Patent Application
20060270032
The disclosure relates to tissue compositions, methods and apparati for culturing tissue. More particularly, the disclosure relates to micropatterned cellular tissue capable of growing and sustaining a desired function in culture.
Historically cell culture techniques and tissue development failed to take into account the necessary microenvironment for cell-cell and cell-matrix communication as well as an adequate diffusional environment for delivery of nutrients and removal of waste products. Cell culture techniques and understanding of the complex interactions cells have with one another and the surrounding environment have improved in the past decade.
While many methods and bioreactors have been developed to grow tissue for the purposes of generating artificial tissues for transplantation or for toxicology studies, these bioreactors do not adequately simulate, in vitro, the mechanisms by which nutrients, gases, and cell-cell interactions are delivered and performed in vivo. For example, cells in living tissue are “polarized” with respect to diffusion gradients. Differential delivery of oxygen and nutrients, as occurs in vivo by means of the capillary system, controls the relative functions of tissue cells and their maturation. Thus, cell culture systems and bioreactors that do not simulate these in vivo delivery mechanisms do not provide a sufficient corollary to in vivo environments to develop tissues or measure tissue responses in vitro.
The ability to develop in vitro tissue, such as hepatic tissue, can provide a supply of tissue for toxicology testing, extracorporeal liver devices as well as tissue for transplantation. For example, liver failure is the cause of death of over 30,000 patients in the United States every year and over 2 million patients worldwide. Current treatments are largely palliative—including delivery of fluids and serum proteins. The only therapy proven to alter mortality is orthotopic liver transplants; however, organs are in scarce supply (McGuire et al., Dig Dis. 13 (6):379-88 (1995)).
Cell-based therapies have been proposed as an alternative to whole organ transplantation, a temporary bridge to transplantation, and/or an adjunct to traditional therapies during liver recovery. Three main approaches have been proposed: transplantation of isolated hepatocytes via injection into the blood stream, development and implantation of hepatocellular tissue constructs, and perfusion of blood through an extracorporeal circuit containing hepatocytes. Investigation in all three areas has dramatically increased in the last decade, yet progress has been stymied by the propensity for isolated hepatocytes to rapidly lose many key liver-specific functions.
Drug-induced liver disease represents a major economic challenge for the pharmaceutical industry since unforeseen liver toxicity and poor bioavailability issues cause more than 50% of new drug candidates to fail in Phase I clinical trials. Also, a third of drug withdrawals from the market and more than half of all warning labels on approved drugs are primarily due to adverse affects on the liver. Therefore, besides pharmacological properties, ADME/Tox (absorption, distribution, metabolism, excretion and toxicity) characteristics are crucial determinants of the ultimate clinical success of a drug. This realization has led to an early introduction of ADME/Tox screening during the drug discovery process, in an effort to select against drugs with problematic properties.
Animal models provide a limited view of human toxicity due to species-specific variations as well as animal-to-animal variability, necessitating 5-10 animals per compound per dose, sometimes in both genders. Incorporating in vitro models into drug development provides several advantages: earlier elimination of problematic drugs, reduction in variability by allowing hundreds of experiments per animal and human models without patient exposure. In the case of the liver, in vitro models can provide valuable information on drug uptake and metabolism, enzyme induction, and drug-drug interactions affecting metabolism and hepatotoxicity.
Several in vitro liver models are used for short-term (hours) investigation of xenobiotic metabolism and toxicity. Perfused whole organs, liver slices and wedge biopsies maintain many aspects of liver's in vivo microenvironment; however, such systems suffer from limited drug availability to inner cell layers, limited viability (<24 h) and are not suitable for enzyme induction studies. Isolated liver microsomes, which are cellular fragments that contain mostly CYP450 enzymes, are used primarily to investigate drug metabolism via the phase I pathways (oxidation, reduction, hydrolysis and the like). However, microsomes lack many important aspects of the cellular machinery where dynamic changes occur (i.e. gene expression, protein synthesis) to alter drug metabolism, toxicity and drug-drug interactions. Besides microsomes, cell lines derived from hepatoblastomas (HepG2) or from immortalization of primary hepatocytes (HepLiu, SV40 immortalized) are finding limited use as reproducible, inexpensive models of hepatic tissue. However, no cell line has been developed to date that maintains physiologic levels of liver-specific functions. Usually such cell lines are plagued by an abnormal repertoire of hepatic functions.
Current in vitro liver models used by the pharmaceutical industry, though useful in a limited capacity, are not fully predictive of in vivo liver metabolism and toxicity. Thus, research has increasingly turned towards using isolated primary human hepatocytes as the gold standard for in vitro studies; however, hepatocytes are notoriously difficult to maintain in culture as they rapidly lose viability and phenotypic functions.
The invention provides methods, systems, and composition that overcome the limitations of current techniques. The invention provides an engineered in vitro model of parenchymal tissue (e.g., human liver) that remains optimally functional for several weeks. More specifically, the invention utilized microfabrication techniques to create 2-D and 3-D cultures that comprise parenchymal cells (e.g., primary human hepatocytes) spatially arranged in a bounded geometry by non-parenchymal cells in a micropatterned coculture. The bounded geometry may be of any regular or irregular dimension (e.g., circular, semi-circular, spheroidal islands of a pre-defined diameter, length, width etc., typically about 250-750 μm). For example, the invention demonstrates that micropatterned human cocultures reproducibly out perform (by several fold) their randomly distributed counterparts, which contain similar cell ratios and numbers. The invention demonstrates that co-cultures require an optimal balance of homotypic and heterotypic interaction to function optimally.
The invention provides an in vitro cellular composition, comprising one or more populations of parenchymal cells defining a cellular island; and a population of non-parenchymal cells, wherein the non-parenchymal cells define a geometric border of the cellular island.
The invention further provides a method of making a plurality of cellular islands on a substrate. The method comprises spotting an adherence material on a substrate at spatially different locations each spot having a defined geometric size and/or shape; contacting the substrate with a population of cells that selectively adhere to the adherence material and/or substrate; and culturing the cells on the substrate to generate a plurality of cellular islands.
The invention also provides an assay system comprising contacting an artificial tissue the tissue comprising parenchymal cells having a bounded geometry bordered by non-parenchymal cells wherein the bounded geometry has at least one dimension from side to side of the bounded geometry of about 250 μm to 750 μm; contacting the artificial tissue with a test agent; and measuring an activity selected from gene expression, cell function, metabolic activity, morphology, and a combination thereof, of the artificial tissue.
The invention provides an artificial tissue comprising islands of parenchymal cells surrounded by stromal cells wherein the islands of parenchymal cells are about 250 μm to 750 μm in diameter or width.
The invention further provides a method of producing a tissue in vitro. The method comprising seeding a first population of cells on a substrate having defined regions for attachment of the first population of cells, wherein the defined regions comprise a bounded geometric dimension of about 250 μm to 750 μm; seeding a second population of cells on the substrate, such that the second population of cells surround or adhere adjacent to the first population of cells; and culturing the cells under conditions and for a sufficient period of time to generate a tissue.
The details of one or more embodiments of the disclosure are set forth in the accompanying drawings and the description below. Other features, objects, and advantages will be apparent from the description and drawings, and from the claims.
Like reference symbols in the various drawings indicate like elements.
As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cellular island” includes a plurality of such cellular islands and reference to “the cell” includes reference to one or more cells known to those skilled in the art, and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein.
The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.
The invention extends parenchymal cell-stromal cell cocultures by utilizing defined bounded geometries defining cell types. In one aspect, the invention extends cocultures such as those previously used for rat and porcine liver models, to a model of human tissue (e.g., human liver). Using microfabrication tools, the invention demonstrates that micropatterned configurations (from single cellular islands to large aggregates) outperform randomly distributed cocultures. Amongst the micropatterned configurations that were engineered, a balance of homotypic and heterotypic interactions can yield functional cocultures having defined or desired phenotypic activity, longevity and proliferative capacity. Such unexpected results demonstrate a different architectural dependence on geometric cocultures as compared with random cocultures. The invention provides characterization of such micropatterned cocultures utilizing antibody-based functional assays as well as DNA microarrays.
The morphology and function of cells in an organism vary with respect to their environment, including distance from sources of metabolites and oxygen as well as homotypic and heterotypic cell interactions. For example, the morphology and function of hepatocytes are known to vary with position along the liver sinusoids from the portal triad to the central vein (Bhatia et al., Cellular Engineering 1:125-135, 1996; Gebhardt R. Pharmaol Ther. 53 (3):275-354, 1992; Jungermann K. Diabete Metab. 18 (1):81-86, 1992; and Lindros, K. O. Gen Pharmacol. 28 (2):191-6, 1997). This phenomenon, referred to a zonation, has been described in virtually all areas of liver function. Oxidative energy metabolism, carbohydrate metabolism, lipid metabolism, nitrogen metabolism, bile conjugation, and xenobiotic metabolism, have all been localized to separate zones. Such compartmentalization of gene expression is thought to underlie the liver's ability to operate as a ‘glucostat’ as well as the pattern of zonal hepatotoxicity observed with some xenobiotics (e.g., environmental toxins, chemical/biological warfare agents, natural compounds such as holistic therapies and nutraceuticals).
Isolated human parenchymal cells (such as hepatocytes) are highly unstable in culture and are therefore of limited utility for studies on drug toxicity, drug-drug interaction, drug-related induction of detoxification enzymes, and other phenomena. In spite of their recognized advantages, primary parenchymal cells are notoriously difficult to maintain in culture as they rapidly lose viability and phenotypic functions upon isolation from their in vivo microenvironment. Isolated hepatocytes rapidly lose important liver-specific functions such as albumin secretion, urea synthesis and cytochrome P450 activity (see, e.g.,
Over the last couple of decades, investigators have been able to stabilize several hepatocyte functions using soluble factor supplementation, extracellular matrix manipulation, and random co-culture with various liver and non-liver derived stromal cell types. Addition of low concentrations of hormones, corticosteroids, cytokines, vitamins, or amino acids can help stabilize liver-specific functions in hepatocytes. Presentation of extracellular matrices of different composition and topologies can also induce similar stabilization. For instance, hepatocytes from a variety of species (human, mouse, rat) secrete albumin when sandwiched between two layers of rat tail collagen-I (double-gel). However, studies have shown that CYP450 activities decline in the double-gel model, and the presence of an overlaid layer of collagen presents transport barriers for drug candidates, thus limiting their use as assay systems. Culture on a tumor-derived basement membrane extract called Matrigel also induces hepatocyte spheroid formation and leads to retention of key hepatocyte functions including P450 activity. While Matrigel can induce functions in rodent hepatocytes, it appears to have fewer effects on human hepatocytes. Though they may find use in specific scenarios during drug discovery and development, most in vitro liver models in use have limited applicability to the development of a robust biomimetic liver platform. For instance, defined media formulations limit the contents of the perfusate, sandwich culture adds a transport barrier and hepatocytes do not express gap junctions, and Matrigel and spheroid culture rely on hepatocyte aggregation with resultant non-uniformity and transport barriers.
The invention overcomes many of these problems by optimizing the homotypic and heterotypic interactions of parenchymal cells with non-parenchymal cells. For example, in the adult liver, hepatocytes interact with a variety of stromal cell types including sinusoidal endothelia, stellate cells, Kupffer cells and fat-storing Ito cells (e.g., heterotypic interactions). These stromal cell types modulate cell fate processes of hepatocytes under both physiologic and pathophysiologic conditions. In vitro, random co-cultivation of primary hepatocytes with a plethora of distinct stromal cell types from different species and organs has been shown to support differentiated hepatocyte function for several weeks in a manner reminiscent of hepatic organogenesis (see
In one aspect, micropatterned cultures comprising cellular islands of parenchymal cells and stromal cells are used. In this aspect, a substrate is modified and prepared such that stromal cells are interspersed with islands of parenchymal cells. Using microfabrication techniques modified, for example, from the semiconductor industry, the substrate is modified to provide for spatially arranging parenchymal cells (e.g., human hepatocytes) and supportive stromal cells (e.g., fibroblasts) in a miniaturizable format. The spatial arrangements can be a parenchymal cell type comprising a bounded geometric shape. The bounded geometric shape can be any shape (e.g., regular or irregular) having dimensions defined by the shape (e.g., diameter, width, length and the like). The dimensions will have a defined scale based upon their shape such that at least one distance from one side to a substantially opposite side is about 200-800 μm (e.g., where the shape is rectangular or oval, the distance between one side to an opposite side is 200-800 μm). For example, parenchymal cells (e.g., hepatocytes) can be prepared in circular islands of varying dimensions (e.g., 36 μm, 100 μm, 490 μm, 4.8 mm, and 12.6 mm in diameter; typically about 250-750 μm) surrounded by stromal cells (e.g., fibroblast such as murine 3T3 fibroblasts) or other materials. For example, hepatocyte detoxification functions are maximized at small patterns, synthetic ability at intermediate dimensions, while metabolic function and normal morphology were retained in all patterns.
In one embodiment, a bioreactor can use primary parenchymal cells (e.g., hepatocytes) alone or in combination with other cell types. Although the examples provided herein utilize hepatocytes, other parenchymal and non-parenchymal cell types that can be used in the bioreactors and cultures systems of the disclosure include pancreatic cells (alpha, beta, gamma, delta), myocytes, enterocytes, renal epithelial cells and other kidney cells, brain cell (neurons, astrocytes, glia), respiratory epithelium, stem cells, and blood cells (e.g., erythrocytes and lymphocytes), adult and embryonic stem cells, blood-brain barrier cells, and other parenchymal cell types known in the art.
In one aspect, the reactor can be used with micropatterned parenchymal (e.g., hepatocytes) co-cultures and stromal cells (e.g., fibroblasts). The scale of the reactor can be altered to allow for the fabrication of a high-throughput microreactor array to allow for interrogation of xenobiotics. In one aspect, a microfluidic device is contemplated that has micropatterned culture areas in or along a fluid flow path.
The invention demonstrates that cell-cell interactions, both homotypic (hepatocyte/hepatocyte) and heterotypic (hepatocyte/stromal), improve viability and differentiated function of parenchymal cells.
The micropatterned cell island cultures of the invention are useful in drug discovery and development including screening for metabolic stability, drug-drug interactions, toxicity and infectious disease. Metabolic stability is a key criterion for selection of lead drug candidates that proceed to preclinical trials.
The invention provides a cellular composition useful for the development of in vitro tissues with desired characteristics and/or the ability to be cultured over long periods of time with minimal de-differentiation. The invention is based, in-part, upon the discovery that distances between homotypic cell populations and their relationship to intervening heterotypic cell populations results in various functional (phenotypic) differences. For example, in one aspect of the invention one or more populations of geometrically defined cellular islands comprising parenchymal cells are generated. As described further herein, the parenchymal cell type may be any parenchymal cell. The specific examples, provided below demonstrate the application of the methods and systems to hepatic parenchymal cells. These parenchymal cell islands are surrounded/separated by a population of non-parenchymal cells.
The cellular islands can take any geometric shape having a desired characteristic and can be defined by length/width, diameter and the like, based upon their geometric shape, which may be circular, oval, square, rectangular, triangular and the like. Furthermore, parenchymal cell function may be modified by altering the pattern configuration (e.g., the distance or geometry of the array of cellular islands). The distance between bounded geometric islands of cells may vary in a culture system (e.g., the distances between islands may be regular or irregular). Using techniques described herein, the spatial distances between cellular islands may be random, regular or irregular. Furthermore, combinations of geometric bounded areas (e.g., cellular islands) of different geometries (e.g., multiple island sizes) may be present on a single substrate with varying distances (e.g., multiple island spacings) or regular distances between the islands. In other words, the invention contemplates the use of cellular islands comprising various geometries and distances on a substrate (e.g., cocultures comprising cellular islands with 250 μm and 400 μm islands that are intermixed and regularly distributed). In one aspect, the cellular islands comprise a diameter or width from about 250 μm to 750 μm. Similarly, where the geometric island comprises a rectangle, the width can comprise about 250 μm to 750 μm. In another aspect, the parenchymal cellular islands are spaced apart from one another by about 2 μm to 1300 μm from center to center of the cellular islands. In yet a further aspect, the parenchymal cell islands comprise a defined width (e.g., 250 μm to 750 μm) that can run the length of a culture area or a portion of the culture area. Parallel islands of parenchymal cells can be separated by parallel rows of stromal cells. In another aspect, the geometric shape may comprise a 3-D shape (e.g., a spheroid). In such instances, the diameter/width and the like, will be from about 250 μm to 750 μm.
As will be recognized in the art, the cellular islands may be present in any culture system including static and fluid flow reactor systems (e.g., microfluidic devices). Such microfluidic devices are useful in the rapid screening of agents where small flow rates and small reagent amounts are required.
The cellular culture of the invention can be made by any number of techniques that will be recognized in the art. For example, a method of making a plurality of cellular islands on a substrate can comprise spotting or layering an adherence material (or plurality of different cell specific adherence materials) on a substrate at spatially different locations each spot having a defined size (e.g., diameter) and spatial arrangement. The spots on the substrate are then contacted with a first cell population or a combination of cell types and cultured to generate cellular islands. Where difference cell-types are simultaneously contacted with the substrate, the substrate, coating or spots on the substrate will support cell-specific binding, thus providing distinct cellular domains. Methods for spotting adherence material (e.g., extracellular matrix material) can include, for example, robotic spotting techniques and lithographic techniques.
Various culture substrates can be used in the methods and systems of the invention. Such substrates include, but are not limited to, glass, polystyrene, polypropylene, stainless steel, silicon and the like. The choice of the substrate should be taken into account where spatially separated cellular islands are to be maintained. The cell culture surface can be chosen from any number of rigid or elastic supports. For example, cell culture material can comprise glass or polymer microscope slides. In some aspect, the substrate may be selected based upon a cell type's propensity to bind to the substrate.
The cell culture surface/substrate used in the methods and systems of the invention can be made of any material suitable for culturing mammalian cells. For example, the substrate can be a material that can be easily sterilized such as plastic or other artificial polymer material, so long as the material is biocompatible. A substrate can be any material that allows cells and/or tissue to adhere (or can be modified to allow cells and/or tissue to adhere or not adhere at select locations) and that allows cells and/or tissue to grow in one or more layers. Any number of materials can be used to form the substrate/surface, including, but not limited to, polyamides; polyesters; polystyrene; polypropylene; polyacrylates; polyvinyl compounds (e.g. polyvinylchloride); polycarbonate (PVC); polytetrafluoroethylene (PTFE); nitrocellulose; cotton; polyglycolic acid (PGA); cellulose; dextran; gelatin, glass, fluoropolymers, fluorinated ethylene propylene, polyvinylidene, polydimethylsiloxane, polystyrene, and silicon substrates (such as fused silica, polysilicon, or single silicon crystals), and the like. Also metals (gold, silver, titanium films) can be used.
As mentioned herein, in some instances the substrate may be modified to promote cellular adhesion and growth (e.g., coated with an adherence material). For example, a glass substrate may be treated with a protein (i.e., a peptide of at least two amino acids) such as collagen or fibronectin to assist cells in adhering to the substrate. In some embodiments, the proteinaceous material is used to define the location of a cellular island. The spot produced by the protein serves as a “template” for formation of the cellular island. Typically, a single protein will be adhered to the substrate, although two or more proteins may be used in certain embodiments. Proteins that are suitable for use in modifying a substrate to facilitate cell adhesion include proteins to which specific cell types adhere under cell culture conditions. For example, hepatocytes are known to bind to collagen. Therefore, collagen is well suited to facilitate binding of hepatocytes. Other suitable proteins include fibronectin, gelatin, collagen type IV, laminin, entactin, and other basement proteins, including glycosaminoglycans such as heparin sulfate. Combinations of such proteins also can be used.
The type of adherence material(s) (e.g., ECM materials, sugars, proteoglycans etc.) deposited in a spot will be determined, in part, by the cell type or types to be cultured. For example, ECM molecules found in the hepatic microenvironment are useful in culturing hepatocytes, the use of primary cells, and a fetal liver-specific reporter ES cell line. The liver has heterogeneous staining for collagen I, collagen III, collagen IV, laminin, and fibronectin. Hepatocytes display integrins β1, β2, α1 , α2, α5, and the nonintegrin fibronectin receptor Agp110 in vivo. Cultured rat hepatocytes display integrins α1, α3, α5, β1, and α6 μ1, and their expression is modulated by the culture conditions.
In one aspect, the invention provides a micropatterned hepatocyte co-culture. Due to species-specific differences in drug metabolism, human hepatocyte cultures can identify the metabolite profiles of drug candidates more effectively than non-human cultures. Although, it will be recognized that non-human cell types may be used in the invention to facilitate identification of properties or metabolisms suitable for further study of human cells. This information can then be used to deduce the mechanism by which the metabolites are generated, with the ultimate goal of focusing clinical studies. Though there are quantitative differences, there is good in vivo to in vitro correlation in drug biotransformation activity when isolated hepatocytes are used. Metabolite profiles obtained via human hepatocyte in vitro models can also be used to choose the appropriate animal species to act as the human surrogate for preclinical pharmacokinetic, pharmacodynamic and toxicological studies. Studies have shown that interspecies variations are retained in vitro and are different depending on the drug being tested.
The invention also provides methods of micropatterning useful to develop tissues with desired characteristics. Although a serial photolithographic based technique was used in the specific examples below to create optimized micropatterned cocultures, the studies indicate that such cocultures can be miniaturized using stencil-based soft lithography in a multi-well format amenable for higher throughput experimentation. Patterning of various combinations and types of extracellular matrix proteins on a single substrate using robotic spotting techniques is also provided by the invention. These matrix arrays coupled with parenchymal (e.g., hepatic) and stromal cocultures are amenable to high-throughput screening in drug development applications. The invention also provides functionally stable 2-D and 3-D cocultures in static and bioreactor settings with closed-loop flow conditions that approximate in vivo conditions. Furthermore, the micropatterning strategy can potentially be used to functionally optimize other systems in which cell-cell interactions are important (e.g., hematopoietic stem cells co-cultivated with stromal cell lines and keratinocytes with fibroblasts).
With regard to placing insoluble and/or soluble factors at specific locations, various micro-spotting techniques using computer-controlled plotters or even ink-jet printers have been developed to spot such factors at defined locations. One technique loads glass fibers having multiple capillaries drilled through them with different materials loaded into each capillary tube. A substrate, such as a glass microscope slide, is then stamped out much like a rubber stamp on each glass slide. Spotting techniques involve the placement of materials at specific sites or regions using manual or automated techniques.
Conventional physical spotting techniques such as quills, pins, or micropipettors are able to deposit material on substrates in the range of 10 to 250 microns in diameter (e.g., about 100 spots/microwell of a 96 well culture plate). In some instances the density can be from 400 to 10000 spots per square centimeter, allowing for clearance between spots. Lithographic techniques, such as those provided by Affymetrix (e.g., U.S. Pat. No. 5,744,305, the disclosure of which is incorporated by reference herein) can produce spots down to about 10 microns square, resulting in approximately 800,000 spots per square centimeter.
A spotting device may employ one or more piezoelectric pumps, acoustic dispersion, liquid printers, micropiezo dispensers, or the like to deliver such reagents to a desired location on a substrate. In some embodiments, the spotting device comprises an apparatus and method like or similar to that described in U.S. Pat. Nos. 6,296,702, 6,440,217, 6,579,367, and 6,849,127.
Accordingly, an automated spotting device can be utilized, e.g. Perkin Elmer BioChip Arrayer™. A number of contact and non-contact microarray printers are available and may be used to dispense/print the soluble and/or insoluble materials on a substrate. For example, non-contact printers are available from Perkin Elmer (BioChip Arrayer™), Labcyte and IMTEK (TopSpot™), and Bioforce (Nanoarrayer™). These devices utilize various approaches to non-contact spotting, including piezo electric dispension; touchless acoustic transfer; en bloc printing from multiple microchannels; and the like. Other approaches include ink jet-based printing and microfluidic platforms. Contact printers are commercially available from TeleChem International (Arraylt™).
Non-contact printing will typically be used for the production of cellular microarrays comprising cellular islands. By utilizing a printer that does not physically contact the surface of substrate, no aberrations or deformities are introduced onto the substrate surface, thereby preventing uneven or aberrant cellular capture at the site of the spotted material.
Printing methods of interest, including those utilizing acoustic or other touchless transfer, also provide benefits of avoiding clogging of the printer aperature, e.g. where solutions have high viscosity, concentration and/or tackiness. Touchless transfer printing also relieves the deadspace inherent to many systems. The use of print heads with multiple ports and the capacity for flexible adjustment of spot size can be used for high-throughput preparation.
The total number of spots on the substrate will vary depending on the substrate size, the size of a desired cellular island, and the spacing between cellular islands. Generally, the pattern present on the surface of the support will comprise at least 2 distinct spots, usually about 10 distinct spots, and more usually about 100 distinct spots, where the number of spots can be as high as 50,000 or higher. Typically, the spot will usually have an overall circular dimension (although other geometries such as spheroids, rectangles, squares and the like may be used) and the diameter will range from about 10 to 5000 μm (e.g., about 200 to 800 μm).
By dispensing or printing onto the surfaces of multi-well culture plates, one can combine the advantages of the array approach with those of the multi-well approach. Typically, the separation between tips in standard spotting device is compatible with both a 384 well and 96 well plates; one can simultaneously print each load in several wells. Printing into wells can be done using both contact and non-contact technology.
The invention can utilize robotic spotting technology to develop a robust, accessible method for forming cellular microarrays or islands of a defined size and spatial configuration on, for example, a cell culture substrate. As used herein, the term “microarray” refers to a plurality of addressed or addressable locations.
In one aspect, the invention provides methods and systems comprising a modified printing buffer used in a spotting device to allow for ECM deposition, and identifying microarray substrates that permit ECM immobilization. The methods and systems of the invention are useful for spotting substantially purified or mixtures of biological proteins, nucleic acids and the like (e.g., collagen I, collagen III, collagen IV, laminin, and fibronectin) in various combinations on a standard cell culture substrate (e.g., a microscope slide) using off-the-shelf chemicals and a conventional DNA robotic spotter.
In another aspect, the invention utilizes photolithographic techniques to generate cellular islands. Drawing on photolithographic micropatterning techniques to manipulate functions of rodent hepatocytes upon co-cultivation with stromal cells, a microtechnology-based process utilizing elastomeric stencils to miniaturize and characterize human liver tissue in an industry-standard multiwell format was used. The approach incorporates ‘soft lithography,’ a set of techniques utilizing reusable, elastomeric, polymer (Polydimethylsiloxane-PDMS) molds of microfabricated structures to overcome limitations of photolithography. In one aspect, the invention provides a process using PDMS stencils consisting of 300 μm thick membranes with through-holes at the bottom of each well in a 24-well mold (
The invention provides methods and systems useful for identifying optimal conditions for controlling cellular development and maturation by varying the size and/or spacing of a cellular island. For example, the methods and systems of the invention are useful for identifying optimal conditions that control the fate of cells (e.g., differentiating stem cells into more mature cells, maintenance of self-renewal, and the like).
The term “adherence material” is a material deposited on a substrate or chip to which a cell or microorganism has some affinity, such as a binding agent. The material can be deposited in a domain or “spot”. The material and a cell or microorganism interact through any means including, for example, electrostatic or hydrophobic interactions, covalent binding or ionic attachment. The material may include, but is not limited to, antibodies, proteins, peptides, nucleic acids, peptide aptamers, nucleic acid aptamers, sugars, proteoglycans, or cellular receptors.
In a specific example, the invention provides methods and compositions useful to optimize hepatocyte function in vitro. The invention extends hepatocyte-fibroblast cocultures, previously used for rat and porcine liver models, to a model of human liver. Using microfabrication tools, the invention demonstrates that micropatterned configurations (from single hepatocyte islands to large aggregates) outperform randomly distributed cocultures. Amongst the micropatterned configurations that were engineered, a clear balance of homotypic and heterotypic interactions can yield functional human cocultures. Such unexpected results demonstrate a different architectural dependence in human cocultures as compared with rat cocultures. The characterization of optimized micropatterned human cocultures is extensive, utilizing antibody-based functional assays as well as DNA microarrays. Studies in cocultured liver tissue indicate that micropatterned human cocultures retain a high level of expression of many important liver-specific genes, while a decline in expression is seen in pure hepatocyte monolayers on collagen, which are commonly used during drug development. The validation of human cocultures as appropriate liver models for drug development includes cell-based acute and chronic toxicity assays using a variety of clinical and non-clinical compounds, as well as induction and inhibition of key CYP450 enzymes.
A cellular island or “spot” refers to a bounded geometrically defined shape of a substantially homogenous cell-type having a defined border. In one aspect, the cellular island or spot is surrounded by different cell-types, materials (e.g., extracellular matrix materials) and the like. The cellular islands can range in size and shape (e.g., may be of uniform dimensions or non-uniform dimensions). Cellular islands may be of different shapes on the same substrate. Furthermore, the distance between two or more cellular islands can be designed using methods known in the art (e.g., lithographic methods and spotting techniques). The distances between cellular islands can be random, regular or irregular. The distance between and/or size of the cellular islands can be modified to provide a desired phenotypic characteristic of morphology to a particular cell types (e.g., a parenchymal cell such as a hepatocyte).
In addition to modulating cellular islands to control heterotypic and/or homotypic interactions the invention can use a bioreactor system that provides the ability to modulate oxygen and nutrient uptake processes of mammalian cells to create a directional gradient in a reactor system. Directional oxygen gradients are present in various biological environments such as, for example, in cancer, tissue development, tissue regeneration, wound healing and in normal tissues. As a result of oxygen gradients along the length of a bioreactor system result in cells exhibiting different functional characteristics based on local oxygen availability. Accordingly, the invention provides methods, reactor systems and compositions that provide the ability of develop human tissues in vitro characteristic of normal tissue, but also to provide similar physiological environments by mimicking oxygen and/or nutrient gradients found in tissues in the body.
The use of the micropattern technology in combination with a bioreactor system allows for the development of microarray bioreactors. Previous bioreactors were not amenable to miniaturization due in part to variable tissue organization due to reliance on self-assembly that underlie variations in nutrient and drug transport, and uncharacterized stromal contaminants (e.g., random cultures). Furthermore, previous random co-cultures have uncharacterized stromal cell population, have difficulty with microscopic imaging, have difficulty assessing cell number (due to proliferating cell populations) and display less cell-specific (e.g., liver specific) function than micropatterned co-cultures. The micropatterning provided by the invention overcomes many of these difficulties.
The bioreactor utilizes co-cultures of cells in which at least two types of cells are configured in a bounded geometric pattern on a substrate. Such micropatterning techniques are useful to modulate the extent of heterotypic and homotypic cell-cell contacts. In addition, co-cultures have improved stability and thereby allow chronic testing (e.g., chronic toxicity testing as required by the Food and Drug Administration for new compounds). Because micropatterned co-cultures are more stable than random cultures the use of co-cultures of the invention and more particularly micropatterned co-cultures provide a beneficial aspect to the cultures systems of the disclosure. Furthermore, because drug-drug interactions often occur over long periods of time the benefit of stable co-cultures allows for analysis of such interactions and toxicology measurements.
In one aspect, the invention provides an in vitro model of human liver tissue that can be utilized for pharmaceutical drug development, basic science research, infectious disease research (e.g., hepatits B, C and malaria) and in the development of tissue for transplantation. The invention provides compositions, methods, and bioreactor systems that allow development of long-term human cultures in vitro. In addition, the compositions, methods and bioreactor systems of the invention provide for the design of particular morphological characteristics by modifying cellular island size and distribution. The compositions, methods and bioreactor systems of the disclosure have been applied to liver cultures and have shown that cellular island size and/or distribution contribute to induction of cellular metabolism that mimics in vivo metabolism. The results demonstrate that cellular distribution modulates gene expression and imply an important role in the maintenance of cell-specific metabolism (e.g., liver specific metabolism). In addition, considerations of the effect of such distribution in the design and optimization of current bioartificial support systems may serve to improve their function.
Characterization using antibody-based functional assays as well as DNA microarrays has demonstrated long-term liver-specific stability (protein and RNA levels) of micropatterned human cocultures of the invention. To demonstrate applications in drug development, acute acute/chronic toxicity assays as well as induction/inhibition of cytochrome P450s (key drug metabolism enzymes) via classic drugs were conducted. For instance, in vitro work with the drug REZULIN (Troglitazone), which was withdrawn from the market in 2000 due to idiosyncratic (1 in 10,000 occurrences) liver toxicity, show that this drug is considerably more toxic than a commonly used analgesic, acetaminophen (active ingredient in Tylenol). Accordingly, the invention is useful to screen for toxicity and drug interactions the may have either positive or negative effects on cellular metabolism.
The tissue cultures and bioreactors of the disclosure may be used to in vitro to screen a wide variety of compounds, such as cytotoxic compounds, growth/regulatory factors, pharmaceutical agents, and the like, to identify agents that modify cell (e.g., hepatocyte) function and/or cause cytotoxicity and death or modify proliferative activity or cell function. For example, the culture system may be used to test adsorption, distribution, metabolism, excretion, and toxicology (ADMET) of various agents. To this end, the cultures are maintained in vitro comprising a defined cellular island geometry and exposed to a compound to be tested. The activity of a compound can be measured by its ability to damage or kill cells in culture or by its ability to modify the function of the cells (e.g., in hepatocytes the expression of P450, and the like). This may readily be assessed by vital staining techniques, ELISA assays, immunohistochemistry, and the like. The effect of growth/regulatory factors on the cells (e.g., hepatocytes, endothelial cells, epithelial cells, pancreatic cells, astrocytes, muscle cells, cancer cells) may be assessed by analyzing the cellular content of the culture, e.g., by total cell counts, and differential cell counts or by metabolic markers such as MTT and XTT. This may also be accomplished using standard cytological and/or histological techniques including the use of immunocytochemical techniques employing antibodies that define type-specific cellular antigens. The effect of various drugs on normal cells cultured in the culture system may be assessed. For example, drugs that affect cholesterol metabolism, e.g., by lowering cholesterol production, could be tested on a liver culture system.
The methods and systems of the invention can be used to make and assay models of both normal and abnormal tissue. For example, surrounding hepatocytes with activated stellate cells would mimic fibrotic liver tissue. In this aspect, hepatocytes islands are generated and are bordered by activated stellate cells. Alternatively, pathologic liver tissue may be used as a source of hepatocytes that are used in the formation of cellular islands. These abnormal hepatocytes can be bordered by normal or abnormal non-parenchymal cell types.
In another aspect, infectious diseases may be monitored in the presence and absence of test agents. For example, in liver cultures of the invention hepatitis B and C may be tested for their effects on heterotypic and homotypic interactions as well as interactions on particular cells. Furthermore, test agents used to treat such diseases may be studied. Similarly, malaria and other infectious diseases and potential therapeutics may be tested.
One advantage of the bioreactor and culture systems of the disclosures (e.g., a single as well as an array of bioreactors of the invention) is that the cells in such a bioreactor or culture system are substantially homogenous and autologous (e.g., the cellular islands are substantially homogenous and autologous) so you can do many experiments on the same biological background. In vivo testing, for example, suffers from animal-to-animal variability and is limited by the number of conditions or agents that can be tested on a given subject.
The cytotoxicity to cells in culture (e.g., human hepatocytes) of pharmaceuticals, anti-neoplastic agents, carcinogens, food additives, and other substances may be tested by utilizing the bioreactor culture system of the disclosure.
In one aspect of the assays system, a stable, growing culture is established within the bioreactor system having a desired size (e.g., island size and distance between islands), morphology and may also include a desired oxygen gradient. The cells/tissue in the culture are exposed to varying concentrations of a test agent. After incubation with a test agent, the culture is examined by phase microscopy or by measuring cell specific functions (e.g., hepatocyte cell indicators) such as protein production/metabolism to determine the highest tolerated dose—the concentration of test agent at which the earliest morphological abnormalities appear or are detected. Cytotoxicity testing can be performed using a variety of supravital dyes to assess cell viability in the culture system, using techniques known to those skilled in the art.
Once a testing range is established, varying concentrations of the test agent can be examined for their effect on viability, growth, and/or morphology of the different cell types by means well known to those skilled in the art.
The bioreactor culture system may also be used to aid in the diagnosis and treatment of malignancies and diseases or in toxicogenomic studies. For example, a biopsy of a tissue (such as, for example, a liver biopsy) may be taken from a subject suspected of having a drug sensitivity, malignancy or other disease or disorder. The biopsy cells can then be cultured in the bioreactor system under appropriate conditions where the activity of the cultured cells can be assessed using techniques known in the art. In addition, such biopsy cultures can be used to screen agent that modify the activity in order to identify a therapeutic regimen to treat the subject to identify genes causing drug sensitivity or toxicology, or disease sensitivity. For example, the subject's tissue culture could be used in vitro to screen cytotoxic and/or pharmaceutical compounds in order to identify those that are most efficacious; i.e. those that kill the malignant or diseased cells, yet spare the normal cells or to identify drugs that do not cause a toxic response due to drug sensitivities (e.g., screening related to personalized medicine). These agents could then be used to therapeutically treat the subject.
Similarly, the beneficial effects of drugs may be assessed using the culture system in vitro; for example, growth factors, hormones, drugs which enhance hepatocyte formation or activity can be tested. In this case, stable micropattern cultures may be exposed to a test agent. After incubation, the micropattern cultures may be examined for viability, growth, morphology, cell typing, and the like as an indication of the efficacy of the test substance. Varying concentrations of the drug may be tested to derive a dose-response curve.
The culture systems of the invention may be used as model systems for the study of physiologic or pathologic conditions. For example, in a specific embodiment, the culture system can be optimized to act in a specific functional manner as described herein by modifying the size or distribution of cellular islands. In another aspect, the oxygen gradient is modified along with the density and or size of a micropattern of cells in the culture system.
A bioreactor useful in the methods of the invention is generally depicted in
In the specific embodiment of
In one aspect, fluid, upon exiting culture device 15 through outlet port 70, contacts a sensor 110 (e.g., an oxygen sensor, metabolite sensor, and the like) that measures an analyte of interest. The data obtained from the sensor 110 can be used to modulate tissue growth and or to obtain data related to the efficacy or toxicity of a particular agent or drug.
In a further embodiment, the bioreactor system 5 may be used in an array of bioreactor systems as depicted in
Referring again to
The top portion 30 of substrate 20 sealingly engages chamber/housing 50 to create a flow space (depicted by the arrows in
Inlet port 60 and outlet ports 70 comprise fittings or adapters that mate tubing to maintain circulation of the fluid in the system. The fittings or adapters may be a Luer fitting, screw threads, or the like. The tubing fittings or adapters may be composed of any material suitable for delivery of fluid (including nutrient media) for cell culture. Such tubing fittings and adapters are known in the art. Typically, inlet port 60 and outlet port 70 comprise fittings or adapters that accept tubing having a desired inner diameter for the size of the reactor and the rate of fluid flow.
Substrate 20 can be made of any material suitable for culturing mammalian cells. Although substrate 20 is depicted in
Certain materials, such as nylon, polystyrene, and the like, are less effective as substrates for cellular and/or tissue attachment. When these materials are used as the substrate it is advisable to pre-treat the substrate prior to inoculation with cells in order to enhance the attachment of cells to the substrate. For example, prior to inoculation with stromal cells and/or parenchymal cells, nylon substrates should be treated with 0.1 M acetic acid, and incubated in polylysine, FBS, and/or collagen to coat the nylon. Polystyrene could be similarly treated using sulfuric acid.
Where the in vitro generated artificial tissue is itself to be implanted in vivo, a biodegradable substrate such as polyglycolic acid, collagen, polylactic acid or hyaluronic acid should be used. Where the tissues are to be maintained for long periods of time or cryo-preserved, non-degradable materials such as nylon, dacron, polystyrene, polyacrylates, polyvinyls, teflons, cotton, and the like, may be used.
After a tissue has been grown, it can be frozen and preserved. In one aspect, the tissue is preserved by reducing the temperature to about 4° C. Where the tissue is to be cryopreserved, cryopreservative is added. Methods for cryopreserving tissue will depend on the type of tissue to be preserved and are well known in the art.
The micropatterned tissues comprising cellular islands of the disclosure can be used in a wide variety of applications. These include, but are not limited to, transplantation or implantation of the cultured artificial tissue in vivo; screening cytotoxic compounds, growth/regulatory factors, pharmaceutical compounds, and the like, in vitro; elucidating the mechanisms of certain diseases; studying the mechanisms by which drugs and/or growth factors operate; diagnosing and monitoring cancer in a patient; gene therapy and protein delivery; the production of biological products; and as the main physiological component of an extracorporeal organ assist device, to name a few. The tissues cultured by means of the bioreactors of the disclosure are particularly suited for the above applications, as the bioreactors allow the culturing of tissues having multifunctional cells. Thus, these tissues effectively simulate tissues grown in vivo.
In one embodiment, the tissue (e.g., in a bioreactor) could be used in vitro to produce biological cell products in high yield. For example, a cell which naturally produces large quantities of a particular biological product (e.g. a growth factor, regulatory factor, peptide hormone, antibody, and the like) or a host cell genetically engineered to produce a foreign gene product could be cultured using the bioreactors of the disclosure in vitro.
For example, to use a bioreactor to produce biological products, a media flow having a concentration of solutes such as nutrients, growth factors and gases flows in through port 60 and out through port 70, over a tissue 10 seeded on substrate 20. The issue is designed with a desired cellular island size and/or distribution that promote the production of a biological product. The concentrations of solutes and nutrients provided are such that the tissue layer produces the desired biological product. Product is then excreted into the media flows, and can be collected from the effluent stream exiting through outlet port 70 using techniques that are well-known in the art.
As indicated above, reactors of different scales can be used for different applications. A large scale reactor can be used to study the effects of nutrient, drugs, and the like on tissue function (e.g., ischemia on the liver and its implications such as cellular hypoxic response and organ preservation). A high throughput reactor can be used for the evaluation of drugs for metabolism, toxicity and adverse xenobiotic interactions. It could also be used for the evaluation of potential cancer drugs and other pharmacological agents in variable oxygen environments. For example, miniaturized bioreactor system can be made into an array such as depicted in
For growth of cells including, for example, hepatocytes and/or stromal cells, media containing solutes required for sustaining and enhancing tissue growth are contacted with the cells. Solutes in the fluid media include nutrients such as proteins, carbohydrates, lipids, growth factors, as well as oxygen and other substances that contribute to cell and/or tissue growth and function. For example, the oxygen gas concentration in the bioreactor system can be regulated to maintain tissue morphology (e.g., zonation in liver tissue cultures). The solutes in the media as well as those produced and released by cells in culture facilitate the development of multifunctional cells.
In another aspect, the invention provides the use of a combination of modified oxygen delivery and micropatterning of co-cultures in order to optimize the tissue culture for specific physiologic functions including, for example, synthetic, metabolic, or detoxification function (depending on the function of interest) in hepatic cell cultures.
Typically, in practicing the methods of the disclosure, the cells are mammalian cells, although the cells may be from two different species (e.g., pigs, humans, rats, mice, and the like). The cells can be primary cells, or they may be derived from an established cell-line. Although any cell type that adheres to a substrate can be used in the methods and systems of the disclosure (e.g., parenchymal and/or stromal cells), exemplary combinations of cells for producing the co-culture include, without limitation: (a) human hepatocytes (e.g., primary hepatocytes) and fibroblasts (e.g., normal or transformed fibroblasts, such as NIH 3T3-J2 cells); (b) hepatocytes and at least one other cell type, particularly liver cells, such as Kupffer cells, Ito cells, endothelial cells, and biliary ductal cells; and (c) stem cells (e.g., liver progenitor cells, oval cells, hematopoietic stem cells, embryonic stem cells, and the like) and human hepatocytes and/or other liver cells and a stromal cell (e.g., a fibroblast). Other combination of hepatocytes, liver cells, and liver precursor cells.
In another aspect, certain cell types have intrinsic attachment capabilities, thus eliminating a need for the addition of serum or exogenous attachment factors. Some cell types will attach to electrically charged cell culture substrates and will adhere to the substrate via cell surface proteins and by secretion of extracellular matrix molecules. Fibroblasts are an example of one cell type that will attach to cell culture substrates under these conditions.
Cells useful in the methods of the disclosure are available from a number of sources including commercial sources. For example, hepatocytes may be isolated by conventional methods (Berry and Friend, 1969, J. Cell Biol. 43:506-520) which can be adapted for human liver biopsy or autopsy material. Typically, a cannula is introduced into the portal vein or a portal branch and the liver is perfused with calcium-free or magnesium-free buffer until the tissue appears pale. The organ is then perfused with a proteolytic enzyme such as a collagenase solution at an adequate flow rate. This should digest the connective tissue framework. The liver is then washed in buffer and the cells are dispersed. The cell suspension may be filtered through a 70 μm nylon mesh to remove debris. Hepatocytes may be selected from the cell suspension by two or three differential centrifugations.
For perfusion of individual lobes of excised human liver, HEPES buffer may be used. Perfusion of collagenase in HEPES buffer may be accomplished at the rate of about 30 ml/minute. A single cell suspension is obtained by further incubation with collagenase for 15-20 minutes at 37° C. (Guguen-Guillouzo and Guillouzo, eds, 1986, “Isolated and Culture Hepatocytes” Paris, INSERM, and London, John Libbey Eurotext, pp. 1-12; 1982, Cell Biol. Int. Rep. 6:625-628).
Hepatocytes may also be obtained by differentiating pluripotent stem cell or liver precursor cells (i.e., hepatocyte precursor cells). The isolated hepatocytes may then be used in the culture systems described herein.
Stromal cells include, for example, fibroblasts obtained from appropriate sources as described further herein. Alternatively, the stromal cells may be obtained from commercial sources or derived from pluripotent stem cells using methods known in the art.
Fibroblasts may be readily isolated by disaggregating an appropriate organ or tissue which is to serve as the source of the fibroblasts. This may be readily accomplished using techniques known to those skilled in the art. For example, the tissue or organ can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage. Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination. These include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase, and/or hyaluronidase, DNase, pronase, dispase and the like. Mechanical disruption can also be accomplished by a number of methods including, but not limited to, the use of grinders, blenders, sieves, homogenizers, pressure cells, or insonators. For a review of tissue disaggregation techniques, see Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-126.
Once the tissue has been reduced to a suspension of individual cells, the suspension can be fractionated into subpopulations from which the fibroblasts and/or other stromal cells and/or elements can be obtained. This also may be accomplished using standard techniques for cell separation including, but not limited to, cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counter-streaming centrifugation), unit gravity separation, countercurrent distribution, electrophoresis, fluorescence-activated cell sorting, and the like. For a review of clonal selection and cell separation techniques, see Freshney, Culture of Animal Cells. A Manual of Basic Techniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp. 137-168.
The isolation of fibroblasts can, for example, be carried out as follows: fresh tissue samples are thoroughly washed and minced in Hanks balanced salt solution (HBSS) in order to remove serum. The minced tissue is incubated from 1-12 hours in a freshly prepared solution of a dissociating enzyme such as trypsin. After such incubation, the dissociated cells are suspended, pelleted by centrifugation and plated onto culture dishes. All fibroblasts will attach before other cells, therefore, appropriate stromal cells can be selectively isolated and grown. The isolated fibroblasts can then be used in the culture systems of the disclosure.
For example, and not by way of limitation, endothelial cells may be isolated from small blood vessels of the brain according to the method of Larson et al. (1987, Microvasc. Res. 34:184) and their numbers expanded by culturing in vitro using the bioreactor system of the disclosure. Silver staining may be used to ascertain the presence of tight junctional complexes specific to small vessel endothelium and associated with the “barrier” function of the endothelium.
Suspensions of pancreatic acinar cells may be prepared by an adaptation of techniques described by others (Ruoff and Hay, 1979, Cell Tissue Res. 204:243-252; and Hay, 1979, in, “Methodological Surveys in Biochemistry Vol. 8, Cell Populations.” London, Ellis Hornwood, Ltd., pp. 143-160). Briefly, the tissue is minced and washed in calcium-free, magnesium-free buffer. The minced tissue fragments are incubated in a solution of trypsin and collagenase. Dissociated cells may be filtered using a 20 μm nylon mesh, resuspended in a suitable buffer such as Hanks balanced salt solution (HBSS), and pelleted by centrifugation. The resulting pellet of cells can be resuspended in minimal amounts of appropriate media and inoculated onto a substrate for culturing in the bioreactor system of the disclosure. The pancreatic cells may be cultured with stromal cells such as fibroblasts. Acinar cells can be identified on the basis of zymogen droplet inclusions.
Cancer tissue may also be cultured using the methods and bioreactor culture system of the disclosure. For example, adenocarcinoma cells can be obtained by separating the adenocarcinoma cells from stromal cells by mincing tumor cells in HBSS, incubating the cells in 0.27% trypsin for 24 hours at 37° C. and further incubating suspended cells in DMEM complete medium on a plastic petri dish for 12 hours at 37° C. Stromal cells selectively adhered to the plastic dishes.
The tissue cultures and bioreactors of the disclosure may be used to study cell and tissue morphology. For example, enzymatic and/or metabolic activity may be monitored in the culture system remotely by fluorescence or spectroscopic measurements on a conventional microscope. In one aspect, a fluorescent metabolite in the fluid/media is used such that cells will fluoresce under appropriate conditions (e.g., upon production of certain enzymes that act upon the metabolite, and the like). Alternatively, recombinant cells can be used in the cultures system, whereby such cells have been genetically modified to include a promoter or polypeptide that produces a therapeutic or diagnostic product under appropriate conditions (e.g., upon zonation or under a particular oxygen concentration). For example, a hepatocyte may be engineered to comprise a GFP (green fluorescent protein) reporter on a P450 gene (CYPIA1). Thus, if a drug activates the promoter, the recombinant cell fluoresces. This is useful for predicting drug-drug interactions that occur due to upregulation in P450s.
The various techniques, methods, and aspects of the invention described above can be implemented in part or in whole using computer-based systems and methods. For example, computer implemented methods can be used in lithography techniques to design cellular islands.
The working examples provided below are to illustrate, not limit, the disclosure. Various parameters of the scientific methods employed in these examples are described in detail below and provide guidance for practicing the disclosure in general.
In these particular working examples, hepatocytes are co-cultured with fibroblasts. Similar methods can be used to co-culture other combinations of cells. These experiments demonstrate that one or more cell types can be cultured in a bioreactor system with controlled oxygen to obtain cells that are phenotypically similar to corresponding cells in vivo as well as tissue that is morphologically similar to tissue in vivo. Although the invention has been generally described above, further aspects of the invention will be apparent from the specific disclosure that follows, which is exemplary and not limiting.
Micropatterning of Collagen. Elastomeric Polydimethylsiloxane (PDMS) stencil devices, consisting of thick-membranes (˜300 μm) with through-holes (500 μm with 1200 μm center-to-center spacing) at the bottom of each well of a 24-well mold were provided by Surface Logix, Inc (Brighton, Mass.). Stencil devices were first sealed (via gentle pressing) to tissue culture treated polystyrene omnitrays (Nunc, Rochester, N.Y.), then each well was incubated with a solution of type-I collagen in water (100 μg/mL) for 1 hour at 37° C. Purification of collagen from rat-tail tendons was previously described. The excess collagen solution in each well was aspirated, the stencil was removed and a PDMS “blank” (24-well mold without stencil membranes) was applied. Collagen-patterned polystyrene was stored dry at 4° C. for up to 2 weeks. In some cases, micropatterned collagen was fluorescently labeled via incubation (1 hour at room temperature) with Alexa Fluor® 488 carboxylic acid, succinimidyl ester (Invitrogen, Carlsbad, Calif.) dissolved in phosphate buffered saline (PBS) at 20 μg/mL. For experiments in
Fibroblast Culture. 3T3-J2 fibroblasts were the gift of Howard Green (Harvard Medical School)1. Cells were cultured at 37° C., 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose, 10% (v/v) calf serum, and 1% (v/v) penicillin-streptomycin. In some cases, fibroblasts were growth-arrested by incubation with 10 μg/mL Mitomycin C (Sigma, St. Louis, Mo.) in culture media for 2 hours.
Microscopy. Specimens were observed and recorded using a Nikon Diaphot microscope equipped with a SPOT digital camera (SPOT Diagnostic Equipment, Sterling Heights, Mich.), and MetaMorph Image Analysis System (Universal Imaging, Westchester, Pa.) for digital image acquisition.
Gene Expression Profiling. Micropatterned hepatocyte-fibroblast co-cultures were washed 3 times with phosphate buffered saline (PBS) to remove traces of serum, followed by treatment with 0.05% Trypsin/EDTA (Invitrogen) for 3 min at 37° C. Fibroblasts were much more sensitive to trypsin-mediated detachment than hepatocytes arranged in clusters (500 μm) via micropatterning. Following incubation with trypsin, plates were shook mildly to remove loosely attached fibroblasts, the supernatant was aspirated and the attached hepatocytes (˜95% purity) were washed 3 times with serum-supplemented hepatocyte medium to neutralize and remove traces of trypsin from the cultures. Hepatocyte RNA was extracted via TRIzol (Invitrogen) and purified using the RNeasy kit (Qiagen) as per manufacturers' instructions. The RNA was labeled, hybridized to an Affymetrix (Santa Clara, Calif.) Human U133 Plus 2.0 Array, and scanned as described previously. Briefly, double-strand cDNA was synthesized using a T7-(dt)24 primer (Oligo) and reverse transcription (Invitrogen) cDNA was then purified with phenol/chloroform/isoamyl alcohol in Phase Lock Gels, extracted with ammonium acetate and precipitated using ethanol. Biotin-labeled cRNA was synthesized using the BioArray™ HighYield™ RNA Transcript Labeling Kit, purified over RNeasy columns (Qiagen), eluted and then fragmented. The quality of expression data was assessed using the manufacturer's instructions which included criteria such as low background values and 3′/5′ actin and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) ratios below 2. All expression data was imported to GCOS (GeneChip Operating System v1.2) and scaled to a target intensity of 2500 to enable comparison across conditions.
Phase I & II Enzyme Activity Assays. Chemicals were purchased from Sigma: Coumarin (CM), 7-Hydroxycoumarin (7-HC), Ethoxyresorufin (ER), Resorufin (RR), Ketoconazole (KC), Sulfaphenazole (SP), Methoxsalen (MS) Salicylamide (SC) or purchased from BD-Gentest: 7-methoxy-4-trifluoromethylcoumarin (MFC), 7-benzyloxy-4-trifluoromethylcoumarin (BFC), 7-hydroxy-4-trifluoromethylcoumarin (7-HFC). Cultures were incubated with substrates (CM, MFC, BFC at 50 μM, ER at 8 μM, 7-HC at 100 μM) dissolved in DMEM without phenol red for 1 hour at 37° C. For inhibition studies, cultures were incubated with substrates in the presence of specific inhibitors (MS at 25 μM with CM, SP at 50 μM with MFC, KC at 50 μM with BFC, SC at 3 mM with 7-HC). The reactions were stopped by collection of the incubation medium. Then, potential metabolite conjugates formed via Phase II activity were hydrolyzed by incubation of supernatants with β-glucuronidase/arylsulfatase (Roche, Ind.) for 2 hours at 37° C. Samples were diluted 1:1 in quenching solution and fluorescent metabolite formation was quantified by means of a fluorescence micro-plate reader (Molecular Devices, Sunnyvale, Calif.) as described elsewhere in detail. Production of 7-HC from CM is a reaction (CM 7-Hydroxylation) mediated by CYP2A6 in humans, production of 7-HFC from BFC or MFC (dealkylation) is mediated by several different CYP450s, and production of RR from ER (ER Odealkylation) is mediated by CYP1A2. Conjugation of 7-HC with glucuronic acid and sulfate groups is mediated by Phase II enzymes, UPD-Glucuronyl-transferase and Sulfotransferase, respectively.
Hepatocyte Isolation and Culture. Primary rat hepatocytes were isolated from 2-3-month old adult female Lewis rats (Charles River Laboratories, Wilmington, Mass.) weighing 180-200. Detailed procedures for rat hepatocyte isolation and purification were previously described. Routinely, 200-300 million cells were isolated with 85%-95% viability and >99% purity. Hepatocyte culture medium consisted of Dulbecco's Modified Eagle's medium (DMEM) with high glucose, 10% (v/v) fetal bovine serum, 0.5 U/mL insulin, 7 ng/mL glucagon, 7.5 μg/mL hydrocortisone, and 1% (v/v) penicillin-streptomycin. Primary human hepatocytes were purchased in suspension from vendors permitted to sell products derived from human organs procured in the United States of America by federally designated Organ Procurement Organizations. Hepatocyte vendors included: In vitro Technologies (Baltimore, Md.), Cambrex Biosciences (Walkersville, Md.), BD Gentest (Woburn, Mass.), ADMET Technologies (Durham, N.C.), CellzDirect (Pittsboro, N.C.) and Tissue Transformation Technologies (Edison N.J.). All work was done with the approval of COUHES (Committee on use of human experimental subjects). Upon receipt, human hepatocytes were pelleted via centrifugation at 50×g for 5 min (4° C.). The supernatant was discarded, cells were re-suspended in hepatocyte culture medium, and viability was assessed using trypan blue exclusion (70-90%).
Hepatocyte-Fibroblast Co-Cultures. In order to create micropatterned co-cultures, hepatocytes were seeded in serum-free hepatocyte medium on collagen-patterned substrates, resulting in a hepatocyte pattern due to selective cell adhesion. The cells were washed with media 2 hours later to remove unattached cells and incubated with serum-supplemented hepatocyte medium overnight. 3T3-J2 fibroblasts were seeded in serum-supplemented fibroblast medium 12-24 hours later to create co-cultures. Culture medium was replaced to hepatocyte medium 24 hours after fibroblast seeding and subsequently replaced daily. For randomly distributed cultures, hepatocytes were seeded in serum-supplemented hepatocyte medium on substrates (glass or polystyrene) with a uniform coating of collagen. In some cases, hepatocytes were fluorescently labeled via incubation (1 hour at 37° C.) with Calcein-AM (Invitrogen) dissolved in culture media at 5 μg/mL. Fibroblasts were fluorescently labeled with CellTracker (Orange CMTMR, Invitrogen) as per manufacturer's instructions.
Biochemical Assays. Spent media was stored at −20° C. Urea concentration was assayed using a colorimetric endpoint assay utilizing diacetylmonoxime with acid and heat (Stanbio Labs, Boerne, Tex.). Albumin content was measured using enzyme linked immunosorbent assays (MP Biomedicals, Irvine, Calif.) with horseradish peroxidase detection and 3, 3′, 5, 5″-tetramethylbenzidine (TMB, Fitzgerald Industries, Concord, Mass.) as a substrate.
Cytochrome-P450 Induction. Stock solutions of prototypic CYP450 inducers (Sigma) were made in dimethylsulfoxide (DMSO), except for Phenobarbital, which was dissolved in water. Cultures were treated with inducers (Rifampin 25 μM, β-Naphthoflavone 30 μM or 50 μM, Phenobarbital 1 mM, Omeprazole 50 μM) dissolved in hepatocyte culture medium for 4 days. Control cultures were treated with vehicle (DMSO) alone for calculations of fold induction. To enable comparisons across inducers, DMSO levels were kept constant at 0.06% (v/v) for all conditions.
Toxicity Assays. Cultures were incubated with various concentrations of compounds dissolved in culture medium for 24 hours (acute toxicity) or extended time periods (chronic toxicity, 1-4 days). Cell viability was subsequently measured via the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma) assay, which involves cleavage of the tetrazolium ring by mitochondrial dehydrogenase enzymes to form a purple precipitate. MTT was added to cells in DMEM without phenol red at a concentration of 0.5 mg/mL. After an incubation time of 1 hour, the purple precipitate was dissolved in a 1:1 solution of DMSO and Isopropanol. The absorbance of the solution was measured at 570 nm (SpectraMax spectrophotometer, Molecular Devices, Sunnyvale, Calif.).
Statistical Analysis. Experiments were repeated at least 2-3 times with duplicate or triplicate samples for each condition. Data from representative experiments is presented, whereas similar trends were seen in multiple trials. All error bars represent standard error of the mean.
Furthermore, without induction, both CYP2B and CYP3A protein was present at low levels after 48 hour perfusion with little distinguishable spatial heterogeneity as compared to not detectable protein under static culture conditions. Next, induction of static cultures with phenobarbital (PB) over the same time period resulted in moderate CYP2B expression and low CYP3A. Dramatic expression of both CYPs over controls was seen after only 36 hours when cultures were perfused with PB. Though expression of CYP2B was increased in all regions, levels were highest in the lower-oxygen outlet regions. Similarly, CYP3A protein showed increasing expression from inlet to outlet. Based on previous studies that showed repression of PB-induced CYP2B expression by epidermal growth factor (EGF), added 2 nM EGF to the perfusion media. At a dose of 200 μM PB, EGF did not significantly alter CYP2B levels along the length of the chamber though maximal levels were noted in the outlet regions. CYP3A levels in response to PB and EGF also showed little difference from PB-only perfusion displaying maximal expression at the outlet.
Experiments were also carried out to evaluate dexamethasone (DEX) as an inducer of CYPs in this perfusion system. DEX induced CYP2B to high levels which were localized to inlet regions of the culture. For CYP3A, induction was mostly uniform, but not detectable in the outlet region. When EGF was added to DEX-perfused cultures, a significant shift in CYP2B spatial distribution was noted from inlet regions to the outlet. CYP3A induction remained uniform in response to DEX and EGF, but was extend across all regions of the culture.
Acetaminophen (APAP) was evaluated for its acute toxic effect on hepatocyte cultures and co-cultures (
For further quantification of regional variations in viability, bright-field images were acquired at low magnification (40×) along the length of the culture for measurement of mean optical density (
Many members of the CYP superfamily responsible for phase I drug and steroid biotransformation are expressed in a zonal pattern in vivo. Among the determinants of the pericentral localization of CYPs under both normal and induced conditions are gradients of oxygen, nutrients, and hormones. Recapitulation of these dynamic gradients in bioreactor cultures resulted in spatial distributions of both CYP2B and CYP3A that mimic those found in vivo. Additionally, CYP induction was potentiated by the perfusion microenvironment of the reactor as shown by the dramatic increase in protein levels over static cultures in response to 200 μM PB. Previous studies demonstrated that the repressive effects of EGF on PB induction are modulated by oxygen.
Addition of EGF with PB in the current study did not significantly alter the spatial CYP2B pattern, but in conjunction with DEX, EGF shifted maximal CYP2B expression from the inlet to the outlet. This shifting effect, also noted to a lesser extent in CYP3A expression, may be due the formation of EGF gradients, thus demonstrating how dynamic gradients of growth factors and hormones regulate CYP zonation.
The proposed mechanism of APAP hepatotoxicity involves the formation of a reactive intermediate, NAPQI, which initiates free-radial damage of intracellular structures. Toxic effects in this study are likely due to the depletion of glutathione, which provides protective inactivation of NAPQI. Though pericentral localization of APAP toxicity in vivo has been attributed to local expression of CYP isoenzymes 2E1 and 3A, reduced oxygen availability in centrilobular regions may also contribute by depleting ATP and glutathione, or increasing damage by reactive species. A combination of these factors likely resulted in the regional toxicity observed in reactor cultures under dynamic oxygen gradients. Demonstration of zonal toxicity in vitro allows decoupling of the effects of CYP bioactivation and glutathione levels on acute APAP toxicity.
Furthermore, this system may allow elucidation of the actions various clinically important compounds such as ethanol or N-acetyl-cysteine and their respective exacerbating or protective effects on APAP toxicity.
As demonstrated by the data, oxygen gradients were applied to cultures of rat hepatocytes to develop and in vitro model of liver zonation. Cells experienced oxygen conditions ranging from normoxia to hypoxia without compromising viability as shown by morphology and fluorescent markers of membrane integrity. The hepatocytes exposed to oxygen gradients exhibited characteristics of in vivo zonation upon induction as shown by PEPCK (predominantly upstream) and CYP2B (predominantly downstream) protein levels. With this in vitro model of liver zonation, the microenvironmental conditions seen in the liver sinusoid that are thought to be responsible for heterogeneous distribution of metabolic and detoxifying functions can be reproduced.
Cell seeding conditions and cell height can be kept uniform within the bioreactor system to insure uniformity of the flow field. The bioreactor experiments carried out in the specific examples herein, were typically conducted at a flow rate of 0.5 mL/min, corresponding to a shear stress of 1.25 dyne/cm2, although higher stress near 7.5 dynes/cm2 may have been present at higher flow rates using validation experiments.
Liver-specific functions in human cocultures can be optimized by varying homotypic and heterotypic interactions. A photolithographic cell patterning technique is provided by the invention which allows study of the relative role of homotypic (hepatocyte-hepatocyte) and heterotypic (hepatocyte-fibroblast) cell-cell interactions in stabilization of liver-specific functions in vitro (see
Due to data showing that rat cocultures can be functionally optimized using micro-patterning, it was hypothesized that a similar optimization can be obtained for human cocultures as well. In order to compare results between species, similar pattern geometries were used to evaluate micropatterned human cocultures. Conventional cocultures contain hepatocytes in a random variety of island sizes from single cell islands to large aggregates (random cocultures). Hence, the expectation was that with a wide array of island sizes, function in random human cocultures would be at a level intermediate of the single island (36 μm) and the large island (4,800 μm) micropatterned configurations. However, micropatterned human cocultures (all configurations) reproducibly outperform (by several fold) their randomly distributed counterparts, which contain similar cell ratios and numbers (see
Such a trend is consistent with the literature, in which phenotypic stabilization of human hepatocytes occurs more effectively with increasing levels of homotypic interactions. As with rat cocultures, the 36 μm human hepatocyte islands reorganized within a day, thereby dissipating the pattern. The micro-patterns with the two large island sizes, however, were intact for the duration of the cultures (3 weeks). Thus, the ‘optimal’ micropatterned configuration was identified as 490 μm islands with 1230 μm center-to-center spacing and a 3:1 fibroblast to hepatocyte ratio. Since reorganization of hepatocyte islands in this optimal configuration is minimal over the length of the culture, real-time tracking of individual islands for morphological and functional changes can be performed using specific reporter systems (i.e. green fluorescent protein).
Use of optimized micropatterned human cocultures to evaluate metabolism and toxicity of xenobiotics. In order to demonstrate that the optimized micropatterned human cocultures are effective liver models for screening of drug candidates, acute and chronic toxicity assays were conducted. Acute toxicity tests involved incubating eight day old cocultures for 24 hrs with various drugs, some with known clinical hepatotoxic potential. After the incubation time period, the cell culture medium was aspirated and a viability assay was conducted (MTT assay—commercially available). Shown as part of
Current in vitro human liver models typically rely on hepatocytes in suspension or as pure monolayers on collagen. Hepatocytes are attachment-dependent cells and thus the ‘cells in suspension’ model is only viable for a few hours, while the pure hepatocyte monolayer typically loses viability and liver-specific function within 24 hrs. Thus, chronic toxicity assays in which the cells are incubated repeatedly for several days or weeks with low doses of a drug cannot be conducted in current liver model systems. Since the optimized micropatterned human cocultures routinely last for 3 weeks, chronic toxicity tests can be performed. In
Besides toxicity testing, induction and inhibition of CYP450 enzymes is quite common during the in vitro testing of a new drug candidate. As can be seen in
One of the major concerns facing current in vitro liver models is the rapid (hrs) decline of expression levels (RNA) of important liver-specific genes. Thus, expression levels of important liver-specific genes in the optimized micropatterned cocultures were compared to those in conventional pure hepatocyte monolayers after several days of culture. Using DNA microarrays (Affymetrix GeneChips), the data demonstrate as shown in
Drawing photolithographic micropatterning techniques to manipulate functions of rodent hepatocytes upon co-cultivation with stromal cells, a microtechnology-based process utilizing elastomeric stencils to miniaturize and characterize human liver tissue in an industry-standard multiwell format was used. The approach incorporates ‘soft lithography,’ a set of techniques utilizing reusable, elastomeric, polymer (Polydimethylsiloxane-PDMS) molds of microfabricated structures to overcome limitations of photolithography. In one aspect, the invention provides a process using PDMS stencils consisting of 300 μm thick membranes with through-holes at the bottom of each well in a 24-well mold (
Collagen island diameter was varied over several orders-of-magnitude. Hepatocyte clustering consistently improved liver-specific functions when compared to unorganized co-cultures (
In order to qualitatively assess the stability of the microscale human liver tissues, hepatocyte morphology and persistence of microscale organization were monitored and found to be maintained for duration of the culture, typically 3-6 weeks (
In order to assess utility of the microscale human liver tissue for drug metabolism studies, CYP450 activity, drug-drug interactions, and phase-II metabolism was characterized. CYP450 activity was assessed using fluorescent substrates and found to be retained in untreated microscale tissues (
To assess utility of the microscale human liver tissue for toxicity assays, the acute and chronic toxicity of model hepatotoxins were quantified. Compounds were characterized by their TC50, defined as the concentration which produced 50% reduction in mitochondrial activity after 24 hr exposure (
Induction of CYP450 activity in the microscale human liver tissues was demonstrated using prototypic inducers and fluorescent substrates (
An advantageous feature of the platform of the invention is its modular design in that various liver or non-liver derived stroma can be used to surround hepatocyte colonies/islands to form micropatterned tissues. 3T3 fibroblasts were chosen because of their ready availability, ease of propagation, and evidence showing that this immortalized cell line can induce high levels of liver-specific functions. Nonetheless, to demonstrate versatility of the platform, co-cultivates of micropatterned human hepatocytes with the non-parenchymal fraction of the human liver also demonstrated stabilization of hepatocyte functions. Furthermore, stencils were used to create a co-culture model of the rat liver that remains functional for over 2 months, allowing chronic studies to be conducted on hundreds of identical rodent liver tissues, thereby reducing noise arising from animal-to-animal variability (
The invention demonstrates that micropatterned clusters of human hepatocytes outperformed their randomly distributed counterparts by several fold, consistent with reports that confluent hepatocyte cultures retain liver-specific functions better than sparse cultures, partly through cadherin interactions. Subsequent introduction of non-parenchymal cells further enhanced hepatocellular functions and longevity of the liver tissues. Thus, the microscale platform described herein uses an order-of-magnitude fewer hepatocytes (10K vs. 200K) and maintains phenotypic functions for a longer time than conventional pure monolayers (weeks vs. days) in similar multiwell formats. Given the high cost of human hepatocytes (˜$80/million), such advantages represent a significant cost savings. The platform, demonstrates induction of liver-specific functions in fresh hepatocytes across donors of multiple age groups, sexes and medical histories (Table 1). The cultures were also capable of being successfully cryopreserved similar to those now widely utilized for short-term cultures, thus providing the potential to generate microscale liver tissue on demand.
*African-American Donors. All other donors were of Caucasian descent.
‘N/A’ - not available at time of purchase.
Liver donor information reported here is specific information (age, sex, cause of death) on liver donors whose freshly isolated hepatocytes were purchased in suspension from multiple vendors for use in experiments of this study.
Several other in vitro models of liver tissue have been proposed. In particular, multilayer or spheroid-based ‘3D’ hepatocellular tissues, some with continuous perfusion, have been reported. As the liver itself is composed of flat, anastomising ‘plates’ that are typically one cell thick, two dimensional (monolayer) platforms of the liver may suffice for many ADME/Tox applications. Furthermore, since monolayer systems (confluent monolayers, collagen sandwich or Matrigel overlay) are still the most commonly utilized platforms in industry 13,14, the microscale tissue proposed here can be mapped easily to existing laboratory protocols including robotic fluid handling, in situ microscopy, and colorimetric/fluorescent plate-reader assays.
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This application claims priority under 35 U.S.C. §119 to U.S. Provisional Application Ser. No. 60/684,508, filed May 24, 2005, the disclosure of which is incorporated herein by reference in its entirety. The disclosures of U.S. Provisional Application Ser. No. 60/450,532, filed Feb. 26, 2003; International Patent Application No. PCT/US2004/006018; U.S. Provisional Application Ser. No. 60/302,879, filed Jul. 3, 2001; and International Patent Application No. PCT/US2002/21207, are also incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60684508 | May 2005 | US |
