Evidence for a secreted chemorepellent that directs glioma cell invasion

Secreted chemotropic cues guide the migration of neuronal and glial cell precursors during neural development. It is not known if chemotropism contributes to directing the invasion of brain tissue by glioma cells. A model system has been developed that allows quantification of invasive behavior using gliomas spheroids embedded in collagen gels. Here we provide evidence that glioma spheroids secrete a chemorepellent factor(s) that directs cells away from the spheroid and into the collagen matrix. The relationship between total invasion, cell number, and implantation distance suggests that glioma cells respond to a gradient of the chemorepellent cue(s) that is well established at 48 h. C6 astrocytoma cells normally invade the collagen at an angle perpendicular to the spheroid edge. In contrast, an adjacent spheroid causes cells to turn away from their normal trajectory and slow their rate of invasion. Astrocytoma cells are repelled by an adjacent glioma spheroid but rapidly infiltrate astrocyte aggregates, indicating that astrocytes do not express the repellent cue. Uniform concentrations of repellent factor(s) in spheroid conditioned medium overwhelm endogenous gradients and render glioma cells less able to exhibit this chemotropic response. Concentration gradients of spheroid conditioned medium in cell migration assays also demonstrate the chemorepellent cue(s)'s tropic effect. Our findings indicate that glioma spheroids produce a secreted diffusible cue(s) that promotes glioma cell invasion. Identification of this factor(s) may advance current therapies that aim to limit tumor cell invasion.     

Mechanical signaling through the discoidin domain receptor 1 plays a central role in tissue fibrosis

ABSTRACT

The preservation of tissue and organ architecture and function depends on tightly regulated interactions of cells with the extracellular matrix (ECM). These interactions are maintained in a dynamic equilibrium that balances intracellular, myosin-generated tension with extracellular resistance conferred by the mechanical properties of the extracellular matrix. Disturbances of this equilibrium can lead to the development of fibrotic lesions that are associated with a wide repertoire of high prevalence diseases including obstructive cardiovascular diseases, muscular dystrophy and cancer. Mechanotransduction is the process by which mechanical cues are converted into biochemical signals. At the core of mechanotransduction are sensory systems, which are frequently located at sites of cell-ECM and cell-cell contacts. As integrins (cell-ECM junctions) and cadherins (cell-cell contacts) have been extensively studied, we focus here on the properties of the discoidin domain receptor 1 (DDR1), a tyrosine kinase that mediates cell adhesion to collagen. DDR1 expression is positively associated with fibrotic lesions of heart, kidney, liver, lung and perivascular tissues. As the most common end-point of all fibrotic disorders is dysregulated collagen remodeling, we consider here the mechanical signaling functions of DDR1 in processing of fibrillar collagen that lead to tissue fibrosis.     

An Off-Lattice Hybrid Discrete-Continuum Model of Tumor Growth and Invasion

We have developed an off-lattice hybrid discrete-continuum (OLHDC) model of tumor growth and invasion. The continuum part of the OLHDC model describes microenvironmental components such as matrix-degrading enzymes, nutrients or oxygen, and extracellular matrix (ECM) concentrations, whereas the discrete portion represents individual cell behavior such as cell cycle, cell-cell, and cell-ECM interactions and cell motility by the often-used persistent random walk, which can be depicted by the Langevin equation. Using this framework of the OLHDC model, we develop a phenomenologically realistic and bio/physically relevant model that encompasses the experimentally observed superdiffusive behavior (at short times) of mammalian cells. When systemic simulations based on the OLHDC model are performed, tumor growth and its morphology are found to be strongly affected by cell-cell adhesion and haptotaxis. There is a combination of the strength of cell-cell adhesion and haptotaxis in which fingerlike shapes, characteristic of invasive tumor, are observed.     

Dependence of invadopodia function on collagen fiber spacing and cross-linking: computational modeling and experimental evidence

Invadopodia are subcellular organelles thought to be critical for extracellular matrix (ECM) degradation and the movement of cells through tissues. Here we examine invadopodia generation, turnover, and function in relation to two structural aspects of the ECM substrates they degrade: cross-linking and fiber density. We set up a cellular automaton computational model that simulates ECM penetration and degradation by invadopodia. Experiments with denatured collagen (gelatin) were used to calibrate the model and demonstrate the inhibitory effect of ECM cross-linking on invadopodia degradation and penetration. Incorporation of dynamic invadopodia behavior into the model amplified the effect of cross-linking on ECM degradation, and was used to model feedback from the ECM. When the model was parameterized with spatial fibrillar dimensions that closely matched the organization, in real life, of native ECM collagen into triple-helical monomers, microfibrils, and macrofibrils, little or no inhibition of invadopodia penetration was observed in simulations of sparse collagen gels, no matter how high the degree of cross-linking. Experimental validation, using live-cell imaging of invadopodia in cells plated on cross-linked gelatin, was consistent with simulations in which ECM cross-linking led to higher rates of both invadopodia retraction and formation. Analyses of invadopodia function from cells plated on cross-linked gelatin and collagen gels under standard concentrations were consistent with simulation results in which sparse collagen gels provided a weak barrier to invadopodia. These results suggest that the organization of collagen, as it may occur in stroma or in vitro collagen gels, forms gaps large enough so as to have little impact on invadopodia penetration/degradation. By contrast, dense ECM, such as gelatin or possibly basement membranes, is an effective obstacle to invadopodia penetration and degradation, particularly when cross-linked. These results provide a novel framework for further studies on ECM structure and modifications that affect invadopodia and tissue invasion by cells.     

The functional cross talk between cancer cells and cancer associated fibroblasts from a cancer mechanics perspective

The function of biological tissues in health and disease is regulated at cellular level and is highly influenced by the physical microenvironment, through the interaction of forces between cells and ECM, which are perceived through mechanosensing pathways. In cancer, both chemical and physical signaling cascades and their interactions are involved during cell-cell and cell-ECM communications to meet requirements of tumor growth. Among stroma cells, cancer associated fibroblasts (CAFs) play key role in tumor growth and pave the way for cancer cells to initiate metastasis and invasion to other tissues, and without recruitment of CAFs, the process of cancer invasion is dysfunctional. This is through an intense chemical and physical cross talks with tumor cells, and interactive remodeling of ECM. During such interaction CAFs apply traction forces and depending on the mechanical properties, deform ECM and in return receive physical signals from the micromechanical environment. Such interaction leads to ECM remodeling by manipulating ECM structure and its mechanical properties. The results are in form of deposition of extra fibers, stiffening, rearrangement and reorganization of fibrous structure, and degradation which are due to a complex secretion and expression of different markers triggered by mechanosensing of tumor cells, specially CAFs. Such events define cancer progress and invasion of cancer cells.A systemic knowledge of chemical and physical factors provides a holistic view of how cancer process and enhances the current treatment methods to provide more diversity among targets that involves tumor cells and ECM structure.     

Colorectal cancer desmoplastic reaction up-regulates collagen synthesis and restricts cancer cell invasion

During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.     

A novel artificial substrate for cell culture: Effects of substrate flexibility/malleability on cell growth and morphology

Summary Gels of glyoxyl agarose (GA) are evaluated as a novel flexible substrate for cell culture with physical properties comparable to extracellular matrix (ECM) gels. We show here that cells adhere well to pure GA gels; in addition, specific interactions involving matrix receptors can be studied when individual matrix molecules are bound to the gel covalently. When cells are grown on such substrates, morphology is comparable to that observed on “natural” matrix gels (reconstituted gels of collagen type I or of Matrigel): rather than being flattened as in monolayer cultures on tissue culture plastic the cells assume a rounded morphology and tend to form tissue-like aggregates. The effects of the artificial matrix gels are discussed in the context of previous publications on cell interactions with the extracellular matrix, suggesting that in addition to specific recognition of matrix molecules the physical properties of ECM by themselves can be decisive for cell differentiation. We conclude that gels of glycoxyl agarose a) provide a useful model to mimic the physical properties of matrix gels without the presence of specific adhesion factors; b) may be useful as a general, non-specific ECM allowing cells to be cultured in vitro under conditions favorable for differentiation; and c) allow to design a variety of “synthetic” ECM models composed of a chemically defined gel matrix, which can be supplemented with covalently bound molecules to be recognized by cell surface receptors.     

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels¹or in biomaterial scaffolds². Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.Download video file.^{(44M, mov)}     

SPARC functions as an inhibitor of adipogenesis

Adipogenesis, a key step in the pathogenesis of obesity, involves extensive ECM remodeling, changes in cell-ECM interactions, and cytoskeletal rearrangement. Matricellular proteins regulate cell-cell and cell-ECM interactions. Evidence in vivo and in vitro indicates that the prototypic matricellular protein, SPARC, inhibits adipogenesis and promotes osteoblastogenesis. Herein we discuss mechanisms underlying the inhibitory effect of SPARC on adipogenesis. SPARC enhances the Wnt/β-catenin signaling pathway and regulates the expression and posttranslational modification of collagen. SPARC might drive preadipocytes away from the status of growth arrest and therefore prevent terminal differentiation. SPARC could also decrease WAT deposition through its negative effects on angiogenesis. Therefore, several stages of white adipose tissue accumulation are sensitive to the inhibitory effects of SPARC.     

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.     

Integrin alpha3beta1 engagement disrupts intercellular adhesion

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of beta1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell-cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell-cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand-collagen type I, fibronectin, or laminin 1-MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell-cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional beta1 integrin and specifically alpha3beta1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial-mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin-ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.     

A Three-Dimensional Computational Model of Collagen Network Mechanics

Extracellular matrix (ECM) strongly influences cellular behaviors, including cell proliferation, adhesion, and particularly migration. In cancer, the rigidity of the stromal collagen environment is thought to control tumor aggressiveness, and collagen alignment has been linked to tumor cell invasion. While the mechanical properties of collagen at both the single fiber scale and the bulk gel scale are quite well studied, how the fiber network responds to local stress or deformation, both structurally and mechanically, is poorly understood. This intermediate scale knowledge is important to understanding cell-ECM interactions and is the focus of this study. We have developed a three-dimensional elastic collagen fiber network model (bead-and-spring model) and studied fiber network behaviors for various biophysical conditions: collagen density, crosslinker strength, crosslinker density, and fiber orientation (random vs. prealigned). We found the best-fit crosslinker parameter values using shear simulation tests in a small strain region. Using this calibrated collagen model, we simulated both shear and tensile tests in a large linear strain region for different network geometry conditions. The results suggest that network geometry is a key determinant of the mechanical properties of the fiber network. We further demonstrated how the fiber network structure and mechanics evolves with a local formation, mimicking the effect of pulling by a pseudopod during cell migration. Our computational fiber network model is a step toward a full biomechanical model of cellular behaviors in various ECM conditions.     

Microregional extracellular matrix heterogeneity in brain modulates glioma cell invasion

The invasion of neoplastic cells into healthy brain tissue is a pathologic hallmark of gliomas and contributes to the failure of current therapeutic modalities (surgery, radiation and chemotherapy). Transformed glial cells share the common attributes of the invasion process, including cell adhesion to extracellular matrix (ECM) components, cell locomotion, and the ability to remodel extracellular space. However, glioma cells have the ability to invade as single cells through the unique environment of the normal central nervous system (CNS). The brain parenchyma has a unique composition, mainly hyaluronan and is devoid of rigid protein barriers composed of collagen, fibronectin and laminin. The integrins and the hyaluronan receptor CD44 are specific adhesion receptors active in glioma-ECM adhesion. These adhesion molecules play a major role in glioma cell-matrix interactions because the neoplastic cells use these receptors to adhere to and migrate along the components of the brain ECM. They also interact with the proteases secreted during glioma progression that degrade ECM allowing tumor cells to spread and diffusely infiltrate the brain parenchyma. The plasminogen activators (PAs), matrix metalloproteinases (MMPs) and lysosomal cysteine peptidases called cathepsins are also induced during the invasive process. Understanding the mechanisms of tumor cell invasion is critical as it plays a central role in glioma progression and failure of current treatment due to tumor recurrence from micro-disseminated disease. This review will focus on the impact of microregional heterogeneity of the ECM on glioma invasion in the normal adult brain and its modifications in tumoral brain.     

Cell jamming: Collective invasion of mesenchymal tumor cells imposed by tissue confinement

Background

Cancer invasion is a multi-step process which coordinates interactions between tumor cells with mechanotransduction towards the surrounding matrix, resulting in distinct cancer invasion strategies. Defined by context, mesenchymal tumors, including melanoma and fibrosarcoma, develop either single-cell or collective invasion modes, however, the mechanical and molecular programs underlying such plasticity of mesenchymal invasion programs remain unclear.

Methods

To test how tissue anatomy determines invasion mode, spheroids of MV3 melanoma and HT1080 fibrosarcoma cells were embedded into 3D collagen matrices of varying density and stiffness and analyzed for migration type and efficacy with matrix metalloproteinase (MMP)-dependent collagen degradation enabled or pharmacologically inhibited.

Results

With increasing collagen density and dependent on proteolytic collagen breakdown and track clearance, but independent of matrix stiffness, cells switched from single-cell to collective invasion modes. Conversion to collective invasion included gain of cell-to-cell junctions, supracellular polarization and joint guidance along migration tracks.

Conclusions

The density of the extracellulair matrix (ECM) determines the invasion mode of mesenchymal tumor cells. Whereas fibrillar, high porosity ECM enables single-cell dissemination, dense matrix induces cell–cell interaction, leader–follower cell behavior and collective migration as an obligate protease-dependent process.

General significance

These findings establish plasticity of cancer invasion programs in response to ECM porosity and confinement, thereby recapitulating invasion patterns of mesenchymal tumors in vivo. The conversion to collective invasion with increasing ECM confinement supports the concept of cell jamming as a guiding principle for melanoma and fibrosarcoma cells into dense tissue.This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Imaging proteolysis by living human glioma cells

Degradation of basement membrane is an essential step for tumor invasion. In order to study degradation in real time as well as localize the site of proteolysis, we have established an assay with living human cancer cells in which we image cleavage of quenched-fluorescent basement membrane type IV collagen (DQ-collagen IV). Accumulation of fluorescent products is imaged with a confocal microscope and localized by optically sectioning both the cells and the matrix on which they are growing. For the studies described here, we seeded U87 human glioma cells as either monolayers or spheroids on a 3-dimensional gelatin matrix in which DQ-collagen IV had been embedded. As early as 24 hours after plating as monolayers, U87 cells were present throughout the 3-dimensional matrix. Cells at all levels had accumulated fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Similar observations were made for U87 spheroids and the individual cells migrating from the spheroids into the gelatin matrix. Both the migrating cells and those within the spheroid contained fluorescent degradation products of DQ-collagen IV intracellularly within vesicles. Thus, glioma cells like breast cancer cells are able to degrade type IV collagen intracellularly, suggesting that this is an important pathway for matrix degradation.     

Cell-Extracellular Matrix Interactions in Normal and Diseased Skin

Mammalian skin comprises a multi-layered epithelium, the epidermis, and an underlying connective tissue, the dermis. The epidermal extracellular matrix is a basement membrane, whereas the dermal ECM comprises fibrillar collagens and associated proteins. There is considerable heterogeneity in ECM composition within both epidermis and dermis. The functional significance of this extends beyond cell adhesion to a range of cell autonomous and nonautonomous processes, including control of epidermal stem cell fate. In skin, cell-ECM interactions influence normal homeostasis, aging, wound healing, and disease. Disturbed integrin and ECM signaling contributes to both tumor formation and fibrosis. Strategies for manipulating cell-ECM interactions to repair skin defects and intervene in a variety of skin diseases hold promise for the future.The focus of this review is the role of cell-ECM interactions in the physiology of normal and diseased mammalian skin. The skin has epithelial and mesenchymal components and contains ECM comprising both fibrillar collagen and basement membrane. Experimentally, it is a highly tractable tissue, and a range of in vitro and in vivo approaches are available to explore cell-ECM interactions. Such studies are of medical importance because of the wide variety of benign and malignant skin diseases. Research on skin therefore provides an integrated, in vivo, context for understanding the functional significance of specific molecular interactions and signaling pathways involved in cell-ECM adhesion.     

Modulation of epithelial morphogenesis and cell fate by cell-to-cell signals and regulated cell adhesion

Cell-cell and cell-extracellular matrix (ECM) interactions control many developmental decisions of epithelial cell fate and morphogenesis. Protein tyrosine kinases are one class of regulatory molecules that have been implicated in the modulation of these processes. Several protein tyrosine kinases co-localize with cell-cell (cadherin) and cell-ECM (integrin) adhesion molecules at specific adhesion domains of epithelial cells. Protein tyrosine kinases may regulate epithelial development by modulating cell-cell and cell-ECM interactions and by relaying signals initiated by these interactions to other cellular components that determine cell structure and function.     

Lymphocyte migration into three-dimensional collagen matrices: a quantitative study

Lymphocytes have been plated onto the surface of three-dimensional gels of native collagen fibers, and their distribution throughout the three- dimensional collagen matrix has been determined in a quantitative fashion at various times thereafter. Information regarding the total number of applied cells may be obtained by this means. Lymphocyte penetration into the collagen gel does not appear to involve the expression of collagenolytic activity, nor does it require the presence of serum. Analysis of the kinetics of lymphocyte penetration into the gel matrix indicates that lymphocytes are migrating in a "random-walk" fashion. Our objective has been to establish a model system for studying the cell-matrix and cell-cell interactions which influence the pattern of lymphocyte recirculation in vivo and the results presented here are discussed in this context.     

Toward single cell traction microscopy within 3D collagen matrices

Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels.     

Effects of degree of cell-cell contact on liver specific functions of rat primary hepatocytes

Cell-cell interaction and the extracellular matrix (ECM) are believed to play essential roles duringin vitro culturing of primary hepatocytes in the control of differentiation and in the maintenance of tissue specific functions. The objective of this study was to examine the effects of degree of cell-cell contact (DCC) on liver specific function of rat primary hepatocytes. Hepatocyte aggregates with various degrees of cell-cell contact,i.e., dispersed cells, longish aggregate, rugged aggregate, and smooth spheroid were obtained at 1, 5–6, 15–20, and 36–48 hrs, respectively in suspension cultures grown in spinner flasks embedded in Caalginate bead and collagen gel in order. The smooth spheroids displayed a decrease in viability and functional activities. This may result from mass transfer limitation and shear damage caused by agitation during aggregation. The rugged aggregate showed a higher viability and albumin secretion rate than the dispersed cells or the other aggregates. This result indicates the possible enhancement of a bioartificial liver's (BAL) performance using primary hepatocytes and the reduction in time to prepare a BAL through optimization of the immobilization time.