2.1. 2D cell culture and differentiation
C2C12 murine myoblast cell line was purchased from ATCC. C2C12 before passage number 16 was cultured on 2D flasks in proliferation medium: Dulbecco’s Modified Eagle’s Medium (DMEM, Multicell) supplemented with 10% fetal bovine serum (FBS, Multicell) and 1% penicillin-streptomycin (PS, Multicell). Pax 7+ Porcine Satellite Cells (PSC) isolated from one-week-old pig dorsal muscle 23 were kindly provided by Prof. Jianyong Han at China Agricultural University. PSCs (before passage number 6) were cultured on 0.05% collagen I-coated 2D flasks in proliferation medium: F10 medium (Multicell) supplemented with 15% FBS, 5 ng/mL FGF (Peprotech) and 1% PS. Once C2C12 and PSCs reach 90% confluence, 2D myogenesis was trigged by replacing growth medium with differentiation medium: DMEM supplemented with 5% FBS, 1% PS and 1µM Erk inhibitor (SCH 772984, Cayman). Myogenic differentiation condition was maintained for 7 days.
3T3L1 murine pre-adipocyte cell line was purchased from ATCC. 3T3L1 was cultured on 2D flasks in expansion medium: DMEM supplemented with 10% FBS and 1% PS. To induce adipogenesis, 3T3L1 was cultured to 100% confluence and kept confluent for 48 hours. Proliferation medium was switched to adipose differentiation medium: DMEM supplemented with 10% FBS, 1.0 µM Dexamethasone (Beyotime), 0.5 mM Methylisobutylxanthine (IBMX, Beyotime), and 1.0 µg/mL Insulin (Macgene). After 3 days, differentiation medium was replaced with adipocyte maintenance medium: DMEM supplemented with 10% FBS and 1.0 µg/mL Insulin. Cells were kept in maintenance medium for 7 days.
2.2. Preparation of vegetable scaffolds
Fresh plants and mushrooms were purchased in a local supermarket. For the fruit, stem and root parts of a plant, the outmost epidermis was removed and the internal part was used. Vegetables were cut longitudinally (Supplementary Fig. 4a) into 0.5–1.2 mm-thick slices using a Leica vibratome (VT1200S) and sterilized by autoclaving. Sterile slices were rinsed with deionized water three times on an orbital shaker. Plant slices were immersed in cell growth media supplemented with 1% gelatin overnight at 37°C. Mushroom slices were first coated with 2 mg/mL Dopamine hydrochloride (Solarbio) in Tris buffer (10 mM, PH 8.5, Meilun bio) for 6 hours at 60°C under constant stirring, and immersed overnight at 37°C in Tris buffer supplemented with 1% gelatin. Dopamine-coated mushroom slices were then washed with deionized water three times before use.
2.3. PH and Contact angle measurement of vegetable scaffolds
PH of as-prepared vegetable slices was measured at room temperature with a PH meter (Smart sensor, PH818M) with a spear-shaped tip to pierce into vegetable samples. At least three replicate measurements were performed for each sample. Prepared plant and mushroom slices were placed on a glass slide and dried in an oven (65°C) for 15 minutes. Water contact angle was measured using an optical contact angle measuring system (DataPhysics Instruments, OCA 15EC) at room temperature. A 1 µ L drop of deionized water was dispensed on the sample surface by a microliter syringe. Images of water droplets were captured within 10 seconds of delivery. The ellipse fitting method was used to calculate static contact angles on both sides of the droplets. At least three replicate measurements were performed for each slice.
2.4. Surface zeta potential measurement of vegetable scaffolds
As-prepared slices were cut into 2 cm long, 1 cm wide sections and loaded on a Surpass 3 electrokinetic analyzer system (Anton Paar) according to the manufacturer’s instructions. Samples were rinsed in 1mM Potassium Chloride solution three times before measurement. The streaming potential method24 was used for the direct analysis of surface zeta potential at the sample/liquid interface.
2.5. Atomic force microscopy (AFM) force measurement of vegetable scaffolds
As-prepared plant and mushroom slices were placed on thin coverslips and mounted on an inverted microscope (Zeiss Observer A1 stand). AFM indentation was performed with a Nanowizard AFM system (JPK Instruments). An AFM cantilever with a nominal spring constant of 0.06 N/m was controlled to approach the sample with a speed of 10 µm/s until it touched the sample. When the force between the cantilever and the sample reached 2 pN, the tip was controlled to detach from the sample to finish a single test for local stiffness. During the indentation process, force–displacement curves were recorded, from which the local Young’s modulus of the samples could be calculated.
2.6. Static cell culture on vegetable scaffolds
To screen appropriate scaffolds, as-prepared vegetable slices were placed in 24 well plates. C2C12 cell suspension containing about 8x106 cells/mL was added dropwise onto vegetable slices. Cells were allowed 2 hours to adhere to the materials before culture media was added.
2.7. Dynamic cell culture on Chinese chives and mushroom scaffolds in spinner flasks
A 3D FloTrix-miniSPIN platform (M1; CytoNiche Biotech, China) was installed inside a cell culture incubator and connected with a spinning rate controller (MP01; CytoNiche Biotech, China). Prepared mushroom and Chinese chives slices with a seeding area of 30 cm2 were placed in sterile 125mL spinner flask bioreactors (SF125; CytoNiche Biotech, China). Cells were harvested from T75 culture flasks and introduced in the spinner flasks at 1500, 1500, 9000 cells/mL, for C2C12, PSC, and 3T3L1 cells respectively.
Spinner flasks were programmed to perform 12 inoculation cycles. Each cycle started from 35 rpm for 5 minutes, followed by 25 rpm for 2 minutes and 1 rpm for 2 hours. After the 12 inoculation cycles, the agitation velocity was switched to a constant rate of 7 rpm. Medium was refreshed every 3–4 days.
2.8. Cell enumeration and viability assessment
Two cultured slices were sampled from each spinner flask daily to monitor cell growth. Cells were dissociated from Chinese chives and mushroom slices with Trypsin for 5 minutes at 37°C. Cell number and viability were evaluated using 0.4% Trypan Blue by Countstar software (ALIT Life Science, China).
2.9. Quantitative reverse transcription PCR (qPCR) analysis
Cultured vegetable slices with a total area of around 30 cm2 were sampled from a spinner flask. Cells were dissociated from the scaffolds with Trypsin, while vegetable debris was removed using a nylon mesh with a 22 µm pore size. Trizol reagent (Vazyme) was added to the collected cells and total RNA was extracted following the manufacturer’s instructions. cDNA was synthesized from 1 µg total RNA using reverse transcriptase (Vazyme). Gene-specific transcription was analyzed by qPCR using AceQ qPCR SYBR Green Master Mix (Vazyme) on a CFX96 instrument (Bio-Rad). All genes were normalized to GAPDH and relative expression levels were evaluated using the 2−ΔΔCT method. Primers are listed in Supplementary Table 4.
2.10. Live-dead and lipid droplet staining
For live-dead cell staining, cultured slices were sampled from the spinner flasks and incubated with Calcein AM and Propidium Iodide dye (Wako, Japan) at 1:1000 dilution in PBS at 37°C for 30 min. Lipid droplets in differentiated 3T3L1 slices were stained with a BODIPY Lipid probe (Thermo Fisher) at 1:1000 dilution in PBS at 37°C for 20 min. Samples were imaged with a Nikon Eclipse fluorescent microscope.
2.11. Immunofluorescent staining
Cultured slices were sampled from the spinner flasks and fixed with 4% paraformaldehyde (PFA) for 30 minutes. Samples were washed three times with PBS and permeabilized in 0.2% Triton X-100 in PBS for 30 minutes. Samples were blocked with 5% bovine serum albumin (BSA, Multicell) in PBS for 1 h at room temperature. Primary antibodies (1:300, in 5% BSA solution) were added and left overnight at 4°C, then washed with PBS three times. Secondary antibodies (1:400, in PBS) were incubated for 2 h at room temperature, and then washed off with PBS. To stain the nuclei, DAPI (1:1000, in PBS) was incubated for 15 minutes at room temperature. Antibody information is listed in Supplementary Table 5. Confocal images were maximum projections of Z-stack images taken by Leica STED confocal microscope at 5 µm step intervals.
2.12. Scanning Electron Microscope (SEM) imaging
Samples were fixed with 2.5% Glutaraldehyde for 2 hours, washed with PBS 3 times, and serially dehydrated with 20%, 40%, 60%, 80%, 100% ethanol. Samples were then serially immersed in 50%, 100%, 100% tert-Butyl alcohol and freeze-dried. Prepared samples were deposited on a Si wafer and gold-coated for 90 s before SEM (FEI Quanta 200) imaging.
2.13. Characterization of vegetable micro-pattern topology and cell morphology on vegetable slices
Topological features of plant and mushroom scaffolds were characterized from SEM images using ImageJ software. Cell aspect ratio was measured from the green channel (representing live cells) of live-dead fluorescent images using ImageJ. Cell alignment angles were statistically quantified using the fast Fourier transformation (FFT) function in ImageJ 25. Briefly, the green channel of a live-dead image was converted into a grayscale FFT image, in which the original spatial-domain pixel intensity was transformed into a frequency domain. The Oval Profile plugin was applied to calculate the radial sum intensity (0-360 degree angle) in the FFT image, which was then plotted in polar coordinates to reflect cell alignment angles in the original fluorescent image. Detailed quantification method is explained in Supplementary Fig. 1.
2.14. Quantification of C2C12 differentiation and fusion indexes
C2C12 fusion and differentiation indexes were quantified based on MHC immunofluorescent staining images using the following criteria:
$$Differentiation index=\frac{Number of {MHC}^{+} nuclei}{Numer of total nuclei }$$
1
$$Fusion index=\frac{Number of {MHC}^{+} nuclei in fused myotubes}{Numer of total nuclei }$$
2
Here, a fused myotube was identified when it contained at least two MHC+ nuclei. MHC+ cells were further classified into 4 subgroups by counting the number of MHC+ nuclei in each myotube: mononucleated (unfused) cell, bi-nucleated myotubes, tri-nucleated myotubes, and myotubes with at least four nuclei.
2.15. Engineered pork-on-vegetables as dumpling stuffing
Differentiation Day-6 porcine myosatellite cells grown on Dopa-mushroom and Chinese chive scaffolds were collected from spinner flasks and washed with PBS. These tissues were then mixed and enveloped in a dumpling wrapper. The dumpling was cooked in boiled water for 10 minutes.
2.16. Nutritional evaluation of engineered pork-on-vegetables
About 3 g of differentiation Day-6 porcine myosatellite cells grown on vegetable scaffolds were harvested and freeze-dried. Nutrition values were tested by Sci-tech Innovation Quality Testing Co., Ltd (Qingdao, China)
2.17. Biofabrication of meat-on-vegetable chips
C2C12 cells were inoculated on Dopa-mushroom scaffolds in spinner flasks at 5000 cells/mL. Cultured slices were stained with live-dead dye on Day-4 to check if the scaffolds were fully covered with myoblasts. Afterward, C2C12 growth media was replaced with myogenic media to allow for myotube development. After 7 days of myogenic differentiation, fully differentiated 3T3L1 adipocytes were then inoculated onto C2C12-mushroom slices to generate meat-on-vegetable chips.
2.18. Cooking loss and taste analysis of engineered Meat-on-mushroom chips
Fresh pork loin and a commercial plant-based minced pork (Beyond Meat) were purchased from a local supermarket. Engineered C2C12-3T3L1 tissues on Dopa-mushroom scaffolds were also harvested. Around 0.4 g of each sample was weighed, grilled with sunflower oil, and weighed again. Cooking loss was calculated as:
$$Cooking loss\left(\%\right)=100 \text{X} (1-\frac{Weight after grilling}{Fresh weight })$$
3
Grilled samples were ground with deionized water in a porcelain mortar, and centrifuged at 10000 rpm for 20 minutes. The supernatant layer containing fat was discarded; the clear liquid in the middle layer was collected, and filtered through 0.45 µm nylon meshes. The filtered liquid was then diluted with deionized water to a final volume of 100 mL for taste analysis using an electronic tongue (Alpha MOS).
2.19. Statistical analysis
Statistical analyses were performed in Origin-Lab 2015 and GraphPad Prism software. For normally distributed data sets with equal variances, a two-sample t-test was used. When normal distribution and equal variance were not met, the Mann-Whitney U test was performed. P values less than 0.05 were considered statistically significant. A Principal Component Analysis (PCA) plot was created using the Clustvis web tool.