Fabrication of cellular constructs with spatial control of cell location (+/-5 microm) is essential to the advancement of a wide range of applications including tissue engineering, stem cell and cancer research. Precise cell placement, especially of multiple cell types in co- or multi-cultures and in three dimensions, can enable research possibilities otherwise impossible, such as the cell-by-cell assembly of complex cellular constructs. Laser-based direct writing, a printing technique first utilized in electronics applications, has been adapted to transfer living cells and other biological materials (e.g., enzymes, proteins and bioceramics). Many different cell types have been printed using laser-based direct writing, and this technique offers significant improvements when compared to conventional cell patterning techniques. The predominance of work to date has not been in application of the technique, but rather focused on demonstrating the ability of direct writing to pattern living cells, in a spatially precise manner, while maintaining cellular viability. This paper reviews laser-based additive direct-write techniques for cell printing, and the various cell types successfully laser direct-written that have applications in tissue engineering, stem cell and cancer research are highlighted. A particular focus is paid to process dynamics modeling and process-induced cell injury during laser-based cell direct writing.

Laser direct-writing provides a method to pattern living cells in vitro, to study various cell–cell interactions, and to build cellular constructs. However, the materials typically used may limit its long-term application. By utilizing gelatin coatings on the print ribbon and growth surface, we developed a new approach for laser cell printing that overcomes the limitations of Matrigel™. Gelatin is free of growth factors and extraneous matrix components that may interfere with cellular processes under investigation. Gelatin-based laser direct-write was able to successfully pattern human dermal fibroblasts with high post-transfer viability (91% ± 3%) and no observed double-strand DNA damage. As seen with atomic force microscopy, gelatin offers a unique benefit in that it is present temporarily to allow cell transfer, but melts and is removed with incubation to reveal the desired application-specific growth surface. This provides unobstructed cellular growth after printing. Monitoring cell location after transfer, we show that melting and removal of gelatin does not affect cellular placement; cells maintained registry within 5.6 ± 2.5 μm to the initial pattern. This study demonstrates the effectiveness of gelatin in laser direct-writing to create spatially precise cell patterns with the potential for applications in tissue engineering, stem cell, and cancer research.

Introduction: Advances in tendon engineering with mesenchymal stem cells (MSCs) are hindered by a need for cues to direct tenogenesis, and markers to assess tenogenic state. We examined the effects of factors involved in embryonic tendon development on adult MSCs, and compared MSC responses to that of embryonic tendon progenitor cells (TPCs), a model system of tenogenically differentiating cells. Methods: Murine MSCs and TPCs subjected to cyclic tensile loading, transforming growth factor-β2 (TGFβ2), and fibroblast growth factor-4 (FGF4) in vitro were assessed for proliferation and mRNA levels of scleraxis, TGFβ2, tenomodulin, collagen type I and elastin. Results: Before treatment, scleraxis and elastin levels in MSCs were lower than in TPCs, while other tendon markers expressed at similar levels in MSCs as TPCs. TGFβ2 alone and combined with loading were tenogenic based on increased scleraxis levels in both MSCs and TPCs. Loading alone had minimal effect. FGF4 downregulated tendon marker levels in MSCs but not in TPCs. Select tendon markers were not consistently upregulated with scleraxis, demonstrating the importance of characterizing a profile of markers. Conclusions: Similar responses as TPCs to specific treatments suggest MSCs have tenogenic potential. Potentially shared mechanisms of cell function between MSCs and TPCs should be investigated in longer term studies.