Original study: Nilsson Hall G. et al.
Published in: Biofabrication (2024)
DOI: 10.1088/1758-5090/ad6e1a
Researchers at KU Leuven and POIETIS combined laser-assisted bioprinting with a custom imaging system to automatically find, target and transfer multiple large cartilaginous spheroids in a single sequence. At 50 µJ, the ‘target and shoot’ system transferred 100% of ~300 µm human periosteum-derived cell (hPDC) spheroids (n = 30), and the team used it to build a four-layered, high-density spheroid construct.
The challenge: placing living building blocks precisely, and fast enough
Multicellular spheroids are attractive building blocks for bottom-up tissue engineering, but assembling them into designed structures is hard. The authors point to three recurring limits of current approaches:
- Extrusion bioprinting: low spheroid density, limited control over where spheroids land, and nozzle clogging.
- Aspiration-assisted bioprinting: precise placement, but one spheroid per operation, with several minutes per transfer.
- Self-assembly in containers: fusion happens without designed architecture, so final shape can differ from what was intended.
Laser-assisted bioprinting (LAB) is nozzle-free, which makes it a candidate to avoid clogging. The open question was whether it could handle large, matrix-rich microtissues rather than single cells, and do so with automated selection.
How 'target and shoot' works
POIETIS developed a custom imaging system and installed it on the LAB workstation; the combination is what the authors call the ‘target and shoot’ system. The workflow runs in four steps:
Scheme 1. Spheroids in a liquid film are imaged, automatically targeted, transferred by laser, and assembled bottom-up into a predesigned pattern. Source: Nilsson Hall et al., Biofabrication 16 (2024) 045029, CC BY 4.0.
- Liquid film loading. Spheroids are suspended in culture medium on the donor. No hydrogel or bioink is used for the transfer itself.
- Image-based targeting. The software distinguishes separated spheroids from agglomerates and aims the laser at the geometric center of each selected spheroid.
- Laser transfer (LIPMO). For large spheroids, the team developed laser induced propulsion of mesoscopic objects (LIPMO). Energy is deposited just below a single spheroid and propels it ballistically to the receiver, using 8 µl of liquid versus 30 µl for conventional laser-induced forward transfer (LIFT).
- Bottom-up assembly. Spheroids are placed in a CAD-defined pattern on the receiver, layer by layer.
Key results
Figure 6(a, b). (a) Automated selection: coloured spheroids are selected and center-targeted; large aggregates are excluded. (b) Spheroids before and after a single laser shoot sequence. Scale bars 500 µm. Source: Nilsson Hall et al., Biofabrication 16 (2024) 045029, CC BY 4.0.
- Automated selection. The imaging system separated single spheroids from agglomerates and targeted the laser on each selected spheroid’s center.
- Multiple spheroids per sequence. Two out of four targeted spheroids were transferred with one laser shoot sequence.
- Energy-dependent transfer efficiency. Transfer rose with laser energy and reached 100% at 50 µJ (n = 30):
| Laser energy (µJ) | Spheroids transferred |
|---|---|
|
50 |
100% |
|
40 |
50% |
|
35 |
20% |
- Cells survive transfer. Large hPDC spheroids printed by LIPMO at 50 µJ showed a live fraction of 0.73 ± 0.03 (normalized fluorescence).
- Four-layered, high-density construct. Spheroids were laser-printed onto bioextruded collagen type I films, with a 6-axis robotic arm moving the construct between the LAB and bioextrusion work areas. H&E staining confirmed the four-layered structure.
- Throughput. Multiple targeted spheroids can be transferred during an 8 ns laser pulse. The authors report an order-of-magnitude gain in process efficiency over aspiration-based methods, which handle one spheroid at a time over several minutes.
Why it matters
The study moves laser-assisted bioprinting from single cells to matrix-rich, pre-differentiated microtissues, with automated selection built in. For researchers building tissues bottom-up, this opens three practical possibilities:
- Designed architecture. Spheroids land where the CAD pattern puts them, supporting studies of how spatial arrangement drives fusion and tissue formation.
- Quality built into each block. Each spheroid carries its acquired phenotype, an approach the authors link to quality-by-design principles for future regulatory translation.
- Mixed niches. Precise placement could combine different spheroid types in one construct, for example, to include vascular structures.
The authors position the work as a proof of concept for tissue-engineered implants and organ-on-a-chip models, and name improved spheroid-suspension films and further multi-targeting automation as next steps.
System Used
NGB-R
3D High-Resolution Multi-Modal Robotic-Assisted Bioprinting System
The NGB-R is a multimodal, 3D bioprinting platform designed and developed to print live tissues and organs. Combining laser-assisted, micro-valve, and extrusion bioprinting, the NGB-R enables true versatility of bioprinting (from cells to spheroids) and offers the possibility of using a large number of biomaterials and hydrogels.
View key capabilities
Multi-Modality, Laser-Assisted, Bio-extrusion, and Micro-valve Bioprinting Techniques
- Robotic-assisted
- High cellular viability (>95%)
Integrated microscopy and image analysis platform
References
Nilsson Hall G, Fan Y, Viellerobe B, Iazzolino A, Dimopoulos A, Poiron C, Clapies A, Luyten F P, Guillemot F and Papantoniou I. Laser-assisted bioprinting of targeted cartilaginous spheroids for high density bottom-up tissue engineering. Biofabrication 16 (2024) 045029. Open access. doi.org/10.1088/1758-5090/ad6e1a
