Sodium alginate powder

The gel solution of A at the concentration of 10% w/v and G solutions at different concentrations of 10–50% w/v were prepared. In a clean and sterilized beaker, 1 g of sodium alginate powder (Sigma-Aldrich, St. Louis, MO, USA) was dissolved into 10 mL of deionized water. Then, 1 g of G powder (Sigma-Aldrich, USA) was dissolved in 10 mL of deionized water under constant stirring at 60 °C. Then, the solution of 10% w/v A and the solution of 10% G were blended and centrifuged at 1200 rpm for 1 min to remove air bubbles. To prepare AG10:20, 2 g of G powder was dissolved in 10 mL of DI water, and 3, 4, and 5 g were dissolved separately in 10 mL of DI water to prepare 30%, 40%, and 50% w/v of G solutions, respectively. Table 1 shows the prepared AG hydrogels with different concentrations of G solutions. The concentration in weight/volume percentage (w/v)% was calculated using Equation (1). The AG solutions were denoted as AG10:10, AG10:20, AG10:30, AG10:40, and AG10:50 according to their corresponding concentrations.
$$(w/v)\%=\frac{weigh of solute in \left(gram\right)}{volume of solution in \left(ml\right)}\times 100$$

Sodium alginate powder (Sigma-Aldrich) was dissolved in phosphate-buffered saline (PBS) without Ca and Mg at 2% (w/v) and filtered through a 0.22 µm filter. 100% Geltrex™ LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Gibco Cat# A1413202), Caco-2 cell culture in DMEM 1X (Wisent Cat# 319-005-CL) to embed the cells, alginate stock solution was mixed to yield final ratios by volume of [50% Matrigel + 0.2% alginate stock]. 30 µL of hydrogel mixture was added per well followed by incubation at 37 °C for 1 h. CaCl₂ was dissolved in PBS without Ca and Mg at 1% (w/v) and filtered, and then diluted in media to a final concentration of 0.1%. [200] µL of CaCl2 media mixture was then added per well followed by incubation at 37 °C for 30 min. Organoids were then cultured in DMEM deprived of glucose or amino acids as described above.

NFC hydrogel and alginate mixtures were prepared to produce NFCA threads. Sodium alginate powder (Sigma-Aldrich, W201502, Finland) was added and mixed briefly with the stock NFC hydrogel (1.47% NFC, GrowDex®, UPM-Kymmene Corporation, Finland). After mixing, the mixture was left to stabilize for 24 hours. The final composition of NFCA prepared for cell studies and suturing performance tests contained 8% (w/v) sodium alginate and 1.35% (w/v) NFC hydrogel. The NFCA threads were prepared by dispensing the mixture with a syringe and a 22G needle into calcium chloride (CaCl₂, anhydrous, Riedel-de-Haen, Germany) and barium chloride (BaCl₂ * 2H₂O, Sigma-Aldrich, Finland) crosslinking solutions (68 mM and 20 mM respectively). Alginate crosslinking method used in this study was implemented from an alginate-based cell microencapsulation study performed by one of our group previously [42 (link)].

Alginate bioink was prepared as described in the literature [52 (link)]. In summary, sodium alginate powder (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in phosphate-buffered saline (PBS 1X, pH 7.4, Sigma-Aldrich, St. Louis, MO, USA) at concentrations of 2%, 4%, 6%, and 8% (w/v). Alginate–gelatin (from bovine skin, type B, Sigma-Aldrich, St. Louis, MO U.S) (Alg-Gel) bioinks were prepared by dissolving varying concentrations of gelatin powder (1%, 2%, and 3% w/v) with 4% w/v alginate powder in PBS. A 4% alginate concentration was selected for the mixture due to its cell viability, as reported in the literature [52 (link)]. The bioinks were vortexed (Cole-Parmer, Vernon Hills, CT, USA) for one minute at 3400 rpm, then restored at 37 °C for one day to fully dissolve the powder(s). For better visualizations post-printing, red food dye was added to the bioink mixture and vortexed for 30 s to increase the contrast, then held for 30–60 min at 37 °C to release any air bubbles before loading it into the syringe barrel. Bioinks were crosslinked in calcium chloride (CaCl₂) (Sigma-Aldrich, St. Louis, MO, USA). This ionic crosslinker was dissolved in PBS at a concentration of 2% (w/v) by vertexing for one minute, followed by loading it into a syringe barrel.

Sodium alginate powder and calcium chloride (CaCI₂) were acquired from Sigma-Aldrich (St. Louis, MO, USA). TEMPO (Anionic type) Cellulose Nanofibrils (T-CNF) Slurry (1% w/v solid in water, Width: 20–50 nm; Length: 0.5 μm–80 μm Surface Group: Carboxyl Hydrophilic) was obtained from Cellulose lab (Fredericton, New Brunswick, Canada). Cellulose Nanocrystals (CNC) spray-dried powder hydrolyzed from wood was purchased from CelluloForce (Montréal, Quebec, Canada). HyClone Dulbecco’s Modified Eagle’s Medium with high glucose (DMEM, GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was purchased from cytiva (Global Life Sciences Solutions USA LLC, Marlborough, MA, USA); Fetal Bovine Serum (FBS, Premium) was obtained from Atlanta Biologicals (Hall County, GA, USA). Penicillin-Streptomycin (Gibco, for mammalian cell culture) and cell viability kit (Invitrogen) for cell viability were obtained from ThermoFisher Scientific (Bohemia, NY, USA).

Gelatin powder (Sigma, USA) was dissolved in DMEM high glucose on a magnetic stirrer at 1250 min⁻¹ (37 °C). After 2 h, sodium alginate powder (Sigma, USA) was added under incessant stirring overnight (37 °C). The hybrid alginate (4.5% w/v) - gelatin (6.5% w/v) hydrogel was mixed with undiluted liquid Matrigel (Corning, USA) in various concentrations, A549 cells (7 * 10⁶ cells/ml), 1.22 M CaSO₄ (Roth, Germany) and DMEM high glucose to obtain the final cell-laden hybrid alginate/gelatin/Matrigel bioink composed of alginate (2% w/v)/gelatin (3% w/v)/Matrigel (0; 5; 20 or 50% w/v)/2.5% CaSO₄/7 * 10⁶ A549 cells/ml. Following CaSO₄-driven initial cross-linking of alginate (8 min after mixing), the cell-laden hydrogel bioink was loaded into the cartridge.

The RBD gene was ordered to BIOMATIK. Plasmid extraction kit and gel recovery kit were purchased from GenAll. Enzymes and buffers for cloning were prepared from Thermofishers. The NTA-Ni resin was purchased from Sigma (USA). Mouse anti-His monoclonal antibody (mAb) HRP-conjugated, HRP-labeled goat anti-mouse IgG and HRP-labeled goat anti-human IgG were obtained from CMG (IRAN). Sodium alginate powder was from Sigma-Aldrich (USA). PT-SARS-CoV-2-Neutralizing-Ab-96 kit was provided by PISHTAZTEB Diagnostics (IRAN). Balb-C mice were purchased from Razi Vaccine & Serum Research Institute (IRAN). Animals have housed in accordance with WHO guidelines and standard laboratory conditions (WHO guidelines on nonclinical evaluation of vaccines 2005 ). Animal research were approved by Iran National Committee by Ethics in Biomedical Research.