. For these experiments using 20% w/w gelatin solutions, three production runs were conducted for each of the three iRJS bath compositions (ethanol:water = 100:0, 80:20, 70:30). The fibrous gelatin production rate using 20% w/w gelatin solutions was ~100 g/h dry weight. For fiber collection, a circulating precipitation bath vortex was maintained during spinning using a rotating collector fixture. The spinning reservoir was lowered into the center of the bath vortex and solution was extruded through the vortex air gap into the circulating bath. Vortex circulation directed fibers to the central rotating collector, where anisotropic fiber scaffolds accumulate by spooling. Unless stated otherwise, gelatin scaffolds were removed from the collector and stored in ethanol:water storage solutions overnight. The scaffolds were then either stored in pure ethanol or crosslinked, washed, freeze-dried, and stored at −20 °C.

Gelatin fiber crosslinking

Fibers were either crosslinked enzymatically by co-spinning gelatin with mTG or chemically crosslinked using EDC-NHS. Unless stated otherwise, chemical crosslinking of gelatin fibers was done using EDC (479 mg/50 mL) and NHS (115 mg/50 mL) in pure ethanol. Chemical crosslinking was performed for 24 h, to ensure complete crosslinking of the gelatin fibers. For enzymatic crosslinking during fiber spinning, we prepared separate 20% w/w gelatin solutions and 50% w/w mTG solutions, both in deionized water maintained at 50 °C. Immediately prior to spinning, gelatin and mTG solutions were mixed at a ratio of 2:1. Based on rheological measurements, the combined gelatin:mTG solutions continue to flow for ~10–20 min after mixing.

Quantification of residual mTG

Residual mTG was quantified in gelatin fiber scaffolds before and after cell culture. Briefly, an enzyme-linked immunosorbent assay (ELISA) was used to detect mTG from Streptomyces mobaraensis (Zedira, Art# E021). Gelatin fiber scaffolds used in cell culture were centrifuged at 200 × g in 5 mL of culture media and the pellet was resuspended at a 1:5 dilution using the sample buffer provided by the manufacturer. Lyophilized gelatin fibers were hydrated in culture media, centrifuged at 200 × g, and resuspended at a 1:5 dilution using provided sample buffer. Resuspended samples were homogenized and centrifuged at 10,000 × g for 5 min. Supernatants were further diluted at a ratio of 1:10 or 1:100, and analyzed using the mTG ELISA assay according to the manufacturer’s protocol. The concentration of mTG in each supernatant was calculated using a standard curve generated by a nonlinear regression of a four-parameter function.

Gelatin fiber fractionation

To produce short-length gelatin fibers, we placed scaffolds measuring ~ 5 cm × 2 cm × 0.5 cm into a commercial blender containing pure ethanol and blended the scaffolds for 10 min using the “ice crush” setting. We transferred the crushed fibers to 50 mL falcon tubes where they were left to sediment overnight. The top fractions were then transferred by pipette to fresh storage tubes. This fractionation procedure resulted in a range of fiber lengths (~10–200 μm) suitable for dispersion on glass coverslips where cell attachment to individual fibers could be observed clearly by optical microscopy.

Fourier transform infrared spectroscopy

FT-IR spectra of gelatin powder and dried fiber scaffolds were obtained using attenuated total reflectance-FT-IR (Lumos, Bruker, MA, USA). The samples were scanned over 600–4000 cm−1 with 16 scans. For data plotting, commercially available software, OriginPro 8.6 (OriginLab Corporation, MA, USA) was used to normalize the original spectra from 0 to 1.

Scanning electron microscopy

The fibers were prepared on SEM stubs and sputter-coated with Pt/Pd (Denton Vacuum, NJ, USA) with a thickness of 5 nm. Field-emission SEM (Zeiss) was used to obtain SEM images of the fibers. Gelatin fibers used for SEM measurements were crosslinked chemically by EDC_NHS to ensure dimensional stability.

Analysis of fiber diameter and alignment

ImageJ software (NIH) with the DiameterJ and OrientationJ plug-ins was used to determine fiber diameter and alignment from the SEM images of the fibers as described in previous studies.66,67 Coherency depicts alignment ranging from 0 (no alignment) to 1 (perfect alignment).

Cell culture

Primary RbSkMC (Rb150-05, Lot #2430, 1st passage) and BAOSMCs (B354-05, Lot #1190, 2nd passage) obtained from a commercial vendor (Cell Applications, San Diego, CA, USA) were cultured according to manufacturer recommendations. Both cell types were thawed and plated in 75 cm2 TCPS flasks at a density of ~2.5 × 103 cells/cm2 (two flasks per cell vial; 0.5 M cells per vial) where they proliferated for 48 h. We passaged the cells one time by trypsinization and centrifugation, replating them at ~2.5 × 103 cells/cm2 into eight flasks (total cell number ~2.0 M cells per original 0.5 M cell vial) where they proliferated to a total volume of ~8.0 M cells. Unless stated otherwise, the resulting cells were seeded at the same density (~2.5 × 103 cells/cm2) in gelatin fiber samples contained in six-well plates. Cell counting was done using a hemocytometer. For adhesion studies, cells were seeded on sparse gelatin fibers for up to 6 days. For culture in gelatin scaffolds that were partially crosslinked enzymatically, cells were cultured for up to 6 days. For culture in chemically crosslinked gelatin scaffolds, cells were cultured for up to 28 days in scaffolds (scaffold thickness ~1.5 mm, scaffold area ~5 cm2). In all cases, the cell culture media used during the first 6 days of culture was manufacturer-supplied proliferation media, Rabbit Skeletal Muscle Cell Growth Medium Kit (Rb151K) for RbSkMC or Bovine Smooth Muscle Cell Growth Medium Kit (B311K) for BAOSMC, replenished daily. For chemically crosslinked gelatin fiber scaffolds seeded with RbSkMC, differentiation media (Rb151D) was supplied every three days for culture days 7–28.

Immunohistochemical staining and imaging

Cells were fixed by 4% paraformaldehyde with 0.05 % Triton-X 100 for 10 min. The fixed samples were washed three times by phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific, USA). The fixed cells were incubated with a primary antibody (anti-vinculin, Abcam, USA) in PBS for 2 h at room temperature, followed by three times PBS wash (10 min per each). Then, the samples were incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Molecular Probes, Thermo Fisher Scientific, USA), Alexa FluoTM 647 Phalloidin (Molecular Probes, Thermo Fisher Scientific, USA), and a secondary antibody (rabbit IgG (H + L) conjugated to Alexa Fluor® 488; Invitrogen, Thermo Fisher Scientific, USA) for 1 h at room temperature. After incubation, the samples were washed by PBS three times (10 mins per each) and mounted on glass slides for imaging. The immunostained samples were imaged by using a spinning disk confocal microscope (Olympus ix83, USA) Andor spinning disk). The 3D reconstruction of z-stacked images was performed by using Zen software (Zeiss, USA).

Cell density measurements

Cell density were determined for 2D and 3D culture using NIH’s ImageJ. Briefly, 3D z-stacks containing DAPI-stained nuclei were projected onto a single field of view, using a maximal intensity projection. To remove fiber autofluorescence, each field of view was background subtracted using a sliding paraboloid reference frame filter, which was applied evenly across each sample. Images were then converted to binaries using global intensity thresholding. To filter out noise, binary images were then “eroded” and “dilated” to remove isolated single pixels and then 2D water shed segmentation was applied to separate convolved nuclei. Each resulting nucleus was then counted using particle analysis and cell densities were normalized over the total area of the field of view. Each nucleus was also fit with an ellipse to measure the semi-major, a, and semi-minor axis, b, of the ellipse, and to determine nuclear eccentricity, ϵ, which is given by the following equation:

$${\it{ \in }} = \sqrt {1 - \frac{{b^2}}{{a^2}}}$$

Histology

Tissue morphology and structure were assessed using H&E staining. Briefly, after fixation in 4% formalin and paraffin embedding, 20 µm-thick slices were cut longitudinally from cultured samples, prosciutto, bacon, turkey, fish balls, rabbit, and ground beef with a sliding microtome at room temperature. Slices were then stained with H&E, imaged with a Leica CM1950 microscope, and processed with Cellsense software.

Shear rheology and TPA of food and cultured tissues

TPA was performed using a Discovery Hybrid 3 Rheometer (TA Instruments) with a 20 mm plate geometry on cultured samples, gelatin fiber scaffolds, and a variety of meat samples. Evaporation plates were used to prevent solution loss during experiments. Tissue-cultured samples were tested on the rheometer after 21 days in culture at 37 °C in 1 cm circular samples (~1.5 mm thick) with the rheometer pre-heated to 37 °C for experiments. Samples were then tested using either a two-cycle compression–relaxation TPA procedure or the same TPA procedure followed by frequency and amplitude mapping on raw samples, a cooking stage, post-cooked frequency and amplitude mapping, and a final TPA step. Initial loading gaps were determined by sample thickness, which was used to set vertical displacement and displacement rates so that the sample would undergo a 25% compression for 50 s, a 50 s withdrawal, a 180 s relaxation, followed by another compression-withdrawal process. Frequency and amplitude mappings were performed at 37 °C and 71 °C in cultured samples, and commercial meats were tested at 23 °C and 71 °C. Frequency maps were sampled logarithmically from 10–1 to 101 Hz at a 1% strain rate and sampled at 10 points per decade. Amplitude mapping was similar from 10–1 to 101 strain % at 10 points per decade. Cultured samples and gelatin fibers were compared to determine whether sample mechanics were scaffold dominated or whether the cultured cell types made a significant contribution to the texture properties of the material. Cultured samples were also compared with real meat samples to study what properties the synthetic samples maintained relative to commercial and butchered products. For TPA parameter analysis, we used manufacturer-supplied software (Trios software v4.5.0.42498, TA Instruments, DE, USA) to obtain values of maximum force during compression (hardness, N), the area under each force curve (N.s), and thereby estimate TPA parameter values following previously published methods.31

Statistical analysis

All data are presented as box plots with all data points overlapping. The edges of the box plots were defined as the 25th and 75th percentiles. The middle bar is the median and the whiskers are the 5th and 95th percentiles. One-way analysis of variance with the post hoc Tukey’s test in OriginPro 8.6 software (OriginLab, MA, USA) was used for statistical comparisons. Statistical significance was determined at *p < 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.