Patent Application
20200115667
The present invention relates generally to the field of tissue engineering. More particularly, it concerns microfluidic techniques.
The continuous development of new fabrication techniques in the field of tissue engineering is leading to the creation of more advanced in vitro models to better understand the underlying influence of the microenvironment on human disease and tissue development. Three dimensional (3D) engineered tissue platforms have the ability to create physiologically representative features with an improved insight into the dynamic intricacies of the pathology of disease that is not possible with traditional two dimensional (2D) models. Some of the features not simulated in 2D culture include but are not limited to cell-cell and cell-matrix signaling, transport studies, and impact of mechanical and chemical gradients on cellular response. In vivo animal models offer more physiologically representative systems but limit control of microenvironmental conditions and the associated capability to determine their influence on physiological response dynamically and minimally invasively. Animal studies are costly for biological investigation and therapeutic refinement, limiting the ability to fully optimize therapeutics. Incorporation of microfluidic technology within 3D in vitro platforms allows long term cell culture and the ability to investigate the influence of flow and transport on dynamic cellular interactions in biological microenvironments. These types of 3D models promote cell growth and migration and are playing a growing role in the study of cancer biology due to their ability to examine the influence of individual factors on tumor progression.
Transport, cell-cell interactions, secretion of angiogenic growth factors, immune response, and other behaviors, that affect cell uptake of therapeutic drugs and play an intricate role in tissue pathology, are all variables of interest to be observed in a representative tumor environment.
Hyperpermeability of the vasculature within the tumor environment along with a lack of lymphatic drainage is responsible for elevated interstitial fluid pressure that can dramatically alter flow patterns as the tumor expands. These hydrodynamic behaviors may lead to increased expression of angiogenic factors and formation of microvessels inside the tumor allowing for tumor growth while transport and drug uptake can be reduced by the fluid dynamics of the tumor vasculature. Macromolecules and nanotherapeutics can fail to reach viable tumor cells due to the irregular extravasation and extravascular convection caused by the conditions of the tumor microenvironment. In order to study the influence of the vasculature on tumor development and transport of drugs, there has been a widespread expansion of in vitro platforms to incorporate channels to simulate vessels.
A number of microfluidic platforms have been developed to study the influence of the vasculature on normal and disease development. Zheng et al. have used additive tissue engineering techniques to develop 3D microfluidic vascular networks (μVN) in a collagen hydrogel for studying angiogenesis and thrombosis (Zheng et al., 2012).
In recent years, different methods have been proposed and discussed for the fabrication of microfluidic based vasculatures constructed within ECM. One method used frequently is to create a housing to encapsulate, polymerize, and pattern collagen to create vasculature using sterile needles (Tourovskai et al., 2014). Another platform was developed to mimic vascular tumor microenvironments that overcomes planar geometries inherent to conventional PDMS based devices to produce a more physiologically representative 3D cylindrical vascular microchannel (Szot et al., 2013). Although fluorinated ethylene propylene (FEP) tubing provided a robust infrastructure for maintaining vessel stability, the dimensions of the platform's tissue chamber could not be easily altered due to limitations set by using off the shelf FEP tubing to form the tissue chamber. The FEP also prevented the platform vasculature and tissue scaffolding from being scalable such that platform could not be built upon to form more advanced tissue systems for further studies. Introducing flow in the microchannel during live cell imaging on microscope stages presented challenges. The platform needed to be filled with water to match refractive indices in order to form an optically clear system for obtaining images. This requirement created the potential for leaks and limited resolution due to slight variations in refractive indices between the multiple mediums that composed the platform. Thus, there is an unmet need for enhanced microfluidic vascularized platforms.
In a first embodiment, there is provided a method of manufacturing a microfluidic device comprising obtaining a base mold with at least one protruding chamber and at least one rod which spans from one edge of the mold through the chamber to the opposite edge of the mold; casting a polymer solution onto the base mold; curing the polymer solution to form a solidified polymer mold; bonding the solidified polymer mold to a surface; inserting extracellular matrix hydrogel into the chamber; and removing the at least one rod once the extracellular matrix hydrogel has polymerized, thereby producing a microfluidic device comprising at least one chamber with at least one channel running through said chamber, wherein the at least one channel comprises an inlet port and an outlet port.
In some aspects, the base mold is an aluminum mold or polydimethylsiloxane (PDMS). In certain aspects, obtaining the base mold comprises performing micro-milling using a computer-numerical-control (CNC) machining system.
In some aspects, the chamber is cylindrical or rectangular. In certain aspects, the rod is a needle. In some aspects, the needle is a 20-30 gauge needle, such as a 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 gauge needle.
In certain aspects, the at least one channel has a diameter of 100 to 1000 μm, such as about 150, 200, 250, 300, 250, 300, 400, 500, 600, 700, 800, or 900 μm. The channel or additional channels in the device may have a diameter of 100-200, 200-300, 300-400, 400-500, 600-700, 700-800, or 900-1000 μm.
In further aspects, the base mold comprises 2, 3, 4, or 5 chambers, wherein each chamber has at least one rod running through said chamber. In some aspects, the chambers are in parallel. In some aspects, the chambers may be separated by the polymer, such as PDMS or a semi-impermeable membrane.
In some aspects, the at least one chamber has two (or 3 or 4) rods running through said chamber. In some aspects, the two rods have different diameters. In some aspects, the two rods have the same diameter.
In some aspects, the polymer solution comprises a silicon-based polymer. In certain aspects, the silicon-based polymer is polydimethylsiloxane (PDMS).
In certain aspects, curing comprises applying heat to the polymer solution. In some aspects, bonding comprises plasma treatment.
In some aspects, the surface is glass. In specific aspects, the glass is further defined a glass coverslip.
In additional aspects, the method further comprises treating the chamber with polyethleneimine (PEI) and/or glutaraldehyde before inserting the extracellular matrix hydrogel.
In some aspects, the extracellular matrix hydrogel comprises elastin, keratin, fibrin, fibronectin, laminin, hyaluronic acid, and/or collagen. In particular aspects, the extracellular matrix hydrogel comprises collagen, such as type I collagen. In some aspects, the extracellular matrix hydrogel comprises collagen and keratin, such as oxidized keratin (also referred to as keratose).
In some aspects, the extracellular matrix hydrogel further comprises one or more populations of cells. In some aspects, the one or more populations of cells are selected from the group consisting of tumor cells, hepatocytes, cardiomyocytes, keratinocytes, fibroblasts, endothelial cells, stem cells, and macrophages. Other cell type may be used depending on the desired model system.
In some aspects, the method further comprises injecting a population of cells into the channel. The rod or needle may be removed from the mold to inject the cells into the channel. The cells may be allowed to (e.g., under pre-conditioning flow) to form a layer, such as a monolayer, along the channel surface. Thus, the channel may be lined with cells to create a vascular structure or lymph vessels and or both. In some aspects, the population of cells comprises endothelial cells, such as TIME cells, HUVECs or lymphatic endothelial cells.
In some aspects, the method further comprises connecting the microfluidic device to a circulation system comprising one or more syringe pumps with controlled flow rates creating one or multiple channels. In some aspects, two or more channels are connected to flow in parallel or series. In some aspects, the flow rate for each of the channels is distinct creating pressure gradients. In some aspects, the flow rate for each of the channels is essentially identical. In other aspects, two or more channels are connected to flow in series. Flow rates can be designed to create fully functional, aligned, and endothelialized vessels.
In a further embodiment, there is provided a microfluidic device comprising a polydimethylsiloxane (PDMS) scaffold; a channel disposed within said PDMS scaffold; and at least one chamber in fluid communication with the channel or channels, wherein the chamber comprises an extracellular matrix hydrogel surrounding the channel.
In some aspects, the chamber is located in an interior region of said PDMS scaffold. In some aspects, the channel extends from the chamber to an external surface of said PDMS scaffold. In some aspects, the channel extends through said PDMS scaffold.
In certain aspects, the device is produced according to the methods of the embodiments.
In some aspects, the extracellular matrix hydrogel comprises elastin, fibrin, fibronectin, laminin, hyaluronic acid, keratin, and/or collagen. In some aspects, the extracellular matrix hydrogel comprises collagen. In some aspects, the collagen is type I collagen. In some aspects, the collagen is present in the hydrogel at a concentration of 5 to 15 mg/mL, such as about 6-7, 7-8, 8-10, 10-12, or 12-15 mg/mL. In some aspects, the collagen is present in the extracellular matrix hydrogel at a concentration of 6 to 12 mg/mL.
In some aspects, the device further comprises a population of cells dispersed within the extracellular matrix hydrogel of the chamber. In some aspects, the population of cells comprises tumor cells, cardiovascular cells, macrophages, kupfer cells, stellate cells, and/or hepatocytes. When modeling skin, keratinocytes, fibroblasts, and/or adipocytes can be incorporated in the extracellular matrix in a single or multi-layer structure.
In some aspects, the device comprise 2, 3, 4, or 5 chambers, wherein each chamber comprises a separate channel.
In some aspects, each chamber comprises a distinct population of cells within the extracellular matrix hydrogel. In some aspects, the at least one chamber comprises two channels. In some aspects, the two channels have distinct diameters. In some aspects, the two channels have essentially identical diameters.
In some aspects, the diameter of a channel is between 100 to 1,000 μm, such as 200 to 500 μm, such as about 150, 200, 250, 300, 250, 300, 400, 500, 600, 700, 800, or 900 μm. The channel or additional channels in the device may have a diameter of 100-200, 200-300, 300-400, 400-500, 600-700, 700-800, or 900-1000 μm.
In some aspects, the channel comprises a population of cells. In some aspects, the population of cells comprises endothelial cells or lymphatic endothelial cells. Immune cells or blood can be circulated through one or multiple channels.
In some aspects, the population of cells or populations of cells in the device comprises at least 1,000 cells, such as at least 5,000, 25,000, 50,000, 100,000, 200,000, 300,000, or 500,000 cells.
In some aspects, the device comprises one population of cells within the extracellular matrix hydrogel of the chamber and a second population of cells within the channel. In some aspects, the device comprises one population of cells within the extracellular matrix hydrogel of the chamber and a second population of cells within the channel. In some aspects, the device comprises tumor cells within the extracellular matrix hydrogel of the chamber and endothelial cells within the channel.
In some aspects, the cells within population comprise detectable markers. In some aspects, the first population of cells comprise a detectable marker distinct from the marker of the second population of cells.
In further embodiments, there are provided network platforms or branching vessels patterned after in vivo architecture. In one embodiment, there is provided a method of producing a network platform comprising filling a base mold, such as a well, with collagen and laying a flat PDMS piece on top to produce a flat collagen surface after polymerization. In some aspects, the well in the top component is aligned with a lithographically produced PDMS stamp that has a designed channel pattern. In some aspects, pins are inserted into the chamber to create an inlet and exit port before the chamber are filled with collagen and polymerized. In some aspects, after polymerization and removal of the PDMS and pins, the top and bottom components of the platform are stacked resulting in a network fully encased in collagen (Zheng et al., 2012; incorporated herein by reference). In some aspects, the process of forming biomaterials about a lithographic pattern produces a square cross-section in the channel. In some aspects, cells are seeded in the channel and a confluent endothelium is established to form a circular cross-section.
A further embodiment provides a method of evaluating a therapeutic or diagnostic agent comprising introducing the therapeutic or diagnostic agent to the flow of the microfluidic device of the embodiments and characterizing the effect of said therapeutic or diagnostic agent. In some aspects, evaluating comprises monitoring transport, uptake, toxicity, and/or efficacy of the therapeutic agent. In some aspects, characterizing is further defined as measuring cell viability, cell morphology, cell proliferation, and/or enzyme secretion.
In another embodiment, there is provided a method of measuring migration of a molecule (e.g., a cell, particle, bacteria, chemical, nanoparticle, or toxicant) comprising obtaining a microfluidic device of the embodiments, wherein the device comprises a chamber with at least two (or multiple) channels running through said chamber and a region of extracellular matrix hydrogel comprising a population of cells between said at least two (or multiple) channels; introducing media to the flow of the device; and monitoring the migration of a cell in the device. In some aspects, the channels comprise endothelial cells or lymphatic endothelial cells and the hydrogel comprises tumor cells and/or fibroblasts. In certain aspects, the hydrogel further comprises macrophages. In some aspects, the hydrogel comprises keratinocytes, fibroblasts, adipocytes, endothelial cells and/or tumor cells. In certain aspects, the media comprises cells, growth factors, cytokines, hormones, antibodies, drugs, and/or enzymes. In some aspects, the media comprises or consists of whole blood.
In a further embodiment, there is provided a multi-layer hydrogel system for modeling skin comprising a first layer of collagen hydrogel comprising keratinocytes (e.g., to mimic the epidermis), a second layer of collagen hydrogel comprising fibroblasts and/or endothelial cells (e.g., cultured in the collagen or single or multiple endothelialized channels forming the dermis), and a third layer of collagen hydrogel comprising adipocytes, endothelial cells, an endothelialized blood vessel, or lymph channels (e.g., to mimic the subcutaneous layer). In some aspects, the first, second, and/or third hydrogel layer further comprises keratin, melanocytes, hair follicles, and/or neural cells. In some aspects, the percentage of collagen and/or additional ECM components, such as keratin, may be varied within the different layers. In some embodiments, the system may be used as a representative skin model. This skin model can be used to assess injury from chemical or thermal insults or transport of chemicals, bacteria, cells, or toxicants across the skin. Assessment of burn or blast injury is also a suitable use of the technology. In some aspects, the extracellular matrix hydrogel comprises collagen and keratin, such as oxidized keratin (also referred to as keratose).
In further embodiments, there is provided a provided an extracellular matrix comprising collagen and keratose. In some aspects, the extracellular matrix is used in a model provided in the present embodiments and aspects thereof.
As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Certain embodiments of the present disclosure provide microfluidic devices, methods of fabricating the devices, as well as methods of using the microfluidic devices. Accordingly, some embodiments provide a physiologically representative three-dimensional in vitro microfluidic vascularized platform of varying complexity that is simple to fabricate and enables high throughput investigation of biological processes and optimization of diagnostics and therapeutics. The in vitro vascularized platforms may consist of single or multiple confluent cylindrical endothelium blood or lymph vessels surrounded by a collagen, keratin, or collagen/keratin blend extracellular matrix seeded with tissue specific cells recreating the tissue-blood vessel interface.
Accordingly, in some embodiments, there is provided herein a method and 3D physiologically representative, high throughput platform for representing vascularized tissues and tumors. The present platform can have the capability to eliminate artificial boundaries, provide a physiologically representative extracellular matrix to facilitate realistic cell growth and response, create a functional endothelium for study of transport and cell migration, and replicate complex vessel and tissue architecture inexpensively. By changing out cells, different tissues can be replicated and the platform can be used for diagnostic, therapeutic, and device development.
Thus, further embodiments provide methods for cardiovascular applications for stent and drug design. The present platform can also be used for cancer drug discovery and optimization is possible by allowing the influence of therapeutic/diagnostic properties on drug localization, specificity, and efficacy, dosing, and time of delivery to be studied. By creating vascularized tumor, liver, and heart in series or parallel toxicity to heart and metabolism by the liver as a function of therapeutic properties and dose can be optimized. Multiple vascularized platforms are provided herein to model the tumor, and distant metastatic sites (bone, brain etc) to enable identification of new molecular diagnostic and therapeutic targets for aggressive, metastatic disease. Also by creating dual vessels in the platform the influence of biochemical/pressure gradients on drug delivery, efficacy, tumor metastasis can be studied in the context of different therapeutics. Multiple layer vascularized skin platforms are also provided, such as for optimizing protective garments to prevent burn injury. In addition, patient specific vascularized tissues can be created for personalized diagnostics and therapeutics
In some aspects, the present platforms are produced by subtractive tissue engineering fabrication methods (e.g., micro-milling techniques) and lithographic techniques with additive tissue engineering methods. The present platform can overcomes restriction to the geometry and size of the tissue chamber while still creating a viable in vitro tumor microenvironment that can easily interface with imaging setups to complete studies of the microenvironment. The bonding of a PDMS chamber directly to a glass cover slip allows the hydrogel to be positioned closer to microscope objectives and eliminates the necessity of using water to create an optically clear system for imaging. The platforms can be customized to represent different tissue types and the complexity of the platform can be varied from single vascularized vessel systems to more intricate vascularized microfluidic networks that capture microvasculature representative of patient tissues for the first time. Since the tissue platform itself may be entirely made of collagen extracellular matrix, cells contained in the system maintain all their biological functions (e.g., growth, migration, gene expression) enabling representative biological and pathological processes to be studied and the response to diagnostics and therapeutics to be optimized in a realistic manner.
In some aspects, the present platforms are functional and physiologically representative endothelium that are created within the vascular channels. In some embodiments, the presence of a functional vasculature in connection with a tissue enables therapeutics and diagnostics to be optimized by honing the properties of the agent (e.g., size, surface properties, shape) to maximize transport/localization in turn diagnostic sensitivity and therapeutic efficacy. The design of this system when coupled with labeled cells can enable dynamic imaging of transport of the diagnostic/therapeutic and real time response (e.g., apoptosis, migration, and necrosis) of the cells targeted enabling optimization of therapeutic features to maximize toxicity. In addition to properties of the therapeutic being optimized, the ideal timing of the drug can be determined based on dynamic understanding of pathological state. Thus, the present system offers a high throughput and inexpensive alternative to animal testing enabling each aspect of therapeutic and diagnostic agent to be optimized.
In one embodiment, there are provided complex vascularized in vitro models to mimic the tumor microenvironment which can be tailored to represent various aggressive breast tumors such as inflammatory breast cancer. Features of these platforms include a continuous, aligned endothelium that allows for cell-cell interactions between vasculature and tumor cells. It was demonstrated the phenotype of the cancer has an influence on the leakiness of the endothelium, initiation of angiogenesis and modulation of the surrounding tumor microenvironment.
In some embodiments, a platform is provided for fabrication of a single endothelialized microchannel encased within a collagen platform hosting tumor cells, such as breast cancer cells. The microfluidic device was developed and utilized to study the influence of cellular interaction on transport phenomenon through vasculature in a hyperpermeable tumor microenvironment. This platform relies on subtractive tissue engineering fabrication techniques. Through confocal imaging, it was demonstrated that the platform produces enhanced leakiness recapitulating physiological features of the tumor microenvironment. The influence of tumor endothelial interactions on transport of particles was also demonstrated.
In certain embodiments, the housing material mold for the platform is fabricated using micro-milling techniques. This method may decrease fabrication time and eliminate the requirement of clean room facilities, multistep fabrication processes, and expensive reagents to produce a vascularized microchannel. The platform can be easily customized to alter geometries of the tissue chamber and vessel size while maintaining a continuous lumen. Additional benefits include reduction in the amount of collagen and reagents required to fabricate the platform. The platform can also establish continuous live cell imaging of particle transport.
Additional embodiments concern fabrication of platforms with vasculature recreated from patient specific data that can be used for developing personalized treatments. The platforms may possess a functional and aligned endothelium without the presence of an artificial boundary as the cells are cultured directly on collagen. This can enable realistic transport of therapeutics and diagnostics to be studied within a tissue and the transport/targeting properties of these agents to be optimized in an inexpensive, and high throughput manner.
Further embodiments enable use of the microfluidic devices provided herein for the identification of new diagnostic/therapeutic targets, optimization of diagnostic/therapeutics agents, and realization of the ultimate goal of personalized treatment plans. In some embodiments, applications include creation of the blood brain barrier for use in homing drug transport and localization.
In further embodiments, multiple vascularized tissues are connected to enable the influence of a diagnostic/therapeutic on the target tissue and toxicity to other collateral tissues/organs (e.g., heart, liver) to be studied dynamically enabling optimization of agent properties and dosing regimens. In one particular embodiment, the device comprises vascularized tumor, liver, and heart that enables study of the transport and therapeutic efficacy and toxicity to the liver and heart.
In some embodiments, the devices provided herein may be used to study the influence that metabolism by the liver has on tissue response and drug effectiveness. This holds particular promise for chemotherapeutic optimization where the goal is to minimize collateral toxicity to the heart and liver while maximizing therapeutic efficacy to the tumor of interest. Another embodiment provides devices with multiple vascularized compartments including the tumor of interest and potential metastatic sites of cancer (e.g., bone, brain, lung) to enable homing of the tumor and diagnostic and therapeutic optimization to effectively diagnose and treat varying stages of disease and identify new molecular markers that signify poor prognosis.
The vascularized platforms can also be adapted to model the interactions between tumors, blood vessels, and lymph vessels which has tremendous promise for tumors such as aggressive tumors that metastasize through the lymph system. In particular embodiments, the system provided herein is capable of replicating complex vascular networks and tissues particularly patterned after patient imaging data (e.g. CT and MRI) to enable personalized treatment plans. The platforms may also be used for cardiovascular applications in which the presence of a vessel and surrounding heart need to be modeled to optimize stent design or placement or drug delivery.
Further, major challenges also arise due to delivery drugs based on adverse pressure and biochemical gradients within a tissue or tumor. Thus, in some embodiments, the present devices may be used as multi-vessel tissue platforms enabling optimization of diagnostic and therapeutics.
Additionally, in some embodiments, there is provided a second platform capable of combining lithographic techniques with additive tissue engineering methods to create intricate endothelialized microfluidic networks that capture the more complex geometries of tumor microvasculature representative of patient tumors. By modeling microvascular networks after in vivo tumors, patient specific in vitro platforms may be created that can be used to develop personalized patient treatments.
In some embodiments, the device comprises a single vessel or multiple vessels, such as dual vessels to study migration of cells. In some embodiments, the collagen used in the present devices is obtained from an animal. For example, the collagen may be obtained from rats, particularly rat tails.
In further embodiments, there are provided multi-layer vascularized skin platform containing blood vessels and lymph vessels, such as to study burn injury characteristics including increased capillary permeability, leakage between dermis and epidermis, and destroyed tissue becomes eschar. The healing of burn wounds comprise an inflammatory phase (e.g., chemotactic factors attract immune cells), proliferative phase (e.g., cell proliferation, tissue regrowth), and a remodeling phase (e.g., fibrous structural proteins and scar tissue formation). The skin platform may be evaluated using live/dead staining, GFP expression, heat shock proteins, such as 27, 47, 60, and 70, CD31 staining, and cell proliferation. Contact burn testing may comprise burning the sample by contact with a heated copper rod. The temperature and exposure time may be controlled. The damage to gels may be visible through live/dead staining and imaging. The gels can remain viable for at least 3 days after burning. The skin platform technology provided herein possesses a functioning vasculature unlike existing skin systems. It enables the dynamic study of skin infection as a function of barrier function, vasculature perfusion, and immune response which will yield key insights for treatment planning. Ultimately, the skin platform can provide a physiologically representative high throughput system for understanding skin diseases and wounds and developing appropriate therapies. The relationship between the skin microenvironment, bacteria, and immune presence on wound evolution and resolution can provide key insights into treatment planning for wounds. This skin platform can be used to further screen drugs for skin infections or other conditions such as atopic dermatitis before proceeding to in vivo models and clinical trials and can easily be customized for any other type of infection, skin disease, or wound. The skin platform can also be used to assess dynamic transport of chemicals particularly toxicants, nanoparticles, or drugs and their response spatially in the skin.
In some embodiments, there is provided a model comprising a tumor with overlying skin, such as a model for breast cancer. In other embodiments, there are provide method for using lymphatic endothelial cells in the vascularized platform provided herein to create lymph blood vessels. These models can be used to study cancel cell invasion into overlying skin and surrounding lymphatics. The tissue may be surrounded by blood vessels and/or lymph vessels. The platform may be used to assess the effect of culture of different cells, drugs, or external stimuli, such as on vessel permeability.
In further embodiments, there is provided a provided an extracellular matrix comprising collagen and keratose. The matrix provided herein can be more thermally and mechanically stable as compared to previous extracellular matrix compositions. The matrix can be used in the platforms provided herein, such as for the growth of fibroblasts. The platform also allows for more accurate testing of temperature response and enables these platforms to respond to thermal insults or therapeutic heating with lasers, RF, and/or ultrasound similar to tissue.
The present disclosure is also directed to methods useful in the analysis of cell behavior. In an embodiment, a method is provided for the measurement of directed migration of cells, bacteria, and viruses in a microfluidic device. Generally a cell, bacteria, or virus is introduced into a first fluid-flow path and may attach to at least one of the surfaces in the flow path. The cell may also adhere to the scaffold. Either at the same time the cell is introduced into the first fluid-flow path or a different time, a biological entity may be introduced into the fluid-flow path. This biological entity may be a cell or sub-cellular component, including growth factors, cytokines, hormones, antibodies, gene expression and enzymes, as well as drugs and other small molecules. At a given time point, the extent of the migration of the cell, e.g., into the scaffold and optionally into the second fluid-flow path, is measured. The cell, bacteria, virus could also be introduced within the extracellular matrix or at the surface as in the case of skin and its transport and response in the platform measured.
In certain embodiments, the methods of the disclosure include the generation of multiple cell type biomaterials, useful in in vitro and in vivo systems such as tissue engineering. The devices described herein are used to fabricate biological or biocompatible materials that contain two or more types of eukaryotic cells. Further, in vitro systems described herein replicate the physiological functions of tissues or organ systems, and are thus useful in, for example, drug testing or toxicity screening of test compounds.
In the present studies, a multi tissue-on-a-chip platform was developed consisting of a vascularized breast tumor and healthy/tumorigenic liver microenvironments connected in series to enable dynamic determination of vessel permeability and transport of nanoparticles/drugs and their associated efficacy and toxicity to the liver. Microenvironments were fabricated from type I collagen of concentrations of 7 mg/ml and 4 mg/ml for tumor and liver respectively to replicate the growth characteristics and compression moduli of these tissues. Wall shear stresses of 4 dyn/cm2 (healthy) and 1 dyn/cm2 (tumor) were employed within each vessel to mimic physiological conditions. Cell morphology was characterized with immunofluorecent staining and the fidelity of liver cells cultured in the platform was demonstrated by measuring albumin release. Dextran particles with sizes of 3 kDa and 70 kDa were perfused in the platform to replicate the hydrodynamic diameters of chemotherapy drugs and nanoparticles conjugated with drugs. The platform was utilized to determine the effect of particle size on the dynamic and spatial diffusion of particles through each microenvironment independently and in response to circulation of particles in varying sequence of microenvironments (tumor to liver or liver to tumor). The results showed that when breast cancer cells were cultured in the microenvironments they had a 2.62-fold (p<0.001) higher vessel porosity compared to vessels within healthy liver microenvironments, which resulted in increased permeability of tumor microenvironment by 2.35- to 2.77-fold (p<0.01) for 3 and 70 kda particles, respectively, compared to healthy liver. Decreased particle accumulation of 2.57-fold (p<0.01) was observed for larger particles compared to smaller particles in the ECM of healthy liver. However, 5.57 (p<0.01) fold greater ECM accumulation of larger particles compared to smaller particles occurred for the breast tumor microenvironment. The particle accumulation within the breast tumor microenvironment decreased by 5.49-fold (p<0.01) if particles were first perfused through the liver and smaller particles demonstrated greater accumulation in the liver. Thus, the platform can be utilized to determine the impact of the tissue/tumor microenvironment or drug/nanoparticle properties on transport, efficacy, selectivity, and toxicity in a dynamic, and high throughput manner for use in treatment optimization.
The tissue on-a-chip microenvironment provided herein can be used to mimic transport in vivo enabling spatial and dynamic assessment of transport of any type of drug/nanoparticle as a function of their size. This device can be used to investigate the influence of other drug/nanoparticle properties including surface charge, dimensionality, targeting ligand, and aspect ratio on transport. By altering the direction of flow the effect of targeting and metabolism on transport kinetics of drugs/chemicals can be simulated in high throughput, inexpensive optimization of nanoparticles or other therapeutics by enabling toxicity, efficacy, and biodistribution measurements as a function of varying microenvironmental conditions and drug/nanoparticle properties. The multi tissue-on-a chip microenvironments can also be utilized for testing a combination of different treatment methods such as hyperthermia, radiation, and a myriad of nanoparticles with unique functionality to create solutions for targeted delivery.
By merging multiple embodiments of the platform including vascularized tumors and skin a first-of-its-kind physiologically representative three-dimensional comprehensive in vitro breast tumor platform for modeling invasion of aggressive breast cancer through the breast tissue, their interactions with nearby blood and lymphatic vessels, as well as invasion into the skin can be used to identify targetable stromal-tumor cell interactions driving the aggressive phenotype of breast tumors. The platform can be used to investigate the tumor stromal interactions of very unique but aggressive and metastatic breast tumors, modeled with SUM149, MDA-IBC3, and MDA-MB-231 cell lines. The platform can consist of functional vascular and lymphatic vessels as opposed to current existing platforms which include only nonfunctional vessels. The skin component will include the hypodermis, dermis, and epidermis. The platform can host stromal cells, MSCs, adipocytes, fibroblasts, keratinocytes, and macrophages, which have been shown to promote skin invasion behavior of breast tumors. The matrix can be composed of collagen ECM representative of breast tumor tissue without the presence of PDMS structural supports maintaining the in vivo tumor architecture. The platform can benefit multiple subtypes of aggressive breast cancer where tumor stromal interactions are also dominant in disease progression and enhance the study of migration phenotypes, both collective emboli migration as well as epithelial to mesenchymal transition.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
An in vitro platform was created as a single vessel vascularized tumor model to study the transport in a hyperpermeable tumor microenvironment resulting from tumor-vasculated interactions. The platform overcomes complications of restrictions to the geometry and size of the tissue chamber due to FEP tubing. The platform utilizes simplified fabrication techniques leading to increased reproducibility (
A confluent endothelium was formed in the tumor microenvironment and then diffusion of particles through the endothelium was investigated. In addition, a microvascular network encased in a collagen hydrogel seeded with breast cancer cells created a physiologically representative vascularized tumor microenvironment (
The platform has increased working distance and creates a viable in vitro tumor microenvironment that can interface with imaging setups to complete studies of the microenvironment. The bonding of a PDMS chamber directly to a glass cover slip allows the hydrogel to be positioned closer to microscope objectives and eliminates the necessity of using water to create an optically clear system for imaging. Water removal from the platform decreased the necessary microscope working distance by over a millimeter (
Linking different size platforms enabled creation of a simple body on a chip platform consisting of multiple chambers with unique vascularized tissue types. Endothelialized channels of different diameters were fabricated using 22, 25, and 30 gauge needles corresponding to vessel diameters of approximately 717, 514, and 311 μm respectively (
Engineered Tumor Microenvironment:
The in vitro tumor platforms were composed of green fluorescent protein (GFP) labeled MDA-MB-231 cells encased in a collagen ECM with a confluent endothelium as shown in
The morphology of the endothelial layer for mono-culture and co-culture platforms was visualized using F-actin staining as evidenced by red fluorescence. Top view of the tissue chamber (
Transport: To add quantitative support to the confocal images showing endothelial behavioral in the single vessel platform,
Others studies have reported similar results that the presence of tumor cells reduces the endothelial barrier function and increases transport of macromolecules through the endothelium into surrounding tissue (Buchnan et al., 2014). Introducing particles larger than dextran (>70 kDA) may decrease the diffusion rate through the endothelium and into the platform. Therefore, particles may congregate along the endothelial wall unable to pass through smaller gaps on the endothelium. Also, increasing the particle size may increase the difference between co-culture diffusion and a non-endothelialized channel, as the co-culture endothelium may be sufficiently confluent to prevent diffusion of larger sized particles.
Network Platforms:
In order to create an in vitro platform that better recapitulates the tumor microenvironment, a microfluidic vascular network was created that enabled improved perfusion and has the capability of scaling the platform to larger tumor sizes. Using soft lithography techniques, a simple geometric pattern was imprinted into a collagen hydrogel to form a network of channels encased in the platform. The network has one inlet and outlet to provide transport through the system. The width of each individual channel was approximately 100 μm (
Evaluation of a top view of the co-culture network platform in
In vitro platforms that re-create physiologically relevant tumor vasculature tailored to an individual allows for evaluation and optimization of patient specific therapies. In these platforms transport of therapeutics could be studied in realistic environments and properties optimized to achieve targeted and efficient drug localization. Following the initial vascular network pattern presented in
For the first time, transport of two different sized particles in an in vivo vascular patterned platform was investigated and presented in
The present disclosure presents devices and methods for the fabrication of microfluidic channels by employing additive and subtractive tissue engineering techniques. Collagen scaffold was used in platforms to accommodate cells culturing and remodeling producing endothelialized vessels of in vitro tumor microenvironments. Vascularized tumor platforms were created with embodiments of scalable channels (single/dual) and networks based upon in vivo vasculature. The single channel platforms allowed the dynamic tracking of tumor-vasculature interactions as well as the spatiotemporal behavior of particle diffusion in a physiologically representative microenvironment. The network platforms can be designed to replicate patient data and allow for the study of transport and drug delivery in conditions that mimic the patient's tissue. All these platforms can be expanded upon to incorporate immune cells, stromal cells, and lymphatic vessels to create a complete tumor microenvironment and be used to evaluate the toxicity of chemotherapeutics and lead to the development of new therapies.
Materials:
Stock solutions of collagen type I (14 mg mL−1) derived from rat tails were prepared. Platforms of varying complexity ranging from single vessel platforms to vascular network platforms were fabricated. Single vessel platforms recreated the tumor endothelial microenvironment of a single blood vessel whereas the vascular networks represented branching blood vessels present in a tumor. Single vessel platforms were formed from polycarbonate and polydimethylsiloxane (PDMS) housing components that interfaced with glass cover slips to produce a tissue chamber with an imaging surface as shown in
Platform Design and Fabrication: All polycarbonate and aluminum components used in fabricating the hydrogel scaffolds were CNC machined. The aluminum mold used for the single vessel platform holds a 22G needle and was used to form a PDMS chamber (
Formation of Collagen Hydrogels: A working collagen solution (6-7 mg mL−1) for use in the platforms was prepared by neutralizing the stock collagen solution. Stock collagen was mixed over ice with 10×DMEM, 1N NaOH, and 1×DMEM. This solution was added to the single vessel channel or vascular network platform chambers and polymerized in an incubator for 25 minutes at 37° C. For the single vessel platform a 22G needle was left encased in the collagen during the polymerization process (
For the vascular network platform, working collagen was prepared in the same manner; the well in the base component of the platform was filled with collagen. Then, a flat PDMS piece was laid on top of the well to produce a flat collagen surface after polymerization (
Cell Culture in Gels: To create a tumor microenvironment, cancer cells were suspended in the working collagen solution to the targeted density of 1×106 cells/mL (Buchanan et al., 2014). The collagen solution containing cells was injected in the chamber followed by incubation of the platform chamber at 37° C. for 25 minutes. After polymerization of the collagen and the formation of vascular channels in the scaffold, TIME cells were injected into the microchannels. For the single vessel platforms, TIME cells (2×107 cells/mL) were injected twice at 10 minute intervals and the platform was slowly rotated during the intervals to promote cell adhesion around the entire channel. To seed endothelial cells into the network platforms 15 μL of media containing TIME cells (5×106 cells/mL) was added to the inlet and allowed to perfuse into the channels for 20 minutes at 37° C. The MDA-MB-231 cells expressed a green fluorescent protein (GFP) signal and TIME cells a red fluorescent protein (RFP) signal.
Formation of Endothelialized Channels:
To form a confluent endothelium the platforms were connected to a syringe pump (Harvard Apparatus) providing a continuous flow of TIME cell media into the channels resulting in a shear stress (τ) of 0.01 dyn/cm2 for 36 hours followed by 36 hours of 0.1 dyn/cm2 (
where Q is the volumetric flow rate, μ is the dynamic fluid viscosity, and r is the radius of the channel. Poiseille flow in the channel was confirmed using μ-PIV. This preconditioning protocol has been previously established by the Rylander group to produce a confluent and aligned endothelium (Buchanan et al., 2014). Air eliminating filters were placed upstream of the inlet to prevent bubbles from entering the channels during perfusion. The outlets fed to a collection reservoir.
An alternate method was also developed to provide flow for the vascular network platform that uses a simplified setup of a reservoir system. The simplified set up is a quicker and easier method to establish flow and operates based on gravity creating a pressure difference at the inlet and outlet to induce a flow through the system (
Viability Analysis:
In order to confirm the utility of the platform the viability was assessed. For this measurement, untagged MDA-MB-231 cells were incorporated in the collagen to form the tumor microenvironment. The viability of MDA-MB-231 cells in the tumor platforms was evaluated using calcein AM (live)/propidium iodine (PI) (dead) (ThermoFisher Scientific) corresponding to green and red stains, respectively (
Endothelium Morphology:
F-actin staining and SEM analysis was completed immediately following endothelial preconditioning to visualize endothelial morphology and orientation in the microchannels. For F-actin staining, the microchannels were perfused with 4% paraformaldehyde and 0.5% triton-X-100 (Sigma Aldrich) for 20 minutes followed by incubation in 1% bovine serum albumin for 30 minutes. Rhodamine Phalloidin (ThermoFisher Scientific) probe was used to label F-actin and DAPI was used to label nuclei and imaged using Leica TCS SP8 confocal laser scanning microscope with HC PL Fluotar 10×/0.30 objective. SEM analysis was performed using a Zeiss Supra40 SEM-Electron Microscope. Microchannels were fixed overnight in an aldehyde mixture osmium treatment for 4 hours. Post fixation, the platforms were dehydrated in a series of ethanol solutions (50-70-95%), then critical point dried by CO2 and coated with a thin layer of platinum-paladium.
Permeability:
Permeability coefficients were obtained to quantify the rate of transport through the endothelium of the microchannel in response to perfusion of 70 kDa dexran particles. Particles with a molecular weight of 70 kDA are commonly used in transport studies and is comparable to large macromolecules. Three conditions of the single vessel platform were evaluated including an acellular microchannel, an endothelialized microchannel without cancer cells in surrounding collagen, and a microchannel containing co-culture of endothelial cells in vessels with cancer cells cultured in the surrounding collagen. Studies were conducted upon completion of the 72 hour preconditioning protocol. Green fluorescent dextran suspended in serum free endothelial basal media (EBM-2) at 10 μg/mL was perfused through the microchannels for 2 hours at a flow rate of 260 μL/min generating a WSS of 1 dyn/cm2 with images being taken every five minutes for evaluation of transport and diffusion. In normal microvessels the average WSS is around 4 dyn/cm2. The abnormal vasculature in tumors can compromise flow resulting in reduced WSS relative to normal blood capillaries and this led to the selection of a lower value of 1 dyn/cm2 for use in particle transport studies.
Imaging for diffusion studies was completed on a widefield inverted Leica DMI 6000 B fluorescence microscope and the tiff images obtained were exported to MATLAB for evaluation. The average fluorescent intensity of the diameter of the collagen was measured and used to determine the diffusion permeability coefficient Pa. This coefficient describes the ability of solute to pass uniformly from the microchannel into the surrounding hydrogel and is calculated with the following equation:
where Ib is the background intensity, I1 is the average initial intensity, I2 is the average intensity after recovery time interval Δt, and d is the diameter of the microchannel (Price et al., 2011). The last five consecutive data points from the 2 hours of flow were used to calculate Pa as these were the time points at which stable and consistent measurements were achieved. Three samples (n=3) were collected for each variation in the diffusion studies. Data is expressed as a mean value ±standard deviation. Significance of the data was verified using Student's t-test and a 95% confidence criteria between groups of data. Blue 0.10 μm fluorescent polymer microspheres were used to visualize particle transport in the network tumor platform to contrast against alternate colors of labeled cancer and endothelial cells. The microspheres were suspended in serum free endothelial media at a density of 10 μg/mL and allowed to perfuse into the system for 1 hour.
Modeling Flow Inside the Vasculature:
WSS is one of the key factors in creating physiological in vitro tumor microenvironments. In order to ensure that continuous flow is sustained throughout the microfluidic platforms, velocity profiles were modeled using Comsol Multiphysics. Stokes law was used to quantify the flow inside the vasculature with the following assumptions: constant fluid viscosity, incompressible fluid, and a low Reynold's number inside the vasculature:
where ∇ is gradient operator and ∇2 is the square of vector Laplacian, P is pressure, u is velocity, μ is viscosity and ρ is density of the fluid.
Flow inside a porous tissue such as the ECM must be modeled. The porosity has a significant impact on flow inside porous membranes and necessitates the use of Darcy's Law, which predicts transport in porous material:
where, κ is the permeability of the material, P is the pressure, u is the velocity and μ is the viscosity. Mass conservation (Equation 5) is also solved simultaneously.
∇·u=0 (5)
Simulations were run on the network platforms with the following conditions: constant flow rate of 260 μl/min, and inlet and outlet boundary conditions of zero gauge pressure. Other ECM properties used in the simulation were permeability of collagen of 10×10−15 m2 and porosity of collagen of 0.49. Resulting flow velocity profile was used to calculate WSS at the vessel walls. WSS of a Newtonian fluid is defined as:
where u is velocity parallel to the vessel, and y is the perpendicular direction to the wall.
A 3D in vitro vascularized tumor platform was developed as a tool for modeling various types of aggressive breast cancer where tumor stromal interactions including tumor-vasculature and tumor-ECM interactions have been shown to direct the disease phenotype. The 3D in vitro vascularized platform was utilized to model both IBC tumors as well as non IBC invasive ductal carcinoma showing the versatility of the platform for studying a wide variety of breast cancers. It replicates conditions that are representative of in vivo tumor vasculature interface such as physiological flow and associated shear stress, a continuous, aligned and functional endothelium while allowing for tumor-endothelial-ECM interactions. The tumor cells of interest were the IBC cells lines SUM149 and MDA-IBC3, and non-IBC invasive ductal carcinoma cell line MDA-MB-231. All these cells lines are hormone receptor negative which correlates with breast cancer of high malignancy and recurrence, and additionally, SUM149 and MDA-MB-231 cells are triple negative indicating they lack amplification of the HER2 receptor. The influence of paracrine signaling was investigated between tumor cells and the vascular endothelium and the in vivo response of a hyperpermeable tumor vasculature was recreated as well as secretion of proangiogenic cytokine VEGF. ECM porosity was characterized as a function of the interactions between the tumor cells and the stroma which revealed a response similar to in vivo migratory behavior of tumor cells. These platforms provide a tool to elucidate disease dynamics of aggressive breast cancer tumors where tumor-stroma interactions are the driving force behind tumor development and progression.
In Vitro 3D Tumor Platform:
The in vitro vascularized tumor platforms consisting of co-culture of either IBC or non-IBC cancer cells with TIME cells was developed. The 78-hour flow protocol with a graded increase in WSS from 0.01 dyne/cm2 to 1 dyne/cm2 resulted in a confluent endothelium as shown in
Following the 78-hour graded flow treatment to establish a confluent endothelium, the resulting in vitro vascularized co-culture platforms are shown in
Endothelium Integrity:
Hyperpermeability and leakiness of tumor blood vessels is characteristic of in vivo breast cancers and studies have revealed this to be influenced by tumor-vasculature interactions.
Staining patterns of PECAM-1 in
Comparison of endothelial coverage of the lumen showed a significant decrease in the endothelium coverage in the triple negative breast cancer platforms of SUM149/TIME and MDA-MB-231/TIME in comparison to MDA-IBC3/TIME and TIME in vitro vascularized platforms as illustrated in
Vascular Permeability:
Intravasation and extravasation of tumor cells for metastasis as well as inefficient delivery of drugs and chemotherapeutics to the tumor are highly influenced by vascular permeability and leakiness. To determine the effect of tumor cells on transport across the endothelium barrier, the platforms were perfused with fluorescently labeled dextran and the resulting changes in florescent intensity from the dextran perfusing across the endothelium were used to determine the effective permeability (
VEGF ELISA:
ELISA measurements for VEGF were performed on flow media collected at 72 hours (following formation of the baseline endothelium) and 78 hours (after exposure to WSS of 1 dyn/cm2 for 6 hrs) as illustrated in
Matrix Porosity:
Invasive tumors modulate the surrounding ECM in order to migrate through the tumor microenvironment and out into the surrounding tissue. To quantify the ability of the tumor cells to modulate the ECM, SEM analysis of the collagen matrix in each of the vascularized breast tumor platforms was used to determine matrix porosity as shown in
Thus, a 3D in vitro vascularized tumor platform is provided herein to model the interactions of multiple aggressive breast tumor with their corresponding stroma and reproduced the in vivo response in terms of vascular permeability and ECM degradation. Utilizing the vascularized breast tumor platforms, it was shown that the type of tumor cell present had a profound impact on the endothelium with the more aggressive and invasive tumor cells creating a leakier vasculature and have provided the first opportunity to spatially observe and quantify this difference. Additionally, it was revealed that the co-culture of tumor and endothelial cells influences the expression levels of the angiogenic cytokine VEGF as well as remodeling of the collagen ECM. Modulation of the ECM and the endothelium in breast cancer are important factors that have been linked to tumor angiogenesis and metastasis and are influential parameters when studying tumor development and progression.
The 3D in vitro vascularized platforms provided herein allow for the investigation and modeling of the tumor-stroma interactions and the influence of the interactions on vascular permeability and matrix porosity for three different highly invasive and aggressive breast cancer phenotypes. Blood vessel leakiness and increased matrix porosity that are representative of in vivo behavior or invasive tumors were recreated. Compared to current 3D in vitro tumor models that focus on recreating specific stages of tumor progression, the platforms provided herein were able to model various stages in cancer progression including early signs of angiogenesis as well as modulation of tumor ECM and vasculature for migration and metastasis. The models can be used as a tool for studying various aggressive breast cancers whose phenotype is driven by tumor stromal interactions. While these platforms do not encompass the entire complexity of the tumor microenvironment, they provide an initial insight into the behavior of aggressive tumors and can be adapted to include other stromal components.
Cell Culture: Human breast carcinoma cell line MDA-MB-231(ATCC® HTB-26™) breast carcinoma, human breast inflammatory cancer cells MDA-IBC3 and SUM149, and telomerase-immortalized human microvascular endothelial (TIME) cells were used in this study. A lentiviral vector system was used to genetically modify MDA-MB-231 and TIME to stably produce either a green fluorescent protein (GFP) or a red fluorescent protein (RFP) respectively for real time imaging studies. Stable fluorescent MDA-MB-231 and TIME cells were from Dr. Shay Soker at the Wake Forest Institute for Regenerative Medicine (Winston-Salem, N.C.). MDA-IBC3 and SUM149 IBC cell lines labeled with GFP were from Dr. Wendy Woodward at MD Anderson Cancer Center (Houston, Tex.).
MDA-MB-231 cells were cultured in Dulbecco's Modified Eagle's medium, nutrient mixture F-12 (DMEM/F12) (Sigma Aldrich) supplemented with 1% penicillin-streptomycin (P/S) (Invitrogen), and 10% fetal bovine serum (FBS). MDA-IBC3 and SUM149 cells were cultured in Ham's F-12 media supplemented with 10% FBS, 1% antibiotic-antimycotic, 1 μg/ml hydrocortisone, and 5 μg/ml insulin. TIME cells were cultured in EBM-2 endothelial growth media supplemented with a growth factor BulletKit (Lonza CC-4176). All cell cultures utilized in this study were maintained in a 5% CO2 atmosphere at 37° C. in an incubator.
In Vitro 3D Tumor Platform Fabrication:
The in vitro 3D tumor microfluidic platforms utilized in this study were composed of collagen type I matrix seeded with either MDA-MB-231, MDA-IBC3, or SUM 149 with a hollow channel seeded with RFP labeled TIME cells housed in a PDMS scaffold. Collagen type I extracted from rat tails was prepared following published protocols to produce stock collagen concentration of 14 mg/ml which was then neutralized with a solution consisting of 10×DMEM, 1N NaOH, and 1×DMEM to produce a final collagen concentration of 7 mg/ml. GFP labeled IBC and non-IBC cells were seeded at a density of 1×106 cells/mL in the 7 mg/ml neutralized collagen solution and polymerized around a needle at 37° C. for 25 minutes. After polymerization, the needle was removed and the resulting hollow void was filled with a solution of 2×105 TIME cells to form an endothelialized vessel lumen. Once TIME cells have had sufficient time to attach to the collagen matrix, flow was introduced using a syringe pump system. A 72 hour graded flow protocol was used to establish a confluent endothelium followed as we have previously published. Briefly, flow was perfused to expose the endothelium to wall shear stress (WSS) (τ) of 0.01 dyn/cm2 for 36 hours followed by 36 hours of 0.1 dyn/cm2. After completion of the 72 hour graded flow protocols, the in vitro vascularized platforms were exposed to 1 dyn/cm2 for 6 hours.
Immunofluorescent Staining:
Endothelial morphology and cell-cell junctions were analyzed by performing immunofluorescent staining for PECAM-1 and F-actin upon completion of the 78 hour graded flow protocol. The staining protocol consisted of perfusing the platforms with 4% paraformaldehyde and 0.5% triton-X for fixation and permeabilization of the cell membranes, respectively. Next, the platforms were incubated in 5% BSA followed by overnight incubation with antibodies for PECAM-1 (Abcam, ab215911) and Rhodamine Phalloidin (ThermoFisher, R415).
Endothelial Permeability:
Endothelial vessel permeability as a function of paracrine signaling between tumor and vasculature was determined by perfusing the channels with 70 kDa GFP labeled dextran. Four conditions of the 3D in vitro vascularized tumor platforms were tested: TIME cell only platform, and platforms consisting of co-culture of TIME cells with either MDA-MB-231, MDA-IBC3, or SUM149 cells. After completion of the flow protocol for establishing a confluent endothelium, green fluorescent dextran suspended in serum free media (10 μg/ml) was perfused through the platforms with images taken every five minutes. The average fluorescent intensity was measured from the images and used to determine the diffusion permeability coefficient Pa as previously published (Buchanan et al., 2014). Three samples (n=3) were used for each platform condition with the resulting permeability factor expressed as a mean value ±standard deviation. Significance of the data was verified using one-way ANOVA and a 95% confidence criteria.
Enzyme-Linked Immunosorbent Assay:
Expression of VEGF was measured using enzyme-linked immunosorbent assays (ELISA) at two points: upon completion of the graded flow protocol (72 hours) for establishing a confluent endothelium and after exposure to WSS of 1 dyn/cm2 (78 hours). 1 ml samples of perfusion media were collected from the flow outlet and ELISA was performed as per manufacturer's protocol (R&D Systems, DVE00).
Scanning Electron Microscopy:
Scanning electron microscopy (SEM) was performed to determine collagen matrix porosity and observe endothelial adhesion to the collagen matrix. After exposure to 78 hour flow protocol, the platforms were fixed in an aldehyde mixture overnight at room temperature followed by fixation with osmium on ice for 4 hours. Post fixation, the platforms were dehydrated in an ascending series of ethanol solutions (50-70-95%) and then critical point dried by CO2. After drying, platforms were coated with a thin layer of platinum-palladium and high-resolution SEM imaging was performed with Zeiss Supra40 SEM-Electron Microscope.
A novel multi tissue-on-a-chip platform was developed to simulate interactions between healthy/tumorigenic liver and breast tumor microenvironments for drug/nanoparticle development and the dynamic transport of fluorescent nanoparticles was assessed in each compartment. The multi tissue-on-a-chip platform consisting of a vascularized breast tumor and healthy liver microenvironments was developed based upon the vascularized platforms described above. To mimic these microenvironments, cell lines of MDA-MB-231 for breast cancer, C3Asub28 for liver cancer, and THLE-3 for healthy liver were used. Microenvironments were fabricated from type I collagen concentrations of 7 mg/ml and 4 mg/ml for tumor and liver respectively to replicate the growth characteristics and compression moduli of these tissues. Fully functional endothelialized vessels within the tumor and liver microenvironments were formed using a graded flow preconditioning protocol. Wall shear stresses of 4 dyn/cm2 (healthy) and 1 dyn/cm2 (tumor) were employed within each vessel to mimic physiological conditions.
To prove feasibility of microenvironments, cell viability was measured for 3 days and native cell morphology was confirmed with SEM imaging and F-actin/DAPI staining. The fidelity of liver cells cultured in the microenvironment was demonstrated by detecting albumin expression and release in response to physiological shear stress. Dextran particles with sizes of 3 kDa and 70 kDa were per-fused in the platform to replicate the hydrodynamic diameters of chemotherapy drugs and drugs conjugated with nanoparticles. The effect of different co-culture conditions on vessel permeability, ECM/vessel porosity and accumulation of nanoparticles were quantified using intensity profiles in response to different interactions between breast tumor and liver microenvironments to simulate the conditions of drugs being metabolized (liver to breast tumor) and non-metabolized (breast tumor to liver). Ultimately, the physiological multi tissue-on-a-chip platforms developed in this study enabled quantification of drug transport and distribution behavior spatially and temporarily.
The first vascularized multi tissue on-a-chip microenvironments was developed herein for modeling cancerous breast and cancerous/healthy liver microenvironments for studying dynamic and spatial transport of particles. Mechanical properties were tuned to mimic native tissues modeled and cell response, vessel permeability, and porosity of vessel and ECM were assessed. Ultimately, the transport kinetics and accumulation of varying sized fluorescent dextran particles representative of chemotherapeutics and nanoparticle conjugated chemotherapeutics within the tumor and liver microenvironments were determined. The influence of particle delivery to specific tissue microenvironments to simulate direct tumor delivery or metabolism of drugs prior to delivery to the tumor was also investigated.
Cell Morphology and Viability:
MDA-MB-231, THLE-3, and C3Asub28 cell lines were cultured in avascular collagen at concentrations mimicking each tissue's mechanical properties without vasculature for 3 days, and cell morphology for each day was characterized.
Unlike the elongated healthy liver morphology, liver cancer cells formed clusters and the size of each cluster increased daily as previously shown for polymer based in vitro platforms by Shuler et al. and Li et al. and in vivo samples by Siveen et al (Wang et al., 2006; Siven, 2014). Moreover, SEM images more clearly denote cell morphology and its interaction with the surrounding collagen matrix. C3Asub28 cells possess a rounded shape, contrary to the epithelial THLE-3 morphology and pleomorphic MDA-MB-231 cells with elongated shape. Cell morphology similarities between day 3 and SEM images showed SEM preparation did not affect cell and matrix properties. The observed morphological elongation for healthy liver cells and aggregation behavior of breast and liver cancer cells is due to cell-cell and cell-ECM interaction as previously mentioned by collagen based in vitro studies (Szot et al., 2011).
Albumin Expression and Release of Healthy Liver Cells:
The functionality of healthy liver cells was determined by detecting albumin expression and release. Albumin expression and release was measured for collagen based vascularized THLE-3/TIME microenvironments for the first time in this study.
First, in
Porosity of Vasculature:
After embedding cells in collagen and successfully preconditioning endothelialized channels for 72 hours to establish a confluent, aligned endothelium, the effect of different co-culture conditions on the vessel confluence was studied as shown in
The last three conditions incorporated different cell types within the collagen ECM in addition to the TIME culture: C3Asub28/TIME, MDA-MB-231/TIME, THLE-3/TIME microenvironments. A tight confluent endothelial lumen in which fluorescence is shown with minimal dark gaps between cells is apparent for the Control −. The vascularized endothelium co-cultured with THLE-3 showed a very similar endothelial confluency compared to Control −.
Moreover, it was observed that artificial modulation of the vessel with TNFα treatment, (Control +), caused vessel permeabilization with significant pore openings compared to Control −. On the other hand, the tumor vessels exhibit a patchy and leaky endothelium with perivascular detachment and non-uniform gaps unlike the uniform, dilated openings of Control +. This strengthened the idea that the cross-talk between cancer and endothelial cells cause a leaky porous domain, leading to the well-known EPR effect, also shown in vascularized tumor microenvironments33. Vessel porosity of varying vascularized tissue microenvironments was reported for the first time in this study and presented in
It was observed that co-culture with THLE-3 did not affect vessel porosity significantly compared to the Control −. Therefore, the change observed for endothelial integrity in cancer microenvironments compared to Control − was mostly likely due to signals provided by the cancer cells. One other reason for the observed patchy endothelial structure could be due to the heterogeneous distribution of cell clumps observed in
Ecm Porosity:
Following transport through the endothelium, nanoparticles or drugs must navigate the ECM to reach the tumor cell, therefore, the ECM structural properties of the microenvironments were characterized. Collagen concentrations of 7 mg/ml and 4 mg/ml were used to create the ECM to replicate the compression modulus of tumor and healthy liver microenvironments. SEM images presented in
Quantified ECM porosity results presented in
Vessel Permeability of Microenvironments:
Permeability was measured using for two different dextran particle sizes (3 and 70 kDa) for 5 different types of microenvironments (acellular (no endothelial cells lining the vessel and no cells in the ECM), TIME monoculture (endothelialized vessel with no cells in the matrix), C3Asub28/TIME, MDAMB-231/TIME and THLE-3/TIME microenvironments). The permeabilities of C3Asub28/TIME and THLE-3/TIME microenvironments were measured for the first time in this study.
Accordingly, permeability of mentioned microenvironments are presented in
However, the presence of cancer cells such as MDA-MB-231 and C3Asub28 with endothelial cells caused higher permeability compared to endothelium monoculture and THLE-3/endothelial microenvironment. The difference between normal and hepatocellular carcinoma was observed as evidenced by cancerous cells increasing permeability by 2.77 (p<0.001) and 2.35 (p<0.05) fold for 70 and 3 kDa particles, respectively. Previous studies on vascularized tumor-endothelial microenvironments also showed similar findings in which an increase in transport of macromolecules occurred due to inclusion of cancer cells (Jain et al., 2014).
There are two underlying reasons for this difference between the two liver cell lines. First, drug has been perfused through normal liver with higher wall shear stress to generate physiological transport. Secondly, due to interaction between cancer and endothelial cells or tumorigenic protein release by cancer cells, endothelial layer porosity decreases, which has been discussed in the previous section. This phenomenon is described as the EPR effect and is more significant compared to high wall shear stress. The presence of tumor cells inside the ECM increased vasculature permeability increasing the likelihood of tumor cell invasion and migration into the endothelial layer. Cancer cells significantly influenced the endothelium as evident by large pores shown in
For all microenvironments, 3 kDa dextran particle were more permeable in the microenvironments than 70 kDa. In vivo drug testing studies have shown that nanoparticle size highly influences permeability (Venkatasubramanian et al., 2008). The relationship between hydrodynamic diameter and permeability coefficient can be explained using Stokes-Einstein Equation of diffusivity (Yuan et al., 1995). By definition, particle size is indirectly proportional to permeability, which is indicated with higher diffusivity of smaller particles. Therefore, more rapid diffusion was observed for 3 kDa particles compared to 70 kDa which results in a higher permeability coefficient. Moreover, the presence of endothelial layer around the vasculature yielded a reduction in permeability coefficient of dextran particle since matrix pore openings were blocked with endothelial monolayer as described in
The validity of permeability measurements were assessed by comparing the fold change between the same Control − and Control + findings in the literature. Collagen based vascularized breast cancer platform developed by Zervantonakis et al. reported the fold change between Control − and Control + as 1.79±0.27, while is similar to our measured value of 1.59±0.13 in this study. Moreover, permeability coefficient of 70 kDa dextran particle was reported as 25.67±1.79 nm/s in vascularized collagen based tumor microenvironments for the same shear stress which compares well with our permeability results (26.83±2.19 nm/s).
Intensity Profiles and Accumulation:
In addition to permeability, intensity profiles of particle fluorescence within the vessel and ECM provided insight regarding the accumulation of each type of particle in the different tissue microenvironments. Spatial and temporal quantification of particle accumulation rate in the ECM and vessel were presented for the first time. 3 and 70 kDa dextran particles were used to mimic chemotherapy and chemotherapy/nanoparticle conjugated drug sizes, specifically for 1.9 and 12.6 nm hydrodynamic diameter, respectively.
Additionally, the increase in maximum intensity over time with smaller particle size at the center of the vessel was observed for tumor cell lines. For microenvironments containing tumor cells, peak intensity of small particle was normalized to large particle sizes and found to be 1.66 and 1.59 fold for MDA-MB-231 and C3Asub28 microenvironments, respectively. This trend can be explained by both collagen based vascularized 3D in vitro tumor microenvironments and modeling studies (Buchanan et al., 2014) for two main reasons. i) Advective transport through the vessel is more dominant than diffusive Brownian motion into the ECM. ii) Particles are diffusing and then leaving the ECM which causes accumulation around the vessel. Given the fact that vessel porosity in tumor microenvironments is significantly higher compared to healthy tissue microenvironments as seen in
Moreover, the peak intensity for liver tumor microenvironments increased by 2.77 and 4.48 fold (p<0.01) for 3 and 70 kDa, respectively. A similar trend was observed for breast tumor microenvironments in which 1.39 and 3.65 fold (p<0.05) increases occurred for 3 and 70 kDa respectively, whereas healthy liver peak intensity did not change significantly compared to Control −.
Connecting the vascularized liver and breast tumor microenvironments in series and perfusing particles in either vessel enabled simulation of the accumulation behavior of metabolization of particles (liver to tumor) or direct delivery to the tumor (tumor to liver). With both microenvironments connected, independent of which microenvironment received particles first, a significant decrease was observed in the magnitude of the intensity in the second microenvironment (
However, as healthy liver was replaced with liver tumor, breast tumor peak intensity increased by 1.31-fold (p<0.05) for 3 kDa and decreased by 2.60 fold (p<0.05) for 70 kDa. To gain further understanding, the intensity rates were determined using data from
This phenomena can be explained with two main reasons: First, the leakiness of endothelial layer causes EPR effect and second, significantly lower shear stress allows more time for particles to diffuse through the ECM similar to work described by Buchanan et al. regarding permeability change with respect to wall shear stress. Although it was concluded that ECM porosity of THLE-3/TIME microenvironment is higher than the ECM of both cancer microenvironments, particle accumulation in the MDA-MB-231/TIME and C3Asub28/TIME microenvironments are 3.45 and 4.81 fold (p<0.05) higher than healthy liver, respectively. This indicates that vessel porosity plays a more dominant role compared to ECM porosity in the accumulation rates of particles in ECM. Particle accumulation in the vessel on the other hand is a factor which should be controlled since this will enhance the likelihood of particles being delivered to other healthy tissues causing toxicity as is shown by in vivo drug distribution studies (Dong et al., 2015).
Also, having higher ECM accumulation in the liver cancer compared to breast cancer was anticipated based on having higher ECM porosity presented in
When multiple microenvironments were connected however, particle accumulation was expected to change due to particles remaining in the microenvironment perfused first before entering the second microenvironment.
In vivo studies with similar particle sizes also emphasized that nanoparticles with hydrodynamic diameter close to 15 nm have greater probability of accumulation in the tumor. Moreover, vessel accumulation under the interaction significantly decreased in secondary microenvironment in all cases as seen in
This outcome was observed for both cases in which the particles being metabolized (liver to tumor) were simulated as being directly delivered to the tumor (tumor to liver). When small particles were perfused through the liver first, breast tumor ECM accumulation was decreased by 5.49 fold (p<0.01) compared to perfusing through the tumor first. Simulated metabolized and non-metabolized cases where healthy liver was replaced with liver tumor cells, particle accumulation was decreased by 1.05 and 3.94 fold (p<0.05) for 3 and 70 kDa particle sizes, respectively.
Thus, it was shown that the multi tissue-on-a-chip devices has greater potential than standard cell culture, static in vitro setups, and if the system is complex enough, it can augment or replace animal testing for advanced drug development before clinical studies. The tissue on-a-chip microenvironment developed in this study provides a system that mimics transport in vivo enabling spatial and dynamic assessment of transport of any type of drug/nanoparticle as a function of their size. This device can be used to investigate the influence of other drug/nanoparticle properties including surface charge, dimensionality, targeting ligand, and aspect ratio on transport. By altering the direction of flow the effect of targeting and metabolism on transport kinetics of drugs/chemicals can be simulated in high throughput, inexpensive optimization of nanoparticles or other therapeutics by enabling toxicity, efficacy, and biodistribution measurements as a function of varying microenvironmental conditions and drug/nanoparticle properties. The multi tissue-on-a chip microenvironments can also be utilized for testing a combination of different treatment methods such as hyperthermia, radiation, and a myriad of nanoparticles with unique functionality to create solutions for targeted delivery.
Human Cell Sources: Human breast cancer cells (MDA-MB-231), healthy liver cells (THLE-3), carcinoma liver cells (C3Asub28), and telomerase immortalized microvascular endothelial cells (TIME) were used in this study. MDA-MB-231 cells (American Type Cell Culture, ATCC, VA, HTB-26) were cultured with Dulbecco's Modified Eagle's medium, nutrient mixture DMEM/F12 (1:1)+LGlutamine, +15 mM HEPES (Invitrogen, CA) supplemented with 10% fetal bovine serum (FBS, Sigma Aldrich, MO), and 1% Penicillin/Streptomycin (P/S, Invitrogen, CA). TIME cells stably transduced with an mKate lentivirus were generously provided by the Wake Forest Institute of Regenerative Medicine, Winston-Salem, N.C. These cells were cultured in Endothelia Basal Medium-2 (EBM-2, Lonza, MD) and supplemented with an Endothelial Growth Media-2 (EGM-2) SingleQuotsO Kit (Lonza, MD), which contains 2% FBS, hydrocortisone, Vascular Endothelial Growth Factor (VEGF-2 ng/mL), Human Fibroblast Growth Factor-basic, (hFGF-B, 4 ng/mL), R3-insulin growth factor, ascorbic acid, human epidermal growth factor, GA-1000, and heparin. Human carcinoma liver, C3Asub28 cells were generously provided by Dr Wei Li from the University of Texas at Austin. These cells were cultured with DMEM/F12 (1:1)+L-Glutamine, +15 mM HEPES with 10% FBS, and 1% P/S. THLE-3 cells (ATCC, VA, CRL-11233) were cultured in BEGM Bullet Kit (Lonza, MD) with additional 5 ng/mL Epiderman Growth Factor (EGF, Invitrogen, CA), 70 ng/mL Phosphoethanolamine (Acros Organics, Belgium), and 10% FBS in a pre-coated flask. All cells were incubated at 37° C. and 95% atmospheric air/5% CO2. Cell growth was monitored every day and cells were used in experiments when they were 70% confluent. All cell lines were used at the first 8 passages.
Tissue Properties and Preparation of Collagen:
Type I collagen was used as the primary extracellular matrix (ECM) component for each tissue microenvironment. Stock solution of type I collagen was prepared by dissolving excised rat tails in an HCl solution at a pH of 2.0 for 12 hours at 23° C. (Buchanan et al., 2013). The solution was then centrifuged at 23° C. for 45 minutes at 30000 g and supernatant was collected and lyophilized. The lyophilized collagen was mixed with diluted 0.1% glacial acetic acid, maintained at 4° C. and mixed every 24 hours for 3 days to create a collagen stock solution. Finally, collagen was centrifuged at 4° C. for 10 minutes at 2700 rpm to remove air bubbles. Since ECM stiffness directly affects cell-matrix interactions, such as cell adhesion/proliferation, and diffusivity of drugs into the tissue, and that collagen concentration dictates ECM mechanical properties, it is critical to select an appropriate final collagen concentration to mimic human desired tissue properties, which also controls tissue porosity. Yeh et al. reported that hepatic tumor microenvironment has stiffness of 3 kPa (Yeh et al., 2002). Similarly, breast cancer tissue stiffness is reported as 4 kPa. Chen et al. reported the healthy human liver compression modulus varies between 0.59-1.73 kPa (Chen et al., 1996). Therefore, the final collagen concentrations for liver and breast carcinomas of 7 mg/ml were employed since previous studies reported corresponding compression modulus of 3-6 kPa (Buchanan et al., 2013). A collagen concentration of 4 mg/ml was used to create the normal liver tissue with corresponding compression modulus of 0.90-1.91 kPa.
Device Design and Fabrication:
An aluminum mold, was fabricated using micromilling techniques, which eliminates multistep fabrication processes and necessity of expensive patterning reagents compared to conventional fabrication technique (photolithography). Well mixed Polydimethylsiloxane (PDMS) with curing agent of 10:1 ratio was poured inside the aluminum mold and baked for 1 hour at 75° C. Solidified PDMS, which is the housing material, consists of inlet and outlet channels, was peeled off from the mold and sterilized under UV for 1 hour with 25×25 glass slide before the bonding process. Then, glass slide and PDMS were plasma treated (Harrick Plasma) and bonded to create the enclosure to surround the tissue microenvironment. To increase adhesion between collagen and PDMS housing, fabricated PDMS housing assembled with glass slide was filled with sterile 1% Polyethylenimine (PEI, Sigma-Aldrich, MO) (diluted with DI H2O) and incubated for 10 minutes. After aspirating PEI, channels were filled with 0.1% glutaraldehyde (Sigma-Aldrich, MO) (diluted with DI H2O) and incubated for another 20 minutes. Glutaraldehyde was removed and the platform was washed twice with sterile DI H2O. Collagen solution was neutralized to pH of 7.4 with 1×DMEM, 10×DMEM and 1N NaOH and mixed with intended cell line at a concentration of 1×106 cells/ml. Collagen-cell mixture was injected into the platform to fill the enclosure. Final concentrations of collagen was selected as 4 and 7 mg/ml for healthy and tumorigenic tissues, respectively, to match human compression modulus of relevant tissue type. A needle was inserted inside the platform to form hollow vessel before the polymerization of collagen. The needle size of 22 and 27G (Jensen Global, CA) were used for tumor and healthy liver tissues, respectively, to provide the relevant physiological wall shear stress (WSS) in the tissues. Applied wall shear stress is a significant phenomenon to mimic human tissue as well as protein release by the cell lines. In a clinical study performed by Karin et al. showed that human wall shear stress in vessel varies between 1-10 dyn/cm2 as well as wall shear stress decreases down to 1 dyn/cm2 for tumor microenvironments. However, wall shear stress higher than 4 dyn/cm2 showed decrease in albumin release according to in vivo study. Therefore, needles at given size were inserted respectively for tumor and healthy liver microenvironments to provide 1 and 4 dyn/cm2 wall shear stresses at the same flow rate. After the incubating the platform for 30 minutes at 37° C. and 5% CO2, the collagen was polymerized and presence of needles created a hollow vessel inside housings.
To create a fully functional aligned endothelium along each channel within each compartment, TIME cell suspension in media (10×106 cells/ml) was introduced in the channel and underwent flow preconditioning for 3 days. Within the first 36 hours, wall shear stress was maintained at 0.01 dyn/cm2 and followed with a linear increase of wall shear stress to 0.1 dyn/cm2 for 1 hour and maintained at this value for the next 36 hours. In the last 6 hours, wall shear stress was linearly increased to physiological wall shear stress. For positive control samples, 20 ng/ml Tumor Necrosis Factor Alpha (TNFα, RnD Systems, MN) was perfused at 0.1 dyn/cm2 for 24 hours after the preconditioning protocol before transport studies. To provide flow into the microfluidic platform, 0.5″ long 22G stainless steel needles were inserted though PDMS ports and partially into the collagen microchannels. Autoclaved Tygon silicon tubing ( 1/16″ ID, Saint Global, France) was connected to the inlet needle and a bubble trap, which is connected to a syringe pump that controls the flow rate. The bubble trap eliminates the likelihood of washing out endothelial cells from the created vessel with the effect of introduced bubble in the platform channel. The outlet needle was similarly connected to silicon tubing that collects the media into a reservoir. Two chambers were connected using 22G pins and the same silicon tubing. Detailed images of the platform before and after assembly and preconditioning are given in
The viability was assessed in avascular platforms to measure growth kinetics of cells located in the ECM. Identical platform preparation protocol was followed as described in the previous section without incorporation of an endothelialized channel. To maintain consistency avascular platforms were cultured with endothelial cell culture media to maintain cell viability. Cell viability was measured using CellTiter-Blue (Promega, WI) Assay over the course of three days. Fluorescent intensity units was converted to cell concentration using the obtained calibration data in this work.
Cell Morphology:
Cell morphology at day 0, 1, and 3 were determined as described previously. Briefly, avascular platforms were fixed with 3.7% paraformaldehyde and permeabilized using 0.1% Trition X-100 (Sigma Aldrich, MO). Then, samples were blocked with 1% BSA (Santa Cruz Biotechnology Inc, CA) for 30 minutes at room temperature followed by an incubation step with rhodamine phalloidin (Invitrogen, CA), a high-affinity probe for F-actin. Samples were counterstained with DAPI (Vector Laboratories, CA), to visualize nuclei. Imaging was performed using Leica SP8 laser scanning confocal microscope. Another set of vascularized platforms were fixed to investigate cell morphology and ECM porosity using Scanning Electron Microscopy (SEM). Aldehyde mixture composed of 0.2 M cacodylate buffer, glutaraldehyde, paraformaldehyde, cation stock, and DI H2O were prepared and fixed at room temperature for 4 hours and washed three times with cacodylate buffer for 15 minutes. Subsequently, reduced osmium solution (1:1) composed of 4% potassium ferrocyanide in 0.2 M cacodylate buffer and 4% aqueous osmium tetroxide was added to samples and maintained on ice for 4 hours. Fixed samples were washed with DI H2O 5 times for 10 minutes afterwards and dehydrated with 50, 70, and 95% ethanol once and twice with 100% ethanol for 15 minutes each. Samples were dried using a critical point drying method, coated with 12 mm thick Pt/Pd layer and imaged with Zeiss Supra40 SEM. All reagents were purchased though Electron Microscopy Sciences, PA.
Assessment of Transport Properties and Quantification:
Transport measurements of varying particle sizes in the multi tissue-on-a-chip microenvironment were conducted for two different scenarios: single microenvironment analysis and microenvironments connected in series to investigate the influence of their interactions and interdependent transport kinetics. When considering only a single microenvironment transport, particles were delivered through the vessel of the microenvironment of interest and transport through the vessel and into the surrounding ECM was measured subsequently and spatially. In these tests, 5 different platform configurations was used: acellular with no cells in the ECM and no cells in the vessel, TIME monoculture consisting only endothelial cells lining the vessel without cells in the ECM, and then vascularized microenvironments denoted as cells in the ECM/cells in the vessel: MDA-MB-231/TIME, C3Asub28/TIME, and THLE-3/TIME microenvironments. Microenvironments were connected in series to consider the influence of interactions between them. Particles were perfused through the first microenvironment's channel with associated diffusion into the corresponding ECM and back into the vessel which resulted in transport to the next tissue compartment. Four different multi tissue-on-a-chip configurations were considered: MDA-MB-231 to C3Asub28, MDA-MB-231 to THLE-3, THLE-3 to MDA-MB-231 and C3Asub28 to MDA-MB-231. Cases when particles were introduced directly in the vessel corresponding to the breast tumor was meant to simulate direct delivery to the breast tumor, where particles are not metabolized, and cases when particles were first introduced into the vessel associated with the liver simulated metabolization by the liver. Passive transport of particles through blood vessels within the previously described microenvironments depends on the permeability of the each vessel endothelialium and the porosity of the vessel and ECM of each tissue. Particle transport begins in the blood vessel, which is surrounded by endothelial cells and ECM. Endothelial integrity controls the barrier function and regulates transport of particles. According to in vivo studies, the gaps between endothelial cells are significantly higher in tumors vessels compared to healthy tissue vessels, and is referred to as the enhanced permeability and retention (EPR) effect. Furthermore, this leakiness of the endothelium may also allow particles to diffuse back into the vessel from the ECM, which creates the vessel accumulation. Additionally, the ECM can act as a sink trapping particles leading to accumulation within the tissue. Therefore, ECM and vessel porosity and permeability, which affect intravasation and extravasation of particles need to be characterized to fully describe expected transport of particles. The effect of porosities on diffusion is also stated by Darcy's Law given in Equation 7: u=K∇P/ζμ=z m (7) where μ is velocity in the porous domain, m is viscosity, ∇P is the pressure gradient vector, and K is hydraulic permeability. This equation suggests porosity (ζ) within the vessel and ECM determines the effectiveness of particle transport through ECMs. The velocity in the porous domain depends on porosities in each domain which will consequently affect permeability and transport of the vessel and ECM.
Therefore, endothelial porosity was determined using fluorescence microscopy images of mKate tagged endothelial cells. ECM porosity is obtained by analyzing SEM images of ECMs as described in the previous section using ImageJ. Selection of particle size is an important factor that controls the circulation time, tumor uptake, and ability of the particle to penetrate the tissue. Common chemotherapy drugs used for breast cancer treatment such as doxorubicin has hydrodynamic diameter in the range of 1.06-1.89 nm, which can also calculated using molecular weight and density of drug. Although the hydrodynamic diameter of nanoparticle chemotherapy conjugated drugs has great variability depending on nanoparticles type, size, and shape, it has been shown that common nanoparticle-chemotherapy conjugated drug size varies between 5-50 nm. In this study, 3 and 70 kDa dextran particle sizes (Sigma-Aldrich, MO), with hydrodynamic diameter of 1.9 nm and 12.6 nm respectively, were selected representing chemotherapy and chemotherapy-nanoparticle conjugated drugs, respectively, to demonstrate the EPR effect on the developed microenvironments. The effect of vessel and ECM porosities and particle size on transport were quantified using two methods, permeability coefficient and intensity profiles of the particles in the vessel and ECM. Fluorescent dextran particles suspended in serum free (to prevent nanoparticle aggregation) endothelial basal media (EBM-2) to the final concentration of 10 mg/ml were perfused through the vascularized microenvironment for 2 hours with a flow rate of 260 mL/min, which yields physiologically representative shear stress in both microenvironments considered with appropriate vessel diameter. Images were taken every 3 minutes using a Leica SP8 Confocal Microscope. Obtained images were exported to Matlab to quantify intensity readings at each time step. For the first method, permeability coefficient was calculated using Equation 8
where Ib is the background intensity, I1 is the average initial intensity, I2 is the average intensity after recovery, time interval Δt(s), and d(μm) is the diameter of the microchannel. By definition, this parameter quantifies the ability of particles to penetrate from the microchannel to vessel wall then to the ECM and allows observation of the EPR effect. The last five or more consecutive data points from the 2 hours of flow were used to calculate Pd. Data was expressed as a mean value ±standard deviation. TNFα stimulation of endothelialized vessels within the microenvironments modulates permeability, which has been shown by Zervantonakis et al., Accordingly, TIME monoculture (Control −) and TNFα treated endothelial vasculature monoculture (Control +) were prepared according to previously published permeability results and fold changes were compared to these studies validating the accuracy of experiments. For the second method, transport was quantified based on intensity profiles across the ECM boundaries when only one microenvironment was considered or when two microenvironments were connected in series with one another. Additionally, the same data was used to quantify the intensity change in the vessel and ECM to observe the rate of accumulation of different particle sizes in each compartment.
Statistical Analysis:
Student's t-test was used to identify the significance level of differences between multiple data sets. In all subsequent figures, p-values are denoted with either asterisks (*) or pound symbols (#) as follows: (*) or pound symbols (#) as follows: * or #: significant at p<0.05, ** or ##: significant at p<0.01, *** or ###: significant at p<0.005, **** or ####: significant at p<0.001. The number of replicates varied depending on the test type: N=4 for viability test, N=5 for ELISA measurements, N=3 for permeability coefficient and accumulation analysis, N=4 for ECM porosity and images were taken at 4 different locations of the ECM, and N=4 for vessel porosity.
Matrix Properties:
The extracellular matrix (ECM) and cellular composition, mechanical, and diffusion properties of the in vitro platform, will be compared to and tuned to match in vivo and patient data for breast tumors, liver, and heart. Collagen concentration influences diffusivity, porosity, and stiffness of the hydrogel, which affects NP-cell-matrix interactions, including matrix remodeling and NP diffusion. Collagen type I will be isolated from rat tail tendons and reconstituted to concentrations of 6-12 mg/mL to form the extracellular matrix for all tissues. This concentration range will yield elastic moduli representative of breast tumors while also maintaining integrity of microchannels under flow and promote cell proliferation. Collagen concentration will be tuned to match Young's modulus for in vivo myocardium (20 kPa-500 kPa) and liver (300 Pa-600 Pa) and verified by measuring the elastic modulus using confined uniaxial compression as we have published.
Cell Culture and Characterization:
The matrix and cellular composition will be tuned to match histology data from respective in vivo tissues. A transformed human microvascular endothelial cell line tagged with mCherry (TIME) will be introduced to form the endothelialized microchannels of all tissues. For the tumor, MDA-MB-231 and inflammatory breast cancer cells (IBC) are labeled with green fluorescent protein, in separate experiments. For the liver, primary hepatocytes and liver sinusoidal endothelial cells or the HepG2 derivative C3A-sub28 cell line with enhanced expression of CYP3A4 mRNA and CYP3A4-mediated activity are cultured in collagen. For the heart, cardiomyocytes (ATCC PCS120010) and 3T3 fibroblasts (ATCC) are used. Prior to inclusion in collagen, hepatocytes, cardiomyocytes, and C3A-sub28 cell lines are stably transfected to express GFP, enabling visualization of cells with confocal laser scanning microscope. All cells (1×106-100×106 cells/ml) are suspended in collagen during polymerization. TIME cells are injected in the microchannel at a density of (10×106 cells/ml), a quantity sufficient to form a confluent lumen.
A vascularized multi-layer skin platform consisting of a dermal layer made of collagen seeded with normal human dermal fibroblasts (NHDFs) and an epidermal layer consisting of a collagen/keratin blend with immortalized keratinocytes was developed (
An IBC in vitro tumor platform was created (
The permeability of blood and lymph vessels was tuned to promote cell migration into and out of these vessels. The influence of vessel leakiness on tumor invasion into the lymph and vascular channels was investigated. Migration of tumor cells, either as a collective migration of tumor emboli as clusters (IBC) or single cell migration (MDA-MB-231) into surrounding vessels is a well described aspect of metastases and treatment resistance in breast cancer with broader implications for other diseases. The single-channel tumor platform was used to study signaling between breast cancer cells within the collagen gel (MDA-MB-231, MDA-IBC3 and SUM 149) and a complete endothelium on the lumen (TIME cells) in response to fluid shear and demonstrated the role of cellular interactions on endothelial permeability as shown in
Combining the embodiments of the multi-layer skin platform and the single layer vascularized platforms as shown in
In order to create the comprehensive tumor platform, each layer was fabricated in succession from the bottom to the top. Each tissue layer was separated by a semipermeable membrane to promote integrity and distinct layers as shown in
Keratose Extraction: Keratose was extracted from human hair obtained from a local barber. Briefly, human hair was chopped into small pieces and soaked in a solution of 2 wt %/vol % paracetic acid (Sigma-Aldrich, St. Louis, Mo.) in DI water for 12 hours. Hair was then filtered from the liquid with a 500 μm sieve (W. S. Tyler, Mentor, Ohio) and rinsed to remove excess oxidant. Free proteins were extracted in excess 100 mM Tris base for 1 hour subsequently followed by DI water for 1 hour and transferred to a shaker at 37° C. at 180 rpm. Extracts were collected with the 500 μm sieve, neutralized, centrifuged, and finally filtered. Extracts were then dialyzed for 24 hours and lyophilized.
Cell Culture:
Primary normal human dermal fibroblasts (NHDFs, PromoCell, Heidelberg, Germany) were used in this study because of their ease of growth and handling and are also the most common and general cell type found in the healthy tissue. MDA-MB-231 breast cancer cells were also used to mimic cancerous tissue. Both cells were seeded in a t-75 Eppendorf HEPA-filtered flask (Eppendorf, Hamburg, Germany) and cultured in fibroblast basal medium 2 (PromoCell, Heidelberg, Germany) supplemented with 10 ml of fetal calf serum, 0.5 ml of human fibroblast growth factor, 2.5 ml of human insulin (PromoCell, Heidelberg, Germany), and 1% penicillin-streptomycin for NHDF and DMEM/F12 50:50 basal medium (Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin for breast cancer cells. Cells were maintained in 5% CO2 atmosphere at 37° C. in a sterile incubator. Media was changed every 2 days. Cells were detached for use in gels by aspirating media, neutralizing with 5 ml of phosphate buffered saline (PBS), and consequently aspirating the PBS. 3 ml of 0.04% trypsin (PromoCell, Heidelberg, Germany) for NHDF and 0.25% trypsin for breast cancer cells were added to the flask and incubated for 3 minutes. Trypsin/cell solution was neutralized with complete media and transferred to a 15 ml conical tube. Cells were centrifuged at 120 g for 5 minutes. Cell pellet was isolated and resuspended in 1 ml of complete media for cell-counting and use.
Collagen Gel Fabrication:
A working collagen solution of 1.5 and 3 mg/ml for NHDF, and 5 and 7 mg/ml for was prepared from the collagen stock solution by neutralizing with 10×DMEM (Sigma-Aldrich, St. Louis, Mo.), 1×DMEM (Gibco™ Gaithersburg, Md.) and 1 N NaOH (Fisher Scientific, Hampton, N.H.) that resulted in a pH of 7.4. For cell culture experiments, cells were embedded in the gel, resulting in a final concentration of 0.3 million and 0.5 million cells/ml for NHDF and breast cancer cells, respectively. The hydrogel solution was then added to cell culture-treated 96 well plates (Sigma-Aldrich, St. Louis, Mo.) and incubated at 37° C. for 40 minutes to allow polymerization. 100 μl of complete media was then added on top of each gel. Media was changed every 2 days.
Collagen/Keratose Gel Fabrication:
50/50 and 20/80 w %/w % collagen/KOS hydrogels were prepared by combining both stock and KOS dissolved in neutralizing buffer of equal volume. For example, to create a 3 mg/ml gel, 6 mg/ml (50/50) and 24 mg/ml (20/80) KOS in neutralizing buffer was prepared to mix with 6 mg/ml collagen stock. These different concentrations were employed for gels using varying stock collagen and C/K gel weight percentages (50/50 vs 20/80). Stock collagen and KOS/neutralilzing buffer solution was combined at 1:1 v:v ratio and mixed thoroughly with a spatula Gels were added to either cell culture-treated 96 well-plates and incubated at 37° C. for 40 minutes to allow polymerization. For cell culture experiments, gels were seeded with 6.5×104 cells/ml and supplied with 100 μl of complete media. Media was changed every 2 days.
Fiber Diameter and Porosity Measured Using SEM Images:
SEM images of various acellular gels were taken to determine if there was any difference in porosity and fiber size. As observed qualitatively in
In order to determine quantitatively the porosity and fiber diameter of the various gels, ImageJ® was utilized. Porosity and fiber size is important in studying cell ECM interactions. Cells grown in a 3D gel can behave differently compared to a 2D plate due to the fiber network, allowing their morphology to change into a spindle shape. Collagen concentration can also influence material properties such as fiber structure, and in turn influencing cellular response. As shown in
Thermally Stability Measurements by DSC and TGA:
DSC was utilized to determine whether addition of keratin increased the protein denaturation temperature when compared to 100% collagen. As shown in the
Cell Viability of Fibroblast in Hydrogels:
Viability of fibroblasts in numerous gel types was measured using CellTiter Blue viability assay. As shown in
Morphology and Growth of Fibroblasts in Hydrogels:
Morphology and growth were analyzed by confocal imaging of fibroblasts stained for actin over the course of 7 days. Fibroblasts proliferated and spread out over the course of 7 days in both the 100% collagen and 50/50 C/K gels. The morphology of the fibroblasts in the 50/50 C/K gel however is more spread out and elongated by day 7. Fibroblasts did not spread out and proliferate in the 20/80 C/K gel as shown in
In an effort to overcome the complex barriers for nanoparticle delivery, recent preclinical and clinical studies have emphasized the significance of the tumor microenvironment as a potential therapeutic target to improve delivery into the tumor. These therapies often use focused physical perturbation of the tumor microenvironment to increase the targeting potential of systemic nanoparticles. More specifically, disrupting the tumor vasculature to enhance extravasation of systemic therapeutics into the tumor has been extensively investigated. While development effort has primarily focused on adjuvant therapeutics such as anti-angiogenesis antibodies or molecular approaches (VEGF, TNFα) to perturb the tumor vasculature and enhance extravasation of anti-neoplastic agents, physical or energy-based solutions (heat, acoustic energy, electroporation) are also currently explored. Despite the challenges associated with nanoparticle photothermal ablations, there remains great promise to utilize these particles for tumor-localized mild hyperthermia (41-45° C.) that enhances nanoparticle transport by modulating the tumor vasculature and dense extracellular matrix (ECM) of the tumor microenvironment. Mild hyperthermia has previously been shown to increase tumor blood flow and tumor microvascular pore size (<400 nm), which can amplify transvascular nanoparticle mass transport into the tumor where particles can accumulate owing to the dysfunctional lymphatic clearance. Additionally, local heating has been shown to strongly affect collagen fiber structure and mechanical properties which improves penetration through the tumor ECM.
In this study, the effects of mild hyperthermia (42° C.) were compared on the mass transport of SWNHs in a traditional 2D cell culture model and a set of relatively simple and high throughput 3D platforms. The results of this study highlight the potential mechanism of synergy between mild hyperthermia and nanoparticle transport and demonstrate the inability of 2D cell cultures, and oversimplified 3D cultures to probe these nanoparticle transport dynamics. After initially characterizing the SWNHs, it was demonstrated that SWNH transport in the tumor microenvironment is primarily enhanced by thermal targeting of an already leaky tumor vasculature, mirroring results in previous in vivo studies. Importantly, as a TE with an internal microfluidic channel was utilized with physiological fluid flows and barriers to nanoparticle drug-delivery (extravasation, diffusion into ECM, and cellular uptake), it was demonstrated that an increase in SWNH permeability exclusively results in higher concentrations into the tumor space in a tumor-vascular co-culture setup. As such, there is potential for further refinement for selective tumor-specific nanoparticle transport enhancement that limits drug delivery to off target tissue. Together, this study highlights the importance of early studies of nanoparticle transport in the tumor microenvironment when nanoparticles are utilized as co-delivery vehicles for generating hyperthermia and delivering drug payloads and demonstrates that these studies can be accomplished using relatively simple and inexpensive 3D TE models.
Increased vascular permeability that occurs as a result of a dynamic endothelial response to mild hyperthermia would enhance nanoparticle extravasation from the vasculature, this increase may not result insignificant enhancement of nanoparticle penetration deep into the tumor interstitial space. It has previously been shown that nanoparticles between 100-300 nm in diameter would be expected to be able to diffuse 100 μm into the tumor after extravasating from the tumor vasculature. As successful nanoparticle-based drug delivery therapies would require sufficient nanoparticle transport to this distance, the present study focused on understanding how mild hyperthermia enhances SWNH-QD penetration 100 μm into the platform.
As no difference was observed between the two groups at different temperatures, it was sought to understand the difference in vascular response between the co-culture and endothelial monoculture microfluidic models by comparing the destabilization of the endothelial monolayer. Representative images after staining for F-actin in samples exposed to 42° C. for 1 h are seen in
All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
The present application claims the priority benefit of U.S. Provisional Application Ser. No. 62/523,004, filed Jun. 21, 2017, the entire contents of which are hereby incorporated by reference.
The invention was made with government support under Grant No. R21 EB019646 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/038734 | 6/21/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62523004 | Jun 2017 | US |
