Alice A. Chen

Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues

In the absence of perfusable vascular networks, three-dimensional (3D) engineered tissues densely populated with cells quickly develop a necrotic core [1]. Yet the lack of a general approach to rapidly construct such networks remains a major challenge for 3D tissue culture [2–4]. Here, we 3D printed rigid filament networks of carbohydrate glass, and used them as a cytocompatible sacrificial template in engineered tissues containing living cells to generate cylindrical networks which could be lined with endothelial cells and perfused with blood under high-pressure pulsatile flow. Because this simple vascular casting approach allows independent control of network geometry, endothelialization, and extravascular tissue, it is compatible with a wide variety of cell types, synthetic and natural extracellular matrices (ECMs), and crosslinking strategies. We also demonstrated that the perfused vascular channels sustained the metabolic function of primary rat hepatocytes in engineered tissue constructs that otherwise exhibited suppressed function in their core.

Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels

We have fabricated a hepatic tissue construct using a multilayer photopatterning platform for embedding cells in hydrogels of complex architecture. We first explored the potential of established hepatocyte culture models to stabilize isolated hepatocytes for pho‐toencapsulation (e.g., double gel, Matrigel, cocultivation with nonparenchymal cells). Using photopolymerizable PEG hydrogels, we then tailored both the chemistry and architecture of the hydrogels to further support hepatocyte survival and Hver‐specific function. Specifically, we incorporated adhesive peptides to ligate key integrins on these adhesion‐dependent cells. To identify the appropriate peptides for incorporation, the integrin expression of cultured hepatocytes was monitored by flow cytometry and their functional role in cell adhesion was assessed on full‐length extracellular matrix (ECM) molecules and their adhesive peptide domains. In addition, we modified the hydrogel architecture to minimize barriers to nutrient transport for these highly metabolic cells. Viability of encapsulated cells was improved in photopatterned hydrogels with structural features of 500 μm in width over unpatterned, bulk hydrogels. Based on these findings, we fabricated a multilayer photopatterned PEG hydrogel structure containing the adhesive RGD peptide sequence to ligate the α5β1 integrin of cocultured hepatocytes. Three‐dimensional photopatterned constructs were visualized by digital volumetric imaging and cultured in a continuous flow bioreactor for 12 d where they performed favorably in comparison to unpatterned, unper‐fused constructs. These studies will have impact in the field of liver biology as well as provide enabling tools for tissue engineering of other organs.—Liu Tsang, V., Chen, A. A., Cho, L. M., Jadin, K. D., Sah, R. L., DeLong, S., West, J. L., Bhatia, S. N. Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels. FASEB J. 21, 790–801 (2007)

Quantum dots to monitor RNAi delivery and improve gene silencing

A critical issue in using RNA interference for identifying genotype/phenotype correlations is the uniformity of gene silencing within a cell population. Variations in transfection efficiency, delivery-induced cytotoxicity and ‘off target’ effects at high siRNA concentrations can confound the interpretation of functional studies. To address this problem, we have developed a novel method of monitoring siRNA delivery that combines unmodified siRNA with seminconductor quantum dots (QDs) as multi color biological probes. We co-transfected siRNA with QDs using standard transfection techniques, thereby leveraging the photostable fluorescent nanoparticles to track delivery of nucleic acid, sort cells by degree of transfection and purify homogenously-silenced subpopulations. Compared to alternative RNAi tracking methods (co-delivery of reporter plasmids and end-labeling the siRNA), QDs exhibit superior photostability and tunable optical properties for an extensive selection of non-overlapping colors. Thus this simple, modular system can be extended toward multiplexed gene knockdown studies, as demonstrated in a two color proof-of-principle study with two biological targets. When the method was applied to investigate the functional role of T-cadherin (T-cad) in cell–cell communication, a subpopulation of highly silenced cells obtained by QD labeling was required to observe significant downstream effects of gene knockdown.

Humanized mice with ectopic artificial liver tissues

“Humanized” mice offer a window into aspects of human physiology that are otherwise inaccessible. The best available methods for liver humanization rely on cell transplantation into immunodeficient mice with liver injury but these methods have not gained widespread use due to the duration and variability of hepatocyte repopulation. In light of the significant progress that has been achieved in clinical cell transplantation through tissue engineering, we sought to develop a humanized mouse model based on the facile and ectopic implantation of a tissue-engineered human liver. These human ectopic artificial livers (HEALs) stabilize the function of cryopreserved primary human hepatocytes through juxtacrine and paracrine signals in polymeric scaffolds. In contrast to current methods, HEALs can be efficiently established in immunocompetent mice with normal liver function. Mice transplanted with HEALs exhibit humanized liver functions persistent for weeks, including synthesis of human proteins, human drug metabolism, drug–drug interaction, and drug-induced liver injury. Here, mice with HEALs are used to predict the disproportionate metabolism and toxicity of “major” human metabolites using multiple routes of administration and monitoring. These advances may enable manufacturing of reproducible in vivo models for diverse drug development and research applications.

Modulation of hepatocyte phenotype in vitro via chemomechanical tuning of polyelectrolyte multilayers

It is increasingly appreciated that since cell and tissue functions are regulated by chemomechanical stimuli, precise control over such stimuli will improve the functionality of tissue models. However, due to the inherent difficulty in decoupling these cues as presented by extracellular materials, few studies have explored the independent modulation of biochemical and mechanical stimuli towards the manipulation of sustained cellular processes. Here, we demonstrate that both mechanical compliance and ligand presentation of synthetic, weak polyelectrolyte multilayers (PEMs) can be tuned independently to influence the adhesion and liver-specific functions of primary rat hepatocytes over extended in vitro culture (two weeks). These synthetic PEMs exhibited elastic moduli E ranging over 200kPa<E<142MPa, as much as one thousand-fold more compliant than tissue-culture polystyrene (E approximately 2.5GPa). The most compliant of these PEM substrata promoted hepatocyte adhesion and spheroidal morphology. Subsequent modification of PEMs with type I collagen and the proteoglycan decorin did not alter substrata compliance, but enhanced the retention of spheroids on surfaces and stabilized hepatic functions (albumin and urea secretion, CYP450 detoxification activity). Decorin exhibited unique compliance-mediated effects on hepatic functions, down-regulating the hepatocyte phenotype when presented on highly compliant substrata while up-regulating hepatocyte functions when presented on increasingly stiffer substrata. These results show that phenotypic functions of liver models can be modulated by leveraging synthetic polymers to study and optimize the interplay of biochemical and mechanical cues at the cell-material interface. More broadly, these results suggest an enabling approach for the systematic design of functional tissue models applied to drug screening, cell-based therapies and fundamental studies in development, physiology and disease.

T-cadherin modulates hepatocyte functions in vitro

Primary hepatocytes from several different species rapidly lose viability and phenotypic functions on isolation from their native microenvironment of the liver. Stromal cells derived from both within and outside the liver can induce phenotypic functions in primary hepatocytes in vitro; however, the molecular mediators underlying this “coculture effect” have not been fully elucidated. We have previously developed a functional genomic screen utilizing cocultures of hepatocytes and 3T3 fibroblasts to identify such candidate hepatocyte‐function‐inducing molecules. In particular, truncated‐cadherin (T‐cadherin) was identified as a potential molecule of interest in induction of hepatic functions. Here we demonstrate that liver‐specific functions of primary rat hepatocytes are induced on cocultivation with Chinese hamster ovary cells engineered to express T‐cadherin on their surface as compared with wild‐type controls. Additionally, culture of cells on substrata presenting recombinant T‐cadherin protein (acellular presentation) enhanced hepatic functions in both pure hepatocyte cultures and in hepatocyte‐stromal cocultures lacking endogenous T‐cadherin expression. Collectively, these data indicate that both cellular and acellular presentation of T‐cadherin can be used to modulate the hepatocyte phenotype in vitro for tissue engineering applications. Our work suggests potential avenues for investigating the role of T‐cadherin on hepatocellular function in vivo in settings such as embryogenesis and liver pathology.— Khetani, S. R., Chen, A. A., Ranscht, B., Bhatia, S. N. T‐cadherin modulates hepatocyte functions in vitro. FASEB J. 22, 3768–3775 (2008)

Hepatic Tissue Engineering

Liver tissue engineering aims to provide novel therapies for liver diseases and create effective tools for understanding fundamental aspects of liver biology and pathologic processes. Approaches range from bio-mimetic in vitro model systems of the liver to three-dimensional implantable constructs. Collectively, these cell-based approaches endeavor to replace or enhance organ transplantation, which is the current standard treatment for liver diseases in most clinical settings. However, the complexity of liver structure and function as well as the limited supply of human hepatocytes pose unique challenges for the field. This chapter reviews advances in the field of liver tissue engineering within the context of current therapies for liver diseases, and clinical alternatives such as cell transplantation strategies and extracorporeal bioartificial liver devices.

3-D Fabrication Technology for Tissue Engineering

Tissue engineering typically involves the combination of cells and biomaterials to form tissues with the goal of replacing or restoring physiological functions lost in diseased organs. The biomaterial scaffolds are designed to provide mechanical support for the cells; however, in practice, the simple addition of cells to porous scaffolds often does not recapitulate sufficient tissue function. Scaffold design previously focused on the incorporation of macroscale features such as interconnected pores for nutrient transport and tissue remodeling. One strategy to further augment the function of tissue-engineered constructs is to mimic the in vivo tissue microarchitecture and cellular microenvirnment. Tissues in the body are divided into repeating functional units (e.g., nephron, islet) [1], whose 3-D architecture coordinates the processes of multiple types of specialized cells. Further, the local environment of these cells presents biochemical and physical stimuli that specifically modulate both cellular functions, e.g. biosynthesis and metabolism, and cellular fate processes such as differentiation, proliferation, apoptosis and migration. Thus, the fabrication of functional 3-D tissue constructs that incorporate both microscale features for appropriate cell functions and macroscale mechanical and transport properties demands control over chemistry and architecture over multiple length scales.