Embedded Electrode Array Plate

Abstract

A device with an embedded electrode array (EEA) capable of testing cell samples includes the embedded electrode array; an interface printed circuit board; a test controller for configuring a stimulus signal and a data channel in the interface printed circuit board to receive and sample a response signal into the data channel; and a data collector for receiving a data set for processing and storage. The embedded electrode array includes tissue culture wells, each having an electrode pad for receiving a tissue cell sample; a signal connector having connector pins for receiving a stimulus signal for each of the tissue culture wells and generating a plurality of response signals from each of the tissue cell samples; and circuit traces embedded in the EEA printed circuit board for connecting one of the connector pins to the electrode pad. The interface printed circuit board includes a signal connector for receiving a signal from a source used as a stimulus signal, and an amplifier-digitizer circuit. TIFF2025502620000002.tif120149

Description

[Technical field]

[0001] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety, and the disclosures of these publications are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art as of the date of the invention described and claimed herein.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves all and any copyright rights whatsoever.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 286,959, entitled "EMBEDDED ELECTRODE ARRAY," filed December 7, 2021, the entirety of which is incorporated herein by reference in its entirety.

[0004] FIELD OF THEINVENTION The present application relates generally to apparatus including biological testing devices, and more specifically to apparatus including embedded electrode arrays (EEA) capable of testing cell samples. [Background technology]

[0005] background The effect of application of pharmacological agents on activity within various cell types is part of most research efforts. One such cellular activity that is tested involves peripheral neurotoxicity of drug introduction into a representative sample of neuronal cells. This test involves growing the cell sample in a test environment, connecting electrodes to various locations within the cell sample, electrically stimulating the cells, and recording the response observed at the sensing electrodes.

[0006] Current testing procedures utilize obtaining single electrode recordings of observed responses to one or more electrical stimuli. Stimulating and sensing electrodes are manually inserted into a cell sample, one or more electrical stimuli are applied to the cell sample, and responses are recorded at the sensing electrodes. The stimulating and sensing electrodes are electrically coupled to electronics that generate the one or more electrical stimuli and to recording electronics that digitally sample the observed responses in the sensing signal. The recorded digital sample data may be stored in a computing system for later analysis.

[0007] Manually inserting electrodes into cell samples and performing cell stimulation and response recording has many deficiencies. Each single recording is performed manually by a technician. The technician must be trained to perform electrode insertions in repeatable locations within the cell sample, which can be difficult to train and perform. The steps performed in the testing process do not result in rapid, repeatable, automated testing, which can lead to variability in test results and ultimately affect the results obtained from a particular test. This testing procedure also uses a set of samples for quality assurance and prevents repeat testing to detect longer term effects on the cell sample from the application of a pharmacological agent.

[0008] Thus, there is a need for a device with an embedded electrode array capable of testing a cell sample.The present invention, in accordance with the principles and exemplary embodiments disclosed herein, seeks to address the limitations and deficiencies in conventional solutions. Summary of the Invention

[0009] overview In accordance with the present invention, the above and other problems are solved by providing a device having an embedded electrode array (EEA) capable of testing a cell sample in accordance with the principles and exemplary embodiments disclosed herein.

[0010] In one embodiment, the invention is an apparatus with an embedded electrode array capable of testing cell samples. The apparatus includes an embedded electrode array; an interface printed circuit board; a test controller for configuring one or more stimulus signals and a plurality of data channels in the interface printed circuit board for receiving and sampling response signals into a plurality of data channels; and a data collector for receiving data sets for the plurality of data channels for processing and storage. The embedded electrode array includes a plurality of tissue culture wells, each having a plurality of electrode pads for receiving a tissue cell sample; one or more signal connectors having a plurality of connector pins for receiving a stimulus signal for each of the plurality of tissue culture wells and generating a plurality of response signals from each of the tissue cell samples; and a plurality of circuit traces embedded in the EEA printed circuit board, each of the plurality of circuit traces connecting one of the plurality of connector pins to an electrode pad in each tissue culture well. The interface printed circuit board includes one or more signal connectors for receiving signals from an external function generator for use as one or more stimulus signals, and a plurality of amplifier-digitizer circuits. Each of the amplifier-digitizer circuits is configured to receive one or more of the plurality of response signals organized into the plurality of data channels and to digitize the response signals for transmission as a data set for each of the plurality of data channels.

[0011] In one embodiment of the present invention, each of the plurality of tissue culture wells includes a cell channel layer for culturing cells into a tissue cell sample, a pair of tissue culture wells disposed at each of the two ends of the cell channel layer, eight electrode pads of the plurality of electrode pads disposed across the cell channel layer, and a single electrode pad of the plurality of electrode pads in each of the pair of tissue culture wells.

[0012] In another aspect of the invention, the embedded electrode array further includes a pair of embedded copper layers in the EEA printed circuit board for defining a plurality of circuit traces, a base polyimide layer between the pair of embedded copper layers, an upper polyimide layer on a surface of one of the two copper layers, and a lower polyimide layer on a surface of one of the two copper layers.

[0013] In another embodiment of the invention, the embedded electrode array is further in a soft gold electroplated layer between one of the two copper layers and the top polyimide layer.

[0014] In another aspect of the invention, each of the tissue culture wells is in a top polyimide layer, exposing the cell channel layer and a plurality of electrodes in the tissue culture well.

[0015] In another aspect of the invention, the test signal is received from an external function generator.

[0016] In another aspect of the invention, the data sets for each of the multiple data channels are generated by multiple amplifier-digitizer circuits in groups of data sets corresponding to data sets from a portion of the tissue culture wells.

[0017] In another aspect of the invention, each of a plurality of amplifier-digitizer circuits receives 16 of the one or more response signals to generate 16 data channels.

[0018] In another aspect of the invention, an interface printed circuit board includes 16 amplifier-digitizer circuits for generating 256 data channels organized into two groups of 128 data channels.

[0019] In another embodiment, the invention is an embedded electrode array including a plurality of tissue culture wells each having a plurality of electrode pads for receiving a tissue cell sample, one or more signal connectors having a plurality of connector pins for receiving a stimulation signal for each of the plurality of tissue culture wells and generating a plurality of response signals from each of the tissue cell samples, and a plurality of circuit traces embedded within the EEA printed circuit board, each of the plurality of circuit traces connecting one of the plurality of connector pins to an electrode pad in a respective tissue culture well.

[0020] In another embodiment, the invention is an interface printed circuit board including one or more signal connectors for receiving test signals for use as one or more stimulation signals to a plurality of tissue culture wells in an attached embedded electrode array, one or more interface signal connectors having a plurality of connector pins for transmitting stimulation signals to each of a plurality of tissue culture wells in the attached embedded electrode array and receiving a plurality of response signals from each of the tissue cell samples, and a plurality of amplifier-digitizer circuits each configured to receive one or more of the response signals organized into a plurality of data channels and digitize the response signals for transmission as a data set for each of the plurality of data channels.

[0021] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described below which form the subject of the claims of the invention.

[0022] It will be appreciated by those skilled in the art that the conception and specific embodiments disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It will also be appreciated by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the present invention, both as to its organization and method of operation, together with further objects and advantages thereof, will be better understood from the following description when considered in connection with the accompanying drawings. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the invention. [Brief description of the drawings]

[0023] Reference is now made to the drawings, in which like reference numbers represent corresponding parts throughout. [Figure 1] FIG. 1 illustrates an exemplary embodiment of cell sample testing using a manual recording aspect in which a cell sample can be tested, according to the present invention. [Diagram 2] FIG. 1 shows a device with an embedded electrode array capable of testing a cell sample according to the present invention. [Diagram 3] FIG. 1 shows a tissue culture plate assembled on a printed circuit board with multiple embedded electrode arrays capable of testing cell samples in accordance with the present invention. [Figure 4A] FIG. 1 shows a close-up view of a connector to an embedded electrode array capable of testing cell samples, in accordance with the present invention. [Figure 4B] FIG. 1 shows a close-up view of a connector to an embedded electrode array capable of testing cell samples, in accordance with the present invention. [Figure 4C] FIG. 1 shows a close-up view of a connector to an embedded electrode array capable of testing cell samples, in accordance with the present invention. [Diagram 5]FIG. 1 shows a printed circuit board having signal lines coupling an embedded electrode array capable of testing cell samples to multiple connectors in accordance with the present invention. [Figure 6] FIG. 1 shows a device with an embedded electrode array capable of testing a cell sample according to the present invention. [Figure 7] FIG. 1 illustrates a printed circuit board with multiple embedded electrode arrays capable of testing cell samples in accordance with the present invention. [Figure 8] FIG. 1 shows a close-up view of an embedded electrode array capable of testing a cell sample, in accordance with the present invention. [Figure 9] FIG. 1 shows a printed circuit board having signal lines coupling an embedded electrode array capable of testing cell samples to multiple connectors in accordance with the present invention. [Figure 10A] FIG. 1 illustrates components within a device with an embedded electrode array capable of testing a cell sample, in accordance with the present invention. [Figure 10B] FIG. 1 illustrates components within a device with an embedded electrode array capable of testing a cell sample, in accordance with the present invention. [Figure 11] FIG. 2 illustrates an exemplary graphical representation of digital channel data collected from multiple tissue culture wells in an embedded electrode array 100, in accordance with the present invention. [Figure 12] FIG. 2 illustrates an exemplary graphical representation of an automated testing process for collecting digital channel data from multiple circuit traces of an embedded electrode array 100 testing a cell sample, in accordance with the present invention. [Figure 13A] FIG. 13 shows an exemplary result of a processed set of digital channel data collected from a printed circuit board having signal lines coupling an embedded electrode array capable of testing a cell sample, in accordance with the present invention. [Figure 13B] FIG. 13 shows an exemplary result of a processed set of digital channel data collected from a printed circuit board having signal lines coupling an embedded electrode array capable of testing a cell sample, in accordance with the present invention. [Figure 14] FIG. 2 illustrates a computer system adapted in accordance with a particular embodiment of a test controller in accordance with the present invention. [Figure 15] FIG. 1 illustrates a computing system of software components of an embedded electrode array controller capable of testing cell samples in accordance with the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Detailed Description The present application relates generally to an apparatus for biological testing devices, and more particularly to an apparatus including an embedded electrode array (EEA) capable of testing cell samples in accordance with the present invention.

[0025] Various embodiments of the present invention will now be described in detail with reference to the above-mentioned drawings attached hereto, in which like reference numerals represent like parts and assemblies throughout the several views. It should be noted, however, that these drawings illustrate only selected embodiments and elements of the apparatus described herein, and thus should not be considered as limiting the scope of the apparatus as described herein, which may acknowledge other equally effective embodiments and applications. The scope of the present invention is limited only by the appended claims. Moreover, any examples described herein are not intended to be limiting, but merely to describe some of the many possible embodiments of the claimed invention.

[0026] In describing embodiments of the present invention, the following terms are used: The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as if each member of the list is individually identified as a separate and unique member. Thus, the individual members of such lists should not be construed as de facto equivalents of any other members of the same list solely based on their presentation in a common group, unless indicated to the contrary. As used herein, the singular forms "a," "an," and "the" are intended to include the plurals unless the context clearly dictates otherwise.

[0027] It will be further understood that the terms "comprises," "including," "comprises," and "comprising" specify the presence of stated features, steps, or components, but do not preclude the presence or addition of one or more other features, steps, or components. It should also be noted that in some alternative implementations, the functions and operations described may occur in an order different from the order depicted in the figures. For example, two figures shown in succession may in fact be executed substantially simultaneously or sometimes in the reverse order, depending on the functions and operations involved.

[0028] The terms "individual" and "user" refer to an entity, e.g., a human being, that uses a device with an embedded electrode array capable of testing a cell sample in accordance with the present invention. The term "user" as used herein refers to one or more users.

[0029] The terms "embedded electrode array" and "EEA printed circuit board" refer to a device having one or more culture wells for testing tissue cell samples using electrodes embedded in the printed circuit board of an array plate that electrically couples the tissue cell sample to a stimulation signal source and a signal recording circuit. The cell sample is grown in a cell channel layer in the printed circuit board that extends outward from the tissue culture well. One or more electrodes are exposed to the cell sample in the cell channel layer at defined intervals that electrically couple the tissue cell sample to a stimulation signal source. The signal recording circuit is electrically coupled to the tissue cell sample in the tissue culture well. In a preferred embodiment, the cell channel layer may be constructed from Cirlex material to generate a Cirlex channel. The terms "embedded electrode array" and "EEA printed circuit board" may be used interchangeably.

[0030] The term "invention" or "present invention" refers to an invention that is being filed via a patent application entitled "Embedded Electrode Array Plate." The term present invention may be used interchangeably with EEA plate.

[0031] The term "printed circuit board" refers to a medium used in electrical and electronic engineering to connect electronic components together in a controlled manner. It takes the form of a laminated sandwich of conductive and insulating layers, each of which is designed with an artwork pattern of traces, planes, and other features resembling wiring on a flat surface, etched from one or more sheet layers of copper laminated on and / or between sheet layers of non-conductive substrate. Electrical components can be secured, typically by soldering, to conductive pads on the outer layers shaped to receive the terminals of the components, both electrically connected and mechanically secured. A separate manufacturing process can add plated through holes that allow interconnection between layers.

[0032] The term "electrode" refers to the non-metallic portion of a circuit, for example an electrical conductor used to contact a tissue cell sample.

[0033] The term "tissue cell sample" refers to neural cells cultured in tissue culture wells and cultured across Cirlex channels in the tissue culture wells in contact with multiple electrodes.

[0034] The term "tissue culture well" refers to a recessed cavity in a printed circuit board for receiving a tissue cell sample to be tested. The tissue cell sample is cultured along the Cirlex channel and extends outward a defined distance from a signal recording electrode located within the tissue culture well.

[0035] The term "Cirlex channel" refers to a recessed cavity or cell channel layer in a printed circuit board for growing a tissue cell sample to be tested. The tissue cell sample extends along the Cirlex channel for a defined distance from a signal recording electrode located in a tissue culture well. One or more stimulating signal electrodes are positioned a defined distance from a signal recording electrode located in the tissue culture well. The terms Circlex channel and cell channel layer may be used interchangeably.

[0036] The term "stimulation signal" refers to an electrical signal that is applied to the tissue cell sample via one of multiple electrodes in a tissue culture well.

[0037] The term "response signal" refers to the electrical signal observed and sampled at one or more electrodes in a tissue culture well in response to application of a stimulus signal to a tissue cell sample.

[0038] The term "Intan RHS amplifier chip" refers to an integrated circuit manufactured by Intan Technologies, Los Angeles, California, that comprises a complete bidirectional electrophysiology interface with multiple independent stimulator / amplifier channels. Each channel integrates a configurable low-noise biopotential amplifier and a programmable constant-current stimulator capable of generating stimulation pulses for extracellular microelectrodes.

[0039] The term "peripheral neurotoxicity" refers to toxic neuropathy or nerve damage caused by exposure to a toxic substance. Peripheral neurotoxicity is a form of peripheral neuropathy, which is damage to nerves away from the brain and spinal cord. Peripheral neuropathy affects the nerves in the arms and hands or legs and feet.

[0040] The term "amplitude distribution" refers to the mathematical distribution of recorded signal values ​​generated from a series of stimulation signals applied to tissue neurons at various distances across the tissue neurons. The mathematical distribution organizes the recorded signal values ​​by the measured amplitudes of signals recorded at known distances across the tissue cell sample.

[0041] The term "velocity distribution" refers to a mathematical distribution of recorded signal values ​​generated from a series of stimulation signals applied to tissue neurons at various distances across the tissue neurons. The mathematical distribution organizes the recorded signal values ​​by calculated signal velocities of the recorded signals at known distances across the tissue cell sample. By measuring the arrival time values ​​of the recorded signal values ​​measured at specific distances from the application location of the stimulation signal, the measured velocity can be calculated as the specific distance divided by the arrival time of the recorded signal.

[0042] Meanwhile, the terms and words used in the present specification and claims should not be interpreted limitedly to their general meaning and dictionary meaning, but should be interpreted based on the meaning and concept corresponding to the technical aspects of the present invention based on the principle that the inventor can appropriately define the terms for the best explanation. Therefore, it should be understood that the embodiment and drawings described in the present specification are only a preferred embodiment of the present invention, do not represent all the technical aspects of the present invention, and there may be various equivalents and modifications that can be substituted for them at the time of filing this application.

[0043] Generally, the present disclosure relates to a device comprising an embedded electrode array (EEA) capable of testing a cell sample according to the present invention. To better understand the present invention, FIG. 1 shows an exemplary embodiment of a cell sample testing using a device for testing a cell sample according to the present invention. The cell sample testing device 100 includes a tissue cell sample 101 cultured in an inner cell-permissive channel 105 running in an outer cell-restrictive layer. The inner cell-permissive channel 105 and the outer cell-restrictive layer 106 can be made of either hydrogel or plastic such as polystyrene or cyclic olefin copolymer. A signal recording electrode 103 is placed in the tissue culture well 104 to record responses observed in the tissue cell sample 101 to an applied stimulation signal. One or more stimulation electrodes 102 are placed in the Cirlex channel 205 at a known distance from the signal recording electrode 103 to introduce an applied stimulation signal to the tissue cell sample 101 and allow observation of a response signal in the tissue cell sample 101 by the signal recording electrode 103. Application of a stimulation signal to the tissue cell sample 101 can be repeated at each of one or more stimulation electrodes 102 to obtain responses at regularly spaced sets along the Cirlex channel 205 to measure transmission of the stimulation signal through the tissue cell sample 101.

[0044] While existing approaches to similar testing of tissue cell samples 101 involve inserting stimulating electrodes 102 and signal recording electrodes 103 into the tissue cell sample 101 grown within an internal cell-permissive channel 105, the present invention allows testing to be performed repeatedly using stimulating electrodes 102 and signal recording electrodes 103 in an external cell-restricting layer 106 without introducing position and distance errors due to the insertion and removal of electrodes from the tissue cell sample 101.

[0045] Examples of such tests involving the growth of tissue cell samples 101 within cell-permissive channels 105 are disclosed in detail in PCT Patent Application No. PCT / US2015 / 050061, filed September 14, 2015, entitled "NEURAL MICROPHYSIOLOGICAL SYSTEMS AND METHODS OF USING THE SAME," and PCT Patent Application No. PCT / US2018 / 063861, filed December 4, 2018, entitled "CELL SYSTEMS USING SPHEROIDS AND METHODS OF MAKING AND USING THE SAME," which are co-owned and are incorporated by reference as if set forth in their entireties herein.

[0046] FIG. 2 illustrates a device with an embedded electrode array capable of testing cell samples, according to the present invention. A single tissue culture well 200 is shown having a pair of tissue culture wells 104a-104b disposed on either end of a Cirlex channel 205. A plurality of stimulating electrodes 212-219 are shown in the Cirlex channel 205, and signal recording electrodes 203, 220 are shown in each of the tissue culture wells 104a-104b. Each of the plurality of stimulating electrodes 212-219 exposed in the Cirlex channel 205 is located at the end of a corresponding set of circuit traces 222-229 in the PCB 206. Similarly, both signal recording electrodes 203, 220 are located at the end of corresponding circuit traces 221, 230.

[0047] The tissue cell sample 101 is placed in one of the tissue culture wells 204a-204b and cultured to span the length of the Cirlex channel 205 while contacting each of the stimulation electrodes 212-219 and signal recording electrodes 211, 220. The set of circuit traces 221-230 are electrically coupled to a stimulation signal source and one or more signal recording circuits, which are described below with reference to Figures 9-10. An automated test system 1000, shown in Figure 10, is connected to the embedded electrode array 100 to automatically generate stimulation signals that are applied to the tissue cell sample 101, while response signals are received and acquired via the signal recording electrodes 211, 220.

[0048] A pair of reference voltage electrodes 201-202 are positioned adjacent to each of the tissue culture wells 204a-204b. These electrodes 201-202 can now apply a voltage or current to the tissue culture wells 200 to stimulate faster growth of the tissue cell sample 101 prior to testing. These electrodes 201-202 are connected to an external BNC connector (connected to the incubator and building ground) to the EEA plate ground to reduce noise.

[0049] FIG. 3 illustrates a printed circuit board (PCB) with multiple embedded electrode arrays capable of testing cell samples in accordance with the present invention. A single tissue culture well 200 may be replicated multiple times on a single PCB 106, as shown in FIGS. 3 and 5. A set of circuit traces 222-229 is shown in FIG. 5 connecting each electrode 211-220 in the tissue culture well 200 shown in FIG. 2 to a set of connectors 561-565 mounted on the PCB array 500. This arrangement of sets of circuit traces 222-229 is repeated for each of the replicated tissue culture wells 501-504, 511-514, 521-524, 531-534, 541-544, and 551-554. Each pin on the connectors 561-565 can be separately controlled within the automated test system 1000.

[0050] 4a-4c show the connection of multiple embedded electrode arrays 100 to corresponding stimulation signal sources and one or more signal recording circuits shown in Fig. 9 to create an automated test system 1000 in accordance with the present invention. The PCB array 500 is electrically coupled to its corresponding interface PCB 900, which includes the stimulation signal source and one or more signal recording circuit PCB boards 900.

[0051] Figures 4a-4b show the coupling of PCB boards via connectors 561-565. Figure 4a shows the connection with the connecting cable 411 removed from the connector 412, and Figure 4b shows the connecting cable 411 inserted into the connector 412. This configuration may be repeated for each of the connectors 561-565 on the PCB array 500. Multiple connecting cables 411 may be combined into a single connector that may be attached to the connectors 561-565 as shown in Figure 4c.

[0052] The PCB array 500 includes a plurality of tissue culture wells 200 having electrodes at the ends of a set of circuit traces 221-230 passed to an interface PCB 900 shown in Figures 9-10 that can sample response signals from each of the signal recording electrodes 103 in each tissue culture well 200. The interface PCB 900 also includes a stimulus source that is applied to the tissue cell samples 101 via the stimulating electrodes 212-219. The interface PCB 900 is controlled by a test computing system 1001 that supports programmatically defining tests to be performed on the tissue cell samples 101 in each tissue culture well 200 of the PCB array 500.

[0053] The automated test system 1000 of Figures 9 and 10a-b can test a subset of the replicated tissue culture wells 501-504, 511-514, 521-524, 531-534, 541-544, and 551-554 at a time, for example, in a row of replicated tissue culture wells 501-504. A typical test would test the top and bottom halves of the PCB array 900 separately. For example, test 1 would be performed on wells (501, 502, 511, 512, 521, 522, 531, 532, 541, 542, 551, 552) and test 2 would be performed on the remaining wells. A set of circuit traces 222-229 are fabricated into a PCB array 500 as disclosed below with reference to Figures 6-8, and the stimulating electrodes 212-219 and signal recording electrodes 211, 220 are completed with a soft gold (Au) layer to eliminate the effects of copper on the tissue cell sample 101.

[0054] The above example describes one of several possible placements and uses of electrodes 211-220. All of the electrodes 211-220 are capable of stimulating and recording. A typical session would involve selecting one electrode (e.g., 215) for stimulation while recording from all of the other electrodes 211-220.

[0055] The number and arrangement of tissue culture wells 200 may be varied as needed by the requirements of any tests being performed, and may be limited by manufacturing-related considerations regarding the size of the PCB array 500, the number and arrangement of connectors 561-565, and the number of channels to be supported by the automated test system 1000. The sets of circuit traces 222-229 on the PCB array 500 from each tissue culture well 200 may be arranged in any number of layers within the PCB array 500, as may be required by the number of channels to be supported.

[0056] 6 is a diagram illustrating a device with an embedded electrode array capable of testing cell samples according to the present invention. An exploded view 600 of the embedded electrode array 500 includes an acrylic well layer 601, a PSA (pressure sensitive adhesive) well layer 602, a Cirlex channel layer 603, a PSA channel layer 604, and a PCB layer 605.

[0057] The acrylic well layer 601 forms isolated medium reservoirs for each of the multiple tissue culture wells 200 on the PCB array 500. This acrylic well layer 601 is laser cut using a CO2 laser to generate the desired pattern. In other embodiments, the embedded electrode array 500 may also include an injection molded array board made from a polycarbonate material.

[0058] The PSA well layer 602 is an adhesive that bonds the well layer 601 to the channel layer 603 and PCB layer 605 to form the media reservoirs for each tissue culture well 200. The PSA well layer 602 is an 81 μm thick pressure sensitive adhesive from Adhesive Research. The PSA well layer 602 is laser cut using a CO2 laser to create the desired pattern.

[0059] The Cirlex channel layer 603 forms a cell-confining template that guides axonal growth across the electrodes 211-220. The Cirlex channel layer 603 is a type of polyimide called Cirlex, with a nominal thickness of 500 μm. Cirlex is manufactured by Fralock, Inc. of Valencia, California, and can be laser cut using a UV laser to produce channels of the desired pattern within the tissue culture well 200.

[0060] The PSA channel layer 604 is an adhesive that bonds the channel layer to the PCB layer 605 to form a cell-restricting mold that guides axonal growth across the electrodes. The adhesive material is a pressure-sensitive adhesive from Adhesive Research that is 81 μm thick. It is laser cut using a UV laser to create the desired pattern.

[0061] PCB layer 605 corresponds to the flexible PCB material of PCB array 500 that contains the actual embedded electrode array. It is a composite device made of multiple layers of polyimide, soft gold electroplated over copper, and adhesive layers. This PCB layer is described in more detail with reference to FIG. 8 described below.

[0062] Each of the above layers 601-605 are manufactured separately with the completed layers being joined into a single embedded electrode array 100. Once the layers are joined into a single device, connectors 561-565 may be inserted and soldered in place before the entire embedded electrode array 100 is connected to an automated test system 1000.

[0063] The plurality of tissue culture wells 200 of the PCB array 500 are created by cutting openings, cavities, and channels in each of these layers. When the above layers 601-605 are bonded together, the electrodes 211-220 are exposed in the tissue culture wells 200 when the openings, cavities, and channels in each of these layers are properly aligned. The Cirlex channels 205 and tissue culture wells 104 in each tissue culture well 200 are also exposed as each opening on any given layer when aligned and configured to allow access to the tissue culture wells 104, Cirlex channels 105, and electrodes 211-220 for use in testing tissue cell samples 101.

[0064] FIG. 7 shows a set of electrode pads in an exemplary tissue culture well of an embedded electrode array in which cell samples can be tested, according to the present invention. A Cirlex channel 701 is shown with a tissue culture well 702a-b at each end. Electrode pads 703a-b, 704a-f are the ends of respective circuit traces 222-229 that extend upward into respective tissue culture wells 200 and are shown disposed within the tissue culture wells 702a-b and the Cirlex channel 701. Each electrode pad 704a-f in the Cirlex channel 701 is 50 μm in diameter and is centered along an axis that spans its length. The Cirlex channel 701 is itself a 200 μm wide tissue culture well. Each of the tissue culture wells 702a-b, which have a diameter of 700 μm, has an electrode pad 703a-b, also having a diameter of 50 μm, at its center.

[0065] The distance between tissue culture wells 701a-701b is 9 mm, and the distance between each adjacent pair of electrode pads 704a-704f in the Cirlex channel 701 is 1 mm. The distance between each electrode pad 703a-703b in tissue culture wells 702a-702b in the PCB array is 500 μm. All of these distances are measured from the center of each electrode pad.

[0066] The position and spacing of these electrode pads 703a-703b, 704a-704f are controlled during manufacturing to ensure that the distance between any pair of two electrode pads is known and consistent in all tissue culture wells 200 of the embedded electrode array 100. When used for testing, a stimulation signal is applied to one of these electrode pads 704a-704f and a response signal is observed at all of the other electrode pads in the tissue culture wells 200. An event occurring in the stimulation signal, for example a voltage change from one extremum of a square wave to another extremum of a square wave, can be identified in the stimulation signal. The response observed in each response signal may occur at a different time relative to the occurrence of the event in the stimulation signal depending on the distance between the electrode introducing the stimulation signal to the tissue cell sample 101 and the electrode 704a-704f that corresponds to the particular response signal. This observed delay in the occurrence of the response in each of the response signals and their distance between each other can be used to calculate the observed velocity of the stimulation signal along the tissue cell sample 101. The position and size tolerances of the electrode pads 703a-703b, 704a-704f constrain the precision possible in any measurement of time and velocity values. These distance, time, and velocity values, and the corresponding amplitude values ​​observed in the response signal, can be used to calculate various metrics related to electrical activity within the tissue cell sample 101, both in the presence and absence of a pharmacological material applied to the tissue cell sample 101 within the tissue culture well 200.

[0067] 8 is a diagram showing a close-up view of an embedded electrode array capable of testing cell samples according to the present invention. The PCB array 500 may be constructed using a multi-layer substrate 800 as shown in FIG. 8. A cross-sectional view of a tissue culture well 200 is shown in the top layer of the multi-layer substrate 800. The multi-layer substrate 800 includes a bottom polyimide cover layer 801, a first adhesive cover layer 802, a bottom copper layer 803, a polyimide base layer 405, a top copper layer 805, a second adhesive cover layer 806, a top polyimide cover layer 807, and a soft gold electroplated layer 810. In an alternative embodiment, an ENIG (electroless nickel immersion gold) layer may be used.

[0068] The bottom polyimide cover layer 801 is a synthetic material layer disposed on the bottom side of the PCB array 500 to seal its internal materials from the environment of the PCB array 500 while it is being used for testing. The polyimide material is a fabric or material made from strings of polyimide monomers having a thickness of 12.5 μm. In a preferred embodiment, the polyimide material corresponds to DuPont Pyralux AP coverlay and DuPont Pyralux LF coverlay manufactured by DuPont Corporation (Wilmington, Del.). Other similar polyimide materials may also be utilized.

[0069] A first adhesive cover layer 802 bonds the bottom polyimide cover layer 801 to the bottom copper layer 803. The first adhesive cover layer 802 has a thickness of 12.5 μm in the preferred embodiment. The adhesive cover layer 802 is part of a DuPont coverlay and functions as a "proprietary B-stage modified acrylic adhesive." A more common class are B-stage acrylic adhesives.

[0070] The bottom copper layer 803 is the first of two copper layers in which a set of circuit traces 221-230 is defined. Each of the circuit traces 221-230 is a separate layer of copper from one of the electrodes 211-220 in the tissue culture well 200 to a connection pin in the connector 561-565. The circuit traces 222-229 are insulated from each other along the polyimide base layer 405 and are configured to reduce or eliminate electrical interference from one circuit trace to another. Where a set of circuit traces 221-230 is not present, for example between a pair of circuit traces, the first adhesive cover layer 802 extends up to the polyimide base layer 405. Each copper layer may be made from 0.5 oz copper that is approximately 17.5 μm thick.

[0071] The polyimide base layer 405 is a synthetic material layer disposed within the PCB array 500 to electrically insulate the bottom copper layer 803 and the top copper layer 805 from each other. The polyimide base layer 405 also provides rigidity and structure to the PCB array 500 to support its use and the tissue cell samples 101 that are grown and tested therein.

[0072] The top copper layer 805 is the second of two copper layers in which the set of circuit traces 221-230 is defined. Each of the circuit traces 221-230 is a separate layer of copper from one of the electrodes 211-220 in the tissue culture well 200 to a connection pin in the connectors 561-565. The circuit traces 222-229 are insulated from one another along the polyimide base layer 405 and are configured to reduce or eliminate electrical interference from one circuit trace to another. All of the circuit traces 222-229 are defined on either the bottom copper layer 803 or the top copper layer 805 which may be connected together, the copper layers being completed through the use of vias, with additional copper being plated into the through holes connecting the two layers. However, the electrodes themselves are not vias, as all of the electrodes are disposed on the top copper layer. One skilled in the art will appreciate that the set of circuit traces 222-229 may be defined on three or more copper layers separated by separate polyimide base layers. Similar to the bottom copper layer 803, the top copper layer 805 may be made of 0.5 oz copper, approximately 17.5 μm thick.

[0073] A second adhesive cover layer 806 bonds the top polyimide cover layer 807 to the top copper layer 805. The second adhesive cover layer 806 has a thickness of 12.5 μm in a preferred embodiment. The adhesive is provided as part of the DuPont coverlay. It is a proprietary B-stage modified acrylic adhesive. Similar adhesives belonging to a more general class, such as B-stage acrylic adhesives, can also be used. The top copper layer 805 extends into the tissue culture well 200 along the Cirlex channel 205 as shown in FIG. 2 and described above. Where there is no set of circuit traces 221-230, the second adhesive cover layer 806 extends down to the polyimide base layer 405, for example, between a pair of circuit traces.

[0074] The top polyimide cover layer 807 is a synthetic material layer disposed on top of the PCB array 500 to seal its interior materials from the environment of the PCB array 500 while it is being used for testing. The polyimide material, like the bottom polyimide cover layer described above, is a fabric material made from strings of polyimide monomers having a thickness of 12.5 μm. Each tissue culture well 200 corresponds to a cut at a specific location of each tissue culture well 200. Cutting through the top polyimide cover layer 807 and the second adhesive cover layer 806 exposes the electrodes 211-220 in the tissue culture well 200.

[0075] The soft gold electroplating layer 810 is a surface plating that is electroplated onto the top copper layer 805. The ENIG layer 810 is exposed to any supporting material that forms the tissue cell sample 101 and the electrical pads of the electrodes 211-220 within the tissue culture well 200, and protects the electrodes 211-212 from corrosion and other anomalies. The ENIG layer 810 has a nominal thickness of about 1 μm, although other thicknesses may be utilized.

[0076] FIG. 9 illustrates a printed circuit board capable of testing cell samples against multiple connectors in accordance with the present invention. As described above, the automated test system 1000 utilizes the PCB array 500 and the interface PCB 900 to perform test operations and record data associated with observed responses to stimuli. The interface PCB 900 is electrically coupled to the PCB array 500 and enables components of the interface PCB 900 to provide stimulus signals and observe corresponding responses. The entire sequence of test operations is performed under the control of a test controller 1001, as described below with reference to FIG. 10. Data associated with observed responses to stimulus signals is collected and stored in a data collector 1010, as described below with reference to FIGS. 10a-b.

[0077] An exemplary layout of the interface PCB 900 includes a stimulation signal source and one or more signal recording circuits for signals observed within the tissue cell sample 101. The interface PCB 900 includes a plurality of function signal toggle switch isolators 901, a reference electrode toggle switch 902, a function generator ground toggle switch 903, a three-position toggle switch 904, a digital multiplexer control connector 905, a function generator connector 906, a power connector 907, a controller connector 908, a plurality of reference electrode jumper connectors 909a-909n each configured to connect to a corresponding one of a plurality of amplifier / AD digitizer circuits 915a-915n, and a connectivity check indicator 910. Each of these toggle switches and jumper signal connectors are used to configure the automated testing system 1000 to perform testing of the tissue cell samples 101 within the tissue culture wells 200 of the PCB array 500.

[0078] The multiple function signal toggle switches 901 allow for the selection of a reference voltage source for applying a voltage to one of two reference electrodes 201-202 in tissue culture wells 200. The multiple function signal toggle switches 901 allow these reference voltage sources to apply separate conditions to six groups of four wells each. Of course, different numbers of multiple function signal toggle switches 901 and different groupings of tissue culture wells 200 for each toggle switch may be used as desired.

[0079] The reference electrode toggle switch 902 allows for the selection of a connection to a common ground used as an amplifier reference voltage to the reference electrode in each tissue culture well 200. Setting the toggle switch 902 to the reference electrode in each tissue culture well 200 eliminates noise in the response signals observed on the electrodes 211-220.

[0080] The function generator ground toggle switch 903 selects the function generator ground signal to a common ground in the embedded electrode array 100 or in the reference electrodes 201-202 in each tissue culture well 200. In a preferred embodiment, the function generator ground toggle switch 903 is set to select common ground during testing.

[0081] The three-position toggle switch 904 selects the source of a multiplexer control signal that selects one of two sets of data channels for use as a data source to be received, processed, and stored for later use. The embedded electrode array 100 organizes the response signals received on the electrodes 211-220 into data channels having two sets of data channels. A control signal received via the digital multiplexer control connector 905 selects which of the two sets is actively connected for storage. The three-position toggle switch 904 selects this source from a first set of data channels, a second set of data channels, and a set of data channels specified in the control signal received by the embedded electrode array 100 via the digital multiplexer control connector 905. The organization of the tissue culture wells 200 into different numbers of sets of data channels may be used in conjunction with an encoded selection value provided to the embedded electrode array 100 to select an active set of data channels from more than two sets of data channels.

[0082] The digital multiplexer control connector 905, which in a preferred embodiment is a BNC type connector, controls the selection of a data channel to be used as a data source that is received, processed, and stored for later use. The embedded electrode array 100 organizes the response signals received on the electrodes 211-220 into data channels having two sets of data channels. A control signal received via the digital multiplexer control connector 905 selects which of the two sets is actively connected for storage. The organization of the tissue culture wells 200 into different numbers of sets of data channels may be used in conjunction with an encoded selection value provided to the embedded electrode array 100 to select an active set of data channels from more than two sets of data channels.

[0083] The function generator connector 906 is a BNC-type connector in a preferred embodiment that allows for the connection of an external function generator to provide a signal that is applied as a stimulation signal to the tissue cell sample 101. The characteristics of the stimulation signal, including the voltage amplitude, frequency, and time between signal cycles, are defined by the external function generator. This external signal is received via the function generator connector 906 and applied to the tissue cell sample 101 using one of the signal recording electrodes 103 in each tissue culture well 200.

[0084] The power connector 907 is a sub-D type connector in the preferred embodiment and receives power from an external power source that supplies the electronics to enable its operation. The power connector 907 may include connections for one or more voltage levels that may be used to power the electronics, as otherwise disclosed herein, and one or more reference voltages used within the implanted electrode array 100. In an alternative embodiment, the power connector 907 may accept an AC voltage that is fed into a transformer-based power supply to generate the necessary power and reference voltages required within the implanted electrode array 100.

[0085] The controller connector 908 is a SPI connector in a preferred embodiment that connects the embedded electrode array 100 to an external test controller 1001, disclosed with reference to FIG. 10 below.

[0086] The plurality of amplifier / AD digitizer circuits 915a-915n are configured into an operational state using a plurality of reference electrode jumper connectors 909a-909n, each of which is electrically coupled to a corresponding one of the plurality of amplifier / AD digitizer circuits 915a-915n.

[0087] The connectivity check indicator 910 corresponds to a button and LED designed to test whether the PCB array 500 and the interface PCB 900 are properly connected to each other. The user presses the button and the system checks to ensure that the ground signal is properly connected. If properly connected, the LED light turns on.

[0088] 10a-10b are diagrams illustrating components within a device with an embedded electrode array capable of testing cell samples according to the present invention. FIG. 10a shows a block diagram of an automated test system 1000 utilizing an embedded electrode array 100. FIG. 10b shows a block diagram of an amplifier / AD digitizer circuit 1003. The automated test system 1000 includes an embedded electrode array 100, an interface PCB 900, a test controller 1001, and a data collector 1010. The embedded electrode array 100 is one of the embedded electrode array PCBs 500 disclosed herein.

[0089] The interface PCB 900 includes a set of EEA connectors 1002, a number of amplifier / AD digitizer integrated circuits (ICs) 1003, a data channel multiplexer 1004, and a control logic circuit 1011, as described below with reference to FIG. 10b.

[0090] The data channel multiplexer 1004 selects one of the two active sets of data channels to enable data collection from the selected data channel. The embedded electrode array 100 organizes the response signals received on the electrodes 211-220 into data channels having two sets of data channels. A control signal received via a digital multiplexer control connector 905 provided to the data channel multiplexer 1004 selects which of the two sets is actively connected for storage. The organization of the tissue culture wells 200 into different numbers of sets of data channels may be used in conjunction with an encoded selection value provided to the embedded electrode array 100 to select an active set of data channels from more than two sets of data channels.

[0091] The control logic circuit 1011 receives a set of control input signals from the test controller 1001 that are used to generate signals that control the various electronic components within the interface PCB 900 and the implanted electrode array 100. The control input signals may be received by the interface PCB 900 using the various connectors described above with reference to Figure 9. The control logic circuit 1011 generates signals that control the operation of the amplifier / DA digitizer circuit 1003 and the data channel multiplexer 1005.

[0092] The test controller 1001 is a programmable computing device that executes operations to perform an automated tissue cell sample 101 testing process. The test controller 1001 implements the automated testing process by instructing the interface PCB 900 to initiate generation of stimulation signals onto one or more stimulation electrodes 102 in tissue culture wells 200 of the embedded electrode array 100, and to retrieve and store digital data sets from data channels associated with the tissue culture wells 200 that receive the stimulation signals. The test controller 1001 may repeat these operations for alternate tissue culture wells 200 using alternate stimulation signals.

[0093] A tissue cell sample 101 must be cultured within the Cirlex channel 205 of each tissue culture well 200 used in the automated testing process. The embedded electrode array 100 may then be configured to operate as otherwise disclosed herein, with the embedded electrode array 100 within the tissue culture well 200 stored in a suitable environment before the automated testing process is initiated. The test controller 1001 may repeat the above sequence of operations until response signal data from each tissue cell sample 101 has been collected using one of the available stimulation signals. The automated testing process may be performed using the tissue cell sample 101 after the required incubation within the Cirlex channel 205 has been completed to generate a baseline data set. One or more chemical and / or pharmacological materials may be applied to one or more tissue cell samples 101, and the automated testing process may be repeated at different time periods following application of the pharmacological materials to obtain a time series of data representative of the effect of the pharmacological materials on the responses observed within the tissue cell sample 101. Automated testing processes can use a variety of pharmacological materials applied using different amounts or concentrations of the pharmacological materials in these testing processes to investigate a wide range of variables as part of a larger set of tests using a particular pharmacological material and a set of similar pharmacological materials.

[0094] The data collector 1010 is a digital electrophysiology test device that generates stimulation signals that are applied to tissue cell samples 101 in each tissue culture well 200 of the embedded electrode array 100. The data collector 1010 also collects and stores digital data sets of time-based response signals generated by tests performed using the embedded electrode array 100. In a preferred embodiment, the data collector 1010 may be a commercially available modular electrophysiology data acquisition system, such as the Intan Stim / Recording System by Intan Technology, Inc. of Los Angeles, Calif. The modular electrophysiology data acquisition system may also be provided by any comparable commercially available system.

[0095] Figure 10b shows a block diagram of the amplifier / AD digitizer circuit 1003. The interface PCB 900 includes a set of EEA connectors 1002, a number of amplifier / AD digitizer integrated circuits (ICs) 1003, a data channel multiplexer 1004, and a control logic circuit 1011, as described below with reference to Figure 10b.

[0096] The set of EEA connectors 1002 allows electrical signals to be passed between the interface PCB 900 and the implanted electrode array 100 using a data cable 1021. This data cable electrically couples each pin on the connectors 561-565 of the implanted electrode array 100 to pins on the set of EEA connectors 1002 to pass power supply voltages, reference voltages, and ground signals, as well as one or more stimulation signals.

[0097] The amplifier / DA digitizer IC 1003, in the form of a block diagram of FIG. 10b, includes a plurality of signal amplifiers 1051a-n, an analog switch 1052, a digital-o-analog circuit 1053, and a data collector interface 1054. In a preferred embodiment, each of the plurality of amplifier / DA digitizer ICs 1003 comprises an Intan Technologies RHS2116 digital electrophysiology stimulator / amplifier integrated circuit manufactured by Intan Technologies, Inc. of Los Angeles, Calif. The Intan RHS2116 processes 16 input signals received from a set of signal recording electrodes 103 in the implanted electrode array 100 to generate digital data associated with 16 separate data channels.

[0098] Each signal from one of the signal recording electrodes 103 is received and amplified by one of the signal amplifiers 1051a-1051n and input to one of 16 signal inputs on an analog switch 1052. The analog switch 1052 cyclically selects each of the 16 signal inputs, one at a time, to pass to a digital-to-analog circuit 1053. The digital-to-analog circuit 1053 samples each of the input signals passed to it to generate a digital representation of the signal in a corresponding one of 16 data channels that is output via a data collector interface 1054.

[0099] The digital-to-analog circuit IC 1053 is a 16-bit DA digitizer that may also use a reference voltage in generating a digital representation of the input signal. The digital-to-analog circuit 1053 typically cycles through each of the 16 input signals repeatedly to generate data for each of the 16 data channels in the amplifier / DA digitizer circuit 1003. The amplifier / DA digitizer circuit 1003 includes 16 signal inputs for generating the 16 data channels, and the interface PCB 900 includes 16 amplifier / DA digitizer circuit IC 1003 that enable the embedded electrode array 100 to provide 256 data channels from the tissue culture wells 200 in the embedded electrode array 100. The number of data channels of the tissue culture wells 200, amplifier / DA digitizer circuit IC 1003, and interface PCB 900, as well as the particular organization of groups of data channels, are described herein for illustrative purposes only. The implanted electrode array 100 should not be limited to any number of each of these items, except as limited by the claims appended hereto.

[0100] FIG. 11 illustrates an exemplary graphical display of digital channel data collected from multiple circuit traces 222-229 of an embedded electrode array 100 testing a cell sample, according to the present invention. FIG. 11 illustrates observed data sets 1101a-1101f from multiple electrodes 211-220 in one tissue culture well 200. In this example, a stimulation signal is applied to electrode 5 1102e and response signals are observed on electrodes 1-5, 6 1102a-1102d, 1102f. The response signals are acquired within the same sampling cycle performed by the amplifier / DA digitizer IC 1003 allowing comparison of the signals. Responses seen within the various observed data sets 1101a-1101f may indicate time differences in similar responses observed within the observed data sets 1101a-1101f as the context of a particular electrode 1102a-1102f from the location of the stimulation signal.

[0101] 2, the location of each particular electrode 1102a-1102f and its distance relative to one another is known and consistent across all tissue culture wells 200 within the embedded electrode array 100. Thus, the velocity of the response to a stimulation signal can be calculated using the time difference between the response to the stimulation signal observed on two particular electrodes 1102a-1102f and the known distance between those two particular electrodes 1102a-1102f. In addition, velocity may be calculated for each pair of two electrodes in a similar manner to determine whether the velocity of the observed response changes as a result of an increase in the distance between the two electrodes 1102a-1102f.

[0102] Many different test studies can be created using the observed data sets 1101a-1101f and the corresponding amplitudes and positions of the corresponding electrodes. Because the observed data sets 1101a-1101f are retained in the data collector 1010, these studies can be generated at a later date using the data sets.

[0103] FIG. 12 shows an exemplary graphical representation of an automated testing process for collecting digital channel data from multiple circuit traces 222-229 of an embedded electrode array 100 testing a cell sample, according to the present invention. In this example, different stimulation signals 1202 are applied to different electrodes 1201 to observe the response at one or more of the remaining electrodes in the tissue culture well 200. Although multiple wells may be stimulated simultaneously, stimulation within each tissue culture well 200 is performed in full sequence. For example, a typical test involves stimulating 12 tissue culture wells 200 in the top half of the PCB array 500. The first stimulation for all 12 tissue culture wells 200 is a low current (1 μA) stimulation at electrode 6 alone for six trials. The current is then increased to 5 μA over six trials, and so on, until a maximum current of 64-80 μA is applied to electrode 6. The stimulation location is then shifted to electrode 5, and the process is repeated. This sequence is typically performed for electrodes 2-6, but can be performed for all ten electrodes within tissue culture well 200. The pressure graph shown in Figure 12 is designed to illustrate the reasoning behind increasing stimulation current, which is similar to increasing pressure in the touch test.

[0104] 13a-b show an exemplary result of a processed set of digital channel data collected from a printed circuit board with signal lines coupling an embedded electrode array on which a cell sample can be tested, according to the present invention. The signal at 1300 shows the processed data in the time domain, and 1320 shows the same signal in the velocity domain along with a record of the noise level. The velocity and amplitude distributions are obtained by performing peak detection on the signal at 1320 to find any peaks that are 6 standard deviations above the noise level (red). For each peak, the velocity and amplitude, e.g., y-height shown, are extracted and collated for distribution. With regard to other metrics, a maximum velocity projection is calculated by taking a signal such as 1320 from all of the responses in each well at a given current level, e.g., 10 electrodes in well A1 stimulated at 5 locations with 48 μA giving 45 unique responses, 9 responses per location excluding the stimulating electrodes. These 45 responses are in the velocity domain and can be superimposed on one another. The maximum value across all 45 responses is calculated to obtain a signal that shows the maximum response, the maximum velocity projection. Different metrics can be extracted from this by taking the area under the curve for different speed regions and comparing the MVP across different current values.

[0105] 14 illustrates a computer system 1400 adapted in accordance with a particular embodiment of a test controller in accordance with the present invention. A central processing unit ("CPU") 1402 is coupled to a system bus 1404. The CPU 1402 may be a general purpose CPU or microprocessor, a graphics processing unit ("GPU"), and / or a microcontroller. The present embodiment is not limited by the architecture of the CPU 1402 so long as the CPU 1402 supports, whether directly or indirectly, the operations described herein. The CPU 1402 may execute various logical instructions in accordance with the present embodiment.

[0106] The computer system 1400 may also include a random access memory (RAM) 1408, which may be a synchronous RAM (SRAM), a dynamic RAM (DRAM), or a synchronous dynamic RAM (SDRAM), etc. The computer system 1400 may utilize the RAM 1408 to store various data structures used by software applications. The computer system 1400 may also include a read only memory (ROM) 1406, which may be a PROM, an EPROM, an EEPROM, an optical storage device, etc. The ROM may store configuration information for booting the computer system 1400. The RAM 1408 and the ROM 1406 hold user and system data, and both the RAM 1408 and the ROM 1406 may be randomly accessed.

[0107] Computer system 1400 may also include an input / output (I / O) adapter 1410, a communications adapter 1414, a user interface adapter 1416, and a display adapter 1422. The I / O adapter 1410 and / or the user interface adapter 1416 may, in particular embodiments, allow a user to interact with computer system 1400. In further embodiments, the display adapter 1422 may display a graphical user interface (GUI) associated with software or web-based applications on a display device 1424, such as a monitor or touch screen.

[0108] The I / O adapter 1410 can couple one or more storage devices 1412, such as one or more of a hard drive, a solid-state storage device, a flash drive, a compact disc (CD) drive, a flash drive, and a tape drive, to the computer system 1400. According to one embodiment, the data storage 1412 may be a separate server coupled to the computer system 1400 via a network connection to the I / O adapter 1410. The communications adapter 1414 may be adapted to couple the computer system 1400 to a network, which may be one or more of a LAN, a WAN, and / or the Internet. The communications adapter 1414 may also be adapted to couple the computer system 1400 to other networks, such as a Global Positioning System (GPS) or a Bluetooth network. The user interface adapter 1416 couples user input devices, such as a keyboard 1420, a pointing device 1418, and / or a touch screen (not shown) to the computer system 1400. The keyboard 1420 may be an on-screen keyboard displayed on a touch panel. Additional devices (not shown), such as a camera, microphone, video camera, accelerometer, compass, and / or gyroscope, may be coupled to the user interface adapter 1416. A display adapter 1422 is driven by the CPU 802 and can control the display on a display device 1424. Any of the devices 1402-1422 may be physical and / or logical.

[0109] Application of the present disclosure is not limited to the architecture of the computer system 1400. Rather, the computer system 1400 is provided as an example of one type of computing device that may be adapted to perform the functions of the test controller 1001 and the data collector 1010. Any suitable processor-based device may be utilized, including, for example, but not limited to, a personal digital assistant (PDA), a tablet computer, a smart phone, a computer game console, and a multiprocessor server. Furthermore, the apparatus of the present disclosure may be implemented on an application specific integrated circuit (ASIC), a very large scale integrated (VLSI) circuit, a state machine digital logic based circuit, or other circuit.

[0110] The embodiments described herein are implemented as logical operations executed by a computer. The logical operations of these various embodiments of the invention are implemented as (1) a sequence of computer-implemented steps or program modules executed on a computing system, and / or (2) as interconnected machine modules or hardware logic within a computing system. The implementation is a matter of choice dependent on the performance requirements of the computing system implementing the invention. Thus, the logical operations making up the embodiments of the invention described herein can be variously referred to as operations, steps, or modules. Thus, one skilled in the art can employ any number of suitable electronic devices and similar structures capable of performing the sequence of logical operations according to the described embodiments. For example, computer system 1400 may be virtualized for access by multiple users and / or applications.

[0111] 15 illustrates a computing system of embedded electrode array controller software components capable of testing cell samples in accordance with the present invention. The embedded electrode array controller 1500 includes a set of software components for initiating the stimulus generation components and recording digital representations of any observed responses to the stimuli. The set of software components includes a test controller component 1501, a test sample sequencer 1502, a signal amplifier control interface 1503, a data receiver interface 1504, a user interface 1501 coupled to a user display 1514 and input device 1514, and a storage interface 1506 coupled to a local memory storage device 1512.

[0112] Test controller component 1501 receives automatic test procedure commands from a user via user interface component 1505. Test controller component 1501 also receives equipment status display data from the electronics in PCB array 500 and interface PCB 900. Test controller component 1501 interacts with the remaining set of processing components 1502-1506 to perform the sequence of operations necessary to implement the automatic test process as necessary. Test controller component 1501 may also generate test status and test result data for a user to view on display device 1513 via user interface component 1505 as operation of the automatic test process progresses.

[0113] The test sample sequencer 1502 generates control signals for configuring the tissue culture wells 200 in the embedded electrode array 100 to perform test operations. The test sample sequencer 1502 with the signal amplifier control interface 1503 defines the characteristics of the stimulation signal to be applied to one of the electrodes 211-220 in each tissue culture well 200 of the embedded electrode array 100. The test sample sequencer 1502 may repeat this configuration for each tissue culture well 200 in use for a particular test operation. The test sample sequencer 1502 initiates the application of the stimulation signal and the recording of all response signals by the data collector 1010 for all tissue culture wells 200 of the embedded electrode array 100 at once. The test sample sequencer 1502 may finish the application of the stimulation signal and the recording of all response signals, reconfigure these settings, and repeat the test procedure operations. The test sample sequencer 1502 may execute as many test procedure operations as required under the control of the test controller component 1501.

[0114] The test controller 1001 communicates with ancillary devices that can configure each stimulation signal that is applied to the tissue cell sample 101 in the tissue culture well 200. The signal amplifier control interface 1503 allows the test controller 1001 to modify any characteristics of the stimulation signal, such as the amplitude, frequency, and inter-event delay, assigned to each electrode 211-220 in each tissue culture well 200 of the embedded electrode array 100. The signal amplifier control interface 1503 in cooperation with the test sample sequencer 1502 can configure each tissue culture well 200 to stimulate the tissue cell sample 101 with one of its electrodes 211-220. The signal amplifier control interface 1503 performs data formatting, inter-computer communication, encryption processing, and all similar operations required by the test controller 1001 to communicate with the components that generate the stimulation signals and address each signal to the electrodes 211-220.

[0115] The Data Receiver Interface 1504 Test Controller 1001 communicates with the Data Collector 1010 and the Interface PCB 900. The Data Receiver Interface 1504 performs data formatting, inter-computer communication, encryption processing, and all similar operations required by the Test Controller 101 to communicate with all connected devices.

[0116] The user interface 1501 coupled to the user display 1514 and input devices 1514 provides input and output processing to provide messages and data to a user necessary to initiate, monitor, control, and terminate the automated test process. This user interface 1501 also accepts commands from a user to instruct the test controller 1001 to perform these tasks as necessary. The user display 1513 can be any computer display device, such as a monitor, LED flat screen, or similar device configured to display data to a user. The input devices 1514 can include pointing devices such as a mouse, trackpad, and trackball, as well as input devices such as a keyboard configured to allow a user to enter data and commands into the test controller 1001.

[0117] The storage interface 1506, coupled to the local memory storage device 1512, handles all data storage operations for the test controller 1001. These operations include writing data to the local memory storage device 1512, deleting data from the local memory storage device 1501, searching and retrieving data from the local memory storage device 1512, and indexing the local memory storage device 1512 to maintain efficient retrieval when needed.

[0118] Although particular combinations of features are described in this application, these combinations are not intended to limit the disclosure of the present invention. Indeed, many of these features may be combined in ways not specifically described in this application. In other words, any of the features mentioned in this application may be included in this new invention in any one or more combinations to enable the functionality required for the desired operation.

[0119] No element, act, or instruction used in this application should be construed as critical or essential to the invention unless expressly described as such. Further, the phrase "based on" is intended to mean "based at least in part on" unless expressly specified otherwise.

Claims

1. 1. A device comprising an embedded electrode array capable of testing a cell sample, said device comprising:

1. An implanted electrode array, comprising: a plurality of tissue culture wells each having a plurality of electrode pads for receiving tissue cell samples; one or more signal connectors having a plurality of connector pins for receiving a stimulus signal for each of the plurality of tissue culture wells and generating a plurality of response signals from each of the tissue cell samples; a plurality of circuit traces embedded within the EEA printed circuit board, each of the plurality of circuit traces connecting one of the plurality of connector pins to an electrode pad in each tissue culture well; and the embedded electrode array comprising: an interface printed circuit board, one or more signal connectors for receiving test signals for use as the one or more stimulation signals to a plurality of tissue culture wells in the attached embedded electrode array; one or more interface signal connectors having a plurality of connector pins for transmitting stimulation signals to each of the plurality of tissue culture wells in the attached embedded electrode array and receiving a plurality of response signals from each of the tissue cell samples; a plurality of amplifier-digitizer circuits, each configured to receive one or more of the response signals organized into a plurality of data channels and digitize the response signals for transmission as a data set for each of the plurality of data channels; the interface printed circuit board; a test controller that configures the one or more stimulus signals and the plurality of data channels in the interface printed circuit board to receive and sample response signals into the plurality of data channels; and a data collector that receives data sets for a plurality of data channels for processing and storage; The device comprising:

2. An embedded electrode array, comprising: a plurality of tissue culture wells each having a plurality of electrode pads for receiving tissue cell samples; one or more signal connectors having a plurality of connector pins for receiving a stimulus signal for each of the plurality of tissue culture wells and generating a plurality of response signals from each of the tissue cell samples; a plurality of circuit traces embedded within the EEA printed circuit board, each of the plurality of circuit traces connecting one of the plurality of connector pins to an electrode pad in each tissue culture well; and Equipped with each of said plurality of tissue culture wells a cell channel layer for culturing cells into said tissue cell sample; a pair of tissue culture wells disposed at each of the two ends of the Circex channel; eight electrode pads of the plurality of electrode pads arranged across a Circex channel; a single electrode pad among the plurality of electrode pads in each of the pair of tissue culture wells; Equipped with The embedded electrode array.

3. a pair of buried copper layers in the EEA printed circuit board for defining the plurality of circuit traces; a base polyimide layer between the pair of buried copper layers; an upper polyimide layer on one of the two copper layers; a bottom polyimide layer on one of the two copper layers; The implanted electrode array of claim 2 further comprising:

4. 4. The embedded electrode array of claim 3, further comprising a soft gold electroplating layer.

5. 5. The embedded electrode array of claim 4, wherein each of the tissue culture wells is in the top polyimide layer exposing the cell channel layer and the plurality of electrodes in the tissue culture well.

6. An interface printed circuit board, comprising: one or more signal connectors for receiving test signals for use as the one or more stimulation signals to a plurality of tissue culture wells in the attached embedded electrode array; one or more interface signal connectors having a plurality of connector pins for transmitting stimulation signals to each of the plurality of tissue culture wells in the attached embedded electrode array and receiving a plurality of response signals from each of the tissue cell samples; a plurality of amplifier-digitizer circuits, each configured to receive one or more of the response signals organized into a plurality of data channels and digitize the response signals for transmission as a data set for each of the plurality of data channels; Equipped with the test signal is received from an external function generator; the interface printed circuit board; 7. An interface printed circuit board, comprising: one or more signal connectors for receiving test signals for use as the one or more stimulation signals to a plurality of tissue culture wells in the attached embedded electrode array; one or more interface signal connectors having a plurality of connector pins for transmitting stimulation signals to each of the plurality of tissue culture wells in the attached embedded electrode array and receiving a plurality of response signals from each of the tissue cell samples; a plurality of amplifier-digitizer circuits, each configured to receive one or more of the response signals organized into a plurality of data channels and digitize the response signals for transmission as a data set for each of the plurality of data channels; Equipped with the data sets for each of the plurality of data channels are generated by the plurality of amplifier-digitizer circuits in groups of data sets corresponding to the data sets from a portion of the tissue culture wells; the interface printed circuit board;

8. 8. The interface printed circuit board of claim 7, wherein each of the plurality of amplifier-digitizer circuits receives 16 of the one or more response signals to generate 16 data channels.

9. 9. The interface printed circuit board of claim 8, wherein the interface printed circuit board comprises 16 amplifier-digitizer circuits for generating 256 data channels organized into two groups of 128 data channels.

10. a plurality of tissue culture wells, each of the plurality of tissue culture wells having a plurality of electrode pads for electrically contacting a tissue cell sample; one or more signal connectors having a plurality of connector pins for receiving a stimulus signal for each of the plurality of tissue culture wells and generating a plurality of response signals from each of the tissue cell samples; a plurality of circuit traces embedded within the embedded electrode array, each of the plurality of circuit traces connecting one of the plurality of connector pins to an electrode pad; 1. An implanted electrode array comprising:

11. each of said plurality of tissue culture wells a cell channel layer for culturing cells into said tissue cell sample; a pair of tissue culture wells disposed at each of two ends of the cell channel layer; Nine electrode pads of the plurality of electrode pads are arranged across the cell channel layer; and a single electrode pad among the plurality of electrode pads in each of the pair of tissue culture wells; 11. The embedded electrode array of claim 10, comprising:

12. a pair of buried copper layers in the EEA printed circuit board for defining the plurality of circuit traces; a base polyimide layer between the pair of buried copper layers; an upper polyimide layer on one of the two copper layers; a bottom polyimide layer on one of the two copper layers; 12. The embedded electrode array of claim 11, further comprising:

13. 13. The embedded electrode array of claim 12, further comprising a soft gold electroplated layer between said top polyimide layer and one of said two copper layers.

14. 14. The embedded electrode array of claim 13, wherein each of the tissue culture wells is in the top polyimide layer exposing the cell channel layer and the plurality of electrodes in the tissue culture well.

15. one or more signal connectors for receiving test signals for use as the one or more stimulation signals to a plurality of tissue culture wells in the attached embedded electrode array; one or more interface signal connectors having a plurality of connector pins for transmitting stimulation signals to each of the plurality of tissue culture wells in the attached embedded electrode array and receiving a plurality of response signals from each of the tissue cell samples; a plurality of amplifier-digitizer circuits, each configured to receive one or more of the response signals organized into a plurality of data channels and digitize the response signals for transmission as a data set for each of the plurality of data channels; An interface printed circuit board comprising:

16. 16. The interface printed circuit board of claim 15, wherein the test signal is received from an external function generator.

17. 16. The interface printed circuit board of claim 15, wherein the data sets for each of the plurality of data channels are generated by the plurality of amplifier-digitizer circuits in groups of data sets corresponding to the data sets from a portion of the tissue culture wells.

18. 18. The interface printed circuit board of claim 17, wherein each of the plurality of amplifier-digitizer circuits receives 16 of the one or more response signals to generate 16 data channels.

19. 20. The interface printed circuit board of claim 18, comprising 16 amplifier-digitizer circuits for generating 256 data channels organized into two groups of 128 data channels.

20. 20. The interface printed circuit board of claim 18, further comprising a digital switch for connecting 128 data channels of one of the two groups at a time to an external data collector.

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