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The ability to precisely pattern embryonic stem (ES) cells in vitro into predefined arrays/geometries may allow for the recreation of a stem cell niche for better understanding of how cellular microenvironmental factors govern stem cell maintenance and differentiation. In this study, a new gelatin-based laser direct-write (LDW) technique was utilized to deposit mouse ES cells into defined arrays of spots, while maintaining stem cell pluripotency. Results obtained from these studies showed that ES cells were successfully printed into specific patterns and remained viable. Furthermore, ES cells retained the expression of Oct4 in nuclei after LDW, indicating that the laser energy did not affect their maintenance of an undifferentiated state. The differentiation potential of mouse ES cells after LDW was confirmed by their ability to form embryoid bodies (EBs) and to spontaneously become cell lineages representing all three germ layers, revealed by the expression of marker proteins of nestin (ectoderm), Myf-5 (mesoderm) and PDX-1 (endoderm), after 7 days of cultivation. Gelatin-based LDW provides a new avenue for stem cell patterning, with precision and control of the cellular microenvironment., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The survival, proliferation, and differentiation of freshly isolated and cultured cells were studied after absorbing film-assisted laser-induced forward transfer. Rat Schwann and astroglial cells and pig lens epithelial cells were used for transfer and the cells were cultured for 2 weeks after laser-pulsed transfer. All three cell types survived, proliferated, and differentiated under cell culture conditions and regained their original phenotype a few days after cell transfer. Time resolution studies have shown that the time required to accelerate the jets and droplets containing the cells was less than 1 micros and that the estimated minimum average acceleration of those ejected cells that reached a constant velocity was approximately 10(7) x g. This suggests that the majority of studied cells tolerated the extremely high acceleration at the beginning of the ejection and the deceleration during impact on the acceptor plate without significant damage to the original phenotype. These results suggest that the absorbing film-assisted laser-induced forward transfer technique appears to be suitable for several potential applications in tissue engineering and the biomedical tissue repair technologies.

A technique by which to print patterns and multilayers of scaffolding and living cells could be used in tissue engineering to fabricate tissue constructs with cells, materials, and chemical diversity at the micron scale. We describe here studies using a laser forward transfer technology to print single-layer patterns of pluripotent murine embryonal carcinoma cells. This report focuses on verifying cell viability and functionality as well as the ability to differentiate cells after laser transfer. We find that when cells are printed onto model tissue scaffolding such as a layer of hydrogel, greater than 95% of the cells survive the transfer process and remain viable. In addition, alkaline comet assays were performed on transferred cells, showing minimal single-strand DNA damage from potential ultraviolet-cell interaction. We also find that laser-transferred cells express microtubular associated protein 2 after retinoic acid stimulus and myosin heavy chain protein after dimethyl sulfoxide stimulus, indicating successful neural and muscular pathway differentiation. These studies provide a foundation so that laser printing may next be used to build heterogeneous multilayer cellular structures, enabling cell growth and differentiation in heterogeneous three-dimensional environments to be uniquely studied.