A method to generate perfusable physiologic-like vascular channels within a liver-on-chip model.

The human vasculature is essential in organs and tissues for the transport of nutrients, metabolic waste products, and the maintenance of homeostasis. The integration of vessels in in vitro organs-on-chip may, therefore, improve the similarity to the native organ microenvironment, ensuring proper physiological functions and reducing the gap between experimental research and clinical outcomes. This gap is particularly evident in drug testing and the use of vascularized models may provide more realistic insights into human responses to drugs in the pre-clinical phases of the drug development pipeline. In this context, different vascularized liver models have been developed to recapitulate the architecture of the hepatic sinusoid, exploiting either porous membranes or bioprinting techniques. In this work, we developed a method to generate perfusable vascular channels with a circular cross section within organs-on-chip without any interposing material between the parenchyma and the surrounding environment. Through this technique, vascularized liver sinusoid-on-chip systems with and without the inclusion of the space of Disse were designed and developed. The recapitulation of the Disse layer, therefore, a gap between hepatocytes and endothelial cells physiologically present in the native liver milieu, seems to enhance hepatic functionality (e.g., albumin production) compared to when hepatocytes are in close contact with endothelial cells. These findings pave the way to numerous further uses of microfluidic technologies coupled with vascularized tissue models (e.g., immune system perfusion) as well as the integration within multiorgan-on-chip settings.

Closed-loop feedback control for microfluidic systems through automated capacitive fluid height sensing.

Precise fluid height sensing in open-channel microfluidics has long been a desirable feature for a wide range of applications. However, performing accurate measurements of the fluid level in small-scale reservoirs (<1 mL) has proven to be an elusive goal, especially if direct fluid-sensor contact needs to be avoided. In particular, gravity-driven systems used in several microfluidic applications to establish pressure gradients and impose flow remain open-loop and largely unmonitored due to these sensing limitations. Here we present an optimized self-shielded coplanar capacitive sensor design and automated control system to provide submillimeter fluid-height resolution (∼250 µm) and control of small-scale open reservoirs without the need for direct fluid contact. Results from testing and validation of our optimized sensor and system also suggest that accurate fluid height information can be used to robustly characterize, calibrate and dynamically control a range of microfluidic systems with complex pumping mechanisms, even in cell culture conditions. Capacitive sensing technology provides a scalable and cost-effective way to enable continuous monitoring and closed-loop feedback control of fluid volumes in small-scale gravity-dominated wells in a variety of microfluidic applications.

Quantitative Assessment of Population Variability in Hepatic Drug Metabolism Using a Perfused Three-Dimensional Human Liver Microphysiological System.

In this work, we first describe the population variability in hepatic drug metabolism using cryopreserved hepatocytes from five different donors cultured in a perfused three-dimensional human liver microphysiological system, and then show how the resulting data can be integrated with a modeling and simulation framework to accomplish in vitro-in vivo translation. For each donor, metabolic depletion profiles of six compounds (phenacetin, diclofenac, lidocaine, ibuprofen, propranolol, and prednisolone) were measured, along with metabolite formation, mRNA levels of 90 metabolism-related genes, and markers of functional viability [lactate dehydrogenase (LDH) release, albumin, and urea production]. Drug depletion data were analyzed with mixed-effects modeling. Substantial interdonor variability was observed with respect to gene expression levels, drug metabolism, and other measured hepatocyte functions. Specifically, interdonor variability in intrinsic metabolic clearance ranged from 24.1% for phenacetin to 66.8% for propranolol (expressed as coefficient of variation). Albumin, urea, LDH, and cytochrome P450 mRNA levels were identified as significant predictors of in vitro metabolic clearance. Predicted clearance values from the liver microphysiological system were correlated with the observed in vivo values. A population physiologically based pharmacokinetic model was developed for lidocaine to illustrate the translation of the in vitro output to the observed pharmacokinetic variability in vivo. Stochastic simulations with this model successfully predicted the observed clinical concentration-time profiles and the associated population variability. This is the first study of population variability in drug metabolism in the context of a microphysiological system and has important implications for the use of these systems during the drug development process.

Quantitative Systems Pharmacology Approaches Applied to Microphysiological Systems (MPS): Data Interpretation and Multi-MPS Integration.

Our goal in developing Microphysiological Systems (MPS) technology is to provide an improved approach for more predictive preclinical drug discovery via a highly integrated experimental/computational paradigm. Success will require quantitative characterization of MPSs and mechanistic analysis of experimental findings sufficient to translate resulting insights from in vitro to in vivo. We describe herein a systems pharmacology approach to MPS development and utilization that incorporates more mechanistic detail than traditional pharmacokinetic/pharmacodynamic (PK/PD) models. A series of studies illustrates diverse facets of our approach. First, we demonstrate two case studies: a PK data analysis and an inflammation response--focused on a single MPS, the liver/immune MPS. Building on the single MPS modeling, a theoretical investigation of a four-MPS interactome then provides a quantitative way to consider several pharmacological concepts such as absorption, distribution, metabolism, and excretion in the design of multi-MPS interactome operation and experiments.

In vitro erythropoiesis from bone marrow-derived progenitors provides a physiological assay for toxic and mutagenic compounds.

The goal of this study was to create an in vitro cell culture system that captures essential features of the in vivo erythroid micronucleus (MN) genotoxicity assay, thus enabling increased throughput and controlled studies of the hematopoietic DNA damage response. We show that adult bone marrow (BM) cultures respond to erythropoietin, the principal hormone that stimulates erythropoiesis, with physiological erythropoietic proliferation, differentiation, and enucleation. We then show that this in vitro erythropoietic system clearly signals exposure to genotoxicants through erythroid MN formation. Furthermore, we determined that DNA repair-deficient (MGMT(-/-)) BM displayed sensitivity to genotoxic exposure in vivo compared with WT BM and that this phenotypic response was reflected in erythropoietic cultures. These findings suggest that this in vitro erythroid MN assay is capable of screening for genotoxicity on BM in a physiologically reflective manner. Finally, responses to genotoxicants during erythroid differentiation varied with exposure time, demonstrating that this system can be used to study the effect of DNA damage at specific developmental stages.

A microscale in vitro physiological model of the liver: predictive screens for drug metabolism and enzyme induction.

In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery -- as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.

Quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes.

An objective of designing molecular vehicles exhibiting virus-like transgene delivery capabilities but with low toxicity and immunogenicity continues to drive synthetic vector development. As no single step within the gene delivery pathway represents the critical limiting barrier for all vector types under all circumstances, improvements in synthetic vehicle design may be aided by quantitative analysis of the contributions of each step to the overall delivery process. To our knowledge, however, synthetic and viral gene delivery methods have not yet been explicitly compared in terms of these delivery pathway steps in a quantitative manner. As a first address of this challenge, we compare here quantitative parameters characterizing intracellular gene delivery steps for an E1/E3-deleted adenoviral vector and three polyethylenimine (PEI)-based vector formulations, as well as the liposomal transfection reagent Lipofectamine and naked DNA; the cargo is a plasmid encoding the beta-galactosidase gene under a CMV promoter, and the cell host is the C3A human hepatocellular carcinoma line. The parameters were determined by applying a previously validated mathematical model to transient time-course measurements of plasmid uptake and trafficking (from whole-cell and isolated nuclei lysates, by real-time quantitative PCR), and gene expression levels, enabling discovery of those for which the adenoviral vector manifested superiority. Parameter-sensitivity analysis permitted identification of processes most critically rate-limiting for each vector. We find that the adenoviral vector advantage in delivery appears to reside partially in its import to the nuclear compartment, but that its vast superiority in transgene expression arises predominantly in our situation from postdelivery events: on the basis of per-nuclear plasmid, expression efficiency from adenovirus is superior by orders of magnitude over the PEI vectors. We find that a chemical modification of a PEI-based vector, which substantially improves its performance, appears to do so by enhancing certain trafficking rate parameters, such as binding and uptake, endosomal escape, and binding to nuclear import machinery, but leaves endosomal escape as a barrier over which transgene delivery could be most sensitively increased further for this polymer.

Nanoscale clustering of RGD peptides at surfaces using Comb polymers. 1. Synthesis and characterization of Comb thin films.

Theoretical and experimental studies were conducted to elucidate the structure and properties of amphiphilic comb polymer thin films presenting nanoscale clusters of Arg-Gly-Asp (RGD) peptides for control of cell adhesion on biomaterials. Combs comprised of a poly(methyl methacrylate) backbone and short poly(ethylene oxide) side chains were synthesized, and peptides were tethered to the side chain ends to create nanoscale peptide clusters. In thin films, comb polymers containing >or = 30 wt % six to nine unit PEO side chains completely resisted adhesion of a model fibroblast cell line in the presence of 7.5% serum over 24 h. These same polymers modified with RGD peptides elicited tunable cell adhesion when mixed with unmodified combs in varying proportion. A self-consistent field lattice model of the interface between comb polymer films and water predicts an organization of the top molecular layer of comb polymer with the backbone oriented parallel to the interface in quasi-two-dimensional confinement and hydrophilic side chains extended in a brushlike layer into solution. This picture of a quasi-2D configuration is consistent with the observed surface properties of comb films in water as well as measurements of the RGD cluster density on mixed comb surfaces using fluorescent nanosphere labeling of ligand clusters.

Nanoscale clustering of RGD peptides at surfaces using comb polymers. 2. Surface segregation of comb polymers in polylactide.

Part 1 of these studies described poly(methyl methacrylate-r-polyoxyethylene methacrylate) P(MMA-r-POEM) comb polymers that present Arg-Gly-Asp (RGD) peptides at a surface in nanoscale clusters on a protein-resistant background for control of cell adhesion. Here in part 2, we examine surface segregation of these peptide-modified and unmodified comb polymers blended with polylactide (PLA) as a self-assembly approach suitable for surface modification of porous tissue engineering scaffolds. Multiple thermodynamic driving forces for surface enrichment of the comb polymer are exploited by annealing PLA/P(MMA-r-POEM) blends above the glass transition of the blend components but below the melting point of PLA, while in contact with water. Predictions of the interfacial composition profiles of annealed blends were made using a self-consistent field (SCF) lattice model. The calculations predict strong enrichment of the comb in the top approximately 50 A of blends, and organization of comb molecules in quasi-2D conformations at the interface, similar to the apparent structure of pure comb surfaces in contact with water described in part 1. Experimentally, PLA/comb blend surfaces were characterized by contact angle measurements, XPS, quantification of ligand-cluster surface density and stability by AFM and fluorescent nanosphere labeling, and cell attachment assays. These data were consistent with SCF predictions, showing significant enrichment of the comb at water-annealed surfaces and RGD cluster densities consistent with 2D conformations for comb molecules in the surface layer. Bulk miscibility of the blends was verified by dynamic rheometry, small-angle neutron scattering, DSC and X-ray diffraction studies. Surface segregation of combs provided tunable cell adhesion on PLA through surface-localized nanoclusters of RGD atop a cell-resistant background.

Effect of pore size and void fraction on cellular adhesion, proliferation, and matrix deposition.

The aim of this study was to determine the influence of two key scaffold design parameters, void fraction (VF) and pore size, on the attachment, growth, and extracellular matrix deposition by several cell types. Disc-shaped, porous, poly(-lactic acid) (L-PLA) scaffolds were manufactured by the TheriForm solid free-form fabrication process to generate scaffolds with two VF (75% and 90%) and four pore size distributions (< 38, 38-63, 63-106, and 106-150 microm). Microcomputed tomography analysis revealed that the average pore size was generally larger than the NaCl used, while VF was at or near the designated percentage. The response of three cell types-canine dermal fibroblasts (DmFb), vascular smooth muscle cells (VSMC), or microvascular epithelial cells (MVEC)-to variations in architecture during a 4-week culture period were assessed using histology, metabolic activity, and extracellular matrix deposition as comparative metrics. DmFb, VSMC, and MVEC showed uniform seeding on scaffolds with 90% VF for each pore size, in contrast to the corresponding 75% VF scaffolds. DmFb showed the least selectivity for pore sizes. VSMC displayed equivalent cell proliferation and matrix deposition for the three largest pore sizes. MVEC formed disconnected webs of tissue with sparse extracellular matrix at 90% VF and >38 to 150 microm; however, when cultured on scaffolds with pores formed with salt particles of <38 microm, MVEC formed a multilayered lining on the scaffolds surface. Culture data from scaffolds with a 75% VF suggests that the structural features were unsuitable for tissue formation. Hence, there were limits of acceptable scaffold architecture (VF, pore size) that modulated in vitro cellular responses.

Advances in biomedical engineering.

The most visible contributions of biomedical engineering to clinical practice involve instrumentation for diagnosis, therapy, and rehabilitation. Cell and tissue engineering also have emerged as clinical realities. In the next 25 years, advances in electronics, optics, materials, and miniaturization will accelerate development of more sophisticated devices for diagnosis and therapy, such as imaging and virtual surgery. The emerging new field of bioengineering-engineering based in the science of molecular cell biology-will greatly expand the scope of biomedical engineering to tackle challenges in molecular and genomic medicine.

Leukemia inhibitory factor (LIF) concentration modulates embryonic stem cell self-renewal and differentiation independently of proliferation.

A major limitation of the widespread use of stem cells in a variety of biotechnological applications is the relatively low level of knowledge about how to maintain these cells in vitro without losing the long-term multilineage growth properties required for their clinical utility. An experimental and theoretical framework for predicting and controlling the outcome of stem cell stimulation by exogenous cytokines would thus be useful. An emerging theme from recent hematopoietic stem cell (HSC)-expansion studies is that a net gain in HSC numbers requires the maintenance of critical signaling ligand(s) above a threshold level. These ligand-receptor complex thresholds can be maintained, for example, by high concentrations of soluble cytokines or by cytokine presentation on cell surfaces. According to such a model, when the relevant ligand-receptor interaction falls below this threshold level, the probability of a differentiation response is increased; otherwise, self-renewal is favored. Taking advantage of the ability of the cytokine leukemia inhibitory factor (LIF) to maintain embryonic stem (ES) cell pluripotentiality at high concentrations, we are testing this model by investigating critical parameters in the control of ES cell responses. We have developed quantitative assays of ES cell differentiation by measuring cell-surface alkaline phosphatase activity, cell-surface stage specific embryonic antigen (SSEA)-1 expression, and the ability of ES cells to form embryoid bodies. Examination of ES cell responses over a range of LIF concentrations shows that LIF supplementation has little effect on ES cell-growth rate but significantly alters the probability of a cell undergoing a self-renewal vs. a differentiation division. In vitro culture parameters such as inoculum cell density, medium exchange, as well as cell-intrinsic processes such as autocrine secretion are shown to affect this decision. In addition to yielding new information on stem cell regulation by exogenous factors, these studies provide important clues about culture of these cells and should stimulate further investigations into the mechanistic basis of stem cell differentiation control.

Cell adhesion and motility depend on nanoscale RGD clustering.

Integrin adhesion receptors play a crucial role in regulating interactions between cells and extracellular matrix (ECM). Integrin activation initiates multiple intracellular signaling pathways and results in regulation of cell functions such as motility, proliferation and differentiation. Two key observations regarding the biophysical nature of integrin-mediated cell-matrix interactions motivated the present study: (1) cell motility can be regulated by modulating the magnitude of cell-substratum adhesion, by varying cell integrin expression level, integrin-ECM binding affinity or substratum ECM surface density; and (2) integrin clustering enables assembly of multiple cytoplasmic regulatory and structural proteins at sites of aggregated integrin cytoplasmic domains, activating certain intracellular signalling pathways. Here, using a minimal integrin adhesion ligand, YGRGD, we test the hypothesis that ligand clustering can affect cell migration in a manner related to its modulation of cell-substratum adhesion. We employ a synthetic polymer-linking method, which allows us to independently and systematically vary both the average surface density and the local (approx. 50 nm scale) spatial distribution of the YGRGD peptide, against a background otherwise inert with respect to cell adhesion. In this system, the ligand was presented in three alternative spatial distributions: singly, in clusters with an average of five ligands per cluster, or in clusters with an average of nine ligands per cluster; for each of these spatial distributions, a range of average ligand densities (1,000-200,000 ligands/micrometer(2)) were examined. Cluster spacing was adjusted in order to present equivalent average ligand densities independently of cluster size. The murine NR6 fibroblast cell line was used as a model because its migration behavior on ECM in the presence and absence of growth factors has been well-characterized and it expresses integrins known to interact with the YGRGD peptide. Using time-lapse videomicroscopy and analysis of individual cell movement paths, we find that NR6 cells can migrate on substrata where adhesion is mediated solely by the YGRGD peptide. As previously observed for migration of NR6 cells on fibronectin, migration speed on YGRGD is a function of the average surface ligand density. Strikingly, clustering of ligand significantly reduced the average ligand density required to support cell migration. In fact, non-clustered integrin ligands support cell attachment but neither full spreading nor haptokinetic or chemokinetic motility. In addition, by quantifying the strength of cell-substratum adhesion, we find that the variation of cell speed with spatial presentation of YGRGD is mediated via its effect on cell adhesion. These effects on motility and adhesion are also observed in the presence of epidermal growth factor (EGF), a known motility-regulating growth factor. Variation in YGRGD presentation also affects the organization of actin filaments within the cell, with a greater number of cells exhibiting stress fibers at higher cluster sizes of YGRGD. Our observations demonstrate that cell motility may be regulated by varying ligand spatial presentation at the nanoscale level, and suggest that integrin clustering is required to support cell locomotion.

Polymer latexes for cell-resistant and cell-interactive surfaces.

Novel polymer latexes were prepared that can be applied in several ways for the control and study of cell behavior on surfaces. Acrylic latexes with glass transitions ranging from -30 to 100 degrees C were synthesized by dispersion polymerization in a water and alcohol solution using an amphiphilic comb copolymer as a stabilizing agent. The comb had a poly(methyl methacrylate) backbone and hydrophilic poly(ethylene glycol) (PEG) side chains, which served to stabilize the dispersion and create a robust hydrophilic coating on the final latex particles. The end groups of the comb stabilizer can be selectively functionalized to obtain latex particles with a controlled density of ligands tethered to their surfaces. Latexes were prepared with adhesion peptides (RGD) linked to the surface of the acrylic beads to induce attachment and spreading of cells. Coalesced films obtained from the RGD-bearing latex particles promoted attachment of WT NR6 fibroblasts, while films from unmodified latex particles were resistant to these cells. Additionally, RGD-linked beads were embedded in cell-resistant comb polymer films to create cell-interactive surfaces with discrete clustered-ligand domains. Cell attachment and morphology were seen to vary with the surface density of the RGD-bearing latex beads.

Biophysical integration of effects of epidermal growth factor and fibronectin on fibroblast migration.

Cell migration is regulated simultaneously by growth factors and extracellular matrix molecules. Although information is continually increasing regarding the relevant signaling pathways, there exists little understanding concerning how these pathways integrate to produce the biophysical processes that govern locomotion. Herein, we report the effects of epidermal growth factor (EGF) and fibronectin (Fn) on multiple facets of fibroblast motility: locomotion speed, membrane extension and retraction activity, and adhesion. A surprising finding is that EGF can either decrease or increase locomotion speed depending on the surface Fn concentration, despite EGF diminishing global cell adhesion at all Fn concentrations. At the same time, the effect of EGF on membrane activity varies from negative to positive to no-effect as Fn concentration and adhesion range from low to high. Taking these effects together, we find that EGF and Fn regulate fibroblast migration speed through integration of the processes of membrane extension, attachment, and detachment, with each of these processes being rate-limiting for locomotion in sequential regimes of increasing adhesivity. Thus, distinct biophysical processes are shown to integrate for overall cell migration responses to growth factor and extracellular matrix stimuli.

Tresyl-mediated synthesis: kinetics of competing coupling and hydrolysis reactions as a function of pH, temperature, and steric factors.

Kinetic parameters have been measured for coupled nucleophilic and solvolytic reactions of 2,2,2-trifluoroethanesulfonyl (tresyl)-modified poly(ethylene glycol) based on a system of coupled differential equations implied by recently proposed elementary reaction mechanisms. Fitted kinetic parameters were found to be strong functions of pH, temperature, and steric factors. To maximize the total yield of coupled amine as well as the fraction of secondary amine linkages, our model predicts that it is desirable to run tresyl coupling reactions at low temperatures at pH approximately 8.0, depending on the amine pKa for primary, unhindered amines. For branched primary amines, our data favor room temperature at a slightly higher pH.

Preparation and use of tethered ligands as biomaterials and tools for cell biology.

The immobilization of biomolecules to various supports has been an important research area for many years. Molecules such as heparin (1,2), as well as various enzymes (3,4), antibodies (5,6), and adhesion ligands (7), have been bound to such supports as silicon or glass, agarose gels, polyethylene oxide (PEO) gels, poly(vinyl alcohol) (PVA) gels, and polymer surfaces. These studies have had important applications in the creation of antithrombogenic surfaces for blood contact, affinity chromatography, and tissue growth and regeneration.

Adhesion-guided in vitro morphogenesis in pure and mixed cell cultures.

The ability to understand and control the morphogenesis of mammalian cells is a fundamental objective of cell and developmental biology and tissue engineering research. Numerous processes, both biochemical and biophysical in nature, have been studied in an attempt to elucidate the mechanisms underlying this behavior. We focus here on the contributions of biophysical phenomena to the morphogenetic behavior of pure and mixed cell populations on solid surfaces in vitro. These principles are illustrated using characteristic liver tissue cells as a model system. The studies discussed demonstrate that cell-substratum and cell-cell adhesive forces are critical determinants of the ultimate morphology, cytoarchitecture, and organization achieved by these cells in vitro.

Microdistribution of substratum-bound ligands affects cell function: hepatocyte spreading on PEO-tethered galactose.

Cell functions are regulated by signal transduction processes triggered by binding interactions with extracellular ligands, including those coupled to insoluble matrices as well as those diffusing in free solution. Whereas receptor interactions with freely diffusible soluble ligands are quantitatively governed by the mean ligand concentration, interactions with spatially constrained substratum-bound ligands may be affected not only by the mean ligand concentration, but also by the ligand spatial microdistribution. To probe this hypothesis we have generated surfaces presenting galactose ligands at different concentrations and different capabilities for micromobility--and thus spatial microdistribution--by means of polyethylene oxide tethering and assayed for hepatocyte spreading mediated by asialoglycoprotein receptor/cytoskeleton linkages. We demonstrate that spreading is not uniquely determined by the mean galactose concentration presented by the culture substrate. Rather, the ability of primary hepatocytes to spread is additionally specified by a combination of ligand concentration and tether length. Our results suggest spreading results when monovalent ligands possess sufficient mobility to form effectively multivalent bonds with the receptor; that is, when the substratum-bound ligands to cluster in spatial microdomains.