Femtosecond laser printing of living human cells

Laser-induced forward transfer (LIFT) has been used in recent years for the flexible and gentle 3D-bioprinting of cells with superior cell survival rates relative to other methods [1]. One drawback of the current state-of-the-art nanosecond laser based cell printing is the fact that material from an inorganic sacrificial layer, which is required for laser energy absorption, is transferred to the printed target structure, where it contaminates the printed construct [2]. Furthermore, existing LIFT based technologies transfer multiple cells at a time, i.e. with a single laser pulse. However, living cells in a functional in vitro microenvironment are exposed to multiple biophysical, biochemical and biological signals. Therefore, a 3D-bioprinting technique providing single cell resolution is desirable for a number of applications, e.g. the investigation of cell-cell interactions and cell niches, which has not yet been achieved using laser based bioprinting. Here we present a new femtosecond laser-based method for the efficient and precise single cell printing and sorting, which avoids the use of non-biological inorganic absorption layers. An ultrashort laser pulse (λ = 1030 nm, 600 fs, few μJ) is focused underneath a cell layer, which is suspended on top of a hydrogel reservoir. Nonlinear absorption leads to plasma ionization and rapid cavitation bubble expansion, which generates a jet of material, transferring cell-laden hydrogel from a gel/cell reservoir to an acceptor stage [3]. In addition, to the effective sacrificial-layer free transfer of multiple cells, individual cells can be selected based on their morphology and phenotype, and transferred to the acceptor slide, isolated from the remaining cells. Furthermore, laser-induced single cell printing efficiencies close to 100% were achieved for the first time.