Patent Application
20240141272
The present subject matter relates to a cell culture exposure system and related methods that enable high-throughput air-liquid interface exposures to multiple doses of particles or volatile compounds while maintaining appropriate cell culture conditions.
Various prior art exposure systems were evaluated in their ability to achieve serial dilution and delivery of volatile organic compounds (VOCs), ultrafine particles/nanoparticles, i.e., typically less than 100 nm, or fine particles, i.e., typically less than 2.5 μm, at an air-liquid interface (ALI) and their ability to maintain physiologically relevant conditions, for example a temperature of 37° C., and relative humidity (RH) of greater than 80%.
Two systems in widespread use are commercially available under the designations CULTEX and VITROCELL. These systems offer dynamic exposure conditions for volatile compounds, such as cigarette smoke, e-cigarette vapor, and nanoparticles, but cannot be used reliably for fine particles, i.e., defined as an aerosol aerodynamic diameter less than 2.5 μm. An exception to this is the VITROCELL CLOUD system which includes an integrated nebulizer and promotes high deposition efficiency for particles via sedimentation within a confined, static chamber. An alternative nebulizer design by Harvard College is also available for larger droplets which are directly applied to a cell surface.
When the above noted exposure apparatuses are used in aerosol toxicity studies, there is often no clear definition of the aerodynamic particle size. In addition, dosimetry metrics are rarely reported which makes it difficult to assess reliability and repeatability of a system under review. As far as is known, all known systems are inadequate to satisfy the requirements of a higher throughput exposure system which can produce up to six doses of VOCs, ultrafine particles, i.e., typically less than 0.1 μm, or fine particles, i.e., typically less than 2.5 μm, with four technical replicates per dose within a standard 24-well cell culture plate to meet toxicity screening demands. In many biological or toxicological applications, such exposure systems must also maintain appropriate cell culture conditions, which are typically a temperature of 37° C., and a relative humidity of greater than 80%. Vehicle controls must also demonstrate minimal cytotoxicity compared to incubator controls.
The difficulties and drawbacks associated with previous approaches are addressed in the present subject matter as follows.
In one aspect, the present subject matter provides a cell culture exposure system which comprises of one or more dilution manifolds including at least one inlet for receiving fluid flow. The system also comprises at least one flow splitter. And, the system comprises an exposure chamber configured to receive a cell culture plate which contains permeable cell culture inserts which maintain cells at the air-liquid interface. Flow splitters are positioned between the outlets of the dilution manifold and the inlets to the exposure chamber to provide flow communication and ensure uniform deposition of delivered test agent across all downstream technical replicates.
In another aspect, the present subject matter provides a cell culture exposure system comprising a dilution manifold having at least one inlet for receiving fluid flow. The system also comprises an assembly of flow splitters. And, the system comprises an exposure chamber configured to receive a cell culture plate which contains permeable cell culture inserts in each well. The assembly of flow splitters is disposed between the dilution manifold and the exposure chamber and provides a flow communication therebetween. The system is configured such that each well of the plurality of wells receives its own independent flow of the fluid flow downstream of the assembly of flow splitters.
In still another aspect, the present subject matter provides a method for exposing a cell culture plate to a test fluid flow. The method comprises providing a cell culture exposure system including at least one dilution manifold including at least one inlet for receiving a fluid flow, at least one flow splitter, and an exposure chamber configured to receive a cell culture plate. The method also comprises providing a cell culture plate having at least one well. The method further comprises positioning cell culture material in the at least one well. The method additionally comprises placing the cell culture plate with cell culture material positioned therein, in the exposure chamber of the cell culture exposure system. And, the method further comprises directing a fluid flow into the at least one inlet of the at least one dilution manifold such that the fluid flow passes through the at least one well and the cell culture material positioned therein.
As will be realized, the subject matter described herein is capable of other and different embodiments and its several details are capable of modifications in various respects, all without departing from the claimed subject matter. Accordingly, the drawings and description are to be regarded as illustrative and not restrictive.
Recently, in vitro airway models grown at the air-liquid interface (ALI) have been used for inhalation toxicity screening. These advanced ALI cell cultures allow direct cell-toxicant interaction to mimic realistic inhalation exposures. In addition, these cultures are uniquely suited to handle methodologically challenging chemicals (MCCs) which include volatile and water-/DMSO-insoluble compounds. However, specialized ALI exposure systems must meet the following specifications to allow regulatory agencies to adopt and validate in vitro assays:
The system must maintain physiological temperature (T) and relative humidity (RH) under dynamic airflow conditions for several exposure types, such as aerosols, volatile compounds, and reactive gases. Vehicle controls, which are typically cells exposed to clean air without test articles, must demonstrate minimal cytotoxicity compared to incubator controls.
The system must also provide serial dilution of a test article with technical replicates to meet higher throughput screening demands and characterize dose-response relationships within a single exposure.
The system must also exhibit flexibility to accommodate inhalable test articles with different physicochemical properties and size metrics, for example, volatile organic compounds (VOCs), ultrafine particles/nanoparticles, i.e., typically less than 100 nm, and fine particles, i.e., typically less than 2.5 μm.
The system must further provide high repeatability. That is, the system should exhibit low standard deviations for particle deposition, which typically are less than 20% between technical replicates and inter-day repeat exposures.
To meet these demands, and in accordance with the present subject matter, a unique aerosol-compatible cell culture exposure system is provided that allow serial dilution and delivery or flow of a wide array of test fluids such as but not limited to aerosols, reactive gases, and VOCs at ALI under physiologically relevant conditions (T=37° C., RH>80%). The present subject matter system meets higher throughout demands for toxicity screening by producing six doses in a single exposure with four technical replicates per dose. Standard deviations of particle deposition between technical replicates are usually less than 10%. Another key feature of the present subject matter system is the direct utilization of standard 24-well cell culture plates with 6.5 mm permeable ALI inserts. This increases throughput, decreases sample handling time, and allows laboratories and evaluation facilities to use commercially available cell culture materials and equipment. Furthermore, the present subject matter system is primarily configured for 3D human airway models grown at ALI, for example, nasal epithelial cells, bronchial epithelial cells, alveolar epithelial cells. However, it is understood that the present systems are also compatible with any cell type grown at ALI, keratinocytes for dermal toxicity studies, or mutagenesis assays, for example an Ames assay.
It is contemplated that the present subject matter systems will be used by various regulatory agencies and other concerns to utilize new approach methods (NAMs) for inhalation toxicity screening in support of the Toxic Substances Control Act (TSCA) and Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).
Current risk assessment strategies are trending toward the reduction and refinement of the use of animal models for toxicity screening and the development and validation of alternative test methods. In vitro NAMs are now being deployed to meet the demands of risk assessment testing in a time- and cost-effective manner. However, appropriate commercially available ALI exposure equipment to support this transition is lacking. The US EPA Office of Chemical Safety and Pollution Prevention (OCSPP), Office of Pesticide Programs (OPP), and Office of Pollution Prevention and Toxics (OPPT) have requested in vitro inhalation data for volatile and non-volatile, water-/DMSO-insoluble compounds that have been nominated by TSCA for further toxicological evaluation. These regulatory partners have a key interest in developing improved in vitro inhalation assays to reduce the need for 90-day animal inhalation studies. This necessitates a specialized ALI exposure system that maintains physiologically relevant conditions, can accomplish serial dosing to achieve benchmark dose modeling of toxicological endpoint assays, such as viability, cytotoxicity, transcriptomics, etc., is flexible to accommodate a diverse range of VOCs and particles, and is repeatable to accomplish replicate studies and repeat-dosing studies.
Repeatable particle delivery is especially difficult because deposition mechanisms, for example, impaction, sedimentation, diffusion, and electrostatic precipitation, vary by particle size and physicochemical properties. This is an important consideration for risk assessment and in vitro study design due to the anatomy of the human respiratory tract and the distinct particle size ranges that penetrate to each region as follows.
Inhalable fraction particle sizes, for example less than 10 μm, generally include the mass fraction of total airborne particles which are inhaled through the nose and mouth. Most large particles from 5-10 μm usually deposit on the upper airways and are successfully cleared by macrophages. The primary deposition mechanism for such particle sizes is impaction. An applicable cell model is Nasal epithelial cells. Generally, the regulatory need is low to moderate. Thoracic fraction particle sizes, for example less than 3 μm in adults, and less than 5 μm in children, are generally the mass fraction of inhaled particles penetrating beyond the larynx and reaching the tracheobronchial region. Particles in the range of 1-2.5 μm are delivered to the terminal bronchiole which is the site of greatest accumulation and tissue destruction. The primary deposition mechanism for these particle sizes is sedimentation. An applicable cell model is Bronchial epithelial cells. Typically, the regulatory need is high. Respirable fraction, for example less than 1 μm of particle sizes, are the mass fraction of inhaled particles that gain access to the gas-exchange region, also referred to the alveolar or pulmonary region. Ultrafine particles and nanoparticles, for example less than 100 nm, deposit in the gas-exchange region. The primary deposition mechanism for such particle sizes is diffusion. An applicable cell model is Alveolar Type I and II epithelial cells. Generally, the regulatory need is high.
Given the wide range of real-world particle populations and deposition mechanisms in the human respiratory tract, an ALI cell culture exposure system must be able to deliver thoracic and respirable particle size ranges to meet risk assessment demands. Both particle size ranges are associated with high toxicity, but it is important to note that these particle groups are subject to different transport physics and deposition mechanisms. Current commercially available ALI exposure systems, for example the VITROCELL system, show strong performance metrics for nanoparticles and volatile compounds. However, the present subject matter systems are unique in their ability to also transport larger thoracic fraction particles, for example less than 2.5 μm, in a repeatable and high throughput manner while maintaining physiologically relevant conditions of relative humidity and temperature. As far as is known, CULTEX RFS is the only other commercially available system with a proven ability to transport inhalable and thoracic fractions of particles with low standard deviations. However, such system exhibits low throughput, for example, n=3, and no serial dilution, and fails to provide physiological relative humidity conditions which leads to high cytotoxicity of vehicle or sham control cells. The present subject matter systems can be broadly used by industry groups, contract research organizations (CROs), and regulatory agencies for chemical screening to aid in regulatory decision making.
The present subject matter provides a unique aerosol-compatible cell culture exposure system that allow serial dilution and delivery of aerosols, reactive gases, and VOCs at ALI under physiologically relevant conditions, for example a temperature of 37° C., and a relative humidity of greater than 80%, to accomplish higher throughput toxicity screening and the incorporation of in vitro NAMs in inhalation risk assessment. Flow parameters can be tuned or selectively adjusted to dilute and deliver VOCs via diffusion with scalar mixing principles, or operational flows can be reduced or otherwise varied to deliver repeatable particle doses via sedimentation and thermophoresis. Vehicle controls demonstrate minimal changes in viability, measured by ATP formation, cytotoxity, measured by lactate dehydrogenase (LDH), and barrier integrity, for example, trans-epithelial electrical resistance (TEER), when compared to incubator controls which is important for toxicological screening of test articles at the ALI.
The present subject matter also provides a modular ALI cell culture exposure system that provides higher throughput toxicological screening of inhaled toxicants by achieving one or more of the criteria described herein.
The systems of the present subject matter comprise modular, leak-proof components which allow the systems to be easily configured for VOCs or aerosols. Generally, the systems comprise at least one dilution manifold having an inlet for receiving a fluid, at least one flow splitter, and an exposure chamber configured to receive a cell culture plate. All of these components are described in greater detail herein.
A wide array of dilution manifolds can be used in the systems of the present subject matter. The dilution manifolds include one or more inner diluters, one or more exhaust diluters, and one or more inlet caps. In certain versions, the dilution manifold may also include a U-turn component. The U-turn component is disposed between two inner diluters and provides flow communication therebetween. The term “flow communication” is used herein and refers to a configuration in which flow of air, gas, or other fluid can readily occur between adjacent and typically adjoining components. Typically, such flow communication occurs through one or more passages defined in the components. As will be understood, the term is not limited to flow of air, but rather includes a wide array of flowable fluids, mists, aerosols, entrained particulates, gas-like slurries, and related collections of matter.
A variety of flow splitters may be used in the systems of the present subject matter. In one version, a VOC-specific flow splitter, herein referred to as a VOC flow splitter, includes a sampling port for real-time monitoring with gas chromatography (GC)-coupled detection methods, for example. In another version, an aerosol-specific flow splitter, herein referred to as an aerosol flow splitter, includes a static mixer to align particle flow paths for uniform deposition. These components are described in greater detail herein.
A combination of a dilution manifold and a flow splitter allows serial dilution and delivery of inhaled toxicants, for example, six (6) doses with four (4) technical replicates, which allows multiplexing endpoint assays.
In many embodiments, the exposure chamber accommodates standard cell culture plates with 6.5 mm ALI inserts and is fully compatible with VOCs or aerosols. In many embodiments, the exposure chamber is also equipped with heating element(s) to enhance deposition via thermophoresis while maintaining ideal cell culture conditions.
The system of the present subject matter can be utilized with different flow parameters to transport VOCs and/or aerosols and target different dose ranges. Each component of the present subject matter systems is configured for easy assembly, operation, and cleaning.
The operation of the system of the present subject matter provides serial dilution of a wide range of test articles, for example, VOCs, reactive gases, and particles, and delivers a plurality of doses with at least four technical replicates per dose to cells cultured at ALI in a standard 24-well plate outfitted with 6.5 mm inserts. In particular applications, the exposure system can be configured to deliver six doses with four technical replicates per dose. Typically, the exposure chamber is sealed and configured to maintain physiological relative humidity and temperature throughout dynamic cell exposure conditions. The dilution manifold is configured to balance inertial and gravitational forces that influence particle movement to minimize losses of fine particles, typically, less than 2.5 μm. A four-way flow splitter coupled with static mixers ensures uniform particle deposition. Technical replicates of particle deposition within a single dose achieve a standard deviation of less than 10%, indicating the repeatability and reliability of the present subject matter systems. A simple substitution of the flow splitter module and alignment of the dilution manifolds enables a system of the present subject matter to deliver volatile compounds and reactive gases. The present subject matter provides a flexible and adaptable exposure system that meet higher throughput demands and risk assessment needs.
Referring to
In another embodiment shown in
The dilution manifold 20 typically includes at least one inlet cap 30, at least one inner dilutor 40, and at least one exhaust dilutor 50 as illustrated in
Referring to
In a particular version of the present subject matter exposure system, the outlet faces of the flow splitter, i.e., the lower faces 76 of the chamber adapters 74 of the aerosol flow splitter 60 or the lower faces 96 of the chamber adapters 94 of the VOC flow splitter 80, provide access to a number of outlet ends of conduits, i.e., conduits 77 or conduits 97, equal to a number of wells in a culture plate used in the exposure system. For example, if the culture plate for use in the cell exposure system provides 24 wells, and so the exposure chamber 100 includes a total of 24 delivery nozzles 136, then the total number of conduit outlets accessible along the lower face of the flow splitter, is also 24. This particular configuration is referred to herein as “an independent well configuration” thus referring to a strategy in which each well of a cell culture plate receives its own independent supply or flow of test fluid from the flow splitter. As a consequence of this particular configuration, no mixing of test fluid occurs downstream of the flow splitter and upstream of the well(s).
The present subject matter also provides various methods, and particularly methods for exposing a cell culture plate to a test fluid flow. In a particular embodiment, such method comprises providing a cell culture exposure system including at least one dilution manifold including at least one inlet for receiving a fluid flow, at least one flow splitter, and an exposure chamber configured to receive a cell culture plate. The method also comprises providing a cell culture plate having at least one well. The method additionally comprises positioning cell culture material in the at least one well. The method further comprises placing the cell culture plate with cell culture material positioned therein, in the exposure chamber of the cell culture exposure system. Then, the method comprises directing a fluid flow into the at least one inlet of the at least one dilution manifold such that the fluid flow passes through the at least one well and the cell culture material positioned therein. In a specific version of the method, the cell culture plate includes 24 wells. And, in this method, a user may wish to utilize particular parameters such as the fluid flow exhibiting a temperature of 37° C. and a relative humidity greater than 80%.
A modular flow splitter configuration creates four technical replicates with uniform deposition for VOCs and fine particles, for example less than 2.5 μm. Alternate embodiments of the flow splitter component include updates to allow real-time sampling of VOCs.
Alternative embodiments include a plenum plate configured to fittingly engage other nozzle assemblies besides a conventional 24 stainless steel nozzle assembly. The plenum plates can be adapted to optimize delivery of inhaled test articles such as but not limited to: (i) altered inner diameter or outer diameter, (ii) altered height of nozzles to optimize delivery parameters, and (iii) shape of nozzle outlet above cells.
Alternative embodiments of the dilution manifold include the use of a plurality and typically two split manifolds, for example each with three dilutors, one full manifold, for example with six dilutors with a U-turn connector that directly attaches to the flow splitters. Alternatively, six dilutors can be connected in series with an external heat source (not shown) to aid transport of reactive gases and volatile compounds that require high heat to remain volatile.
Alternative embodiments of the system of the present subject matter include adapted designs for 6- and 12-well plates which achieve serial dilution. Such system may include one or more of the following modifications: shortened dilution manifolds to provide 3-4 doses per plate, updated flow splitters which feature a two-way split with an integrated static mixer sleeve or sampling port, and an updated plenum plate with a reduced number of nozzles centered over 6- or 12-well inserts.
Two technical replicates per dose in the exposure chamber can be achieved by an isolated plenum space over two wells which balances the vacuum flow rates experienced by each well.
Contrary to prior art systems, the present subject matter provides a cell culture exposure system that are flexible and compatible with fine particles (<2.5 μm) as well as VOCs, reactive gases, and ultrafine particles (<0.1 μm) under dynamic airflow conditions. The modular dilution manifold, flow splitters, and sealed exposure chamber accomplish serial dilution and produce six doses with four technical replicates per dose within a standard 24-well cell culture plate while maintaining appropriate cell culture conditions (T=37° C., RH>80%). Notably, standard deviation of technical replicates of particle deposition is often less than 10% with liquid fluorescein particles with a median mass aerodynamic diameter (MMAD) of 1.5 μm. The geometry of this system is optimized for low flow rates which minimize cell cytotoxicity due to shear stress.
In a particular embodiment, a system of the present subject matter provides serial dilution of VOC and aerosols to achieve six (6) doses with four (4) technical replicates per dose.
Although the system described in US Patent Publication No. 2018/0171280 claims the same achievement, the plenum design fails to achieve balanced flows so four technical replicates cannot be achieved downstream of each dose for VOCs. Moreover, that system cannot transport aerosols and fails to achieve serial dilution of particles.
In another particular embodiment, a system of the present subject matter utilizes standard 24-well cell culture plates with 6.5 mm inserts to increase throughput and minimize sampling handling time. Therefore, inserts do not need to be individually transferred from cell culture plate to the exposure apparatus.
As far as is known, the system described in US Patent Publication No. 2018/0171280 is also compatible with standard 24-well plates and achieves serial dilution under dynamic airflow conditions, but the geometry of the exposure unit is incompatible with particles. The shared plenum space creates undesirable pressure gradients and variable airflow rates which prevents repeatable particle deposition or VOC delivery within technical replicates.
Many other benefits will no doubt become apparent from future application and development of this technology.
All patents, applications, standards, and articles noted herein are hereby incorporated by reference in their entirety.
The present subject matter includes all operable combinations of features and aspects described herein. Thus, for example if one feature is described in association with an embodiment and another feature is described in association with another embodiment, it will be understood that the present subject matter includes embodiments having a combination of these features.
As described hereinabove, the present subject matter solves many problems associated with previous strategies, systems and/or devices. However, it will be appreciated that various changes in the details, materials and arrangements of components, which have been herein described and illustrated in order to explain the nature of the present subject matter, may be made by those skilled in the art without departing from the principle and scope of the claimed subject matter, as expressed in the appended claims.
