1. Introduction

In the last decade we have witnessed the booming of additive manufacturing (AM, also 3D printing), including its related fabrication technologies, materials, and applications. The biotechnology and bioprocessing fields have been significantly influenced by AM, with reports spanning upstream and downstream processing, including sorting and selection of cell strains (Lin et al., 2016), bioreactors (Saha et al., 2018), harvesting (Shakeel Syed et al., 2017), filtration (Tan & Franzreb, 2020), chromatography (Salmean & Dimartino, 2019), and extraction (H. Wang et al., 2017). One of the most popular AM methods employed in bioengineering is digital light processing (DLP) where a three-dimensional model is built, layer upon layer, by selectively curing a photo-sensible liquid resin. Reasons for the success of DLP in biotechnology include its relatively low cost, fast speed (litre sized objects can be printed overnight), and high resolution (generally in the order of 50 μm).
Historically, AM was primarily in the domain of the automotive, aerospace, and biomedical industries which favoured materials with mechanical properties over chemical characteristics and fabricated non-porous structures where strength and stiffness are key. On the other hand, bioprocess applications often require porous materials to maximize the total surface area available for cell adhesion, adsorption, and allow intraparticle mass transfer. Besides, materials in the biotechnology industry heavily exploit chemical characteristics such as electrostatic charge and hydrophobic behaviour to appropriately modulate their interactions with species as diverse as cells, proteins, and small metabolites. This requirement contrasts the status quo where the composition of commercially available AM materials is IP protected, making it impossible to rationally design material-species interactions of interest. Proprietary compositions also complicate compliance with FDA or EMEA requirements, hindering adoption of 3D printing in the biomanufacturing industry.
Here, we present a novel polymeric formulation for DLP 3D printing which can be easily tuned to adjust its chemical, porous, and mechanical properties of printed parts. The formulation consists of a few simple ingredients, including monomers and crosslinkers to create the polymeric network (Figure 1a), a UV photoinitiator to trigger the photopolymerization reaction, a photoabsorber to increase the resolution of the printed model, and porogenic components. The key feature of the proposed formulation lays in the use of bifunctional monomers bearing both a (meth)acrylate functionality for photopolymerization and a suitable chemical moiety for biomolecular interactions, e.g. charged groups, alkyl or aryl groups, or reactive groups for successive covalent immobilization of a desired ligand. By appropriate selection of the bifunctional monomers, different materials with different surface derivatizations to suit a range of applications in bioprocess engineering can be obtained. Furthermore, the nature and relative concentration of the components making up the overall formulation will directly impact on the propagation kinetics of the free-radical polymerization reaction, in turn affecting the morphology of the resulting polymeric network and its porous microstructure (e.g. surface area, average pore size, pore size distribution) (Barner-Kowollik et al., 2014; Buback, 2009). In this work, we manipulated these characteristics to produce different formulations for DLP printing, and obtain porous monoliths with different chemical and macroporous properties for use in chromatography, immobilized enzyme bioreactors, and biofilm bioreactors.
A Schoen gyroid topology was selected for fabricating the structured monoliths (Figure 1b,d,f). Gyroids are members of Triply Periodic Minimal Surfaces (TPMS), highly versatile geometries with maximized surface area for mass transfer (Femmer et al., 2015) and excellent load-bearing stiffness. TPMS are described by simple equations and their properties in terms of size, surface area, hydraulic diameter, bed porosity, tortuosity, wall thickness, etc. can be tuned by altering the equation parameters (Schoen, 2012). We first simulated fluid flow and solute dispersion to validate the suitability of the gyroid topology for packed beds. The gyroid structure showed 5 fold higher efficiency (measured in terms of minimum reduced plate height, Figure 1b) and 4 fold higher permeability (6.4 X 10-14m2) compared to traditional random packing of spherical particles (1.61 X 10-14m2) (Schure et al., 2004). In particular, the interconnected gyroid lattice ensured appropriate radial intermixing, in turn reducing axial dispersion and band broadening, while showing minimal flow resistance with lower pressure drops than random beds.
Use of porogens enabled formation of a highly interconnected porous network defined by polymeric globules (Figure S1). The final porous microstructure was principally determined by polymer chemistry, with acrylate-based formulations producing smaller pores (271 ± 120 nm for the AETAC and 289 ± 112 nm for the CEA materials, Figure S1,d) than methacrylate-based formulations (905 ± 410 nm for the MAETAC material). This is consistent with the higher reactivity of acrylates over methacrylate groups (Barner-Kowollik et al., 2014), with acrylates generating a higher number of polymerization nuclei, in turn leading to smaller globules and smaller pores than methacrylates. Fine tuning of the porous characteristics of the 3D printed matrices can be achieved by adjusting the overall composition as its nature and concentration directly affects the kinetic and thermodynamic properties of the mixture. This ultimately enables modulation of diffusional mass transfer within the 3D printed scaffold and the surface area available for adsorption and reaction.
We first demonstrate use of the proposed formulation for chromatography applications. In particular, strong anion and weak cation exchange monoliths were fabricated using resins with bifunctional acrylates bearing positive quaternary amine (AETAC monomer) and negative carboxyl groups (CEA monomer) respectively (Figure 1a). Ion-exchangers with varying ligand densities were obtained by altering the concentration of the functional monomers in the formulation, enabling adjustment and optimization of the adsorption characteristics towards the target solute. The ion exchangers were initially 3D printed as hollow cylinders (Figure 1c-e), and adsorption of pure Bovine Serum Albumin (BSA) and lysozyme (LYS) was measured in batch conditions on the AETAC and CEA materials, respectively. Maximum binding capacities of 104.2 ± 10.6 mg of BSA per mL of AETAC-based support, and 108.1 ± 25.9 mg of LYS per mL of CEA-based material were recorded (Figure 2a and 2b), about 5 fold higher than for commercial monoliths (Hahn et al., 2002) and chromatographic membranes (Boi et al., 2020) and in line or above standard chromatographic resins (Staby et al., 2005). Testing in dynamic conditions was carried out using Schoen gyroid columns (Figure 1f-h) by loading BSA and myoglobin (MYO) onto the AETAC material (Figure 2c, Simon et al., 2020) and BSA and LYS on the CEA material (Figure 2d). The chromatograms reveal elution pattern in line with the electrostatic interactions established at the buffer’s pH, thus confirming the availability of the surface quaternary amine and carboxyl groups to establish appropriate electrostatic interactions with the protein models. Also, approximately 90% of the proteins adsorbed were recovered during elution for both materials, demonstrating that the strength of the electrostatic interactions can be appropriately tuned to enable bind and elute operation of the ion-exchangers.
The potential to fabricate immobilized enzyme bioreactors for biotransformations was tested by covalently immobilizing trypsin onto the free carboxylic groups of the CEA material. In particular, 3D printed supports were exposed to trypsin solutions having different enzyme concentrations, leading to materials with progressively increasing immobilized trypsin, up to a maximum density of 7.5 ± 0.04 mg of trypsin per g of support, corresponding to 66% utilization of the theoretical carboxyl groups. Enzymatic activity was tested in batch experiments by introducing N-α-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) to 3D printed hollow cylinders, and monitoring the formation of N-α-benzoyl-D,L-arginine (BA) as the product of the enzyme catalyzed reaction. Results indicated that the immobilized trypsin retained its hydrolytic activity (Figure 3a). Dynamic experiments were carried out using 3D printed gyroids to demonstrate the operation of the bioreactor in steady-state, continuous mode. Five flow rates in the range of 0.5 – 8 mL/min were tested and product formation was verified for all of them. As the flow rate increased, the concentration of product in the effluent stream descreased due to a combination of mass transport phenomena and actual reaction kinetics (Figure 3b). Yet, productivity, expressed in terms of product formed per unit time, showed a proportional growth with increased flow rates due to higher throughputs. This is consistent with other case studies for continuous packed bed reactors bearing immobilized enzymes (Halim et al., 2009).
The bioreactor concept was extended to whole-cell biocatalysis. We selected the biodesulfurization reaction catalyzed by immobilizedRhodococcus opacus as a case study for an emerging eco-friendly alternative to traditional desulfurization techniques employed in oil refineries (Mohebali & Ball, 2016). R. opacushas negatively charged cell membrane rich in mycolic acid. Accordingly, the support material was engineered with positively charged and hydrophilic bifunctional monomers displaying quaternary amine (MAETAC) and hydroxyl groups (HEMA, Figure 1a) to enhance cell immobilization. A methacrylate functionality was chosen to enable autoclaving of the supports for repeated use. Immobilization experiments with standard culture medium revealed that R. opacus bacteria formed stable and healthy biofilms on the 3D printed supports (Figure 4a). In particular, a biofilm covering the external surface of the supports was obtained, with rod-like morphology typical of filamentous aggregates of matureRhodococcus cells (approximately 4.0 and 0.5 microns in length and width, respectively). Since the pore size of the matrix is between 0.4 and 1.0 microns, the biofilm could penetrate into the porous architecture, aiding cell adhesion and biofilm stability. The gyroid-based bioreactor was tested for the biodesulfurization of benzothiophene (BT) in perfusion mode. Results reveal conversion of BT into phenolic end products (Figure 4b,c), proving the biodesulfurization potential of the 3D printed bacterial biofilm bioreactor. In addition, production of phenolic compounds increases over time, indicating adaptation of the biofilm to the reacting conditions and confirming the biocompatibility of the support material with a thriving bacterial population.
Taken together, the versatile material formulation here presented could be used to create complex three-dimensional matrices for chromatography and bioreactors. The chemical, physical, mechanical and microporous properties of the supports can be easily tuned by changing the composition of the DLP resin formulation. For example, a combination of bifunctional monomers bearing alkyl chains and polar groups could be employed to tune the hydrophobic/hydrophilic as well as charged state of the matrices. We expect this versatility, coupled with cost-effective and rapid DLP 3D printing (all models were 3D printed in a few hours), will truly enable fabrication of complex three dimensional architectures to suit a range of diverse experimental requirements for a multitude of applications in bioprocess engineering.