Abstract:
Devices, systems, and methods for facilitating placement of cells and materials in culture plates configured for high-throughput applications are provided. A culture system is provided with a culture plate having a lid for guiding placement of cells and materials in each individual culture well of a culture plate. The lid may provide for coupling to an electrophysiology culture plate comprising a biosensor plate and a biologic culture plate, where the biosensor plate underlies and is coupled to the culture well plate such that each biosensor is operatively coupled to one culture well of the plurality of culture wells. A containment device that physically influences the positioning of fluid received in the culture plate is also provided herein.

Description:
BACKGROUND OF THE INVENTION 
       [0001]    In vitro testing is often used in the earlier stages of pre-clinical testing to eliminate unsafe compounds prior to advancement to the later animal stages. For example, interconnected cellular networks of cardiomyocytes may be formed on a substrate for the testing of potential new heart therapies. Primary cardiomyocytes harvested from an animal, or animal or human stem-cell-derived cardiomyocytes, form interconnected cellular networks when cultured on a cell culture substrate. The individual cardiomyocytes within a network are connected through gap junctions that allow ions to flow from one cell to another. This electrical connection allows an electrical action potential, which is first generated by a pacemaker cell, to propagate from one cell to the next. 
         [0002]    Formation of an electrical action potential starts with a buildup of charge across a cell membrane. This buildup occurs spontaneously in cardiac cells, and more frequently in pacemaker cardiomyocytes than non-pacemaking cardiomyocytes. When the transmembrane charge reaches a threshold value, ions rush into the first cell (the depolarization phase). This triggers an action potential, which is a sharp influx of additional ions into the cytoplasm. The gap junctions distributed across the cell membrane allow ions to flow into neighboring cells, enabling the spread of the action potential. 
         [0003]    Molecular processes within the cell tie the electrical action potential to the physical contraction of the cardiomyocytes. The propagation of the cardiac action potential across an in vitro cellular network, and the resulting contraction, resembles the propagation and contraction observed within the human heart and thus is often referred to as a “beat”. Many in vitro cardiomyocyte networks exhibit spontaneous beating, where each cardiac action potential propagation (and corresponding physical beat) is followed by a brief pause and then another cardiac action potential propagation and beat. 
         [0004]    In vitro diagnostics allow researchers to analyze non-electrical properties of many types of cells, such as cell viability, density, and proliferation rates. However, electrically active cultures, such as cardiomyocytes, enable researchers to test additional properties related to electrical activity. For example, a cardiomyocyte culture may be assessed by the gap junction distribution, or degree of electrical connectivity between cells. This property may relate to the ability of a beat to be transmitted homogenously throughout a culture. 
         [0005]    In another example, electrical measurements taken from a cardiomyocyte give researchers an indication of the cell health, quality, and level of maturity. For example, patch-clamp techniques provide measures of the action potential of an individual cell. A patch-clamp uses an electrode inserted into the cell membrane to measure transmembrane voltage. For a healthy cell, the cardiac action potential is initiated with a depolarization phase, where sodium rushes into the cell. The depolarization phase is followed by a plateau phase, dominated by the influx of calcium, where the cells remains depolarized, and ultimately a repolarization phase characterized by an outflux of potassium and a return to the starting transmembrane potential. Patch-clamp technologies can be used to detect abnormalities in the action potential within a single cell, which may point to functional problems. However, performing testing on individual cells is difficult and time-consuming. Furthermore, cardiomyocytes may behave differently when separated from their network, thus calling experimental results into question. 
         [0006]    Other technologies, such as impedance measurement systems, can provide information about the physical beating of the cells, but do not reveal important functional information associated with the electrical action potential. Finally, optical imaging of the network electrophysiology can be performed using secondary voltage sensitive optical reporters. However, these protocols may be time consuming and cytotoxic, eliminating the ability to perform multiple experiments on the same culture. 
         [0007]    Microelectrode arrays (MEAs) having a plurality of microelectrodes situated within each well enable researchers to measure signals from electrically active cells cultured on their surfaces. Herein, “microelectrode” and “electrode” will be used interchangeably. Cells are cultured across the array of electrodes within a well such that signals are detected from multiple electrically active cells, such as cardiomyocytes, simultaneously. These signals, called field potential signals, may change shape in response to the addition of a candidate compound to the cardiomyocyte culture. The changes may be used to evaluate the cardiac safety risk of a compound. Additionally, these measures may be used to develop and characterize new stem cell lines, to compare the electrophysiology of the cells to in vivo signals from native cardiomyocytes, and/or to evaluate in vitro models of disease. 
         [0008]    In vitro electrophysiology culture systems having biosensors, MEAs, can provide important insights into networks of electrically active cells. MEA-based electrophysiology culture systems can be configured to concurrently monitor single-cell and network-level activity over extended periods of time and without affecting the cell culture under investigation. Since their instrumental role in the landmark discovery of spontaneous waves in a developing retina, the variety and scope of MEA-based electrophysiology applications has dramatically expanded. Recently, for example, MEA-based electrophysiology culture systems have been used to investigate the suppression of epileptic activity and in the study of novel plasticity mechanisms in cultured neural networks. Advances in cell culture preparations have similarly led to applications for MEA-based electrophysiology culture systems in the fields of drug screening, safety pharmacology, and biosensing. 
         [0009]    Working with MEA-based systems requires physical access to the culture plate itself—for example, to setup, modify, or verify an ongoing experiment. One potential downside to physical access is the exposure of the culture plate and its contents to the ambient atmosphere. Typical ambient conditions are not ideal for cell growth and maintenance. For example, many cells prefer a Carbon dioxide (CO 2 )-rich environment that does not exist in the ambient atmosphere. Therefore, a scientist may inadvertently jeopardize the health of the cells any time they interact with the culture plate. A need exists for a system that allows physical and/or chemical interaction with cells in an MEA-based system while maintaining the accuracy of results (e.g., by preventing beat-period drift) and also minimizing harm to the cells themselves. 
         [0010]    In order to accomplish these and other goals, Applicant has created various devices and systems for creating and maintaining a localized environment for a cell culture plate. A localized environment may describe any environment in proximity to the cell culture plate, and may include, for example, temperature, CO 2  concentration, oxygen concentration, and so on. 
       SUMMARY 
       [0011]    In one example embodiment, a system is provided for creating a localized environment for a cell culture plate. The system includes a dock shaped to receive the culture plate and a gas distribution device positioned on the dock. The gas distribution device includes a frame having a plurality of sides connected to form an opening, an internal channel within the frame for directing the gas mixture within the gas distribution device, an inlet port in fluid communication with the internal channel for receiving a gas mixture, and an aperture in fluid communication with the internal channel and configured to direct the gas mixture toward the opening. 
         [0012]    The gas distribution device may include a plurality of apertures spaced apart from one another along the frame and configured to direct the gas mixture toward the opening. The gas mixture may comprise a CO 2  concentration of about 2-100%. The gas distribution device may provide a localized environment proximate the culture plate having a CO 2  concentration of about 0.5-20%. The plurality of apertures may be non-uniform in size, and/or the spacing between at least some of the plurality of apertures may be non-uniform. 
         [0013]    The system may further include a heater arranged to provide heating energy to the culture plate. The heater may be used to support the culture plate within the dock. The system may also include a lid arranged to enclose the culture plate and at least a portion of the gas distribution device. The lid can include magnets configured to secure the lid to at least one of the gas distribution device or the dock. The lid can also include a gripping feature configured to mate with a robotic gripper. The dock and the gas distribution device respectively may include complementary magnets, and wherein the magnets are positioned to align the gas distribution device on the dock. Alternatively or in addition, the gas distribution device may mechanically engage the dock. 
         [0014]    In some embodiments, a cross sectional area of the internal channel varies along a length of the channel of the gas distribution device. The system may also include a processor configured to determine a desired flow rate for the gas distribution device. The gas distribution device may allow a user to physically interact with the culture plate without disrupting the localized environment proximate the culture plate. 
         [0015]    In another example embodiment, a method is provided for creating a localized environment for a cell culture plate. The method can include introducing a pressurized gas mixture into a gas distribution device, channeling the gas mixture within an internal channel of the gas distribution device, and expelling the gas mixture via a plurality of apertures in fluid communication with the internal channel, wherein the plurality of apertures are spaced apart from one another along the internal channel and are configured to direct the gas mixture at least partially toward a portion of the gas distribution device 
         [0016]    The method can also include providing a heater configured to provide heating energy to the culture plate and a dock shaped to receive the culture plate and the gas distribution device. The method may also include expelling a gas mixture having a CO 2  concentration of about 2-100% and/or forming a localized environment proximate the culture plate having a CO 2  concentration of about 0.5-20%. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0017]      FIG. 1  is a schematic of an example embodiment of a gas distribution device for use with a culture plate. 
           [0018]      FIG. 2  is a cross sectional view of a portion of the example gas distribution device of  FIG. 1 . 
           [0019]      FIG. 3  is a schematic of an example embodiment of a gas distribution device having a lid affixed to the device. 
           [0020]      FIG. 4  is a schematic of an example embodiment of a gas distribution device arranged on a dock. 
           [0021]      FIG. 5  is a schematic of an example embodiment of a gas distribution device having a lid affixed to the device, with the device arranged on a dock. 
           [0022]      FIG. 6  is a graphical representation of cardiomyocyte beat period stability over time when utilizing an example embodiment of a gas distribution device for an initial time period. 
       
    
    
     DETAILED DESCRIPTION 
       [0023]    The present invention can be understood more readily by reference to the following detailed description, examples, drawing, and claims, and their previous and following description. However, before the present devices, systems, and/or methods are disclosed and described, it is to be understood that this invention is not limited to the specific devices, systems, and/or methods disclosed unless otherwise specified, as such can, of course, vary. The terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. 
         [0024]    Reference will be made to the drawings to describe various aspects of one or more implementations of the invention. The drawings are diagrammatic and schematic representations of one or more implementations, and are not limiting of the present disclosure. Moreover, while various drawings are provided at a scale that is considered functional for one or more implementations, the drawings are not necessarily drawn to scale for all contemplated implementations. The drawings thus represent an example scale, but no inference should be drawn from the drawings as to any required scale. 
         [0025]    In the following description, numerous specific details are set forth in order to provide a thorough understanding described herein. It will be obvious, however, to one skilled in the art that the present disclosure may be practiced without these specific details. In other instances, well known aspects of electrophysiology culture systems, machining techniques, injection molding methodologies, and microelectromechanical systems (MEMS) have not been described in particular detail in order to avoid unnecessarily obscuring aspects of the disclosed implementations. 
         [0026]    Turning to  FIG. 1 , a gas distribution device  110  is depicted. Gas distribution device  110  includes a frame  120  having various sides connected to one another. In the embodiment of  FIG. 1 , the frame  120  includes four sides connected to one another forming an opening  130 . Gas distribution device  110  may also include a frame  120  having fewer, or greater than, four sides. In addition, the sides of frame  120  are not necessarily connected to one another. For example, one side of the frame  120  may have a break with no material present. The frame  120  may also have non-uniform walls. For example, the frame  120  of gas distribution device  110  includes cutouts  140  on two sides of the frame  120 . These cutouts may be useful in, for example, accessing an MEA plate underneath the gas distribution device  110 . In any event, the central space between the portions of the frame  120  form opening  130 . 
         [0027]    Gas distribution device  110  is configured to receive and expel a gas mixture or any other type of fluid. Gas distribution device  110  can receive a gas mixture via inlet port  150 , which is configured to attach to a hose or other device for providing the gas mixture. Inlet port  150  is in fluid communication with an internal channel (labeled  210  in  FIG. 2 ) within the frame  120 . By attaching a pressurized source of gas to inlet port  150 , the gas is provided to the entire internal channel. 
         [0028]    Also in fluid communication with the internal channel  210  is at least one aperture  160 . As shown in  FIG. 1 , the gas distribution device  110  may have a plurality of apertures  160 . The apertures  160  are formed within the frame  120  and connect to the internal channel  210 . Any number of apertures  160  may be used according to the particular needs of the implementation. The size and/or shape of the apertures  160  may be varied as well, preferably in a manner that corresponds to the number of apertures  160  being used. For example, using a larger number of apertures  160  within the frame  120  may call for using smaller apertures  160 , while using a smaller number of apertures  160  within the frame  120  may call for using larger apertures  160 . 
         [0029]    Apertures  160  may take any shape. For example, they may be round, square, rectangular, or triangular. In addition, apertures  160  may have a variety of sizes. In one example embodiment, each aperture  160  is approximately 0.5 mm×1.5 mm in size. However, apertures  160  may be smaller or larger. For example, apertures  160  may be approximately 0.25 mm×0.25 mm. As another example, apertures  160  may be approximately 100 mm×100 mm. Of course, apertures  160  may also be any sizes in between these example sizes. In some embodiments, aperture  160  may extend around a portion of the gas distribution device. In other embodiments aperture  160  may extend around the entire gas distribution device. Different sizes of apertures  160  may be mixed and matched to achieve the desired flow rate and flow pattern. 
         [0030]    As shown in  FIG. 2 , the apertures  160  may connect to the internal channel  210  via passages  220  within the frame  120 . Passages  220  may be formed by, for example, providing a portion of the internal channel  210  that is cut out such that the gas mixture can reach the aperture  160 . In  FIG. 2 , the passage  220  has a generally rectangular shape and is generally perpendicular to the direction of the internal channel  210 . This orientation may be useful for directing gas flow in a direction that is generally perpendicular to the internal channel  210 . In some embodiments, the passage  220  may be cut at an angle to the internal channel  210 , which may provide gas flow at a similar angle. The sizes, orientations, and alignments of passages  220  may be mixed and match to achieve uniform flow properties across all apertures  160  and provide suitably even coverage across opening  130 . 
         [0031]      FIG. 3  shows an embodiment of the gas distribution device  110  that includes a lid  310  that fits within the frame  120  of the device. The lid  310  may be used to temporarily cover a culture plate associated with the gas distribution device  110 , allowing the localized environment to be maintained while providing a lower flow rate of the gas mixture. Use of the lid  310  provides additional containment of the environment by effectively eliminating a major exit path for the gas distributed via the gas distribution device  110 . The lid  310  may be formed such that it is compatible with automated machines such as the Hamilton Microlab NIMBUS. For example, it may include one or more gripping features  320  configured to mate with a robotic gripper. As shown in  FIG. 3 , the gripping features  320  may comprise portions of the lid  310  arranged perpendicular to the plane defined by the frame  120 . The gripping features  320  may also include ridges or other textual features that enhance the ability of a robotic gripper to grip the lid  310  securely. These features allow for an automated process that involves removing and/or installing the lid  310  on the gas distribution device  110 . 
         [0032]      FIG. 4  illustrates an example embodiment of a dock  410  shaped to receive a culture plate  420 , with a gas distribution device  110  installed on the dock  410 . The dock  410  may also include a heater located underneath the culture plate  420  and potentially supporting the culture plate  420  from below. 
         [0033]    The gas distribution device  110  may be loosely positioned on the dock  410  or may be secured in some manner. For example, in some embodiments the gas distribution device  110  is mechanically attached to the dock via, for example, a press-fit connection, interlocking connection, or some other mechanical connection. In another embodiment, however, the gas distribution device  110  contains magnets corresponding to magnets with the dock  410 . The corresponding sets of magnets can operate to position the gas distribution device  110  in the correct location and while also allowing for easy removal of the device. As shown in  FIG. 5 , a lid  310  may be used in conjunction with the gas distribution device  110  being attached to the dock  410 . 
         [0034]    When the dock  410  includes a culture plate  420  positioned therein and the gas distribution device  110  is attached and operable, the gas distribution device  110  is configured to provide a localized environment for the culture plate  420 . The particular localized environment may vary depending on the particular needs to the medium within the culture plate. The term “localized environment” refers to the area proximate the culture plate  420  and its contents. For example, the localized environment of a culture plate may include the gas that is in direct contact with the culture plate  420  or its contents. As another example, the localized environment may include the area directly above the culture plate  420 . As yet another example, the localized environment may include the opening  130  generally formed by frame  120  and shown in  FIG. 1 . 
         [0035]    Generally speaking, the gas distribution device  110  can be configured to provide a localized environment providing an optimal concentration of any necessary element. For example, the gas distribution device  110  can be configured to provide a localized environment proximate the culture plate having a CO 2  concentration of about 0.5-20%. Higher or lower concentrations may be used, but a concentration of about 0.5-20% has been shown to improve cell activity and lifespan and increase the success rate of certain types of experiments. 
         [0036]    In order to achieve and maintain the desired localized environment, the gas distribution device  110  may be configured to provide a gas mixture rich with whatever elements are desired for the culture being studied. For example, the gas distribution device  110  may provide a gas mixture having a CO 2  concentration of about 2-100%. The optimal CO 2  concentration of the gas mixture may vary based on many factors, such as: the flow rate of the gas mixture, the size/number/orientation of apertures, and whether a lid is being used. A lower flow rate may require a higher concentration of CO 2  in the gas mixture, while the use of a lid can lower the required concentration of CO 2 . The concentration may be controlled and changed dynamically to meet the needs of the culture at issue. For example, a processor may be located within the dock  410  and may control the flow rate automatically. 
         [0037]    Gas distribution device may be used in a variety of scenarios and circumstances. For example,  FIG. 6  shows a graphical representation of cardiomyocyte beat period stability over time when utilizing an example embodiment of a gas distribution device  110  for an initial time period. Generally speaking, Cardiomyocytes are electrically active cells that are able to spontaneously contract. When cultured in vitro (e.g., on an MEA), cardiac cells form a functional beating syncytium, in which the entire culture contracts at the same time due to rapid conduction of electrical signals (cardiac action potentials) from cell to cell. 
         [0038]    The field potential waveform captured by MEA recordings provides information on the depolarization, repolarization, and propagation of the cardiac action potential. For example, a depolarization spike on the field potential is due to current through the ion channels of the cell, and corresponds to the elements of the signal in a clinical Electro Cardio Graph (ECG) signal. From the depolarization spike, we also derive the onset of beat timing. The beat period is defined as the time interval between two consecutive beats. Although beat period is variable across cell types, any deviation from a culture&#39;s baseline may be indicative of poor culture health. Furthermore, cell sensitivity to compounds depends on beat period as well. 
         [0039]    As a culture develops, the beat rate and beat period stabilize. Maintaining these stabilized rates depends, in part, on external factors such as pH and temperature. Temperature can be maintained through the use of heating elements integrated into the system or the MEA itself. 
         [0040]    With respect to pH, careful control may be necessary to counteract pH changes caused by waste products of the cells. One way to control pH is to provide bicarbonate buffering tuned to a particular CO 2  level. In some embodiments a CO 2  level of about 5% may be used to maintain an appropriate pH. While other CO 2  levels may be used as well,  FIG. 6  shows data based on 5% CO 2 . 
         [0041]      FIG. 6  shows a chart of the beat period, measured as a percent change from a baseline beat period, over a time period of about 110 minutes. During the stage labeled “A” a gas distribution device was utilized to provide CO 2 -rich air to the cultures, thereby maintaining a localized environment. As shown in the graph, the beat period did not differ substantially from the baseline during stage A, fluctuating up to only approximately 3%. Stage B begins at the roughly 55-minute mark, and signifies the time when the CO 2  distribution ceased and the cultures were exposed to the ambient environment. The results show a sharp change in beat period stability, decreasing over 50% in about 30 minutes. This experiment demonstrates the utilization of an example embodiment of a device according to the present disclosure in a practical setting and shows excellent performance with respect to signal (in this case beat period) stability.