Scientists at Germany’s RWTH Aachen University and DWI Leibniz-Institute for Interactive Materials have used Nanoscribe’s two-photon polymerisation (2PP)-based 3D printing technology in a study to develop an in-flow, channel-integrated, continuous microfabrication process for cell scaffold particles of any shape.
The process, known as continuous two-photon vertical flow lithography, allows for the creation of particles of any shape, in micrometre size, with submicron features, in various materials, in large quantities and at high throughput. It has proved capable of producing 150,000 particles in a single continuous run of 72 hours, which is 2,083 particles per hour.
Background
Millimetre- and micrometre-sized particles are useful and flexible building blocks for scaffolds that are needed in chemical and biochemical reactors. Bioreactors serve to immobilise and analyse enzymes, cells or microorganisms on the surface of the reactor’s internal structure. Key to the performance of bioreactors is the capability to tune the properties of the particles that affect the bioreaction characteristics. It was with this application in mind that RWTH Aachen University and DWI Leibniz-Institute for Interactive Materials scientists set out to develop the continuous two-photon vertical flow lithography process.
The study aimed to apply this particle production approach to tissue engineering applications, expanding the scope for producing adaptive, responsive and permeable cell scaffolds. It was found that 3D printed particles exhibited surface interactions that were conducive to their self-assembling into scaffolds. Scaffolds of various particle shapes, sizes and materials are fabricated. The scaffolds' hydraulic resistances and packing densities are analysed via permeation experiments.
The continuous two-photon vertical flow lithography process. The 2PP laser is scanned in a single X, Y plane while the flow of the liquid resin transports the printed X, Y slice continuously in the Z direction. Image courtesy of RWTH Aachen University.
In-flow printing of particles
The concept relies on 2PP-based high-precision 3D printing in a microfluidic channel. The 2PP laser is scanned in a single X, Y plane while the flow of the liquid resin transports the printed X, Y slice continuously in the Z direction. As soon as one particle is complete, the next one is printed the same way. Using Nanoscribe’s DeScribe software and adapting the output file with a phyton script, various designs with complex geometries can be sliced and prepared for in-flow printing. The in-flow printing process is continuous and therefore allows many thousands of particles to be produced in hours. The particles can be produced in any shape, as continuous two-photon vertical flow lithography benefits from the enormous design freedom that is characteristic of 2PP-based 3D printing.
In-flow printing of particles has advantages when compared with on-substrate printing because the continuous fabrication allows for printing one particle after the other, with no waiting time between the two. The in-flow printing approach also circumvents the Z-movement of the stage when printing layer by layer because it is the flow that transports the X, Y slice in the Z-direction. Thus, in-flow printing represents a speed increase in the fabrication of small, complex-shaped particles.
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The envisioned layer-by-layer self-assembly process of 3D printed any shape particles into scaffolds. The interaction of the particles and how they assemble depend on the particles’ properties, including shape, material, charge, softness and solvent wettability. The self-assembly process still represents a challenging task. Image courtesy of RWTH Aachen University.
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confocal micrograph of in-flow 3D printed particles (red) with holes as cell culture scaffolds. First signs of self-assembly of the particles are visible although this process remains difficult to control. There are signs of cells (blue) infiltrating the porous particles and actin filaments (green) percolating the cells. Image courtesy of RWTH Aachen University.
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A confocal micrograph of the 3D printed particles (green). The particles tend to self-assemble, although they are not yet providing optimal results. The image shows interactions between the particles and the cells (blue).
3D biohybrid tissue
Particles below 100 µm tend to self-assemble into complex scaffolds. The reacting characteristics of the scaffolds can be adjusted when tuning the particles’ shape, size, porosity and material properties. This translates to high flexibility when creating scaffolds for cell cultures and tissue engineering. Tuning the particle geometry affects the surface-to-volume ratio, tailoring the hydrodynamics through and around the assembly. Nevertheless, there are still challenges to control the organisation process of the particles when producing scaffolds by in-flow self-assembly.
To investigate 3D biohybrid tissue viability, the scientists examined the interaction between mouse fibroblast cell culture and in-flow printed particles. The particles were printed with Nanoscribe’s non-cytotoxic IP-Visio resin, which affords low fluorescence for better cell microscopy analysis. At four days of cultivation, the cells interacted with the printed scaffolds; they proliferated, adhered to the scaffolds, infiltrated them and interconnected the particles, building a new form of tissue.
The scientists have written a paper on the study, which has been published in the journal Small1.
RWTH Aachen University
DWI Leibniz-Institute for Interactive Materials
Nanoscribe
Reference1Lüken, A., Stüwe, L., Rauer, S., Oelker, J., Linkhorst, J. and Wessling, M. (2022) Fabrication, flow assembly, and permeation of microscopic any shape particles. Small, volume 18, issue 15, p. 2107508.Available at: bit.ly/3J5b4c0