Single Cell Bioprinting with Ultrashort Laser Pulses

Abstract

Tissue engineering requires the precise positioning of mammalian cells and biomaterials on substrate surfaces or in preprocessed scaffolds. Although the development of 2D and 3D bioprinting technologies has made substantial progress in recent years, precise, cell‐friendly, easy to use, and fast technologies for selecting and positioning mammalian cells with single cell precision are still in need. A new laser‐based bioprinting approach is therefore presented, which allows the selection of individual cells from complex cell mixtures based on morphology or fluorescence and their transfer onto a 2D target substrate or a preprocessed 3D scaffold with single cell precision and high cell viability (93–99% cell survival, depending on cell type and substrate). In addition to precise cell positioning, this approach can also be used for the generation of 3D structures by transferring and depositing multiple hydrogel droplets. By further automating and combining this approach with other 3D printing technologies, such as two‐photon stereolithography, it has a high potential of becoming a fast and versatile technology for the 2D and 3D bioprinting of mammalian cells with single cell resolution.

Extending Single Cell Bioprinting from Femtosecond to Picosecond Laser Pulse Durations

“…Under appropriate conditions, single cells can be reliably transferred with a high survival rate after transfer through the entire pulse duration range. This agrees very well with our previous findings obtained for a laser pulse duration of 0.6 ps [22,23]. Nevertheless, it is mandatory to investigate the long-term cell viability of ps-laser printed cells in a future study.…”

“…Due to the lower mass-density, living human mesenchymal stem cells (hMSC, SCP1 cell line) were suspended at the hydrogel surface (Figure 1 left). To visualize and investigate the process dynamics, including cavitation bubble expansion and subsequent jet propagation, pump-probe [30] time-resolved imaging observation was carried out, as described previously [23]. Briefly, the transfer process was illuminated by a 28 ns white-light pulse (Nanolite KL-L, High-Speed Photo-Systeme, Wedel, Germany) and then was captured with an imaging system comprising a microscope objective (Mitutoyo Plan Apo 5×/0.14, Japan), a tube lens (TTL200-A, Thorlabs, Bergkirchen, Germany), and a CCD1 camera (PCO, Pixefly USB, Kelheim, Germany).…”

“…With increasing laser pulse duration, we observed less efficient energy conversion and comparable, but slower jet dynamics. In our earlier work the cell survival rate obtained immediately after the transfer showed no significant difference in their ability to migrate and proliferate over a 66 h time scale [22,23]. Thus, to test the principal bioprinting compatibility at different pulse durations, only the cell survival rate 15 min after transfer was determined.…”

“…In our previous work [22,23], we demonstrated that by focusing a 600 fs laser pulse with a wavelength of 1030 nm into a hydrogel underneath a layer of cells, small droplets of hydrogel containing living cells can be ejected from the reservoir and transferred to an acceptor stage. The transferred cells showed a survival rate up to 95% without DNA double strand breaks and maintained their ability to migrate and proliferate even 66 h after the cell transfer.…”

“…Furthermore, the single cells can be transferred with a position precision of ±11.8 µm. By integrating this film-free cell transfer approach into an inverted epifluorescence microscope, this new approach allowed the precise and efficient selection and positioning of individual mammalian cells from a complex cell mixture (based on morphology or fluorescence) onto a 2D target substrate or a 3D pre-processed scaffold with single cell spatial precision [23]. It was found that the transfer process requires less than 100 µs per cell, manually searching for a cell takes ≈5 s, setting the laser focus another ≈5 s, and positioning of the acceptor surface takes further ≈10 s. To date the technique can be used for single cell studies.…”