Laser printing based on laser-induced forward transfer (LIFT) is a new biofabrication technique for the arrangement of biological materials or living cells in well-defined patterns. In the current study, skin cell lines (fibroblasts/keratinocytes) and human mesenchymal stem cells (hMSC) were chosen for laser printing experiments due to their high potential in regeneration of human skin and new application possibilities of stem cell therapy. To evaluate the influence of LIFT on the cells, their survival rate, their proliferation and apoptotic activity, and the DNA damages and modifications of their cell surface markers were assessed and statistically evaluated over several days. The cells survived the transfer procedure with a rate of 98% ± 1% standard error of the mean (skin cells) and 90% ± 10% (hMSC), respectively. All used cell types maintain their ability to proliferate after LIFT. Further, skin cells and hMSC did not show an increase of apoptosis or DNA fragmentation. In addition, the hMSC keep their phenotype as proven by fluorescence activated cell sorting (FACS) analysis. This study demonstrates LIFT as a suitable technique for unharmed computer-controlled positioning of different cell types and a promising tool for future applications in the ex vivo generation of tissue replacements.

Laser-assisted bioprinting of multi-cellular replicates in accordance with CAD blueprint may substantially improve our understandings of fundamental aspects of 3 D cell-cell and cell-matrix interactions in vitro. For predictable printing results, a profound knowledge about effects of different processing parameters is essential for realisation of 3 D cell models with well-defined cell densities. Time-resolved imaging of the hydrogel jet dynamics and quantitative assessment of the dependence of printed droplet diameter on the process characteristics were conducted. The existence of a counterjet was visualised, proving the bubble collapsing theory for the jet formation. Furthermore, by adjusting the viscosity and height of the applied hydrogel layer in combination with different laser pulse energies, the printing of volumes in the range of 10 to 7000 picolitres was demonstrated. Additionally, the relationship between the viscosity and the layer thickness at different laser pulse energies on the printed droplet volume was identified. These findings are essential for the advancement of laser-assisted bioprinting by enabling predictable printing results and the integration of computational methods in the generation of 3 D multi-cellular constructs.

Research on human induced pluripotent stem cells (hiPSCs) is one of the fastest growing fields in biomedicine. Generated from patient’s own somatic cells, hiPSCs can be differentiated towards all functional cell types and returned to the patient without immunological concerns. 3D printing of hiPSCs could enable the generation of functional organs for replacement therapies or realization of organ-on-chip systems for individualized medicine. Printing of living cells was demonstrated with immortalized cell lines, primary cells, and adult stem cells with different printing technologies and biomaterials. However, hiPSCs are more sensitive to handling procedures, in particular, when dissociated into single cells. Both pluripotency and directed differentiation are influenced by numerous environmental factors including culture media, biomaterials, and cell density. Notably, existing literature on the effect of applied biomaterials on pluripotency is rather ambiguous. In this study, laser bioprinting of undifferentiated hiPSCs in combination with different biomaterials was performed and the impact on cells’ behavior, pluripotency, and differentiation was investigated. Our findings suggest that hiPSCs are indeed more sensitive to the applied biomaterials, but not to laser printing itself. With appropriate biomaterials, such as the hyaluronic acid based solutions applied in this study, hiPSCs can be successfully laser printed without losing their pluripotency.

For more than a decade, living cells and biomaterials (typically hydrogels) are printed via laser-assisted bioprinting. Often, a thin metal layer is applied as laser-absorbing material called dynamic release layer (DRL). This layer is vaporized by focused laser pulses generating vapor pressure that propels forward a coated biomaterial. Different lasers with laser wavelengths from 193 to 1064 nanometer have been used. As a metal DRL gold, silver, or titanium layers have been used. The applied laser pulse durations were usually in the nanosecond range from 1 to 30 ns. In addition, some studies with femtosecond lasers have been published. However, there are no studies on the effect of all these lasers parameters on bioprinting with a metal DRL, and on comparing different wavelengths and pulse durations – except one study comparing 500 femtosecond pulses with 15 ns pulses. In this paper, the effects of laser wavelength (355, 532, and 1064 nm) and laser pulse duration (in the range of 8 to 200 ns) are investigated. Furthermore, the effects of laser pulse energy, intensity, and focal spot size are studied. The printed droplet volume, hydrogel jet velocity, and cell viability are analyzed.

Bioprinting is seen as a promising technique for tissue engineering, with hopes of one day being able to produce whole organs. However, thick tissue requires a functional vascular network, which naturally contains vessels of various sizes, down to capillaries of ~10 µm in diameter, often spaced less than 200 µm apart. If such thick tissues are to be printed, the vasculature would likely need to be printed at the same time, including the capillaries. While there are many approaches in tissue engineering to produce larger vessels in a defined manner, the small capillaries usually arise only in random patterns by sprouting from the larger vessels or from randomly distributed endothelial cells. Here, we investigated whether the small capillaries could also be printed in predefined patterns. For this purpose, we used a laser-based bioprinting technique that allows for the combination of high resolution and high cell density. Our aim was to achieve the formation of closed tubular structures with lumina by laser-printed endothelial cells along the printed patterns on a surface and in bioprinted tissue. This study shows that such capillaries are directly printable; however, persistence of the printed tubular structures was achieved only in tissue with external stimulation by other cell types.