Padmavathy Rajagopalan

3D Hepatic Cultures Simultaneously Maintain Primary Hepatocyte and Liver Sinusoidal Endothelial Cell Phenotypes

Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.

Biocompatible, detachable, and free-standing polyelectrolyte multilayer films.

Self-assembled polyelectrolyte multilayers have gained tremendous popularity over the past decade and have been incorporated in diverse applications. However, the fabrication of detachable and free-standing polyelectrolyte multilayers (PEMs) has proven to be difficult. We report the design of detachable, free-standing, and biocompatible PEMs comprised of hyaluronic acid (anionic PE) and chitosan (cationic PE). These PEMs can be detached from an underlying inert substrate without any postprocessing steps. Our approach enables the fabrication of detachable PEMs from a wide range of polyelectrolytes. Cross-linked PEMs exhibited greater than 95% weight retention when maintained in phosphate buffered saline at 37 °C over a seven day period. The PEM thickness was approximately 3 μm for dried films and increased 2-fold under hydration. A unique feature of the detachable, free-standing PEMs is their optical transparency in the 400-900 nm range under hydrated conditions. The Youngs modulus of the cross-linked films ranged from 300-400 MPa, rendering these detachable free-standing multilayers ideal for biomaterial applications. BALB/c 3T3 fibroblasts adhered on the PEMs and colonized the entire surface over a six day period. The cellular responses, as well as the physical properties, demonstrate that the detachable PEM films exhibit tremendous potential for applications in biomaterials and tissue engineering.

Nano- and sub-micron porous polyelectrolyte multilayer assemblies: Biomimetic surfaces for human corneal epithelial cells

In vivo, corneal epithelial cells adhere on basement membranes that exhibit porosity on the nanoscale with the diameters of pores and fibers ranging from 20 to 200 nm. Polyelectrolyte multilayers with porosity ranging from the nano to the microscale were assembled to mimic the pore sizes of corneal membranes in vivo. The average pore diameter was found to be 100 nm and 600 nm for the nanoporous and sub-micron porous films respectively. In this study, a purely physical feature, specifically, porosity, provided cues to human corneal epithelial cells. Porous surfaces that exhibited either 100 nm or 600 nm pore diameters supported corneal cell adhesion, however, nanoscale porosity significantly enhanced corneal epithelial cellular response. Corneal epithelial cell proliferation and migration speeds were significantly higher on nanoporous topographies. The actin cytoskeletal organization was well defined and vinculin focal adhesions were found in cells presented with a nanoscale environment. These trends prevailed for fibronectin-coated surfaces as well suggesting that for human corneal epithelial cells, the physical environment plays a defining role in guiding cell behavior.

The Design of In Vitro Liver Sinusoid Mimics Using Chitosan–Hyaluronic Acid Polyelectrolyte Multilayers

Interactions between hepatocytes and liver sinusoidal endothelial cells (LSECs) are essential for the development and maintenance of hepatic phenotypic functions. We report the assembly of three-dimensional liver sinusoidal mimics comprised of primary rat hepatocytes, LSECs, and an intermediate chitosan-hyaluronic acid polyelectrolyte multilayer (PEM). The height of the PEMs ranged from 30 to 55 nm and exhibited a shear modulus of approximately 100 kPa. Hepatocyte-PEM cellular constructs exhibited stable urea and albumin production over a 7-day period, and these values were either higher or similar to cells cultured in a collagen sandwich. This is of significance because the thickness of a collagen gel is approximately 1000-fold higher than the height of the chitosan-hyaluronic acid PEM. In the hepatocyte-PEM-LSEC liver-mimetic cellular constructs, LSEC phenotype was maintained, and these cultures exhibited stable urea and albumin production. CYP1A1/2 activity measured over a 7-day period was significantly higher in the hepatocyte-PEM-LSEC constructs than in collagen sandwich cultures. A 16-fold increase in CYP1A1/2 activity was observed for hepatocyte-PEM-10,000 LSEC samples, thereby suggesting that interactions between hepatocytes and LSECs are critical in enhancing the detoxification capability in hepatic cultures in vitro.

Cell Migration at the Interface of a Dual Chemical-Mechanical Gradient

Cell migration plays a critical role in numerous physiological processes, such as wound healing, response to inflammation, and cancer metastasis. In recent years, accumulating evidence indicates that cell movement is regulated not only by chemical signals but also by mechanical stimuli. In this study, the primary goal is to identify whether a chemical or mechanical stimulus plays the decisive role in directing cell migration. Measuring the motility of cells when they are presented with a combination of chemical and mechanical cues will provide insight into the complex physiological phenomena that guide and direct migration. A novel polyacrylamide hydrogel was designed with an interfacial region where the chemical and mechanical properties varied in opposing directions. One side of the interface was stiff (high Youngs modulus) with a low protein concentration, whereas the other side of the interface was compliant (low Youngs modulus) with a high protein concentration. The chemical gradient was created by varying the collagen (type I) concentration and the mechanical gradient was introduced by changing the extent of cross-linking in the polymer. The length of the interface with opposing chemical-mechanical profiles was found to be approximately 100 mum. Our results demonstrate that when Balb/c 3T3 fibroblasts were presented with a choice, they either migrated preferentially toward the high-collagen-compliant (low Youngs modulus) side of the interfacial region or remained on the high-collagen region, suggesting a more dominant role for chemical stimuli in directing fibroblast locomotion.

Designing a fibrotic microenvironment to investigate changes in human liver sinusoidal endothelial cell function.

The deposition of extracellular matrix (ECM) proteins by hepatic cells during fibrosis leads to the stiffening of the organ and perturbed cellular functions. Changes in the elasticity of liver tissue are manifested by altered phenotype in hepatic cells. We have investigated changes in human liver sinusoidal endothelial cells (hLSECs) that occur as the elastic modulus of their matrix transitions from healthy (6kPa) to fibrotic (36kPa) conditions. We have also investigated the role played by Kupffer cells in the dedifferentiation of hLSECs. We report the complete loss of fenestrae and the expression of CD31 at the surface as a result of increasing elastic moduli. LSECs exhibited a greater number of actin stress fibers and vinculin focal adhesion on the stiffer substrate, as well. A novel finding is that these identical trends can be obtained on soft (6kPa) substrates by introducing an inflamed microenvironment through the addition of Kupffer cells. hLSEC monocultures on 6kPa gels exhibited fenestrae that were 140.7±52.6nm in diameter as well as a lack of surface CD31 expression. Co-culturing hLSECs with rat Kupffer cells (rKCs) on 6kPa substrates, resulted in the complete loss of fenestrae, an increase in CD31 expression and in a well-organized cytoskeleton. These results demonstrate that the increasing stiffness of liver matrices does not solely result in changes in hLSEC phenotype. Even on soft substrates, culturing hLSECs in an inflamed microenvironment can result in their dedifferentiation. Our findings demonstrate the interplay between matrix elasticity and inflammation in the progression of hepatic fibrosis.

Discovering Networks of Perturbed Biological Processes in Hepatocyte Cultures

The liver plays a vital role in glucose homeostasis, the synthesis of bile acids and the detoxification of foreign substances. Liver culture systems are widely used to test adverse effects of drugs and environmental toxicants. The two most prevalent liver culture systems are hepatocyte monolayers (HMs) and collagen sandwiches (CS). Despite their wide use, comprehensive transcriptional programs and interaction networks in these culture systems have not been systematically investigated. We integrated an existing temporal transcriptional dataset for HM and CS cultures of rat hepatocytes with a functional interaction network of rat genes. We aimed to exploit the functional interactions to identify statistically significant linkages between perturbed biological processes. To this end, we developed a novel approach to compute Contextual Biological Process Linkage Networks (CBPLNs). CBPLNs revealed numerous meaningful connections between different biological processes and gene sets, which we were successful in interpreting within the context of liver metabolism. Multiple phenomena captured by CBPLNs at the process level such as regulation, downstream effects, and feedback loops have well described counterparts at the gene and protein level. CBPLNs reveal high-level linkages between pathways and processes, making the identification of important biological trends more tractable than through interactions between individual genes and molecules alone. Our approach may provide a new route to explore, analyze, and understand cellular responses to internal and external cues within the context of the intricate networks of molecular interactions that control cellular behavior.

Synthesis and near infrared properties of rare earth ionomers

Fully exchanged, anhydrous ionomers of ethylene-co-acrylic acid (EAA) copolymers and ethylene-co-methacrylic acid (EMAA) copolymers containing Dy +3 , Er +3 , Sm +3 , Tb +3 , Tm +3 , and Yb +3 , and mixtures of them, were synthesized and studied in the near infrared region by reflection and Fourier Transform Laser Raman spectroscopies. The EAA copolymers ranged from 1.4 to 8.7 mol % acid and the EMAA copolymers were 7.3 and 16.2 mol % acid. The ionomers were shown to be essentially free of carboxylic acid groups, water, or other forms containing O-H groups and were characterized by infrared and other methods. They are light and heat stable, and become thermoplastic and moldable at ca. 220°C under pressure. When excited at 1.064 μ with a Nd : YAG laser, these ionomers exhibit novel, lanthanide-dependent near infrared luminescence and strong Raman scattering in the near infrared region. The strongest luminescence is observed with Sm +3 ionomers. The Dy +3 ionomer Raman-shifts this source to emit light most strongly in the 1.53-1.55 μ range where the ionomer also transmits light well.

The promise of organotypic hepatic and gastrointestinal models

Advances in the design and assembly of in vitro organotypic liver and gastrointestinal (GI) models can accelerate our understanding of metabolism, nutrient absorption, and the effect of microbial flora. Such models can provide comprehensive information on how of environmental toxins, drugs, and pharmaceuticals interact with and within these organs. Information obtained from such models could elucidate the complicated cascades of signaling mechanisms that occur in vivo. Because experiments on large-scale animal models are expensive and resource intensive, the design of organotypic models has renewed significance. The challenges and approaches to designing liver and GI models are similar. Because these organs are in close proximity and interact continually, we have described recent design considerations to guide future tissue models.

Systems biology characterization of engineered tissues.

Tissue engineering and molecular systems biology are inherently interdisciplinary fields that have been developed independently so far. In this review, we first provide a brief introduction to tissue engineering and to molecular systems biology. Next, we highlight some prominent applications of systems biology techniques in tissue engineering. Finally, we outline research directions that can successfully blend these two fields. Through these examples, we propose that experimental and computational advances in molecular systems biology can lead to predictive models of bioengineered tissues that enhance our understanding of bioengineered systems. In turn, the unique challenges posed by tissue engineering will usher in new experimental techniques and computational advances in systems biology.