Patent Application
20130344501
The present invention relates generally to the fields of microbiology, infectious disease, immunology, cell biology, toxicology, cancer, environmental microbiology, bioengineering, biotechnology, vaccine/adjuvant/therapeutic and drug development.
Conventional two-dimensional (2-D) cell culture involves growing cells as monolayers on solid, impermeable surfaces (plastic or glass) or in uniform suspension. However, this “flat biology” 2-D approach, in which key phenotypic and functional characteristics are lost, often does not accurately predict in vivo tissue responses. (See, e.g., Bissell et al., Curr. Opin. Cell Biol. 15, 753-762 (2003)).
One key reason for the loss of differentiation that occurs in monolayers is the dissociation from the native in vivo three-dimensional structure to 2-D propagation on flat, impermeable substrates in vitro, which also prevents cells from responding to chemical and molecular gradients in three dimensions (reflecting the apical, basal and lateral cell surfaces) (Abbott, A. Nature 424, 870-872 (2003); Freshney, R. Culture of Animal Cells: a Manual of Basic Technique (Wiley-Liss, New York, 2000); Schmeichel, K. L. & Bissell, et al., J. Cell Sci. 116, 2377-2388 (2003); O'Brien, L. E., et al., Nature Rev. Mol. Cell. Biol. 3, 531-537 (2002)). Because 2-D monolayers lack the complexity, and often the physiological relevance, of the tissues that are encountered by a pathogen during the natural course of infection in vivo, they are often unfaithful predictors of the infection process.
Thus, to overcome some of the inherent limitations associated with 2-D monolayers, as well as the high cost, availability and variability of animal models, a need exists for three-dimensional tissue systems. A three-dimensional tissue system can help bridge the gap between cell-based discovery research and animal models for studying both host-pathogen interactions and disease progression, as well as for the development of novel drugs and therapeutics.
One challenge in developing physiologically relevant tissue systems, such as an immunocompetent tissue system, is in recapitulating the dynamic integrated three-dimensional network of lymphoid cells, innate immune cells, epithelial cells, effector molecules, and cellular microenvironments, all of which are vital for host protection and defense. Conventional cell culture technologies, including alternative three-dimensional tissue culture systems, do not provide the comprehensive structural and functional aspects that are required to produce physiologically relevant equivalents of the immune system.
This disclosure describes methods for producing three-dimensional, physiologically relevant immune tissue systems under low fluid shear conditions.
In one aspect, a method of producing a three-dimensional, physiologically relevant immune tissue system is disclosed. The method includes a) introducing an immune cell and at least one other cell type into a low fluid shear environment; and b) co-culturing the immune cell and the at least one other cell type under conditions selected to produce a three-dimensional immune tissue system with one or more physiologically relevant characteristics.
In one or more embodiments, the one or more physiologically relevant characteristics are selected from the group consisting of one or more differentiated and functional cells, assembly into relevant three-dimensional aggregates, production of extracellular matrix components, and physiologically relevant cell type ratios.
In one or more embodiments, the immune cells are selected from the group consisting of monocytes, astrocytes, neuronal cells, macrophages, dendritic cells, B cells, T cells, natural killer cells, basophils, eosinophils, and neutrophils from healthy and/or diseased subjects.
In one or more embodiments, the immune cells are astrocytes and neuronal cells.
In one or more embodiments, the immune cells further comprise monocytes.
In one or more embodiments, the method further includes culturing the immune cell and/or the at least one other cell type in a monolayer before placing in the low fluid shear environment.
In one or more embodiments, the method further includes developing the immune cell and/or at least one other cell type into three-dimensional cells before placing in the low fluid shear environment.
In one or more embodiments, the method includes first placing the immune cell in the low fluid shear environment, first placing the at least one other cell type in the low fluid shear environment, or placing the immune cell and the at least one other cell type simultaneously in the low fluid shear environment.
In one or more embodiments, the method includes developing the cells into three-dimensional cells on a scaffold. In one or more embodiments, the scaffold is made of microcarrier beads.
In one or more embodiments, the at least one other cell type is an immune or epithelial cell.
In one or more embodiments, the one or more physiologically relevant characteristics are selected from the group consisting of a differentiated epithelium, one or more functional macrophage-like cells, a localization of macrophage-like cells on or beneath the epithelial surface, production of one or more extracellular matrix components, and a physiologically relevant macrophage-to-epithelial cell ratio.
In one or more embodiments, the physiologically relevant macrophage-to-epithelial cell ratio ranges from about 1:30 to about 1:40.
In one or more embodiments, the epithelial cells are selected from the group consisting of alveolar, bronchial, small intestinal, large intestinal, cervical, urogenital, gastrointestinal tract, respiratory tract, and vaginal epithelial cells from healthy and/or diseased subjects and vaginal epithelial cells from healthy and/or diseased subjects.
In one or more embodiments, the epithelial cells and the immune cells are derived from human cell lines.
In one or more embodiments, the epithelial cells are small intestinal epithelial cells, and the immune cells are monocytes.
In one or more embodiments, the epithelial cells are large intestinal epithelial cells, and the immune cells are monocytes.
In one or more embodiments, the immune cell is a monocyte, and the at least one other cell type is an alveolar epithelial cell.
In one or more embodiments, the method further includes culturing the alveolar epithelial cells in a monolayer and developing the alveolar epithelial cells into three-dimensional cells in the low fluid shear environment.
In one or more embodiments, the ratio of the monocytes to the three-dimensional alveolar epithelial cells ranges from about 1:100 to about 100:1.
In one or more embodiments, the low fluid shear conditions ranges from about 0 dynes/cm2 to about 10.0 dynes/cm.2
In one or more embodiments, the time period of step b) ranges from about 1 day to about 40 days.
In one or more embodiments, the low fluid shear environment is provided by one or more bioreactors.
In one or more embodiments, the bioreactor is a rotating wall vessel (RWV).
In one or more embodiments, the bioreactor has a rotation speed that ranges from about 10 rpm to about 30 rpm.
In one or more embodiments, the RWV is a slow transfer lateral vessel (STLV).
In one or more embodiments, the RWV is a high-aspect rotating vessel (HARV).
In one or more embodiments, the low fluid shear environment is a spaceflight environment.
In one or more embodiments, the conditions appropriate for producing a three-dimensional immune tissue system with physiologically relevant characteristics are selected from the group consisting of appropriate culture medium, temperature, oxygen level, pH, composition of the extracellular matrix, and time in the low fluid shear environment.
In one or more embodiments, the culture medium is GTSF-2.
In one or more embodiments, the time in the low fluid shear environment ranges from about 24 hours to about 1 year.
In one or more embodiments, the method further includes conducting one or more biochemical analyses to determine that the three-dimensional tissue system has one or more physiologically relevant characteristics.
In another aspect, the disclosure includes a kit for producing a three-dimensional, physiologically relevant tissue system, comprising an immune cell line and at least one other cell line; and informational material for producing a three-dimensional, physiologically relevant tissue system.
The foregoing and other objects of the present invention, the various features thereof, as well as the invention itself, may be more fully understood from the following description, when read together with the accompanying drawings. The following drawings are presented for the purpose of illustration only, and are not intended to be limiting.
All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein.
The disclosure is based, in part, on the discovery of methods for producing three-dimensional, physiologically relevant immune tissue systems under low fluid shear conditions. The disclosure also relates to methods of assessing disease pathogenesis and disease treatment using three-dimensional, physiologically relevant immune tissue systems, such as related to, for example, infectious disease, cancer, inflammation, chemosensitivity, toxicology, reactogenicity, vaccine/adjuvant/therapeutic and drug design, development and screening, immunogenicity, and tissue homeostasis and transition to disease. The disclosure also relates to kits for producing three-dimensional, physiologically relevant immune tissue systems under low fluid shear conditions.
The present disclosure presents methods that improve upon conventional cell culture methods and animal models to better predict in vivo human responses to infectious pathogens, toxins, drugs vaccines/adjuvants, chemotherapeutic agents, cosmetics, and other chemicals. The method serve as preditive platforms for identifying bisignatures for the transition from normal homeostasis to disease development.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.
The abbreviation “RWV” refers to rotating wall vessel. The RWV is an optimized suspension culture method in which cells are grown on extracellular matrix-coated microcarrier beads in cylindrical bioreactors, called slow turning lateral vessels (STLV) or high aspect ratio vessels (HARV), in physiologically relevant low fluid-shear conditions (Nickerson et al., 2004b).
The term “cell lines” means cells that are derived from an organism and maintained in an environment that provides the conditions necessary for division (typically referred to as culturing). “Cell lines” can be maintained indefinitely in a constantly dividing state (i.e., immortalized) or maintained in culture up to cell senescence (e.g., approximately 50 cell divisions). “Primary cells” are cells taken directly from living tissue (e.g., biopsy material) and established for growth in vitro.
“Fluid shear” is shear stress created by a fluid along a solid boundary. In biological systems, a solid boundary can be a tissue, such as an epithelial layer. Biomechanical forces such as fluid shear are known to influence cellular differentiation and development.
As used herein, “low fluid shear environment” means an environment with low fluid shear along with other physical and biological factors that affect the metabolism, signaling, and/or other interactions of a cell. A “low fluid shear environment” includes shear stress rates ranging from about 0 dynes/cm2 to about 10.0 dynes/cm.2 A “low fluid shear environment” simulates the environment encountered during spaceflight, in certain spaceflight analogs including in a bioreactor, such as a RWV bioreactor, and in certain areas of the body, including mucosal tissues of the gastrointestinal, lung and urogenital tracts, which are primary sites of infection.
As used herein, the term “about” is used to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 10 percent up or down (higher or lower).
As used herein, the term “tissue system” means a dynamic, integrated group of cells in a definite organization that carry out a function. For example, an immune tissue system is a dynamic, integrated group of cells, including, but not limited to, lymphoid cells, innate immune cells, epithelial cells, and effector molecules, which carry out the function of host protection and defense.
Methods of Producing Three-Dimensional Tissue Systems with Physiologically Relevant Characteristics
The methods and systems disclosed herein relate to producing three-dimensional, physiologically relevant tissue systems. In one embodiment, the tissue system is an immunocompetent tissue system. In one embodiment, the methods comprise introducing an immune cell and at least one other cell type, including immune cells, into a low fluid shear environment. Next, the immune cell and the at least one other cell type are co-cultured under conditions selected to produce a three-dimensional immune tissue system with one or more physiologically relevant characteristics.
The term “physiologically relevant characteristics” refer to characteristics of tissue systems that are similar both structurally and functionally to those found in in vivo tissues, including human tissues. The methods produce three-dimensional tissue systems with similar cellular organization, morphology, and histology to in vivo tissue systems. Physiologically relevant characteristics include, but are not limited to, one or more differentiated and functional cells; production of extracellular matrix components; assembly into relevant three-dimensional aggregates; and physiologically relevant cell type ratios. Physiologically relevant characteristics can differ depending on the specific tissue system. In one embodiment, for an immunocompetent tissue system, physiologically relevant characteristics further include differentiated immune cells (for example, macrophages) that conduct immune cell functions, such as phagocytosis and production of inflammatory mediators.
In another embodiment, for an immunocompetent tissue system with epithelial cells, physiologically relevant characteristics can include a differentiated epithelium, one or more functional macrophage-like cells, a localization of macrophage-like cells on (or beneath) the epithelial surface, production of one or more extracellular matrix components, and a physiologically relevant macrophage-to-epithelial cell ratio. The methods produce tissue systems that are structurally and functionally similar to endogenous mucosa.
The low fluid shear environment enables the development of three-dimensional tissue systems, including immunocompetent co-culture systems containing human immune cells, that display physiologically relevant characteristics similar to in vivo human tissues. Low shear environments can be generated in several ways. For example, the optimized suspension culture RWV bioreactor (including the high-aspect rotating vessel (HARV) and slow turning/transfer lateral vessel (STLV)) generates such a low shear environment. In addition, the conditions encountered during spaceflight and in certain spaceflight analogs provide the appropriate low fluid shear environment. Use of one or a combination of these low shear environments in the disclosed methods is contemplated.
Embodiments of the methods include culturing immune cells and at least one other cell lines from particular tissues, including additional immune cells and epithelial cells. Examples of epithelial cells or cell lines include, but are not limited to alveolar, bronchial, small intestinal, large intestinal, cervical, vaginal, urogenital tract, gastrointestinal tract, and respiratory tract epithelial cells from healthy and/or diseased subjects. The term “epithelial” encompasses cells derived from developmental lineages that are epithelial, endothelial, or mesothelial. Examples of immune cells or cell lines include, but are not limited to, monocytes, astrocytes, neuronal cells, macrophages, dendritic cells, B cells, T cells, natural killer cells, basophils, eosinophils, and neutrophils from healthy and/or diseased subjects.
In one embodiment, the methods include culturing the immune cell and/or the at least one other cell type in a monolayer before placing in the low fluid shear environment. In another embodiment, the methods include developing the immune cell and/or at least one other cell type into three-dimensional cells before placing in the low fluid shear environment.
In one embodiment, the methods include first placing the immune cell in the low fluid shear environment, first placing the at least one other cell type in the low fluid shear environment, or placing the immune cell and the at least one other cell type simultaneously in the low fluid shear environment.
In one embodiment, methods of producing a three-dimensional, physiologically relevant immune tissue system includes first culturing epithelial cells in a monolayer. Next, the epithelial cells are placed in a low fluid shear environment for a time period such that the epithelial cells develop into mature three-dimensional epithelial cells. Then, the mature three-dimensional epithelial cells are co-cultured with undifferentiated immune cells in the low fluid shear environment under conditions selected to produce a three-dimensional immune tissue system with one or more physiologically relevant characteristics. In some embodiments, the undifferentiated immune cells (e.g., monocytes) are pre-differentiated into macrophages with chemical treatment prior to placement in the low fluid shear environment. In other embodiments, the undifferentiated immune cells autonomously differentiate into macrophages in the low fluid shear environment. Physiologically relevant characteristics include one or more differentiated cells, production of extracellular matrix components, and physiologically relevant cell type ratios. In certain embodiments, physiologically relevant characteristics include of a differentiated epithelium, one or more functional macrophage-like cells, a localization of macrophage-like cells on (or beneath) the epithelial surface, production of one or more extracellular matrix components, and a physiologically relevant macrophage-to-epithelial cell ratio.
In some embodiments, undifferentiated immune cells (e.g., monocytes) are first cultured in the low fluid shear environment, and epithelial cells are added to the low fluid shear environment. In other embodiments, the epithelial cells are first cultured in the low fluid shear environment, and the undifferentiated immune cells are added to the low fluid shear environment. In other embodiments, the undifferentiated immune cells and epithelial cells are placed simultaneously in the low fluid shear environment.
In other embodiments, immune cells including astrocytes and neuronal cells are co-cultured under conditions selected to produce a three-dimensional immune tissue system with one or more physiologically relevant characteristics. In one embodiment, neuronal cells are placed in the low fluid shear environment, and then astrocytes are placed in the low fluid shear environment. In another embodiment, astrocytes are first placed in the low fluid shear environment, and then neuronal cells are added. In another embodiment, neuronal cells and astrocytes are placed in the low fluid shear environment simultaneously.
In other embodiments, astrocytes, neuronal cells, and monocytes are co-cultured under conditions selected to produce a three-dimensional immune tissue system with one or more physiologically relevant characteristics.
In additional embodiments, the methods comprise placing the epithelial cells into a low fluid shear environment for a time period. The time period allows the cells to develop into a mature three-dimensional group of cells or tissues. The period of time that the cells can be cultured in the low shear environment is from about 24 hours to about 1 year, from about 24 hours to about 10 months, from about 24 hours to about 6 months, from about 24 hours to about 2 months, from about 24 hours to about 1 month, from about 24 hours to about 1 week, from about 24 hours to about 120 hours, from about 36 hours to about 108 hours, from about 48 hours to about 96 hours, from about 60 hours to about 84 hours, and is about 72 hours. In certain embodiments, the period of time is about 5 days to about 25 days. In other embodiments, the period of time is about 9 days to about 20 days. In particular embodiments, the period of time is about 9 days.
Embodiments of the methods disclosed herein also include co-culturing three-dimensional epithelial cells with undifferentiated immune cells in the low fluid shear environment, and the undifferentiated immune cells then undergo spontaneous differentiation to become immunocompetent. The immune cells can be undifferentiated monocytes, macrophages (i.e., differentiated monocytes), dendritic cells (i.e., differentiated monocytes), astrocytes, neuronal cells, neutrophils, eosinophils, basophils, and lymphocytes, including T lymphocytes, B lymphocytes, and natural killer cells from healthy and/or diseased subjects. In other aspects, the immune cells are differentiated using well-known chemical treatments prior to incorporating into the three-dimensional tissue system. In other embodiments, the methods include co-culturing three-dimensional epithelial cells with differentiated immune cells in the low fluid shear environment.
In certain embodiments, the cells are immune cells, epithelial cells, and other cell types, such as stem cells. In some embodiments, the cells are derived from primary cells from healthy mammals (e.g., humans and non-human primates). In particular aspects, the epithelial cells and the immune cells are derived from human cell lines. For example, epithelial cell lines include, but are not limited to, A549, Int-407 (small intestinal epithelial cells), HT-29, V19I, 5637, HT29 (colonic cells) and HCT-8. Immune cell lines include, but are not limited to, U937 (monocytes), THP-1 (monocytes), neuronal cells such as SH-SY5Y (neuroblastoma), and astrocytes (HTB-14/U87-MG astrocytoma-glioblastoma).
In certain aspects, the methods further include co-culturing the three-dimensional epithelial cells and the undifferentiated immune cells in the low fluid shear environment under conditions selected to produce a three-dimensional immune tissue system with physiologically relevant characteristics. The conditions include, but are not limited to, the culture medium, temperature, pH, the fluid shear, oxygen levels, composition of the extracellular matrix, and time in the low fluid shear environment. In some embodiments, the culture medium is GTSF-2 with and without antibiotics, MEM, DMEM, EME, and RPMI (all with or without antibiotics). In another embodiment, the culture medium is with or without Fungizone. In another embodiment, the culture medium is with or without serum. Extracellular matrix components are collagen-based biodegradable matrices that include, but are not limited to, collagens, glycoproteins laminin, fibronectin, tenascin, elastin, a number of proteoglycans and glycosaminoglycans. Extracellular matrix material, such as submucosal tissue, can be obtained from various sources known in the art. Commercially available ECM products, such as MaxGel Human ECM, may be used in the disclosed methods.
In another embodiment, the method further includes conducting biochemical analyses on the three-dimensional immune tissue system to determine that the tissue system has one or more physiologically relevant characteristics. These biochemical analyses are well-known in the art and include, but are not limited to, microscopy, flow cytometry analysis, cell viability assay, phagocytosis assay, inflammatory response profiling of cytokines/chemokines, Toll-like receptors, NODs, etc, antibody production (sIgA, IgG), Th1 and Th2 type responses, T helper and cytotoxic T cell responses, biosignature profiling at the transcriptomic, proteomic and metabolomic levels.
In another embodiment, the time period for introducing the monolayer epithelial cells into an environment with low fluid shear conditions to develop into mature three-dimensional epithelial cells ranges from about 1 day to about 40 days, from about 2 days to about 29 days, from about 5 days to about 25 days, from about 9 days to about 20 days, or about 9 days.
In another embodiment, the ratio of the undifferentiated immune cells and the mature three-dimensional epithelial cells can range broadly from about 1:1×109 to about 1×109:1, 1:1×108 to about 1×108, 1:1×107 to about 1×107, 11:1×106 to about 1×106:1, 1:1×105 to about 1×105, 1:1×104 to about 1×104, 1:1×103 to about 1×103, 1:1×102 to about 1×102, 1:10 to about 10:1, 1:5 to about 5:1, 1:4 to about 4:1, 1:3 to about 3:1, from about 1:2 to about 2:1, or is about 1:1. Similar ratio ranges are contemplated for other cell types, including immune cells, epithelial cells, stem cells, etc.
In another embodiment, the low fluid shear environment is provided by a bioreactor. Suitable ranges for the low fluid shear environment ranges from about 0 dynes/cm2 to about 10.0 dynes/cm2, from about 0.1 dynes/cm2 to about 1.9 dynes/cm2, from about 0.2 dynes/cm2 to about 1.8 dynes/cm2, from about 0.3 dynes/cm2 to about 1.7 dynes/cm2, from about 0.4 dynes/cm2 to about 1.6 dynes/cm2, from about 0.5 dynes/cm2 to about 1.5 dynes/cm2, from about 0.6 dynes/cm2 to about 1.4 dynes/cm2, and from about 0.7 dynes/cm2 to about 1.3 dynes/cm2, from about 0.8 dynes/cm2 to about 1.2 dynes/cm2, and from about 0.9 dynes/cm2 to about 1.1 dynes/cm.2
In one embodiment, the bioreactor can be a RWV bioreactor. In one embodiment, the RWV bioreactor can be a STLV. In another embodiment, the RWV bioreactor can be a HARV.
In one embodiment, the rotation speed of the bioreactor ranges from about 10 rotations per minute (rpm) to about 30 rpm, from about 15 rpm to about 25 rpm, and is about 20 rpm.
In one embodiment, the time for co-culturing different cell types (e.g., epithelial cells with monocytes) in the bioreactor ranges from hours to years. In one embodiment, the time ranges from about 24 hours to about 1 year, from about 24 hours to about 10 months, from about 24 hours to about 6 months, from about 24 hours to about 2 months, from about 24 hours to about 1 month, from about 24 hours to about 1 week, from about 24 hours to about 120 hours, from about 36 hours to about 108 hours, from about 48 hours to about 96 hours, from about 60 hours to about 84 hours, and is about 72 hours.
In another embodiment, the culture medium is GTSF-2 with and without antibiotics, MEM, DMEM, EME, and RPMI (all with or without antibiotics). In another embodiment, the culture medium is with or without Fungizone.
In another embodiment, the one or more physiologically relevant characteristics are selected from the group consisting of a highly differentiated epithelium, one or more functional macrophage-like cells, a localization of macrophage-like cells on (or beneath) the epithelial surface, and a physiologically relevant macrophage-to-epithelial ratio.
In another embodiment, the one or more functional macrophage-like cells were autonomously differentiated.
In another aspect, the physiologically relevant macrophage-to-epithelial cell ratio ranges from about 1:30 to about 1:40, from about 1:32 to about 1:38, from about 1:33 to about 1:37, and is about 1:32.
In one aspect, the tissue system is comprised of both lung epithelial cells and immune cells co-cultured in the low fluid shear environment. The lung epithelial-immune cell tissue equivalent is characterized as having cellular organization, morphology, and histology similar to in vivo lung epithelial tissue. For example, the tissue system includes immunocompetent cells in the epithelial tissue, which results in immunocompetence of the tissue system. In another aspect, the tissue system is comprised of both intestinal epithelial cells and immune cells co-cultured in the low fluid shear environment. The intestinal epithelial-immune cell tissue equivalent is characterized as having cellular organization, morphology, and histology similar to in vivo intestinal tissue. In another aspect, the tissue system is comprised of astrocytes, neuronal cells, and/or monocytes that develop into a three-dimensional immune tissue system with one or more physiologically relevant characteristics.
Embodiments of the methods disclosed herein improve the predictive capability of tissue culture systems to mimic human and patient-specific responses, decreasing expensive animal use (through reduction, replacement and refinement) and human clinical testing. By serving as better predictors of human and patient-specific responses, these three-dimensional tissue systems allow personalized medicine to better predict in vivo human responses to, for example, infectious pathogens, toxins, drugs/therapeutics, vaccines/adjuvants, chemotherapeutic agents, cosmetics and other chemicals and identify biomarkers for transition from normal homeostasis to disease.
Through more optimal cell-matrix and cell-cell interactions, three-dimensional models of various tissues display significant morphological, phenotypic and molecular aspects of the parental tissue. These characteristics include, but are not limited to, establishment of apical and basolateral polarity, enhanced expression of tight junctions, extracellular matrix and brushborder proteins, highly localized mucin production, and pluripotent properties. Since cellular differentiation and tissue architecture is more in vivo-like in three-dimensional models as compared to conventional two-dimensional monolayers, three-dimensional tissue systems have the potential to mimic and predict cellular responses following exposure to foreign agents such as pathogens, candidate drugs and toxins (Mueller-Klieser, 1997, Abbott, 2003, Schmeichel et al., 2003, Nickerson et al., 2004a).
In one aspect, the methods disclosed include co-culturing immune cells and at least one other cell type, including additional immune cells, into three-dimensional, functional cells. In one embodiment, the cells are developed into three-dimensional cells by using a scaffold or support surface. Scaffolds may be made of any suitable material, such as glass, plastic, foam, fiber meshes or bioscaffolds, including decelularized bioscaffolds. Other suitable support surfaces include tubes, sutures, membranes, films, microparticles, and microcarrier beads. The microcarriers may include, but are not be limited to, Cytodex 1, Cytodex 3, Microhex, Cultisphere, Solohill collagen, Solohill FACT, Solohill hillex II, Solohill pronect or Solohill plastic.
In one embodiment, the scaffold is coated with one or more extracellular matrix components. Extracellular matrix components are collagen-based biodegradable matrices that include, but are not limited to, collagens, glycoproteins laminin, fibronectin, tenascin, elastin, a number of proteoglycans and glycosaminoglycans. Extracellular matrix material, such as submucosal tissue, can be obtained from various sources known in the art. For example, intestinal tissue can be harvested from animals raised for meat production, including pigs, cattle and sheep or other warm-blooded vertebrates. Commercially available ECM products, such as MaxGel Human ECM, may be used in the disclosed methods.
The RWV is an optimized suspension culture method in which cells are grown on extracellular matrix-coated microcarrier beads in cylindrical bioreactors, called slow turning lateral vessels (STLV) or high aspect ratio vessels (HARV), in physiologically relevant low fluid-shear conditions (Nickerson et al., 2004b) (
The HARV and the STLV differ in their aeration source. The low sedimental physiological fluid shear conditions are relevant to those encountered in vivo and during spaceflight culture, and the conditions mimic the major characteristics that are operative in the human host and drive differentiated form and functionality of human tissues, e.g., three-dimensional architecture, multi-cellular complexity, cell shape and biomechanical forces. RWV-derived three-dimensional immunocompetent co-culture models allow the contribution of every different cell type in a tissue to be studied in the disease response to pathogen and toxin challenge (biological or chemical) or drug, vaccine/adjuvant, therapeutic, cosmetic or other treatments—and thus serve as predictive human surrogate platforms to understand the transition from normal to disease development.
RWV-derived three-dimensional tissue models have been developed, some of which have been used for infectious disease studies. Examples include the human lung (Carterson et al., 2005, Crabbe et al, 2011), small intestine (Nickerson et al., 2001; Straub et al, 2007), colon (Carvalho et al., 2005, Honer zu Bentrup et al., 2006; Radtke et al, 2010), liver (Sainz et al., 2009) and bladder (Smith et al., 2006). The present disclosure uses the bioreactor environment to co-culture an immune cell line and at least one other cell line to produce three-dimensional, physiologically relevant tissue systems. In one embodiment, the three-dimensional, physiologically relevant tissue system is immunocompetent.
The design of the RWV bioreactor (
The RWV is a cylindrical, rotating bioreactor that is filled with culture medium. The sedimentation of cells in the vessel is offset by the rotating fluid, creating a constant, gentle fall of cells through the medium under conditions of physiologically relevant fluid shear (Nickerson, C., et al. (2004) Microbiol. Mol. Biol. Rev. 68, 345-361).
Fluid shear is a biomechanical force known to influence cellular differentiation and development in mammals. The dynamic culture conditions in the RWV allow cells to grow in three dimensions, to aggregate based on natural cellular affinities (facilitating co-culture of multiple cell types) and to differentiate into three-dimensional tissue-like systems (Nickerson, C. A. & Ott, et al. (2004) ASM News 70, 169-175; Unsworth, B. R. & Lelkes, et al. (1998) Nature Med. 4, 901-907). The RWV design also allows easy manipulation of culture conditions, including, for example, the addition or removal of cells and media at various time points.
Three-Dimensional Co-Culture in the RWV
In one aspect, to initiate three-dimensional cell culture in the RWV, cells can be first grown as conventional monolayers in standard tissue culture flasks (
Various cell culture media can be used in the disclosed methods, including, but not limited to, GTSF-2 with and without antibiotics (and with or without fungizone, serum, ITS, short chain fatty acids, etc.), MEM, DMEM, EMEM, RPMI, AIMS, IMDM (all with or without antibiotics). Both chemically defined media and media that is serum-supplemented can be used with the disclosed methods. In some embodiments, a combination of different types of media or one specific media, such as GTSF-2 without antibiotics or fungizone, can be used.
In one aspect, a method of assessing disease pathogenesis in a three-dimensional, physiologically relevant immune tissue system is provided.
The method comprises introducing an infectious agent, such as a pathogen, or a compound into a three-dimensional, physiologically relevant tissue system. The three-dimensional, physiologically relevant immune system is produced under low fluid shear conditions and has one or more physiologically relevant characteristics. Next, the disease effects of the pathogen or compound on the three-dimensional, physiologically relevant immune tissue system are tested using methods known in the art. The compound includes, but is not limited to, toxins, antimicrobial drugs, adjuvants, and vaccines.
In one embodiment, after the three-dimensional, physiologically relevant immune tissue system described herein are established, the tissue system is exposed to infectious agents and compounds. In one embodiment, this exposure can be accomplished by removing the three-dimensional cells from the low fluid shear environment and distributing them evenly in multi-well plates or other convenient formats for testing. Cells removed from the low fluid shear environment retain their differentiated state long enough to be amenable to a wide range of experimental manipulations, as determined empirically for each cell type by immunohistochemical, histological and functionality assessments. In another embodiment, a pathogen or compound is introduced into the low fluid shear environment simultaneously with the three-dimensional, physiologically relevant immune tissue system.
The disclosed three-dimensional, physiologically relevant immune tissue systems can therefore be incorporated into existing assays used for infection studies, such as assays testing microbial adherence, invasion and intracellular survival; microscopic examination; transcriptomic, proteomic and metabolomic analyses; expression profiling for cytokines and other inflammatory mediators; and flow cytometry (Nickerson, C. A., et al. (2007) J. Neuroimmune Pharmacol. 2, 26-31). For studies that require homogeneous cell suspensions, such as flow cytometry and cell viability assays (Carterson, A. J. et al. (2005) Pseudomonas aeruginosa pathogenesis. Infect. Immun. 73, 1129-1140; Nickerson, C. A. et al. (2001) Infect. Immun. 69, 7106-7120), three-dimensional cell cultures can be removed from the microcarrier beads using conventional enzymatic and non-enzymatic treatments (for example, trypsin or EDTA, respectively).
Although the treatments for flow cytometry analysis can disrupt the delicate three-dimensional architecture, the expression of cellular markers such as those for differentiation and apoptosis can still be quantified at the single-cell level.
In the context of vaccines and adjuvants, for example, embodiments are used to predict the efficacy of a vaccine/adjuvant and its clinical correlates of protection by means of an in vitro challenge with disease agents. In other embodiments, the tissue systems are prepared using human cell lines or primary cells from healthy (i.e., not diseased, uninfected, naive) individuals or from individuals suffering from diseases or infections. “Diseased cells” include virally infected cells, bacterially infected cells, tumor cells, cells from patients with genetics disorders, and cells and tissues affected by a pathogen or involved in an immune-mediated disease, such as, e.g., autoimmune disease.
Embodiments can be used to assess the interaction of substances with the immune system, and thus can be used to accelerate and/or improve the accuracy, predictability, safety and efficacy, for example, of vaccines, adjuvants, drugs, biologics, immunotherapy, cosmetic and chemical development.
In another aspect, a method of assessing a disease treatment in a three-dimensional, physiologically relevant tissue system is disclosed. The method comprises establishing a disease state in a three-dimensional, physiologically relevant tissue system, which was produced under fluid shear conditions and has one or more physiologically relevant characteristics. Next, a disease treatment is introduced into the three-dimensional, physiologically relevant tissue system. Then, the effect of the disease treatment on the disease is tested using methods known in the art.
Mechanisms of Microbial Pathogenesis.
There are many pathogens that lack practical and representative cell culture or animal models which accurately reflect the host response to infection, and the RWV has enabled researchers to study such pathogens (Alcantara Warren, C. et al. (2008) J. Infect. Dis. 198, 143-149; Duray, P. H. et al. (2005) Invasion of human tissue ex vivo by Borrelia burgdorferi. J. Infect. Dis. 191, 1747-1754; Long, J. P., et al. (1999) In vitro Cell Dev. Biol. Anim. 35, 49-54; Margolis, L. B. et al. (1997) AIDS Res. Hum. Retroviruses 13, 1411-1420; Straub, T. M. et al. (2007) Emerg. Infect. Dis. 13, 396-403). In this regard, three-dimensional models of the intestine have been shown to have in vivo-like expression levels and distribution patterns of key biological surface markers that are directly accessible to pathogens (Nickerson, C. A. et al. (2001) Infect. Immun. 69, 7106-7120; Carvalho, H. M., et al. (2005) Cell. Microbiol. 7, 1771-1781; Honer zu Bentrup, K. et al. (2006) Microbes Infect. 8, 1813-1825; Radtke et. al, 2010, Analysis of interactions of Salmonella type three secretion mutants with 3-D intestinal epithelial cells. PLoS ONE, 2010 Dec. 29; 5(12):e15750) (Table 1), and this contributes to the ability of these models to support productive pathogen infection and replication.
The complexity and in vivo-like characteristics of the disclosed three-dimensional tissue systems make them not only useful systems to investigate the mechanisms involved in, for example, microbial pathogenesis, but also valuable tools to study the host response to, for example, microbial infection. The intestinal and lung mucosa are two major portals of entry for microorganisms. Both tissues have been modelled using the RWV and subsequently used to study enteric and respiratory pathogens, respectively (Carterson, A. J. et al. (2005) Infect. Immun. 73, 1129-1140; Nickerson, C. A. et al. (2001) Infect. Immun. 69, 7106-7120; Honer zu Bentrup, K. et al. (2006) Microbes Infect. 8, 1813-1825; Vertrees, R. A. et al. (2008) Cancer Biol. Ther. 7, 404-412; Straub et al, 2007, Emerg. Infect. Dis. 13, 396-403; Radtke et al, 2010, PLoS ONE, 2010 Dec. 29; 5(12):e15750). The previous models do not use co-culture methods with an immune cell and at least one other cell type to produce three-dimensional immune tissue systems with one or more physiologically relevant characteristics.
A protective and regulated inflammatory response is also crucial for host defense and survival during infection (Imler, J. L. & Hoffmann, et al. (2001) Trends Cell Biol. 11, 304-311; Kabelitz, D. & Medzhitov, et al. (2007) Curr. Opin. Immunol. 19, 1-3; Akira, S., et al. (2006) Cell 124, 783-801; Medzhitov, R. & Janeway, et al. (1997) Curr. Opin. Immunol. 9, 4-9).
The disclosed three-dimensional tissue system is a meaningful predictor of the outcomes of and host responses to in vivo infections.
The disclosure relates to a kit for producing a three-dimensional, physiologically relevant tissue system. In one aspect, the kit comprises immune cell line and at least one other cell line; and informational material for producing a three-dimensional, physiologically relevant tissue system.
In one embodiment, the kit comprise an epithelial cell line; monocytes; and informational material for producing a three-dimensional, physiologically relevant tissue system. In other embodiments, the kit comprise astrocytes, neuronal cells, and informational material for producing a three-dimensional, physiologically relevant tissue system. Examples of intestinal cell lines include, but are not limited to alveolar, bronchial, small intestinal, large intestinal, cervical, and vaginal epithelial cells. Immune cell lines include, but are not limited to, undifferentiated monocytes, macrophages (i.e., differentiated monocytes), dendritic cells (i.e., differentiated monocytes), astrocytes, neuronal cells, neutrophils, eosinophils, basophils, and lymphocytes, including T lymphocytes, B lymphocytes, and natural killer cells from healthy and/or diseased subjects.
In other embodiments, the kit further comprises monocytes. The informational material can be descriptive, instructional, marketing or other material that relates to the methods described herein.
The informational material of the kits is not limited in its form. In some instances, the informational material can include information about how to produce a three-dimensional, physiologically relevant tissue system, including conditions of the bioreactor appropriate for differentiation of the cells and one or more biochemical analyses to determine that the three-dimensional tissue has one or more physiologically relevant characteristics.
In some cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. The informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In other instances, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about the nanoparticles therein and/or their use in the methods described herein. The informational material can also be provided in any combination of formats.
In addition to the cell lines (for example, an epithelial cell line and monocytes), the kit can include other ingredients, including but not limited to cell culture medium, extracellular matrix components, and scaffolds. Extracellular matrix components are collagen-based biodegradable matrices that include, but are not limited to, collagens, glycoproteins laminin, fibronectin, tenascin, elastin, a number of proteoglycans and glycosaminoglycans. Extracellular matrix material, such as submucosal tissue, can be obtained from various sources known in the art. Commercially available ECM products, such as MaxGel Human ECM, may be used in the disclosed methods. Scaffolds may be made of any suitable material, such as glass, plastic, foam, fiber meshes or bioscaffolds, including decellularized bioscaffolds. Other suitable support surfaces include tubes, sutures, membranes, films, microparticles, and microcarrier beads. The microcarriers may include, but are not be limited to, Cytodex 1, Cytodex 3, Microhex, Cultisphere, Solohill collagen, Solohill FACT, Solohill hillex II, Solohill pronect or Solohill plastic.
The study described in this Example developed a lung tissue model that reflected the parental tissue with regard to three-dimensional architecture, differentiation and multi-cellular complexity to incorporate the role of alveolar epithelium and macrophages, and their interactions, in the overall response of each cell type to bacterial virulence factors, such as QS signals.
A RWV-derived three-dimensional co-culture model of epithelial cells and immune cells was developed. The previously developed three-dimensional monotypic alveolar A549 lung epithelium model (Carterson et al., 2005) only included one cell type. In this disclosure, the methods produce three-dimensional tissue systems with physiologically relevant characteristics; this system is a multi-cellular immunocompetent model that includes differentiated immune cells, such as macrophages. Macrophages are an important innate immune defense cell of the lung. The expression of specific cell surface markers and phagocytic activity demonstrated the presence of naturally differentiated macrophages in the co-culture model.
This three-dimensional co-culture model was applied to study the cytotoxic effects of 3-oxo-C12 HSL on macrophages and epithelial cells in a more in vivo-like tissue model, showing surprising discrepancies with previously reported in vitro studies using single cell types. The multi-cellular complexity and biologically relevant cellular organization of this three-dimensional co-culture model, which integrated the pivotal role of mononuclear phagocytic cells in the antibacterial defense and mediation of inflammation, more adequately mimicked the in vivo lung response to respiratory pathogens and their virulence factors.
Quorum sensing (QS) signals mediate inter-cellular communication among bacteria, which coordinates population gene expression and triggers the production of virulence factors. The opportunistic pathogen Pseudomonas aeruginosa, responsible for terminal lung infections in patients with cystic fibrosis, predominantly produces the QS molecules N-(3-oxododecanoyl)-
QS regulates P. aeruginosa virulence determinants, such as biofilm formation and the production of pigments, proteases and exotoxins, all contribute to lung pathology (Williams et al., 2009, Winstanley et al., 2009). In addition to inter-species gene regulation, 3-oxo-C12 HSL acts across kingdoms, as it was shown to exert immunomodulatory and cytotoxic effects in eukaryotes (Telford et al., 1998, Tateda et al., 2003, Williams et al., 2004, Kaufmann et al., 2008).
A significant loss in viability of pure cultures of monocytes and macrophages was demonstrated, when exposed to high concentrations of 3-oxo-C12 HSL, while a variety of epithelial cell lines, including cultures of alveolar epithelial cells, were not affected (Tateda et al., 2003). Indeed, pure cultures of airway epithelial cells were found to produce paraoxanase enzymes that inactivate 3-oxo-C12 HSL through a lactonase mechanism (Ozer et al., 2005). However, these past studies were performed on single cell type cultures of either monocytes/macrophages or epithelial cells, respectively, which do not reflect the multi-cellular complexity of the parental tissue in vivo.
This becomes of particular importance given the fact that mutual interactions and cytokine networking between alveolar epithelium and macrophages are essential for the innate defense of the lung to bacterial infection (Standiford et al., 1991, Krakauer, 2002, Amano et al., 2004, Kannan et al., 2009). Besides the role of alveolar macrophages in the elimination of the daily-inhaled bacterial load, secretion products from macrophages trigger immune responses in alveolar epithelial cells, such as cytokine production (Standiford et al., 1991, Krakauer, 2002).
On the other hand, alveolar epithelial cells increase the immune function of alveolar macrophages in response to infection with P. aeruginosa and other opportunistic pathogens, which is partly mediated through the intercellular adhesion molecule ICAM-1 (Amano et al., 2004, Kannan et al., 2009). The expression of ICAM-1 on the apical surface of alveolar epithelial cells induces phagocytic capabilities of alveolar macrophages and facilitates their migration along the epithelial cell surface (Paine et al., 2002). P. aeruginosa virulence factors, such as lipopolysaccharides, lipopeptides, porins and phenazins, up-regulate ICAM-1 expression in different host cell types, including airway epithelium (Perfetto et al., 2003, Greene et al., 2005).
The three-dimensional co-culture tissue system of alveolar epithelium and macrophages using the rotating wall vessel (RWV) bioreactor in this Example was developed by adding undifferentiated monocytes to RWV-derived alveolar epithelium. This three-dimensional tissue system expressed important architectural/phenotypic hallmarks of the parental tissue, as evidenced by highly differentiated epithelium, spontaneous differentiation of monocytes to functional macrophage-like cells, localization of these cells on the alveolar surface, and a macrophage-to-epithelial cell ratio relevant to the in vivo situation. Co-cultivation of macrophages with alveolar epithelium counteracted 3-oxo-C12-HSL-induced cytotoxicity via removal of quorum sensing molecules by alveolar cells. Furthermore, 3-oxo-C12-HSL-exposed macrophages should be able to mediate innate immunity since 3-oxo-C12-HSL induced the intercellular adhesion molecule ICAM-1 in both alveolar epithelium and macrophages. This Example demonstrates the importance of multi-cellular organotypic models to integrate the role of different cell types in overall lung homeostasis and disease development in response to external factors.
Expression of Epithelial Differentiation Markers.
Three-dimensional A549-U937 co-culture aggregates were stained with selected markers for the assessment of epithelial cell differentiation. In
Architectural Organization of Three-Dimensional Co-Cultures.
The three-dimensional alveolar epithelial cells were present as a unilayer on the surface of the microcarrier beads, which is physiologically relevant to the parental tissue (
After 72 hour co-cultivation of three-dimensional A549 cells with U937 monocytes, CD45 positive cells lining the surface of the alveolar epithelium were detected with confocal laser scanning microscopy (CLSM) (
ICAM-1 Expression and Phagocytic Activity of Adherent U937 Cells.
ICAM-1 was chosen as a marker to assess natural differentiation of monocytes to macrophages in the three-dimensional coculture model. Indeed, when monocytes were cultured alone, a majority stained weakly positive for ICAM-1, while macrophages differentiated with phorbol 12-myristate 13-acetate (PMA) showed strong ICAM-1 labeling (FIG. 2A2-3). FIGS. 2A1-3 show ICAM-1 expression on co-cultured alveolar epithelium and monocytes/macrophages in the three-dimensional A549-U937 co-culture (A1) compared to undifferentiated U937 monocytes (A2) and PMA-differentiated macrophages (A3). White arrows point out macrophage-like cells. Cell nuclei are stained blue, shown as 3 (DAPI), and the images are based on 400× magnifications. Imaging of three-dimensional A549 aggregates co-cultured with monocytes for 72 hours indicated that cells adhering to the epithelial surface expressed the ICAM-1 antigen more abundantly than undifferentiated control monocytes (FIG. 2A1). Dual labeling of the co-cultures for CD45 and ICAM-1 confirmed that adherent cells were monocytes/macrophages (
Surprisingly, U937 cells (CD45 positive) co-cultured with A549 cells in three-dimensional were capable of phagocytosing 2 μm beads (FIG. 2B1). No phagocytic activity was observed in three-dimensional A549 monotypic cultures, except for occasional adherence of beads to the epithelial surface.
Effective Ratio of Macrophages to Epithelial Cells in the Three-Dimensional Co-Culture.
The effective ratio of CD45-positive macrophage-like cells to the total cell population was determined by flow cytometry, after 72 hours of co-cultivation. On average, 3.1±1.5% macrophage-like cells were found in the co-culture, corresponding to an effective ratio of 1 monocyte per 32 epithelial cells.
Response of Monotypic and Co-Cultures to 3-oxo-C12 HSL
Cell Viability.
As presented in
Assessment of 3-oxo-C12 HSL Concentrations as a Function of Time.
In three-dimensional monotypic (A549) and co-cultures (A549-U937), the concentration of 3-oxo-C12 HSL rapidly decreased with time (
ICAM-1 Expression.
ICAM-1 expression of A549 cells (derived from the three-dimensional A549-U937 co-culture) exposed to 100 μM 3-oxo-C12 HSL significantly increased when compared to the solvent control (1.66±0.11-fold, p<0.01) (
Lower concentrations of the QS molecule did not affect ICAM-1 expression. Similar trends were observed with three-dimensional monotypic cultures of A549 cells. A significant increase (2.93±0.79-fold, p<0.01) in the ICAM-1 signal of the macrophage-like cells was observed when the three-dimensional A549-U937 co-culture was exposed to 100 μM 3-oxo-C12 HSL, compared to the control samples (
The alveolar epithelial cell line A549 (ATCC CCL-185) and the monocytic cell line U937 (ATCC CRL-1593.2) originated from the American Type Culture Collection (ATCC, Manassas, Va.). All eukaryotic cells were cultured in GTSF-2 medium (Hyclone, Logan, Utah) supplemented with 10% fetal bovine serum, 2.5 mg/l insulin transferring sodium selenite (Sigma-Aldrich), and 1 ml/l Fungizone (Invitrogen) and were incubated at 37° C. under 5% CO2.
Three-Dimensional A549 Monotypic Culture.
The three-dimensional A549 monotypic cultures were made as previously described (Carterson et al., 2005).
Three-Dimensional A549-U937 Co-Culture.
A549 three-dimensional aggregates were grown as described above and after 9 days of three-dimensional A549 cultivation, undifferentiated U937 monocytes were added to the HARV as follows. three-dimensional A549 cells were counted by trypsinization of an aggregate sample and U937 cells (grown in T75 flasks) were added in a 1:1 ratio to the three-dimensional A549 culture. A549 and U937 cells were co-cultured in the HARV at 20 rpm for 72 h. After addition of the monocytes, culture medium was changed after 48 hours of co-cultivation where after medium was replenished every 24 hours.
To obtain differentiated macrophages, U937 monocytes were exposed to 10−8M PMA (originating from a 10−3M stock in DMSO) (Sigma-Aldrich) for 48 hours. Exposure of monocytes to phorbol esters induces morphological, physiological and molecular characteristics of terminally differentiated macrophages (Rovera et al., 1979, Prieto et al., 1994). For CLSM staining purposes, monocytes were differentiated with PMA and A549 cells were grown in 6-well plates containing sterile coverslips. For CLSM staining of undifferentiated U937, cytodex-3 beads (5 mg/ml) were added to the culture medium, allowing adhesion of U937 to the beads and facilitation of the staining procedure.
Antibodies.
The antibodies used for CLSM imaging in the present study were from mouse origin and targeted the human CD45 (Abcam), ICAM-1 (Abcam), MUC5AC (Invitrogen), ZO-1 (Invitrogen) and β-catenin (Chemicon). All primary antibodies were used in a dilution of 1:50. ICAM-1 was the only directly labeled antibody used for CLSM staining and was conjugated with phycoerythrin (PE). A goat anti-mouse secondary antibody labeled with Alexa Fluor 555 (Invitrogen) was used to detect the bound primary antibodies and was diluted 1:500 in blocking solution (8% bovine serum albumin, 0.05% Triton-X100 in DPBS). For dual labeling purposes of ICAM-1 and CD45 or MUC5AC, a rabbit anti-mouse secondary antibody was used conjugated with Alexa Fluor 488 (Invitrogen) to label CD45 and MUC5AC primary antibodies. Phalloidin conjugated with Alexa Fluor 633 (Invitrogen) was used to stain the F-actin fraction of the cytoskeleton. The cell nucleus was visualized with 4′,6-diamidino-2-phenylindole hydrochloride (DAPI) (Invitrogen).
Fixation and Staining.
three-dimensional aggregates of monotypic and co-culture models, undifferentiated U937 cells (adherent to microcarrier beads) and coverslips containing A549 monolayers or PMA-differentiated macrophages were rinsed 3 times with DPBS (Invitrogen) and fixed with 4% paraformaldehyde (in PBS) (Electron Microscopy Services) for 30 min at ambient temperature. Non-specific binding sites were blocked with blocking solution (8% bovine serum albumin, 0.05% Triton-X100 in DPBS) for 30 min. Cells were washed 3 times with Tween-PBS (T-PBS) (0.1% Tween 20 in DPBS) and incubated with the primary antibody (diluted in blocking solution) for 1 hour at room temperature. After rinsing 3 times with T-PBS, cells were incubated, when needed, with the secondary antibody (diluted in blocking solution) for 30 min in a darkened environment. Control samples were stained with the secondary antibody alone and indicated the absence of non-specific binding. For dual staining of cells with ICAM-1 and CD45 or MUC5AC, the above-mentioned protocol was first performed for the CD45 or MUC5AC primary and secondary antibody followed by staining with the directly labeled ICAM-1 antibody. For staining of the cytoskeleton, cells were incubated 30 min with phalloidin-Alexa Fluor 633 (Invitrogen). Aggregates or coverslips were mounted on a glass slide containing Prolong Gold with DAPI mounting solution (Invitrogen).
Imaging.
Optical sections of the three-dimensional aggregates and monolayers were obtained using a Zeiss LSM 510 Duo laser scanning microscope equipped with detectors and filter sets for monitoring emissions of the selected fluorophores. Images were acquired using a Plan-Neofluar 40×/1.3 oil DIC objective and were analyzed with the Zeiss LSM software package. Z-stacks of aggregates (200-300 μm) were made with a 1 μm interval and post-acquisition reconstruction of three-dimensional images was performed with the Zeiss LSM software package. Axiovision 4.7 software from Carl Zeiss was used to further process collected images.
Antibodies.
Mouse anti-human CD45 (Invitrogen) and ICAM-1 (Invitrogen) conjugated with phycoerythrin (PE)-Cy5 and FITC respectively were used for flow cytometry staining. Sample preparation and fluorescent staining of cells. Cells were rinsed 2 times with pre-warmed HBSS (Invitrogen), detached from microcarrier beads/culture flasks with 0.25% trypsin-EDTA (Invitrogen) and counted using a hemocytometer. For inclusion of cells detached from the microcarrier beads (three-dimensional A549 and three-dimensional A549-U937 co-cultures) in the frame of QS exposure studies (see below), whole cultures (both aggregates and bulk liquid) were centrifuged at 1200 rpm for 6 min for each washing step. Next, microcarrier beads were removed (if needed) by filtering through a stainless steel cell dissociation sieve (pore size 104 μm) (Sigma-Aldrich). Approximately 1-2×106 cells were used for staining. Cells were washed in ice cold staining buffer (10% FBS, 1% sodium azide in DPBS), resuspended in 3% BSA (in DPBS) containing the antibody (concentrations according to manufacturer's instructions) and incubated at 4° C. in the dark. For dual labeling, the described protocol was consecutively repeated for each antibody. Subsequently, cells were rinsed 2 times with ice cold staining buffer and resuspended in fresh staining buffer for analysis.
Cell Viability Assay.
Assessment of live-, apoptotic-, and necrotic cell populations was performed with the annexin V-propidium iodide (PI) kit for flow cytometry analysis (ImmunoSource) according to the manufacturer's instructions.
Flow Cytometry Analysis.
Stained cell suspensions were analyzed with an EPICS XL flow cytometer (Beckman-Coulter) or a Cytomics FC 500 system (Beckman-Coulter), equipped with an argon laser and filters for adequate excitation of the chosen fluorophores. Forward scatter, side scatter, FITC-, PI and PE-Cy5 emissions were measured and a total of 10,000 cells were recorded for each sample. The respective cell populations were delimited to eliminate background signals originating from cell debris. To assess background fluorescent signals from the tested cell populations, non-stained samples were included.
Determination of the U937-A549 Ratio for Three-Dimensional Co-Cultures.
three-dimensional co-cultures of A549 and U937 cells (as described above) were stained for CD45, since this marker specifically stains leukocytes. The ratio of CD45-positive cells (U937) to the total cell population [i.e. CD45 positive and -negative cells (A549)] revealed the effective fraction of monocytes/macrophages in the co-culture.
Table 2 below presents the different variables that were tested for the successful generation of the three-dimensional A549-U937 co-culture model.
The phagocytosis activity of monocytes and macrophages cultured alone or co-cultured with A549 cells in three-dimensional was assessed based on the uptake of bacterial-sized 2 μm fluorescent polypropylene beads (Fluoresbrite plain YG, Polysciences). The phagocytosis assay was performed as previously described (Gao et al., 2010).
Exposure of Monocytes, Three-Dimensional Monotypic, and Three-Dimensional Co-Cultures to 3-oxo-C12 HSL
After 72 hours of co-cultivation, aggregates containing 2×106 cells were seeded into 6-well plates containing fresh medium (5 ml total volume). Subsequently, aggregates were spiked with 5, 10 or 100 μM 3-oxo-C12 HSL (Sigma-Aldrich) (starting from a 20 mM stock solution in DMSO, stored at −20° C.). Equal volumes of DMSO, containing the respective amounts of 3-oxo-C12 HSL, were added to each well (25 μl) and a solvent control (25 μl DMSO) was included in each experiment. Six-well plates were placed at 37° C. in a 5% CO2 environment and incubated for 20 hours. For exposure assays of monocytes to 3-oxo-C12 HSL, 2×106 U937 cells/well were transferred in 6-well plates. To assess the influence of 3-oxo-C12 HSL on monocytes cultured on microcarrier beads, collagen I-coated beads were added to the cells at the same concentration as for seeding three-dimensional cultures (1 bead per 2500 cells). Subsequently, monocyte cultures (with or without microcarrier beads) were spiked with 3-oxo-C12 HSL (see above). A solvent control was included. After 20 hours incubation, cells were processed for flow cytometry analysis. Cell viability was assayed with trypan blue (0.4%) (Sigma) and/or with the annexin V-PI kit (ImmunoSource) (see above). However, since both annexin V-PI and trypan blue staining indicated that cell death was associated with an alteration in cell size, as reflected through the forward scatter signal of the flow cytometer (
Assessment of 3-oxo-C-12 HSL Concentrations
Following exposure of three-dimensional cultures and monocytes to 3-oxo-C12 HSL, cell-free samples were taken and stored at −20° C. at indicated time points. The remaining 3-oxo-C12 HSL concentration was assessed as previously described (Crabbé et al., 2008) using the indicator strain E. coli harboring the reporter plasmid pUCP22NotI-PlasB::gfp(ASV)Plac::lasR (Hentzer et al., 2002). A 3-oxo-C12 HSL standard series was included ranging from 1.63 to 100 μM. Culture medium containing microcarrier beads was used as a control to normalize for bioadsorption.
All experiments were performed at least in biological duplicate and technical triplicate. The statistical determination of significance (α=0.05) was done with Microsoft Office Excel 2003 using a two-sample Student t-test on the biological repeats of each experimental condition.
Epithelial ICAM-1 Expression Following Infection with P. Aeruginosa
3-D monotypic and co-cultures were subjected to P. aeruginosa PAO1 infection after 72 hours of co-cultivation. After 20 hours of infection, the bacterial load in both culture types was approximately 1.4×108 CFU/ml.
CLSM imaging revealed that most of the detected P. aeruginosa cells were present in the mucus layer (based on MUC5AC staining) and did not invade the epithelial cells (
ICAM-1-expression was enhanced in both monotypic and co-cultures following infection, when compared to non-infected controls. While the non-infected A549-U937 cultures only displayed a slightly higher, non significant, ICAM-1 expression than the A549 cultures (cf. ICAM-1 after 4 days of co-cultivation, figure not shown), the increase in ICAM-1 signal intensity after infection was 1.6-fold higher in the co-cultures compared to the monotypic cultures (p<0.05) (
Prior to infection with P. aeruginosa, both monotypic and co-cultures produced background levels of the tested cytokines except for RANTES, IP-10, ICAM-1, IL-6 and IL-8 (
The simultaneous presence of alveolar epithelium and functional macrophages in an in vitro model of alveolar tissue was important to adequately investigate the response of the lung to bacterial virulence factors. The RWV technology was adopted to develop a three-dimensional organotypic co-culture model of alveolar epithelium and macrophages. This three-dimensional RWV-derived co-culture model of epithelium and immune cells showed the presence of naturally differentiated (i.e. no chemical treatment) macrophage-like cells in the mucus layer of the alveolar epithelium, as evidenced by their increased expression of the macrophage surface marker ICAM-1 and enhanced phagocytosis of bacterial-sized beads, when compared to monocytes cultured alone. These data indicated that the adopted culture conditions, in which monocytes were placed in contact with highly differentiated alveolar epithelium, induced the spontaneous transition to a phenotype with characteristics of normal alveolar macrophages. Indeed, contact of monocytes with the extracellular matrix, epithelial cells and their secreted factors play a role in their in vitro differentiation to macrophages (Spottl et al., 2001, Striz et al., 2001, Spoettl et al., 2007). Co-cultivation of monocytes with alveolar epithelium in three-dimensional also increased the expression of ICAM-1 on the cell surface of the alveolar epithelial cells.
These data confirmed the role of alveolar macrophages in the mediation of the epithelial innate immune response, since ICAM-1 activates the phagocytic activity of macrophages (Paine et al., 2002). Consequently, the effective ratio of mononuclear cells to alveolar epithelial cells in the non-infected lung is important for the basal ICAM-1 level of the epithelium. The effective ratio of macrophages-to-alveolar epithelial cells in the three-dimensional co-culture models was determined to be on average 1:32 after 3 days of co-cultivation, comparable to the healthy non-infected lung (one alveolar macrophage to approximately 40 epithelial cells) (Crapo et al., 1983, Paine et al., 2002).
The secreted mucin, MUC5AC, presumably played a role in the adherence of the macrophage-like cells to the surface of the alveolar epithelium. These results are consistent with the architecture of the parental tissue in vivo, since alveolar macrophages are localized in the alveolar surfactant film lining the alveolar epithelium (Jonsson et al., 1986). The presence of a well-defined mucus layer on the surface of the alveolar epithelium reflects more closely the in vivo situation of patients with chronic obstructive pulmonary diseases, such as cystic fibrosis, since hypersecretion and reduced clearance of mucus in these patients results in the presence of a thick, viscous mucus layer in both the conductive and respiratory zones of the lung (Foweraker, 2009).
While this and other studies have shown that 3-oxo-C12 HSL induced apoptosis in pure cultures of monocytes and macrophages at concentrations above 25 μM (Tateda et al., 2003, Kravchenko et al., 2006, Li et al., 2009), exposure of the three-dimensional co-cultured monocytes/macrophages with alveolar epithelium to concentrations up to 100 μM did not induce cell death in either of the cell populations. Thus, this Example shows that alveolar epithelial cells can protect monocytes/macrophages against the detrimental effects of 3-oxo-C12 HSL. Although not bound by any theory, the protection of macrophages in the three-dimensional co-culture model presumably occurred through enzymatic degradation of the QS molecules by the alveolar epithelium (Chun et al., 2004, Ozer et al., 2005). This Example indicates that alveolar epithelium removes 3-oxo-C12 HSL from the cell environment before it has time to exert any cytotoxic effects on macrophages. This Example further suggests that the initial exposure of macrophages to 3-oxo-C12 HSL, i.e. before removal by the A549 cells, did not trigger mechanisms of apoptosis or necrosis in the former population.
The tested 3-oxo-C12 HSL concentrations were in a physiologically relevant range since P. aeruginosa biofilms, typically found in the lung mucus of cystic fibrosis patients, can locally produce 3-oxo-C12 HSL concentrations up to 600 μM (Charlton et al., 2000), while planktonic cultures produce 3-oxo-C12 HSL concentrations from 1 to 10 μM. This Example indicates that, due to protection by the alveolar epithelium, macrophages should still be able to trigger host immune responses, e.g. by means of ICAM-1 induction, despite the presence of high concentrations of 3-oxo-C12 HSL. Indeed, this Example demonstrated that 3-oxo-C12 HSL enhanced the expression of ICAM-1 in both alveolar epithelial and macrophage-like cells. In accordance with these results, Wagner and colleagues reported that the ligand of ICAM-1, CD11b, was induced in response to 3-oxo-C12 HSL in polymorphonuclear neutrophils (Wagner et al., 2007). The 3-oxo-C12 HSL molecules influence mammalian gene expression through, among others, selective impairment of the NF-κB pathway and phosphorylation of p38 and eIF2α protein kinases (Kravchenko et al., 2006, Kravchenko et al., 2008). Although not bound by any theory, since p38 has been demonstrated to mediate the expression of ICAM-1 in macrophages (Cui et al., 2009), the activation of the p38 pathway by 3-oxo-C12 HSL was presumably at the origin of the observed increase in ICAM-1.
In conclusion, this Example developed an advanced three-dimensional multi-cellular alveolar co-culture model of epithelium and macrophage-like cells that displayed physiologically relevant morphological characteristics. These in vivo-like hallmarks included (i) highly differentiated alveolar epithelium, (ii) the autonomous differentiation of monocytes to functional macrophage-like cells, (iii) the localization of macrophage-like cells on the alveolar surface, and (iv) a macrophage-to-epithelial cell ratio of approximately 1:32.
This tissue system was used to investigate the host response to the QS molecule 3-oxo-C12 HSL, allowing the profiling of key aspects of the complex networking between the epithelium and the prominent innate immune cell of the lung. We demonstrated for the first time that the deleterious effects of the 3-oxo-C12 HSL QS signal of P. aeruginosa on mononuclear phagocytes could be counteracted by three-dimensional co-cultivation with alveolar epithelium and could trigger mechanisms of innate immunity in both cell populations.
Collectively, these data indicate that different cell types in the lung orchestrate the overall response of each single cell type to bacterial virulence factors; these interactions need to be taken into careful consideration when predicting the host response to infection with regard to potential therapeutic applications. Furthermore, this study stressed the importance of highly differentiated multi-cellular organotypic models to assess overall organ homeostasis and disease development.
The studies described in Example 2 established and characterized a biologically relevant functional three-dimensional co-culture model using human intestinal epithelial cells cultured with macrophages.
Cell-Line Derived Model:
Three-dimensional co-culture models derived from human cell lines with i) large intestinal epithelial cells (HT29 colonic cells) and monocytes (U937), and with ii) small intestinal epithelial cells (Int-407) and monocytes were established and optimized. Extensive optimization parameters empirically tested and outcomes determined to date for this three-dimensional co-culture model development are shown in Table 2. Separate 3-D monotypic cultures of each of these cell lines were also established as controls for the co-culture model. For the parameters listed in Table 2, sample aliquots of three-dimensional cultures (monotypic and co-culture models) were removed from the bioreactor at the times indicated below and monitored for i) kinetics of aggregate formation (using light microscopy), and ii) conditions for addition of macrophages to the intestinal cells using different macrophage-specific markers and differentiation for cell-specific markers (confocal microscopy) throughout the course of development of this model at the indicated timepoints to determine optimal time for harvesting. Once a procedure was established with the HT29 and U937 co-culture model, this procedure was also used to develop the small intestinal (Int-407) co-culture model with U937 cells.
Validation of the 3-D Co-Culture Model:
Models were characterized by a) light microscopy for kinetics of 3-D aggregate formation (
Specific markers were profiled to ensure optimal differentiation of each cell type in the three-dimensional co-culture aggregates, and include: Intestinal cells—ZO-1, MUC5AC; Macrophages—CD54, CD45, CD68, CD84. Markers specifically profiled at this stage of the study include: a) ICAM-1, CD54 expressed both in monocytes and macrophages (more so in the latter) as well as on the apical cell side of the intestinal epithelium co-cultured with differentiated U937 cells; b) CD45, expressed in all mononuclear phagocytic cells but not in epithelial cells; c) CD68, known as microsialin, predominantly expressed in cytoplasmic granules of monocytes/macrophages, dendritic cells, and granulocytes, and d) CD84, a Cd2 subset of the Ig superfamily, expressed on mature B cells, B cell lines, monocytes, and it strongly stains tissue macrophages. Each of these markers was also profiled in the monotypic three-dimensional models of each cell type. Immunohistochemical profiling showed important physiological differences in expression and distribution of these markers between the 3-D co-culture models as compared to 3-D monotypic cultures. Representative immunohistochemical profiling comparisons for select markers between 3-D co-cultures and 3-D monotypic cultures of each cell type, are shown in
In
Summary.
Example 2 demonstrated that co-cultures of human colon and monocyte cell lines cultured in the RWV bioreactor aggregate based on natural cellular affinities and self-assemble into biologically relevant three-dimensional aggregates. Example 2 also demonstrated that a single media is compatible with establishment and differentiation of both cell types in the three-dimensional co-culture models. In general, expression patterns of cell specific markers in the three-dimensional co-culture models revealed tissue organization and differentiation relevant to that found in the normal tissue in vivo, as opposed to the same cells grown as monotypic three-dimensional cultures. In addition, the expression of the different macrophage/phagocytic markers validated the incorporation of macrophages in this model, and the uptake of beads and bacteria by this immunocompetent model further validated the functionality. These results demonstrated the physiological relevance of this cell culture system, which is useful as a valuable tool for developing drugs and testing various conditions of pathophysiological relevance associated with the intestine.
The studies in this Example 3 established and characterized a biologically relevant three-dimensional multicellular co-culture model with human neuronal cells cultured in combination with macrophages and astrocytic cells. Furthermore, this Example identified HIV-associated dementia complex (HAD) molecular markers in the three-dimensional multicellular co-culture model.
Establishment of Multicellular Organotypic Co-Cultures. Cell-Line Derived Model 1:
The studies in Example 3 established and optimized three-dimensional multi-cellular co-culture models derived from human cell lines with neuronal cells (SH-SY5Y neuroblastoma) and astrocytes (HTB-14/U87-MG astrocytoma-glioblastoma). Extensive optimization parameters empirically tested and the outcomes determined to date for this three-dimensional co-culture model development are shown in Table 3. Separate 3-D monotypic cultures of each of these cell lines were also established as controls for the multicellular co-culture model. For parameters 1-3 listed in Table 3, sample aliquots of 3-D cultures (monotypic and co-culture models) were removed from the bioreactor at times indicated below and monitored for viability (trypan blue exclusion), kinetics of aggregate formation (light microscopy—
Model 1 development was completed by refining parameters listed in Table 1: a) timing of addition of different cell types—i.e. addition of astrocytes prior to neuronal cells—and also simultaneous cell addition; b) ratio of different cell types—10:1 ratio of astrocytes to neuronal cells, c) immunohistochemical validation of model differentiation under conditions a-b, including double-staining with cell-specific markers; d) addition of third cell type, the human monocytic cell line, THP-1, to 3-D co-culture model of neuronal cells and astrocytes; e) immunohistochemical and morphological validations.
Validation of the Three-Dimensional Co-Culture Model:
Models were characterized by a) light microscopy for kinetics of 3-D aggregate formation (
Summary.
Example 3 demonstrated that co-cultures of human neuronal, astrocyte and monocytic cell lines cultured in the RWV bioreactor aggregate based on natural cellular affinities and self-assemble into biologically relevant three-dimensional aggregates. This Example also demonstrated that a single media is compatible with establishment and differentiation of both cell types in the 3-D co-culture models. In general, expression patterns of cell specific markers in the 3-D co-culture models revealed tissue organization and differentiation relevant to that found in the normal tissue in vivo, as compared to the same cells grown as monotypic 3-D cultures and the poor organization and abnormal/diffuse expression observed in monolayers suggestive of tumor cell de-differentiation. These results demonstrate the physiological relevance of this cell culture system for model development and application toward, for example, HIV infection studies.
Although the invention has been described and illustrated in the foregoing illustrative embodiments, it is understood that the present disclosure has been made only by way of example, and that numerous changes in the details of implementation of the invention can be made without departing from the spirit and scope of the invention, which is limited only by the claims that follow. Features of the disclosed embodiments can be combined and rearranged in various ways within the scope and spirit of the invention.
This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 61/408, 528, filed Oct. 29, 2010, the contents of which are hereby incorporated by reference.
The present invention was made with United States government support under Grant No. HHS-NIH-NIMH R21MH080702 awarded by the National Institutes of Health (NIH) and NCC2-1362, NNJ04HF75F, and NNJ06HE92 awarded by the National Aeronautics and Space Administration (NASA). The United States government may have certain rights in this invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US11/58625 | 10/31/2011 | WO | 00 | 9/6/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61408528 | Oct 2010 | US |
