Micropatterning of living cells by laser-guided direct writing: application to fabrication of hepatic– endothelial sinusoid-like structures Yaakov Nahmias 1 & David J Odde 2 1 Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, 51 Blossom Street, Boston, Massachusetts 02114, USA. 2 Department of Biomedical Engineering, University of Minnesota, 7-132 Nils Hasselmo Hall, 312 Church Street SE, Minneapolis, Minnesota 55455, USA. Correspondence should be addressed to D.J.O. (oddex002@umn.edu). Published online 21 December 2006; doi:10.1038/nprot.2006.386 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. INTRODUCTION The liver is one of the largest internal organs in the body, accounting for 2% of the weight of an adult 1,2 . It has been ascribed over 500 functions including albumin secretion, urea production, bile clearance and glycogen storage. The liver also serves as a hub for carbohydrate, lipid and amino-acid metabolism 3 . In addition to its metabolic activity, the liver is one of the body’s first lines of defense, inactivating toxins and xenobiotics, clearing foreign particles and producing acute-phase proteins in response to injury or infection 4–10 . Finally, the liver has the unique capacity to regenerate from massive injuries, restoring to its original mass even if less than 20% of the organ is undamaged 3,11,12 . Loss of liver function causes 25,000 deaths per year in the United States alone, and is one of the most frequent causes of death worldwide 13 . The only known treatment for liver failure is ortho- topic liver transplantation, but unfortunately only a fraction of the organs needed are available (less than 7,000 organs per year in the United States). This organ shortage has spurred research in the field of hepatic tissue engineering aiming to create functional liver tissue to support or even cure patients awaiting transplantation 14,15 . Similar liver tissue engineering technology is also used in the pharmaceutical industry, screening potential new drugs before in vivo animal and human studies 6,7 , and for basic science including the study of liver development 16 , regeneration 12 , ischemia/reperfu- sion injury 17 , fibrosis 18 , viral infection 19 and inflammation 20 . One of the principal limitations to hepatic tissue engineering is oxygen and nutrient transport. Primary hepatocytes are highly metabolic cells, consuming oxygen at rates as high as 0.9 nmol per second per 10 6 cells 21,22 . This requirement has limited the size of engineered liver tissue to the oxygen diffusion limit, B200 mm (see ref. 23). One possible solution to this problem is to directly write microscale endothelial vascular structures within the tissue engi- neered liver, reconstructing the tissue cell-by-cell according to its native architecture 24 . This technique has the advantage of recaptur- ing the heterotypic cell–cell interactions found in the adult liver and is shown to be important in maintaining liver function 25 and differentiated phenotype 26,27 and in mediating its response to infection and toxic challenge 28,29 . LGDW LGDW is a unique cellular patterning technique able to deposit cells with micrometer accuracy on arbitrary matrices, including soft gels such as collagen or Matrigel 30–33 . The unique ability of LGDW to micropattern cells on the basement membrane Matrigel allows us to use the intrinsic ability of endothelial cells to self-assemble into vascular structures 34,35 for the assembly of the liver tissue. At its core, LGDW is a variation of the commonly used optical trap (laser tweezers, optical tweezers) 30,36 . Like an optical trap, the system is composed of a single-mode (TEM 00 ) laser, which is focused by a lens onto a target area. Cells in the laser beam path experience radiation forces owing to the scattering of photons by the cell–media interface 36 . As cells have a higher refractive index than that of their surrounding media, most of the photons will be scattered away from the axis of the laser beam, causing an opposite and equal force trapping the cells in the beam axis 36 . In contrast to an optical trap, in LGDW, the laser beam is weakly focused to a spot about 5 mm in radius, allowing cells to be pushed along the beam axis until they are deposited on the surface 30 . By translating the surface relative to the beam, one can write arbitrary patterns using living cells 31 . In this case, LGDW resolution is only limited by the size of the cells (B10 mm in diameter) and the magnitude of the opposing thermal forces. As an added advantage, weakly focused beams have a long working distance, several centimeters on average, allowing the remote patterning of cells, under sterile conditions, in standard tissue culture wells and dishes. To avoid optical damage to cells, LGDW uses the near-infrared part of the electromagnetic spectrum (700–1,000 nm), which is past the absorption spectra of most proteins and before the infrared absorption of water. Photons in this range of the spectrum lack the energy to create free radicals (as in the ultraviolet) and are not absorbed by DNA, suggesting that the laser has little chance of p u o r G g n i h s i l b u P e r u t a N 6 0 0 2 © natureprotocols / m o c . e r u t a n . w w w / / : p t t h 2288 | VOL.1 NO.5 | 2006 | NATURE PROTOCOLS PROTOCOL