Nahmias, Y. & Odde, D.J. Micropatterning of living cells by laser-guided direct writing: application to fabrication of hepatic-endothelial sinusoid-like structures. Nat. Protoc. 1, 2288-2296

Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, 51 Blossom Street, Boston, Massachusetts 02114, USA.
Nature Protocol (Impact Factor: 9.67). 02/2006; 1(5):2288-96. DOI: 10.1038/nprot.2006.386
Source: PubMed


Here, we describe a simple protocol for the design and construction of a laser-guided direct writing (LGDW) system able to micropattern the self-assembly of liver sinusoid-like structures with micrometer resolution in vitro. To the best of our knowledge, LGDW is the only technique able to pattern cells "on the fly" with micrometer precision on arbitrary matrices, including soft gels such as Matrigel. By micropatterning endothelial cells on Matrigel, one can control the self-assembly of vascular structures and associated liver tissue. LGDW is therefore uniquely suited for studying the role of tissue architecture and mechanical properties at the single-cell resolution, and for studying the effects of heterotypic cell-cell interactions underlying processes such as liver morphogenesis, differentiation and angiogenesis. The total time required to carry out this protocol is typically 7 h.

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    • "MAPLE DW provides CAD/CAM control and processing along with selective cell patterning. LG DW [81] [82] [83] [84] and laser guidance [85] [86] [87] typically use a laser operating at an ∼800 nm wavelength. The laser beam is weakly focused into a liquid suspension of cells and the force of the light moves the cells from the suspension onto a translating receiving substrate (figure 5(a)), but the working distance is usually limited to less than 300 μm. "
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    ABSTRACT: 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.
    Biofabrication 09/2010; 2(3):032001. DOI:10.1088/1758-5082/2/3/032001 · 4.29 Impact Factor
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    • "High-throughput laser printing of cells and biomaterials for tissue engineering. Acta Biomater (2009), doi:10.1016/j.actbio.2009.09.029 merizing, micromachining, laser catapulting [30], LGDW [10] [11]) could thus be combined with bioprinting when the microarchitecture and the cell microenvironment of some tissue parts or layers do not require precise positioning of components. Consequently, a coefficient expressing the integrability or the interacting capacity of the bioprinting method has to be introduced in Eq. (2), leading to: BEC ¼ integrability  speed  volume resolution ð3Þ "
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    ABSTRACT: In parallel with ink-jet printing and bioplotting, biological laser printing (BioLP) using laser-induced forward transfer has emerged as an alternative method in the assembly and micropatterning of biomaterials and cells. This paper presents results of high-throughput laser printing of a biopolymer (sodium alginate), biomaterials (nano-sized hydroxyapatite (HA) synthesized by wet precipitation) and human endothelial cells (EA.hy926), thus demonstrating the interest in this technique for three-dimensional tissue construction. A rapid prototyping workstation equipped with an IR pulsed laser (tau=30 ns, lambda=1064 nm, f=1-100 kHz), galvanometric mirrors (scanning speed up to 2000 mm s(-1)) and micrometric translation stages (x, y, z) was set up. The droplet generation process was controlled by monitoring laser fluence, focalization conditions and writing speed, to take into account its mechanism, which is driven mainly by bubble dynamics. Droplets 70 microm in diameter and containing around five to seven living cells per droplet were obtained, thereby minimizing the dead volume of the hydrogel that surrounds the cells. In addition to cell transfer, the potential of using high-throughput BioLP for creating well-defined nano-sized HA patterns is demonstrated. Finally, bioprinting efficiency criteria (speed, volume, resolution, integrability) for the purpose of tissue engineering are discussed.
    Acta biomaterialia 10/2009; 6(7):2494-500. DOI:10.1016/j.actbio.2009.09.029 · 6.03 Impact Factor
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    • "An exemplification of this is none other than flow cytometry (Bennewith and Durand, 2004; Wongchaowart et al., 2006; MacLennan et al., 2007). There are several other physical sciencebased techniques which are undergoing research and development for assessing their utility within the life sciences (Singh and Hillier, 2007; Nahmias and Odde, 2006; Jayasinghe 2006a, 2006b). However, techniques such as ink-jet printing and laser-guided cell writing have several obstacles which limit their exploration. "
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    ABSTRACT: Bio-electrosprays, pioneered in 2005, have undergone several developmental studies which have seen this technique evolve as a novel direct in vivo tissue engineering and regenerative medicinal strategy. Those studies have been a hallmark for electrosprays; however, in this communication we report our on-going developmental investigations for exploring bio-electrosprays as a potential medical device and diagnostic protocol. The studies reported here demonstrate the ability to directly jet whole human blood without affecting the genetic make-up, which has been interrogated by way of reverse transcription-polymerase chain reaction (RT-PCR) in comparison to controls (p = 0.7337). These studies demonstrate bio-electrosprays as a possible diagnostic protocol.
    Journal of Tissue Engineering and Regenerative Medicine 10/2009; 3(7):562-6. DOI:10.1002/term.185 · 5.20 Impact Factor
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