Sacrificial-layer free transfer of mammalian cells using near infrared femtosecond laser pulses

Abstract

Laser-induced cell transfer has been developed in recent years for the flexible and gentle printing of cells. Because of the high transfer rates and the superior cell survival rates, this technique has great potential for tissue engineering applications. However, the fact that material from an inorganic sacrificial layer, which is required for laser energy absorption, is usually transferred to the printed target structure, constitutes a major drawback of laser based cell printing. Therefore alternative approaches using deep UV laser sources and protein based acceptor films for energy absorption, have been introduced. Nevertheless, deep UV radiation can introduce DNA double strand breaks, thereby imposing the risk of carcinogenesis. Here we present a method for the laser-induced transfer of hydrogels and mammalian cells, which neither requires any sacrificial material for energy absorption, nor the use of UV lasers. Instead, we focus a near infrared femtosecond (fs) laser pulse (λ = 1030 nm, 450 fs) directly underneath a thin cell layer, suspended on top of a hydrogel reservoir, to induce a rapidly expanding cavitation bubble in the gel, which generates a jet of material, transferring cells and hydrogel from the gel/cell reservoir to an acceptor stage. By controlling laser pulse energy, well-defined cell-laden droplets can be transferred with high spatial resolution. The transferred human (SCP1) and murine (B16F1) cells show high survival rates, and good cell viability. Time laps microscopy reveals unaffected cell behavior including normal cell proliferation.

Single Cell Bioprinting with Ultrashort Laser Pulses

“…Our approach avoids these shortcomings by using fs NIR laser pulses in the biological optical window, which do not require inorganic sacrificial layers and do not induce DNA double strand breaks. [ 33 ] Only in the laser focus, the photon density is sufficient to initiate nonlinear absorption and an optical breakdown. [ 44 ] Outside of the focus, where no multiphoton absorption occurs, the 1030 nm laser pulses hardly interact with the cells.…”

“…This process can be used to transfer small gel‐droplets containing ≈10–30 cells to a target substrate with high cell viability. [ 33 ] Based on these findings, we have now elaborated a method, which allows the identification and selection of individual mammalian cells from a cell reservoir as well as the subsequent transfer of these cells to a target surface with single cell precision and a 93–99% cell‐survival rate, depending on cell type and target substrate (see Figure a,b). We have integrated this setup into an inverted optical microscope (Figure 1a), which allows us to use cell morphology (e.g., cell shape or size) or fluorescence to select and sort individual cells from a heterogeneous cell population.…”

“…[ 36 ] The resulting hydrogel jet [ 30 ] can be used to transfer single or multiple cells from the hydrogel/cell reservoir to the target substrate. [ 33 ]…”

Laser‐Induced Forward Transfer: Fundamentals and Applications

“…One potential niche market is the printing of biological materials, which are usually found in aqueous solutions and suspensions, and which are therefore transparent to most popular laser wavelengths. Thus, it has been proved that the technique is feasible for printing biomolecules like DNA and some proteins, and therefore for biosensor fabrication (Sections and ), and more recently it has been shown that FF‐LIFT is especially suited for printing living cells for tissue engineering applications (Section ). Another set of materials very attractive to be printed with the film‐free approach are optical grade adhesives, normally used in the fabrication of micro‐optical components …”

Precision 3D‐Printed Cell Scaffolds Mimicking Native Tissue Composition and Mechanics

“…This quest, to ultimately recreate the in vivo matrix in all its detail, continues to inspire new fabrication method developments. [15][16][17] Initially limited to 2D micro-patterning, such as elastomere stamps, [18,19] spotted DNA microarrays, [20] or polyacrylamide gels, [21] additive manufacturing increasingly expanded the capacity to engineer cell and tissue environments in vitro. [22] In particular, one-photon stereolithography and volumetric bioprinting achieved fast fabrication of clinically relevantly sized tissue constructs of protein-based resins at two-digit µm scale resolution.…”