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Stretchable zein-coated alginate fiber for aligning muscle cells to artificially produce cultivated meat – npj Science of Food

Reagents

Sodium alginate was purchased from Junsei Chemical Co. (Tokyo, Japan). A live/dead cell viability kit was purchased from Invitrogen (Carlsbad, CA, USA). Tartrazine was purchased from GreenTech (Daejeon, South Korea). Horse serum and fungizone were purchased from Gibco (Grand Island, NY, USA). Dulbecco’s Modified Eagle’s medium (DMEM), penicillin-streptomycin (P/S), fetal bovine serum (FBS), L-glutamine, and phosphate-buffered saline (PBS; pH 7.4) were purchased from WelGene (Daegu, Gyeongbuk, South Korea). Zein powder, calcium chloride dihydrate, AESO, PEGDA (molecular weight: 575 g/mol), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, ≥95%), collagenase I, entactin-collagen-laminin (ECL), insulin, linoleic acid, Nile red, bovine serum albumin, and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals used in this study were of analytical grade.

Zein-coated alginate fiber preparation

The zein-coated ink was prepared by solubilizing zein in 70% ethanol containing 5% CaCl2 aqueous solution. The ZA fibers were prepared using a wet-spinning technique (Fig. 1). Briefly, sodium alginate was dissolved in PBS (1% w/v) and prepared in a 10 mL syringe. After the zein-coated ink was placed in a coagulation bath, alginate was injected into the bath at a rate of 0.05 mL/min using a syringe pump (Legato 101; KD Scientific, Holliston, MA, USA). The ZA fibers were collected on the take-up roller and washed several times with PBS to remove residual ethanol and Ca2+.

Mechanical measurements

A CT3 Texture Analyzer (Brookfield, Toronto, ON, Canada) with a 4500 g load cell (Brookfield, Toronto, ON, Canada) in tensile mode was used to measure the mechanical properties of A and ZA fibers. To determine the difference in stress/strain, the fibers were prepared in four states (A, soaked A, ZA, and soaked ZA). The A and ZA fibers were prepared and investigated after 1 h. Soaked A and ZA fibers were prepared and immersed in PBS for 48 h. The samples were cut to a length of 20 mm, and the crosshead velocity was kept at 0.1 mm/s in the tensile measurement. The elastic modulus of the fiber was calculated from the slope over a strain ratio of 5–15%.

Fabrication of the 3D printed stretching device

Three dimensional printing was performed as previously described46. A mixture of AESO/PEGDA (ratio 80:20), 0.5% (w/v) LAP, and 1% (v/v) tartrazine pigment was used as a bioink for the 3D printing of the stretching device34. The stretching device model was created using Fusion 360 software (Autodesk, San Rafael, CA, USA) and printed using an IM2 DLP 3D printer (Carima, Seoul, Korea). After 3D printing, the stretching devices were washed several times with PBS prior to UV sterilization.

Scanning electron microscopy

A and ZA fibers were frozen in a deep freezer at –70 °C for 24 h and lyophilized for 24 h to prevent structural collapse. After lyophilization, the samples were secured to a stub using carbon tape and coated with platinum. Superficial crust morphologies were imaged using an SU-8010 Scanning Electron Microscope (Hitachi, Tokyo, Japan).

Cell culture and differentiation

C2C12 cells were propagated in DMEM supplemented with 10% heat-inactivated FBS and 1% P/S. The cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C. For muscle fibers, C2C12 cells were seeded onto the fibers at 2 × 106 cells/mL. To induce myogenic differentiation, the medium was replaced with myogenic differentiation medium (MDM; DMEM supplemented with 2% horse serum and 1% P/S), and the cells were cultured for 6 days. MDM was refreshed every 2 days.

Primary cell isolation, culture, and differentiation

The isolation, cell culture, and differentiation of primary BSCs and adipocytes were performed as previously described40,47. Muscle and adipose tissue samples were obtained from Hanwoo cattle at Gyeongbuk Livestock Research Institute. Ethics approval was not required for the acquisition of muscle and fat samples from autopsy of cattle donated for educational purposes. Research of Gyeongbuk Livestock Research Institute is approved by the Ministry of Agriculture, Food and Rural Affairs to handle animal materials. A 0.5 cm biopsy punch was used to isolate a piece of muscle from the longissimus of a 3-year-old cow to obtain primary BSCs. Additionally, primary adipocytes were isolated from 50 g peri-renal fat tissue obtained during an autopsy performed on a 4-year-old cow for anatomical education at University Animal Hospital (Seoul National University, Seoul, South Korea). For primary satellite cells, muscle tissue was immersed in 70% ethanol for 5 min for disinfection and washed with PBS. The fat and connective tissues were carefully removed using forceps and scissors. The tissue was minced and digested with 0.25% collagenase I for 1 h at 37 °C with shaking. The tissue was further digested with 0.25% trypsin/EDTA for 25 min at 37 °C with shaking. The samples were centrifuged for 10 min at 200 × g, and the supernatant was discarded. To remove fibroblasts, cell pellets were incubated in uncoated culture flasks for 1 h at 37 °C in a 5% CO2 incubator in DMEM supplemented with 10% FBS, 1% P/S, and 250 μg/mL fungizone. Non-adhering primary satellite cells were collected and transferred to cell culture flasks coated with 1 mg/mL ECL. When the cells reached 70% confluence, the growth medium was changed every 2 days, and the cells were passaged at 70% confluence. Primary satellite cells were differentiated or cryopreserved at passage 0 using a CELLBANKER 1 (ZENOAQ, Fukushima, Japan) for subsequent investigation. For myogenic differentiation, primary satellite cells were cultured until 90% confluence, and the medium was replaced with MDM. The cells were then cultured for 6 days, and MDM was refreshed every 2 days.

For primary adipocytes, the adipose tissue was first immersed in 70% ethanol for 5 min for disinfection and washed with PBS. The blood vessels and connective tissues were carefully removed using forceps and scissors. The adipose tissue was then minced and digested with 0.25% collagenase I for 1 h at 37 °C with shaking. The cell suspension was filtered through a 40 μm cell strainer and centrifuged for 5 min at 450 × g to obtain cell pellets. The preadipocytes were grown in growth medium (DMEM supplemented with 10% horse serum and 1% P/S). The growth medium was changed every 2 days, and the cells were passaged at 70% confluence. Preadipocytes were differentiated or cryopreserved at passage 0 using a CELLBANKER 1 for subsequent investigation. For adipogenic differentiation, preadipocytes were cultured, and the medium was replaced with adipogenic differentiation medium (ADM; DMEM supplemented with 10% FBS, 1% P/S, 10 μg/mL insulin, and 300 μM linoleic acid). The cells were then incubated, and ADM was refreshed every 2 days.

Cell viability assay

To investigate the cytotoxicity of ZA fibers compared with that of 2D culture dishes and A fibers, cell viability was investigated using a live/dead cell viability kit containing Calcein–AM and ethidium homodimer-1. Cells were seeded at 2 × 106 cells/mL on 2D culture dishes, A fibers, and ZA fibers, respectively, and incubated for 6 days in a humidified atmosphere of 5% CO2 at 37 °C. After 1, 3, and 6 days of incubation, the samples were stained with 500 μL of staining reagent according to the manufacturer’s protocol. Samples were imaged using a Lionheart FX automated microscope (BioTek, Winooski, VT, USA). Cell viability was calculated as the ratio of live cells to the total number of cells.

Immunocytochemistry

For immunostaining, differentiated cell-laden fibers or cells were fixed with 4% paraformaldehyde at room temperature for 15 min and washed thrice with PBS. For permeabilization, the differentiated cell-laden fibers or cells were incubated with 0.25% Triton X-100 in PBS for 15 min. Then, the samples were blocked in 5% bovine serum albumin in 0.05% Triton X-100 (prepared in PBS) for 1 h and treated with an anti-α-tubulin primary antibody (1:100 in PBS; Developmental Studies Hybridoma Bank, Iowa City, MA, USA), desmin primary antibody (1:200 in PBS; ABclonal, Woburn, MA, USA) or VE-cadherin (1:200 in PBS; Abcam, Cambridge, MA, USA) at room temperature for 1 h. Subsequently, the samples were washed with PBS and stained with Alexa Fluor 488, 555, or 594-labeled secondary antibodies (1:300 in PBS; Invitrogen, Carlsbad, CA, USA) for 1 h and counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (300 nM; Invitrogen, Carlsbad, CA, USA). The samples were visualized using a Lionheart FX automated microscope.

Quantification of myoblasts/myotubes structure characteristics

Fluorescence images of myoblasts and myotubes were analyzed using Fiji/ImageJ software (National Institutes of Health, Bethesda, MD, USA). Fluorescence was quantified by analyzing the area, circularity, and length of the myoblasts/myotubes corresponding to the short and long axes of the ellipse. The alignment angles of the cells were categorized from −90° to 90° in 10° increments.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was isolated from C2C12 cells or primary adipocytes using the TRIzol reagent (iNtRON, Seongnam, Gyeonggi, Korea) according to the manufacturer’s instructions. RNA quality was determined at an absorbance of 260 nm (A260) using a BioTek plate reader and Take3 plate reader (BioTek, Winooski, VT, USA). cDNA was synthesized from the total RNA using HKGscript™ 5X RT Premix (HK Genomics, Daejeon, Korea) at 50 °C for 15 min and 70 °C for 15 min. RT-qPCR was performed on a LightCycler® 96 system (Roche, Basel, Switzerland) using the SYBR Green qPCR Master Mix (Genetbio, Daejeon, Korea). The primer sequences are listed in Table 1. Thermal cycling conditions were: 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s, and 60 °C for 40 s.

Table 1 Primer sequences used in real-time RT-qPCR analysis

Nile red staining

For lipid droplet staining, the differentiated adipocyte-laden fibers were fixed with 4% paraformaldehyde at room temperature for 15 min and washed thrice with PBS. Samples were incubated for 30 min at 1:1,000 dilution in PBS containing 1 mg/mL Nile red. Samples were then washed thrice with PBS, and the cell nuclei were stained with DAPI. Samples were visualized using a Lionheart FX automated microscope.

Statistical analysis

Data are presented as means ± standard deviation (s.d.), and means were compared using one-way analysis of variance (ANOVA) followed by Tukey’s test or a two-tailed paired and unpaired Student’s t test. Statistical significance was set at P < 0.05. All analyses were performed using GraphPad Prism 8.0.2 (GraphPad Software, La Jolla, CA, USA).

Reporting summary

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