Composition of material formulations
All chemicals employed are listed in the Supplementary Information (SI). All formulations included 48% v/v cyclohexanol and 12% v/v dodecanol as porogens, and 1% w/v Omnirad 819 as photoinitiator. A mixture of poly(ethylene glycol) diacrylate (PEGDA, 12% v/v) and alkoxylated pentaerythritol tetraacrylate (SR494, 12% v/v) as crosslinkers, and 0.125% w/v Tinuvin 326 as photoabsorber was employed for the acrylate formulations. The relative concentration of the monomers, namely [2-(acryloyloxy)ethyl]trimethylammonium chloride (AETAC) and 2-carboxyethyl acrylate (CEA), was varied to adjust the ligand density (0, 4, 8, 12, 16% vol), with di(ethylene glycol) ethyl ether acrylate (DEGEEA) as non-functional monomer to obtain a total monomer concentration of 16% vol. The methacrylate formulation was composed of [2-(methacryloyloxy)ethyl]trimethylammonium chloride (MAETAC, 12% v/v) and hydroxyethyl methacrylate (HEMA, 12% v/v) functional monomers, ethylene glycol dimethacrylate (EDMA, 16% v/v) crosslinker, and Tinuvin 326 (0.1% w/v) photoabsorber.
Model design, fabrication, and characterization
Computer-Aided Design (CAD) models of hollow cylinders and gyroidal columns were created on Fusion 360 (Autodesk, USA), exported as Standard Tessellation Language (STL) files, and sliced using Netfabb 2017 (Autodesk, USA). A Solflex 350 (W2P Engineering, Austria) DLP printer was employed to fabricate all parts. Post-printing, the parts were washed three times in isopropyl alcohol in an ultrasonic bath (Allendale Ultrasonics, UK) and then fully cured in water with a xenon Otoflash G171 unit (NK-Optik, Germany). The parts were stored in sterile 0.1 M phosphate buffer until use. A TM4000Plus scanning electron microscope (SEM, Hitachi, Japan) and a Zeiss Crossbeam 550 focused ion beam (FIB) SEM (Jena, Germany) were used for imaging, with samples prepared by freeze fracturing with liquid nitrogen and drying in ethanol, followed by a final wash in hexamethyldisilazane before sputter coating using an Emscope SC500 (Bio-Rad, UK). Mean pore sizes and distributions were evaluated from the SEM images (see section 2 in the Additional file 1 for additional details).
Computational model and data analysis
Computational fluid dynamics was performed in COMSOL Multiplysics 5.4 (COMSOL, USA). A series of six gyroidal unit cells extending along the axial direction was built and the Navier Stokes equation for fluid motion was employed to determine the steady-state velocity field and estimate the pressure drop per unit length across the structure (no slip boundary conditions at the gyroidal walls, periodic boundary conditions at the cell periphery). The advection–diffusion model for mass transfer was then employed to model the dispersion of a pulse injection of an inert tracer (Schure et al. 2004). The average concentration profile over time at two different cross sections was employed to determine the plate height of the gyroidal column according to residence time distribution analysis (RTD) (Dolamore et al. 2018). Column permeability was calculated from the estimated pressure drop over the column.
Chromatography
3D printed hollow cylinders were employed in batch experiments by inserting them into 96-well plates and reading the absorbance using a Modulus II microplate reader (Turner BioSystems, USA). Batch adsorption on the anion exchange chromatography (AEX) material (based on the AETAC monomer) involved an initial equilibration in phosphate buffer (20 mM, pH 7.4) for a minimum of 48 h, followed by addition of a bovine serum albumin solution (BSA, 0–32 mg/mL) in phosphate buffer. Similarly, cation exchange chromatography (CEX) materials (based on the CEA monomer) were equilibrated in binding buffer (20 mM phosphate, pH 7.4) before loading a lysozyme solution (LYS, 0–4 mg/mL). Flow experiments were carried out feeding protein mixtures of BSA, LYS, and myoglobin (MYO) onto gyroidal columns (50% external porosity, 500 µm wall thickness) slotted into 10 mm i.d. SNAP® glass housing (Essential Life Solutions, USA) and connected to an ÄKTA™ Purifier 10 system (GE Healthcare, Sweden) equipped with a UV detector to record absorbance at 280 nm. The flow rate was kept at 1 mL/min (75 cm/h linear velocity) as generally employed in lab-scale experiments.
Immobilized enzyme bioreactor
Trypsin was immobilized on CEA supports via the 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC) protocol. Briefly, the 3D printed materials were equilibrated in a 0.1 M sodium phosphate (pH 7.4) activation buffer, followed by a 35-min immersion on activation buffer containing 1:10 molar excess of EDC with respect to carboxylic groups. After extensive washing in activation buffer, coupling of the enzyme was obtained by soaking the 3D printed models in trypsin solutions (1–10 mg/mL) in 0.1 M phosphate buffer pH 7.4 for 2 h at room temperature. Non-bound trypsin was removed by washing with 0.1 mM Tris buffer (pH 8). The amount of trypsin immobilized on the 3D printed materials was calculated as the difference of the initial and final concentration of trypsin using the bicinchoninic acid (BCA) assay (Smith et al. 1985). A control experiment was run by adding trypsin solutions to non-activated cylinders. Similar to the chromatography runs, the activity of the immobilized trypsin was tested both in batch (hollow cylinders in multi-well plate format) and dynamic conditions (gyroids with 50% external porosity, 500 μm wall thickness, 25 mm diameter, 10 mm bed height, flow rate ranging from 0.5 to 8 mL/min). In both cases, after equilibration in 50 mM Tris buffer pH 8, a 1 mM N-α-benzoyl-l-arginine ethyl ester hydrochloride (BAEE) substrate solution in 50 mM Tris buffer pH 8 was fed to the 3D printed models, and formation of the N-α-Benzoyl-L-arginine (BA) hydrolysis product was monitored at 253 nm.
Bacterial biofilm bioreactor
Biofilms of Rhodococcus opacus IEGM 248 cells were obtained by perfusing fresh cultures (exponential growth phase) for 3 days in recirculation mode (1 mL/min) through gyroidal supports (50% external porosity, 2 mm wall thickness, 10 mm diameter, 40 mm height) in a glass column, followed by column washes with quarter strength Ringer’s solution to remove non-adsorbed biomass (free cells). The obtained biofilms were then grown by continuous feed (2 mL/min) of a mineral salts medium (MSM, 2.0 g/L sucrose, 7 g/L Na2HPO4, 6 g/L KH2PO4, 2 g/L NH4Cl, 0.2 g/L MgCl2·6H2O, 0.03 g/L CaCl2·2H2O, 0.001 g/L FeCl3·6H2O) spiked with 0.2 mM benzothiophene (BT) as sole sulphur source. According to the biodesulphurization reaction, BT is converted into phenolic compounds (principally hydroxyphenylacetaldehyde) whose presence in the perfusate was confirmed using the Gibbs test (Gibbs 1927; Wang et al. 2013).