Sodium acrylate

Nutrient browth (CM0001) was purchased from OXOID LTD. Basingstoke, Hampshire, England, and agar, Bacteriological (CAS: 9002-18-0) purchased from bio plus Chemicals. Dextrose, Ethanol, Silver nitrate(AgNO₃), Sodium acrylate, Butyl acrylate (BA), methyl methacrylate (MMA) and methacrylic acid (MAA), Potassium persulfate (KPS, Sigma-Aldrich), Titanium oxide were purchased from Sigma-Aldrich, ammonia water (E.Merk), Acrylic Dispersant,Natrosol 250-HR,NP-9(sigma-Aldrich) Texanol were purchased from Dow Chemical Company (Thailand), Mergal, propylene glycol,Antifoam,brighty 425 mesh, P-820, China Clay, Talcum powder, Laponite Gel were purchased from Guangdong Weng Jiang Chemical Reagent Co., Ltd from China and 2-(methacryloyloxy)ethyl acetoacetate (AAEM) were purchased from Shanghai, China.

Neurons were fixed and incubated with primary and secondary antibodies as described above. After secondary antibody incubations, neurons were anchored with succinimidyl ester of 6-((Acryloyl)amino) hexanoic acid (AcX) (Thermofisher) in PBS (0.1 mg/ml) for 6 h at 4 °C. Samples were then incubated for 30 min at 37 °C in a gelling chamber containing the polymer solution (2 M NaCl, 8.625% sodium acrylate (Sigma), 2.5% acrylamide (Sigma), 0.15%N,N’-methylenebisacrylamide, 0.02% ammonium persulfate (Biorad), 0.02% TEMED (Biorad) in PBS) as described in [51 ] and [52 ]. To allow equidistant expansion, gelled coverslips were transferred into digestion solution (50 mM Tris, 1 mM EDTA, 1% Triton X-100, 0.8 M guanidine HCl, pH8) containing proteinase K (800 u/mL, NE) and incubated for 6 h at room temperature. After digestion, gel-embedded neurons were transferred to glass bottom imaging plates (Cellvis) and rinsed with PBS to allow for an approximately 2.4-fold expansion of the sample followed by immediate confocal imaging. The diameters of the gelled coverslips were measured pre- and post-expansion with a ruler to calculate the expansion factor.

Sodium acrylate (Sigma-Aldrich, Søborg, Denmark), sn-Glycero-3-phosphocholine, >98% (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), cefadroxil 95–105%, (VWR, Søborg, Denmark), 2-methylpyrazine (VWR, Søborg, Denmark), and 2-benzoxazolinone, ≥98.0% (VWR, Søborg, Denmark), 2,5-furandicarboxylic acid (TCI Europe N. V., Zwijndrecht, Belgium) were used in the study. Xenopus oocytes were incubated in either HEPES-based Kulori buffer (90 mM NaCl, 1 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5 mM HEPES pH 7.4) supplemented with gentamycin (100 μg/mL), or MES-based Kulori buffer (90 mM NaCl, 1 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 5 mM MES pH 5.0).

For stereolithographic printing, two bioinks based on gelatin and PEG were used. Both bioinks were synthesized, as previously shown [40 (link),41 (link)]. In short, 10 wt % gelatin (porcine skin Type B, Sigma) was dissolved in phosphate buffered saline (PBS) at 50 °C. After, 20-fold molar excess methacrylic anhydride (Sigma) was added and the reaction continued for 3 h. After reaction, the product (GelMA) was dialyzed against distilled water. Products were freeze dried and lyophilized for precise bioink preparation. Degradable PEG-bis-(acryloyloxy acetate) was synthesized in a two-step reaction. First, PEG-bis-chloroacetate was synthesized by reacting PEG (Sigma) with chloroaceryl chloride (Sigma). In the second step, acrylic groups were added by reacting the product with sodium acrylate (Sigma). Products were recovered by precipitation in cold ethylether (Sigma), dialyzed against distilled water and freeze-dried for long-term storage. The photoinitiator lithium phenyl-2,4,6-trimethylbenzoy phosphinate was used at 0.1 wt % in all bioinks. For the bioprinting process, cells were mixed with bioink-solutions containing the photoinitiator to form a bioink cell suspension ready for photopolymerization.

Solvents and reagents were purchased from commercial sources and used as received, unless otherwise specified. Methanol (ACS reagent grade) was obtained from Fisher Scientific. Molecular biology grade acrylamide (catalogue number A9099), sodium acrylate (catalogue number 408220), 19:1 acrylamide/bis-acrylamide (catalogue number A2917) and ammonium persulfate (APS; catalogue number A3678) were purchased from Sigma-Aldrich. Ultrapure N,N,N′,N′-tetramethylethylenediamine (TEMED; catalogue number 15524010) and SYBR Gold Nucleic Acid Gel Stain (catalogue number S11494) were obtained from Thermo Scientific. Desalted oligonucleotides were purchased from Integrated DNA Technologies (IDT). Nitrogen gas (>99.999%) was used under inert conditions and supplied by an in-house gas generator. To ensure an inert condition, it was purified through a Model 1000 oxygen trap from Sigma-Aldrich (catalogue number Z290246). Reagents with unreacted acrylamide groups were stored at 4 °C or −20 °C, protected from unnecessary exposure to light. Actin protein (>95% pure, rabbit skeletal muscle, catalogue number AKL95) was obtained from Cytoskeleton and DNase I (catalogue number M0303) was obtained from New England Biolabs. Matrigel was obtained from Corning (catalogue numbers 354277 and 356231). DNA ladders were purchased from Thermo Fischer (catalogue number SM1211).

Sodium acrylate, 30 nm IONP, N,N’-methylenebisacrylamide (MBA), potassium persulfate, N,N,N’,N’-tetramethylethylenediamine (TEMED), PBR, sodium dodecyl sulphate (SDS), liquid paraffin, and chloroform were obtained from Sigma–Aldrich Company.