This work describes the selective targeting of pigmented retinal pigment epithelial (RPE) cells by a single pulsed laser irradiation. We observed: (1) single pulsed laser irradiation caused cellular damages on pigmented, and not on non-pigmented RPE cells at laser radiant exposure up to 2550 mJ/cm2; (2) in the mixture of pigmented and non-pigmented RPE cells, single pulsed laser-induced damage was confined to pigmented RPE cells. This study demonstrates that the pigmented RPE cells can be selectively damaged, using a single pulsed laser irradiation, without thermal coagulation to adjacent non-pigmented RPE cells. © 2008 Optical Society of America.

A major goal of liver tissue engineering is to understand how the constituent cell types interact to achieve liver-specific structure and function. Here we show that hepatocytes migrate toward and adhere to endothelial vascular structures formed on Matrigel in vitro, and that hepatocyte recruitment is dependent on endothelium-derived hepatocyte growth factor. The hepatocyte-decorated endothelial vascular structures resemble in vivo sinusoids containing plate-like structures, bile canaliculi, and a lumen. The sinusoid-like structures retained cytochrome P450 expression and activity, in addition to stable albumin expression and secretion rate for over 2 months in vitro. The stability of the sinusoid-like structures was dependent on the presence of vimentin-positive fibroblasts in culture. The sinusoid-like structures formed by hepatocytes and pure populations of endothelial cells collapsed after 10 days in culture. In contrast, culture of hepatocytes with fibroblast-contaminated human dermal microvascular endothelial cells or a combination of human umbilical vein endothelial cells and normal human dermal fibroblasts resulted in stable sinusoid-like structures surrounded by a fibroblastic capsule that maintained liver specific functions for several months in vitro. These results demonstrate that specification of endothelial cell position ultimately determines hepatocyte position in vitro, suggesting that similar interactions might occur in vivo. The novelty of the culture's sinusoid-like organization and long-term function suggest a new model for the study of liver toxicity, iscaemia/reperfusion injury, and fibrosis. © Mary Ann Liebert, Inc.

The liver is the largest internal organ in the body, responsible for over 500 metabolic, regulatory, and immune functions. Loss of liver function leads to liver failure which causes over 25 000 deaths/year in the United States. Efforts in the field of hepatic tissue engineering include the design of bioartificial liver systems to prolong patient's lives during liver failure, for drug toxicity screening and for the study of liver regeneration, ischemia/reperfusion injury, fibrosis, viral infection, and inflammation. This chapter will overview the current state-of-the-art in hepatology including isolated perfused liver, culture of liver slices and tissue explants, hepatocyte culture on collagen "sandwich" and spheroids, coculture of hepatocytes with non-parenchymal cells, and the integration of these culture techniques with microfluidics and reactor design. This work will discuss the role of oxygen and medium composition in hepatocyte culture and present promising new technologies for hepatocyte proliferation and function. We will also discuss liver development, architecture, and function as they relate to these culture techniques. Finally, we will review current opportunities and major challenges in integrating cell culture, bioreactor design, and microtechnology to develop new systems for novel applications. © Springer-Verlag 2006.

Low-density lipoprotein (LDL) is an important carrier of plasma cholesterol and triglycerides whose concentration is regulated by the liver parenchymal cells. Abnormal LDL regulation is thought to cause atherosclerosis, while viral binding to LDL has been suggested to facilitate hepatitis C infection. Primary hepatocytes quickly lose the ability to clear LDL during in vitro culture. Here we show that the coculture of hepatocytes with liver sinusoidal endothelial cells (LSEC) significantly increases the ability of hepatocytes to uptake LDL in vitro. LDL uptake does not increase when hepatocytes are cocultured with other cell types such as fibroblasts or umbilical vein endothelial cells. We find that LSECs induce the hepatic expression of the LDL receptor and the epidermal growth factor receptor. In addition, while hepatocytes in single culture did not take up hepatitis C virus (HCV)-like particles, the hepatocytes cocultured with LSECs showed a high level of HCV-like particle uptake. We suggest that coculture with LSECs induces the emergence of a sinusoidal surface in primary hepatocytes conducive to the uptake of HCV-like particles. In conclusion, our findings describe a novel model of polarized hepatocytes in vitro that can be used for the study of LDL metabolism and hepatitis C infection. Copyright © 2006 by the American Association for the Study of Liver Diseases.

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.

Oxygen is an important component of the cellular microenvironment, mediating cell survival, differentiation, and function. Oxygen supply is a limiting factor during culture of highly metabolic cells such as hepatocytes. Here we present a simple formulation of a fluorocarbon-based oxygen carrier embedded in collagen gel that increases oxygen concentration in culture 6-fold. Rat hepatocytes cultured on oxygen carrier-collagen showed a significant increase in viability and function. Cytochrome P450IA1 activity was increased by 140% in serum-free cultures and by 820% in serum-containing cultures. The significantly higher hepatocellular function on oxygen carrier-collagen matrix persisted and increased during long-term culture. Long-term albumin secretion was increased by 350% in serum-free cultures and by 166% in serum-containing culture. Long-term urea secretion was increased by 79% in serum-free cultures and by 76% in serum-containing cultures. We conclude that oxygen supply may limit hepatocyte function in vitro. This limitation can be overcome by addition of an oxygen carrier to the extracellular matrix. Culture of hepatocytes on oxygen-carrying matrix mimics the oxygen-rich environment of the liver and provides a simple method for enhanced long-term function. © FASEB.

We present an easily applicable and inexpensive method for patterning cells on arbitrary surfaces including biological gels with little loss of viability or function. Single-cell suspensions of human umbilical vein endothelial cells and NIH 3T3 fibroblasts were sprayed with an off-the-shelf airbrush through a mask to create 100-μm scale patterns on collagen gels. Three-dimensional patterns were created by layering a collagen gel on top of the first pattern and patterning the top gel. Coculture of rat hepatocytes with NIH 3T3 patterns on collagen gels resulted in localized increased activity of cytochrome P-450 along the pattern. These results suggest that cell spraying is a useful tool for the study of heterotypic cellular interactions and tissue-engineering applications on biologically relevant matrices, and for the creation of three-dimensional cell patterns in vitro. © Mary Ann Liebert, Inc.

Ischemia-reperfusion (I/R) injury is the main cause of primary dysfunction or nonfunction after liver transplantation. Steatotic (i.e. fatty) livers comprise up to 25% of the liver donor pool. Steatotic livers are more susceptible to cold or warm ischemia, suffer greater hepatocellular damage during reperfusion, and have impaired regeneration. These factors contribute to the increased postoperative complications and poor survival rates after transplantation or after resection surgery in experimental animals and humans with fatty livers. For example, patients receiving livers with mild-to-moderate steatosis, which are usually considered "marginally acceptable," have a 77% survival 2 years posttransplantation compared with a 91% survival in patients receiving nonsteatotic livers. The mechanisms underlying the high sensitivity of fatty livers to I/R injury are poorly understood. The motivation of this work is to develop an in vitro model of hepatic I/R so that the many potential factors involved can be decoupled. We cultured rat hepatocytes in the collagen sandwich configuration and made them fatty by exposure to fatty acid-supplemented culture medium. Cultures were exposed to defined periods of ischemia followed by normoxia and cell viability was determined. In some experiments, we co-cultured hepatocytes with nonparenchymal cells (NPCs) to determine the impact of these cells on the response. Results with cultured hepatocytes were consistent with in vivo studies. Fatty hepatocytes were more sensitive to I/R injury, as indicated by higher lactate dehydrogenase release when compared to lean hepatocytes. Hepatocyte damage increased as a function of duration of the ischemic period, and was highest during the reperfusion phase. Co-culture with Kupffer cells, which are known to be activated during I/R in vivo, exacerbated the damaging effect of I/R. This cell culture model will be useful to elucidate the mechanisms of I/R in the liver at the cellular level. Furthermore, this system may be a useful platform for high-throughput screening of treatments which could enhance the resistance of lean and fatty liver cells to I/R.

One of the principal limitations to the size of an engineered tissue is oxygen and nutrient transport. Lacking a vascular bed, cells embedded in an engineered tissue will consume all available oxygen within hours while out branching blood vessels will take days to vascularize the implanted tissue. One possible solution is to directly write vascular structures within the engineered tissue prior to implantation, reconstructing the tissue according to its native architecture. The cell patterning technique, laser-guided direct writing (LGDW), can pattern multiple cells types with micrometer resolution on arbitrary surfaces, including biological gels. Here we show that LGDW can pattern human umbilical vein endothelial cells (HUVEC) in two- and three-dimensions with micrometer accuracy. By patterning HUVEC on Matrigel, we can direct their self-assembly into vascular structures along the desired pattern. Finally, co-culturing the vascular structures with hepatocytes resulted in an aggregated tubular structure similar in organization to a hepatic sinusoid. This capability can facilitate studies of tissue architecture at the single cell level, and of heterotypic interactions underlying processes such as liver and pancreas morphogenesis, differentiation, and angiogenesis. © 2005 Wiley Periodicals, Inc.

Optical traps are routinely used for the manipulation of neutral particles. However, optical trap design is limited by the lack of an accurate theory. The generalized Lorenz-Mie theory (GLMT) solves the scattering problem for arbitrary particle size and predicts radial forces accurately. Here we show that the GLMT predicts the observed radial and axial forces in a variety of optical manipulators. We also present a dimensionless parameter β for the prediction of axial forces. Coupled with our correlation for radial escape forces, we now have a set of two simple correlations for the practical design of radiationforce-based systems. © 2004 Optical Society of America.

Radiation forces allow the remote manipulation of physical objects. Two radiation force-based systems have been used, in particular: 1) laser trapping, which uses a strongly convergent beam to form a micrometer-sized focal point in which particles can be trapped and manipulated in three dimensions, and 2) laser guidance, which uses a weakly convergent beam to achieve radial confinement of particles about the beam axis coupled with pushing along the beam axis, which allows the high precision delivery of particles over hundreds of micrometers. Biological applications of laser trapping include high-precision molecular motor force measurement, and those of laser guidance include the direct writing of living cells in two and three dimensions for tissue engineering applications. The results presented here show that a general electromagnetic theory, the Generalized Lorenz-Mie Theory, is able to accurately predict experimental results for both schemes without any assumptions regarding the size of the particle relative to the wavelength of the radiation. In addition, radial forces are found to be directly correlated to the dimensionless particle size (a/ω) where the particle radius (a) is normalized over the beam radius (ω). The dimensionless particle size can be used to theoretically estimate radiation forces in any arbitrary setup, and facilitate the design of radiation force-based systems for a variety of applications in biology and medicine.