Impedance-based determination of metrics derived from cytopathic effects

Abstract

Impedance-based determination of metrics derived from Cytopathic Effects (CPE), such as Tissue Culture Infections Dose (TCID) values, is provided via determining a first plurality of cellular impedances (CI) corresponding to a plurality of cell samples while performing the impedance assay; inoculating the plurality of cell samples with a plurality of different dilutions of the virus such that a first and second subsets of the plurality of cell samples are respectively inoculated with first and second dilutions of a virus; determining, after inoculating the plurality of cell samples, a second plurality of CIs; determining, from the first plurality of CIs and the second plurality of CIs, cytopathic effects (CPE) experienced by the plurality of cell samples; calculating, a CPE-derived metric.

Description

IMPEDANCE-BASED DETERMINATION OF METRICS DERIVED FROMCYTOPATHIC EFFECTSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present disclosure claims the benefit of and priority to U.S. Provisional Patent Application No.: 63 / 654,742 titled “IMPEDANCE-BASED DETERMINATION OF METRICS DERIVED FROM CYTOPATHIC EFFECTS” and filed on May 31, 2024, which is incorporated herein it its entirety.BACKGROUND

[0002] Viruses may cause devastating diseases; however, viruses are simple systems that can be manipulated to be beneficial and useful for many purposes in different areas, such as the mcdical / thcrapcutical fields for vaccine development and gene therapy. The concentration of viruses in a sample - the virus titer - is often used as a measurement in such fields. There are two common types of virus titers - physical and functional.

[0003] In a physical virus titer, an operator determines particle concentration by measuring corresponding nucleic acid or protein amounts, while in a functional virus titer, an operator counts infective virus particles. Generally, the functional titer is more accurate than physical titer because functional titer measures how much virus actually gets into the target cell, but may be more time consuming and challenging to perform. Examples of functional titer methods include plaque assays, focus-formings, and Tissue Culture Infections Dose-50 (TCID50) assays.

[0004] In a TCID50 assay, an operator quantifies the number of viruses needed to produce cytopathic effects (CPE) in 50% of inoculated cells. TCID50 assays may be particularly useful in assaying viruses that to not form plaques (cf. plaque assays). Similarly to plaque assays, TCID50 assays use an incubation time with several samples that include different dilutions of the virus are observed to identify when different samples exhibit signs of infection. These assays generally take two to fourteen days, depending on the virus and the host cells. The signs of infection, also referred to as the cytopathic effects (CPE), include cell death or morphological changes to the host cells, which may be determined by various techniques, including colorimetric measurement of compounds or dyes introduced to the sample or other optical-based measurements. The dilution inthe samples in which 50% of the host cells cultures are infected is calculated as the TCID50, which is expressed as 50% infectious dose per milliliter (ml) - TCID50 / ml.SUMMARY

[0005] The present disclosure provides for methods and systems for impedance-based determination of metrics derived from Cytopathic Effects (CPE), such as Tissue Culture Infections Dose (TCID) values.

[0006] In some embodiments, a method for performing an impedance assay is provided, the method comprising: preparing a plurality of cell samples; determining a first plurality of cellular impedance (CI) values corresponding to the plurality of cell samples while performing the impedance assay; preparing a plurality of different dilutions of a virus; inoculating, after determining the first plurality of CI values, the plurality of cell samples with the plurality of different dilutions of the vims such that a first subset of the plurality of cell samples is inoculated with a first dilution of the plurality of different dilutions of the virus and a second subset of the plurality of cell samples is inoculated with a second dilution of the plurality of different dilutions of the virus; determining, after inoculating the plurality of cell samples, a second plurality of CI values corresponding to the plurality of cell samples, wherein each Cl value of the second plurality of CI values is associated with one CI value of the first plurality of CI values; determining, from the first plurality of CI values and the second plurality of CI values, a plurality of cytopathic effect (CPE) statuses experienced by the plurality of cell samples; and calculating, based on the plurality of CPE statuses, one or more of a TCID for a predefined percentage of the samples experiencing the CPE statuses and a TCID / ml (milliliter).

[0007] In some such methods, preparing the plurality of cell samples includes incubating the cell samples for a predefined period of time after adding the cell samples and growth medium to a sample container.

[0008] In some such methods, preparing the plurality of cell samples includes adding a candidate therapeutic agent to the cell samples.

[0009] In some such methods, preparing the plurality of cell samples includes placing each cell sample prepared in a corresponding well of a wellplate having one of: 6 wells, 8 wells, 16 wells, 32 wells, 96 wells, and 384 wells.

[0010] In some such methods, preparing the plurality of cell samples includes: adding host cells with growth medium to a plurality of separate containers for each cell sample of the plurality of cell samples; incubating the host cells for a predefined incubation time period; and obtaining a base CI reading for each cell sample of the plurality of cell samples when the predefined time period concluded. room In some such methods, incubating includes controlling one or more operational settings during the predefined incubation time period, including at least one of: nutrient and gas exchange; oxygen control; pH control; a feeding regime of media exchange versus media addition; mixing or shear force; device O2 permeability; vessel size; removal of activation beads; or enrichment / selection of a particular subpopulation.

[0012] In some such methods, the predefined incubation time period is selected from one of: 4, 8, 12, 24, 36, 48, 60, or 72 hours, or 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days.

[0013] In some such methods, inoculating the plurality of cell samples with the plurality of different dilutions of the vims is a last time an operator physically interacts with the plurality of cells samples until the impedance assay concludes.

[0014] In some such methods, inoculating the plurality of cell samples with the plurality of different dilutions of the virus includes: aspirating culture media from the plurality of cell samples; adding the plurality of different dilutions of the virus to the cell samples according to a predefined layout; and adding fresh cell growth medium into each of the plurality of cell samples.

[0015] In some such methods, approximately 200 microliters of the fresh growth medium is added to each of the plurality of cell samples.

[0016] In some such methods, the plurality of different dilutions include dilution factors of 1:2, 1:3, 1:5, and 1:10.

[0017] In some such methods, the methods further comprise: determining, at a predefined interval, a plurality of CI measurements corresponding to the plurality of cell samples at different times between determining the first plurality of CI values and the second plurality of CI values, which are included as an initial set of the plurality of CI measurements and a final set of the plurality of Cl measurements, respectively.

[0018] In some such methods, determining the plurality of CPE statuses experienced by the plurality of cell samples is based on any two sets of pluralities of CI measurements taken at different times from the plurality of CI measurements.

[0019] In some such methods, the plurality of CI measurements are output in real-time.

[0020] In some such methods, the plurality of CI measurements are stored for historical review.

[0021] In some such methods, the plurality of cell samples include one or more control samples in which a given concentration of the virus to which the control samples are inoculated is no virus.

[0022] In some such methods, the methods further comprise: comparing cell growth of the one or more control samples against historic cell growth patterns previously collected from a quality control sample in which no virus was inoculated; and in response to the cell growth of the one or more control samples falling outside of quality threshold relative to the historic cell growth patterns, rejecting the TCID and the TCID / ml milliliter calculated.

[0023] In some such methods, the control sample was collected from a previous impedance assay of a different vims from respective control samples in the impedance assay.

[0024] In some such methods, each individual cell sample of the plurality of cell samples is included in an individual well of a plurality of wells, each well of the plurality of wells including a first electrode and a second electrode across which each CI value of the first plurality of CI values and the second plurality of CI values is calculated for a corresponding cell sample of the plurality of cell samples.

[0025] In some such methods, the first electrode and the second electrode in each well of the plurality of wells are spaced apart to define an imaging window in a base of the well, the method further comprising: capturing an image of the individual cell sample.

[0026] In some such methods, an imaging device used for capturing the image of the individual cell sample is located below the individual well and a light source is located above the individual well, such that light from the light source passes first through the individual cell sample and then through the imaging window.

[0027] In some such methods, an imaging device used for capturing the image of the individual cell sample is located above the individual well and a light source is located below the individual well, such that light from the light source passes first through the imaging window and then through the cell sample.

[0028] In some such methods, a duration of the impedance assay lasts between determining the first plurality of Cis and determining, from the first plurality of CI values and the second plurality of CI values, the plurality CPE statuses, wherein the duration is at least one of: 15, 30,60, 75, 90, 105, 120, 135, 150, 165, 180, or 195 minutes, 4, 8, 12, 24, 36, 48, 60, or 72 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0029] In some such methods, the methods further comprise receiving a user-defined CPE definition, selected from among: a change in impedance between a first time and a second time greater than a timed CPE threshold; a trend in impedance change between the first time and the second time above a trending CPE threshold; and an impedance value outside of an expected CPE window.

[0030] In some such methods, the methods further comprise receiving a user-defined calculation formula for the TCID, selected from among: a Reed-Muench method; a Spearman- Karber method; and an Improved Karber Method.

[0031] In some such methods, the TCID is a TCID50.

[0032] In some such methods, the methods further comprise after calculating the TCID and the TCID / ml: adding an optical reagent to the plurality of cells; and performing an optical TCID assay on the plurality of cell samples.

[0033] In some such methods, the plurality of Cis are determined via an impedance analysis device electrically connected to electrodes corresponding to a plurality of wells that hold the plurality of cell samples, and wherein the impedance analysis device is an RCTA system (offered by Agilent Technologies, Inc. of Santa Clara California).

[0034] In some embodiments, a method is provided, comprising: receiving selection of a virology mode for analyzing a plurality of cell samples held in corresponding wells of a wellplate for use by an impedance analysis device; receiving dilution or concentration information for a virus used to inoculate the plurality of cell samples; monitoring impedances of the cell samples over a duration of an assay; determining cytopathic effect (CPE) status experienced by the plurality of cell samples based on the impedances of the cell samples over the duration of the assay; and calculating, based on the CPE status, a TCID for a predefined percentage of the samples experiencing the CPE status and a TCID / ml (milliliter).

[0035] In some such methods, the methods further comprise: receiving volume information for the virus and the cell samples; and calculating a TCID / ml value based on the TCID and the volume information.

[0036] In some such methods, the methods further comprise calculating a time dependent TCID based on the CPE status determined over the duration of the assay.

[0037] In some such methods, the methods further comprise receiving a user-defined calculation formula for the TCID, selected from among: a Reed-Muench method; a Spearman- Karber method; and an Improved-Karber Method.

[0038] In some such methods, the methods further comprise receiving a user-defined defined percentage of tissue infectious dose.

[0039] In some such methods, the user-defined defined percentage of tissue infectious dose is 50%, and the TCID is a TCID50.

[0040] In some such methods, the methods further comprise calculating a virus reduction neutralization test (VRNT) value based on the CPE.

[0041] In some such methods, the CI for the plurality of cell samples is output in real-time.

[0042] In some such methods, the methods further comprise storing the CI for the plurality of cell samples for historical analysis.

[0043] In some such methods, the methods further comprise comparing a series of CI values for a cell sample of the plurality of cell samples indicated to be a control sample in which the virus was not inoculated against a historical series of CI values for a historical control sample; and in response to the series of CI values for the control sample deviating more than an allowed amount from the historical series of Cl values, marking the TCID as compromised.

[0044] Additional features and advantages of the disclosed method and apparatus are described in, and will be apparent from, the following Detailed Description and the Figures. The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the figures and description. Moreover, it should be noted that the language used in the specification has been principally selected for readability and instructional purposes, and not to limit the scope of the inventive subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 illustrates an example titer setup, according to embodiments of the present disclosure.

[0046] Figures 2A-2C illustrate example circuit layouts in an impedance sensing well, according to embodiments of the present disclosure.

[0047] Figure 3 is a flowchart of an example operating procedure for impedance-based determination of Tissue Culture Infections Dose (TCID) values, according to embodiments of the present disclosure.

[0048] Figure 4 is a flowchart of an example method for impedance-based determination of Tissue Culture Infections Dose (TCID) values, according to embodiments of the present disclosure.

[0049] Figure 5 illustrates a computing device, according to embodiments of the present disclosure.

[0050] Figure 6 is a chart displaying experimental results collected from a plurality of cell samples monitored via the impedance assay, according to embodiments of the present disclosure.

[0051] Figures 7-11 illustrate several example graphical user interfaces (GUIs), according to embodiments of the present disclosure that may be used to control and analyze the results of the impedance assay.DETAILED DESCRIPTION

[0052] The present disclosure provides for impedance-based determination of metrics derived from Cytopathic Effects (CPE), such as Tissue Culture Infections Dose (TCID) values, which may supplement (or be supplemented by) traditional methodologies for determination of TCID (e.g., TCID50). Additionally, the teachings of the present disclosure may be used in various other assays and tests, such calculating a virus reduction neutralization test (VRNT50) value. Similarly, although generally discussed in relation to a TCID50 assay, various other percentages may be specified by an operator other than 50 (e.g., 25% for a TCID25 assay or n% for a TCIDn assay); 50 is selected as an example that persons of ordinary skill in the art will readily recognize. In various embodiments, the determination is provided via various hardware and software components particularly adapted for the collection, calculation, and readout of the inputs and outputs of a TCID50 determination. To differentiate traditional optical TCID50 assays and other methodologies of performing TCID50 assays from the presently described methodologies, the novel impedance-based assays described herein may be referred to as IMP assays. Advantageously, the presently described systems and methods for IMP assays offer increased sensitivity (resulting in a reduced false negative rate for CPE), does not require additional reagents or dyes for CPE detection, has reduced labor inputs, reduced biohazard / exposure risks foroperators and contamination risks for the samples, and reduced chance for operator error by offering a more streamlined process than was previously possible.Advantages offered by IMP Assays over Traditional Assays

[0053] Generally, the count of infection events in an optical TCID50 assay is lower than in a comparable plaque assay, which can lead to lower sensitivity or a higher false negative rate in an optical TCID50 assay than a plaque-based TCID50 assay. In addition, for obvious infection phenotypes like cell lysis, a cell can be identified as CPE-positive vs. negative reliably without additional reagents, while more subtle phenotypes may require more expensive reagents, such as antibodies against viral antigens to reliably detect infection. Even though the traditional optical TCID50 assay provides an estimated titer range, due to the limited sensitivity to small differences in infectivity can cause promising lead compounds or vaccines to be missed. In contrast to traditional optical TCID50 assays, the presently described impedance-based TCID50 assay (IMP assay) offers greater sensitivity to detect cell viability, cell number, degree of cell adhesion, morphology, and even subtle cellular changes, which could be missed by the traditional optical TCID50 assays. The sensitivity of the presently described IMP assay to cellular morphology exceeds conventional optical light microscopy by more than 200-fold in various experimental results.

[0054] The colorimetric measurement for traditional optical TCID50 assays uses compounds / dyes for CPE detection after viral inoculation. For the optical TCID50 assay, expensive reagents may be used to increase sensitivity, such as fluorescent immuno staining reagents. In some cases, the reagents may not be commercially available, which may result in operators having to custom-produce the desired reagents, which can further introduce variability and complexity in the assay process. In contrast, the IMP assay is label-free, which reduces the cost associated with additional reagents and eliminates complications of using detection dyes (e.g., MTS((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium))), enzymatic reactions (e.g., MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)), recombinant-tagged target proteins (e.g., fluorescent viral particles), or other related detection approaches (e.g., fluorescent immunostaining). Moreover, the IMP assay does not require an operator to add a reagent at an intermediate time between culture start and measurement; thereby reducing the complexity of the assay process.

[0055] Additionally, for traditional optical TCID50 assays, the image processing relies on choosing parameters that are sufficiently tolerant of the variability without adversely affecting the assay's sensitivity or precision. The parameter choice is typically in the hands of the operator, which can lead to the assay results being dependent on the parameters chosen or the person choosing those parameters. In contrast, the IMP assay provides an objective readout that is reported without any processing or input from the operator, thereby resulting in reduced variation of TCID50 values between operators and across different assays.

[0056] The traditional optical TCID50 assay is an endpoint assay, in which one reading for each sample is taken at the conclusion of an assay period. Because the addition of dyes and reagents may affect the virality of the vims, efficacy of various compounds under test, etc., an operator needs to determine the optimal inoculation time, which could be any day after the addition of viruses to the cell culture and termination time when the reagents are added and the optical assay is performed. If multiple readings arc desired, operators ran multiple optical assays that arc terminated on different days post-inoculation, which is a laborious and costly process. In contrast, IMP assay is non-invasive, which allows continuous real-time monitoring rather than destructive terminal endpoint assays. During the validation of the viral titer method, the operator can run the assay and collect data at various timepoints until the operator decides to terminate the assay. The earliest time points that yet can provide reliable CPE-positive detection for TCID50 calculation could be easily determined from one real-time experiment. In addition, unlike the conventional optical TCID50 assay, which may require additional cell washing and staining steps after viral infection for CPE detection, the IMP assay uses a simple workflow, which only needs a one-time manual addition of the virus to the host cell plate. The real-time virus-induced CPE is automatically determined by the software. The software facilitates the ease of use and automatic calculation of TCID50 / ml (milliliter).

[0057] Additionally, because the described IMP assay does not require multiple exposures of an operator to the virus as the traditional optical TCID50 assay (e.g., at the inoculation step, the cell washing and staining steps, and even during the data analysis steps), the IMP assay reduces the risk of biohazards and exposure. In contrast, the IMP assay described herein only requires onetime exposure to the virus at the inoculation step, and the biohazards / viruses remain in the assay plate. Because no additional washing and staining steps are involved in the IMP assay, fewer biohazards are generated from the IMP assay compared to traditional assays.

[0058] Due to the quantitative and real-time nature of IMP measurement, the time course of the IMP readout can be used as an internal measure for cell health status used in various experiments. For example, in a given experiment, and operator may specify that the IMP cell index must reach a certain threshold value so that the cells can be treated with viruses for the assay, and the operator can be informed of then that threshold value is reached. Additionally, when the assay is performed across several wells or samples, the variation among the repeat wells for positive and / or negative control wells could also be used as built-in quality control measures. For example, a criterion may be set so that the CV for the IMP cell index at each time of the assay for all the negative control wells has to be met - e.g. <15%, to qualify for the given assay as producing a reliable or repeatable output.Definitions

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0060] As used herein, the articles "a" and "an" refer to one or to more than one (e.g., to at least one) of the grammatical object of the article.

[0061] 'About" and "approximately" as the term used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent(%), typically, within 10%, and more typically, within 5% of a given value or range of values.

[0062] “Above” and “below” are used herein to refer to orientations with respect to how an element is positioned during operation. Because many elements described in the present disclosure include liquids, and may omit caps or coverings, the level and orientation of the elements holding those liquids may need to be considered for proper liquid containment. Accordingly, although a wellplate with several openings to several wells may be stored in various orientations, one of ordinary skill in the art will recognize the orientation that a wellplate is to be held in during operation, and will recognize which elements of the wellplate would be “above” or “below” each other in the intended operational orientation, regardless of the current orientation of the wellplate.

[0063] "Acquire" or "acquiring" as the term used herein refers to obtaining possession of a physical entity, or a value, e.g., a numerical value, by "directly acquiring" or "indirectly acquiring" the physical entity or value. "Directly acquiring" means performing a process (e.g., performing asynthetic or analytical method) to obtain the physical entity or value. "Indirectly acquiring" refers to receiving the physical entity or value from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Directly acquiring a physical entity includes performing a process that includes a physical change in a physical substance, e.g., a starting material. Exemplary changes include making a physical entity from two or more starting materials, shearing or fragmenting a substance, separating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond. Directly acquiring a value includes performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process which includes a physical change in a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as "physical analysis"), performing an analytical method, e.g., a method which includes one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from another substance; combining an analyte, or fragment or other derivative thereof, with another substance, e.g., a buffer, solvent, or reactant; or changing the structure of an analyte, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the analyte; or by changing the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond, between a first and a second atom of the reagent. In an embodiment, directly acquiring encompasses a direct measurement. In an embodiment, indirectly acquiring encompasses an inference.

[0064] "Acquiring a sample" as the term used herein refers to obtaining possession of a sample, e.g., a sample described herein, by "directly acquiring" or "indirectly acquiring" the sample. "Directly acquiring a sample" means performing a process (e.g., performing a physical method such as a surgery or extraction) to obtain the sample. "Indirectly acquiring a sample" refers to receiving the sample from another party or source (e.g., a third-party laboratory that directly acquired the sample). Directly acquiring a sample includes performing a process that includes a physical change in a physical substance, e.g., a starling material, such as a tissue, e.g., a tissue in a human patient or a tissue that has was previously isolated from a patient. Exemplary changes include making a physical entity from a starting material; dissecting or scraping a tissue; separating or purifying a substance; combining two or more separate entities into a mixture; or performing a chemical reaction that includes breaking or forming a covalent or non-covalent bond.

[0065] “Biological subject” as the term used herein refers to a human or non-human animal from which a sample is obtained, or to which a cultured cell population is delivered as part of a therapy for the treatment or prophylaxis of a medical condition, such as a cancer or a tumor. In various embodiments, the biological subject from which the sample is obtained may be the same or different than the biological subject to which a cultured cell population derived from the sample is provided.

[0066] "Cancer" and "tumor" as the terms used interchangeably herein refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells are often in the form of a tumor, but such cells can exist alone within an animal, or can be a non-tumorigenic cancer cell, such as a leukemia cell. These terms include a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term "cancer" includes prcmalignant, as well as malignant cancers.

[0067] 'Cell sample" as the term used herein refers to a sample that comprises a cell. In an embodiment, the cell sample comprises a plurality of cells. In various embodiments, the cell is disposed in a medium.

[0068] A "Cell Index" or "Cl" is a parameter that can derived from measured impedance values and that can be used to reflect the change in impedance values. There are a number of methods to derive or calculate Cell Index.

[0069] “Connection pad” is used herein to refer to an area on an apparatus or a device which is electrically connected to at least one electrode or all electrode elements within at least one electrode structure on an apparatus or a device and which can be operatively connected to external electrical circuits (e.g., an impedance measurement circuit or a signal source). The electrical connection between a connection pad and an impedance measurement circuit or a signal source can be direct or indirect, through any appropriate electrical conduction means such as leads or wires. Such electrical conduction means may also go through electrode or electrical conduction paths located on other regions of the apparatus or device.

[0070] 'Critical Quality attribute", “Quality attribute”, “Quality parameter” and similar terminology as the terms used interchangeably herein to describe a measurable characteristic of the cell product that can include potency, metabolic fitness or any other quantifiable parameters of the cell.

[0071] A “delta cell index” at a given time point is calculated by subtracting the cell index at a standard time point from the cell index at the given time point. Thus, the delta cell index is the absolute change in the cell index from an initial time (the standard time point) to the measurement time.

[0072] A “detectable change in impedance between or among the electrodes” is used herein to refer to the impedance between or among the electrodes would have a significant change that can be detected by an impedance analyzer or impedance measurement circuit when molecule binding reaction or cell attachment or cell adhesion or cell presence occurs on the electrode surfaces. The impedance change refers to the difference in impedance values when cell attachment or cell adhesion or cell presence or molecule binding reaction occurs on the electrode surface of the apparatus and when no molecular reaction occurs on the electrode surface or no cell is present on the electrode surface. The impedance change may occur upon the presence or decay of a beat from a cardiomyocytc. Alternatively, the impedance change refers to the difference in impedance values when cells are attached to the electrode surface and when cells are not attached to the electrode surface, or when the number, type, activity, or morphology of cells attached to the electrodecomprising surface of the apparatus changes. In most cases, the change in impedance is larger than 0.1% to be detectable. Preferably, the detectable change in impedance is larger than 1%, 2%, 5%, or 8%. More preferably, the detectable change in impedance is larger than 10%. Impedance between or among electrodes is typically a function of the frequency of the applied electric field for measurement. “Detectable change in impedance between or among the electrodes” does not require the impedance change at all frequencies being detectable. “Detectable change in impedance between or among the electrodes” only requires a detectable change in impedance at any single frequency (or multiple frequencies). In addition, impedance has two components, resistance and reactance (reactance can be divided into two categories, capacitive reactance and inductive reactance). “Detectable change in impedance between or among the electrodes” requires only that either one of resistance and reactance has a detectable change at any single frequency or multiple frequencies. In the present disclosure, impedance is the electrical or electronic impedance. The method for the measurement of such impedance is achieved by, (1) applying a voltage between or among the electrodes at a given frequency (or multiple frequencies, or having specific voltage waveform) and monitoring the electrical current through the electrodes at the frequency (or multiple frequencies, or having specific waveform), dividing the voltage amplitude value by thecurrent amplitude value to derive the impedance value; (2) applying an electric current of a single frequency component (or multiple frequencies or having specific current wave form) through said electrodes and monitoring the voltage resulted between or among the electrodes at the frequency (or multiple frequencies, or having specific waveform), dividing the voltage amplitude value by the current amplitude value to derive the impedance value; (3) other methods that can measure or determine electric impedance. Note that in the description above of “dividing the voltage amplitude value by the current amplitude value to derive the impedance value”, the “division” is done for the values of current amplitude and voltage amplitude at same frequencies. Measurement of such electric impedance is an electronic or electrical process that does not involve the use of any reagents.

[0073] Electrode” is used herein to refer to a structure having a high electrical conductivity, that is, an electrical conductivity much higher than the electrical conductivity of the surrounding materials.

[0074] An “electrode structure” is used herein to refer to a single electrode, particularly one with a complex structure (as, for example, a spiral electrode structure), or a collection of at least two electrode elements that are electrically connected together. All the electrode elements within an “electrode structure” are electrically connected.

[0075] “Electrode element” is used herein to refer to a single structural feature of an electrode structure, such as, for example, a fingerlike or branched projection of an interdigitated electrode structure.

[0076] ‘Electrode structure unit” is used herein to refer to two or more electrode structures that are constructed to have dimensions and spacing such that they can, when connected to a signal source, operate as a unit to generate an electrical field in the region of spaces around the electrode structures. Preferred electrode structure units of the present disclosure can measure impedance changes due to cell attachment to an electrode surface. Non-limiting examples of electrode structure units are interdigitated electrode structure units and concentric electrode structure units.

[0077] ‘Electrode traces” is used herein to refer to electrically conductive paths that extend from electrodes or electrode elements or electrode structures toward one end or boundary of a device or apparatus for connecting the electrodes or electrode elements or electrode structures to an impedance analyzer. The end or boundary of a device may correspond to the connection pads on the device or apparatus.

[0078] “Electrodes have substantially same surface area” is used herein to refer to the surface areas of the electrodes referred to are not substantially different from each other, so that the impedance change due to cell attachment or growth on any one of the electrodes referred to will contribute to the overall detectable change in impedance to a same or similar degree as the impedance change due to cell attachment or growth on any other of the electrodes referred to. In other words, where electrodes have substantially the same surface area, any one of the electrodes can contribute to overall change in impedance upon cell attachment or growth on the electrode. In most cases, the ratio of surface area between the largest electrode and the smallest electrode that have “substantially the same surface area” is less than 10. Preferably, the ratio of surface area between the largest electrode and the smallest electrode of an electrode structure is less than 5, 4, 3, 2, 1.5, 1.2 or 1.1. More preferably, the at least two electrodes of an electrode structure have nearly identical or identical surface area.

[0079] ‘Immune cell” as the term is used herein refers to cells of an immune system of a biological subject, which may include, but are not limited to: an immune effector cell, a primary immune cell, an immortalized immune cell (e.g., a THP1 cell), a genetically engineered immune cell, a Natural Killer (NK) cell (e.g., a primary NK cell, a naive NK cell, a primary naive NK cell, etc.), a T cell (e.g., a primary T cell, a naive T cell, a T helper cell, cytotoxic T cell, memory T cell, virtual memory T cell, regulatory T cell, innate-like T cell, NK T cell, mucosal associated in variant T cell, gamma delta T cell, etc.) e.g., as the term used herein and refers to an NK cell that has been genetically engineered to express a T cell receptor (TCR).

[0080] "Impedance value" is the impedance measured for electrodes in a well with or without cell present. Impedance is generally a function of the frequency such that impedance values depend on frequencies at which the measurement was conducted. For the present disclosure, impedance value refers to impedance measured at either single frequency or multiple frequencies. Furthermore, impedance has two components, one resistance component and one reactance component. Impedance value in the present disclosure refers to resistance component, or reactance component, or both resistance and reactance component. Thus, when “impedance value” was measured or monitored, we are referring to that, resistance, or reactance, or both resistance and reactance were measured or monitored. In many embodiments of the methods of the present disclosure, impedance values also refer to parameter values that are derived from raw, measuredimpedance data. For example, cell index, or normalized cell index, or delta cell index could be used to represent impedance values.

[0081] “Impedance measurement at millisecond time resolution,” as the term is used herein, refers to the ability to perform a series of at least two consecutive impedance measurements within milliseconds of one another for each and every well of the system where each well is associated with an electrode array for impedance measurement. In each instance the time between two consecutive impedance measurements are less than 300 milliseconds from one another. Preferably, consecutive impedance measurements are less than 200 milliseconds from one another. More preferably impedance measurements are less than 100 milliseconds from one another. For example, the time between two consecutive impedance measurements are 40 milliseconds from one another for each and every well of the system. To be clear, that is to say, in a time period of 40 milliseconds, the system has the ability to measure two impedance data points for each and every well of the system.

[0082] “Interdigitated” is used herein to refer to having projections coming from one direction that interlace with projections coming from a different direction in the manner of the fingers of folded hands (with the caveat that interdigitated electrode elements preferably do not contact one another).

[0083] “Microelectrode strip or electrode strip” is used herein to refer to a non-conducting substrate strip on which electrodes or electrode structure units are fabricated or incorporated. The non-limiting examples of the non-conducting substrate strips include polymer membrane, glass, plastic sheets, ceramics, insulator-on-semiconductor, fiber glass (like those for manufacturing printed-circuits-board). Electrode structure units having different geometries can be fabricated or made on the substrate strip by any suitable microfabrication, micromachining, or other methods. Non-limiting examples of electrode geometries include interdigitated electrodes, circle-on-line electrodes, diamond-on-line electrodes, castellated electrodes, or sinusoidal electrodes. Characteristic dimensions of these electrode geometries may vary from as small as less than 5 micron, or less than 10 micron, to as large as over 200 micron, over 500 micron, over 1 mm. The characteristic dimensions of the electrode geometries refer to the smallest width of the electrode elements, or smallest gaps between the adjacent electrode elements, or size of a repeating feature on the electrode geometries. The microelectrode strip can be of any geometry for the present disclosure. One exemplary geometry for the microelectrode strips is rectangular shape - havingthe width of the strip between less than 50 micron to over 10 mm, and having the length of the strip between less than 60 micron to over 15 mm. An exemplary geometry of the microelectrode strips may have a geometry having a width of 200 micron and a length of 20 mm. A single microelectrode strip may have two electrodes serving as a measurement unit, or multiple such two- electrodes serving as multiple measurement units, or a single electrode structure unit as a measurement unit, or multiple electrode structure units serving as multiple electrode structure units. In one exemplary embodiment, when multiple electrode structure units are fabricated on a single microelectrode strip, these electrode structure units are positioned along the length direction of the strip. The electrode structure units may be of squared-shape, or rectangular- shape, or circle shapes. Each of electrode structure units may occupy size from less than 50 micron by 50 micron, to larger than 2 mm x 2 mm.

[0084] A “Normalized Cell Index” or “NCI” is used herein to refer to a parameter that divides the Cell Index for every selected well by the Cell Index at the Normalization time point: NCIt. = CIti / CInmi time, where CIt= CI at a specific time point “i”, and CInmi_time = CI at a user-defined normalization time point.

[0085] “Optimize” and variations thereof, is used in a sense understood by data scientists to refer to actions taken for continual improvement of a system relative to a goal. An optimized value will be understood to represent “near-best” value for a given reward framework, which may oscillate around a local maximum or a global maximum for a “best” value or set of values, which may change as the goal changes or as input conditions change. Accordingly, an optimal solution for a first goal at a given time may be suboptimal for a second goal at that time or suboptimal for the first goal at a later time.

[0086] Or" is used herein to mean, and is used interchangeably with, the term "and / or", unless context clearly indicates otherwise. The use of the term "and / or" in some places herein does not mean that uses of the term "or" are not interchangeable with the term "and / or" unless the context clearly indicates otherwise.

[0087] "Primary cell" as the term used herein refers to a cell isolated or harvested directly from a subject, organ, or tissue. For example, primary cells can be isolated from blood obtained from a living subject. Primary cells can be isolated or harvested using enzymatic or mechanical methods. Once isolated or harvested, primary cells can be cultured in media containing essential nutrients and growth factors to support proliferation. Primary cells can be suspension cells that do notrequire attachment for growth (e.g., anchorage-independent cells) or adherent cells that require attachment for growth (e.g., anchorage-dependent cells).

[0088] Percent Cytopathic Effect” or “% CPE” is defined herein as [1- (NCIt. / NClnc t.)] xlOO. Where NCIt.= NCI at a specific time point “i”, and NCInc t. = Average NCI of negative controls (nc) at a user-defined specific time point “i”.

[0089] "Sample" as the term is used herein refers to a biological sample obtained or derived from a source of interest. In an embodiment, the source of interest comprises an organism, such as an animal or human. The source of the sample can be blood or a blood constituent; a bodily fluid; a solid tissue as from a fresh, frozen and / or preserved organ, tissue, biopsy, resection, smear, or aspirate; or cells from any time in gestation or development of a subject. In an embodiment, the source of the sample is blood or a blood constituent. In an embodiment, the sample is a primary sample, e.g., obtained directly from a source of interest by any appropriate means. In an embodiment, the sample is a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample.

[0090] “Serially different concentrations” as the term is used herein means that each well contains a test compound (e.g., a virus) with a serially diluted concentrations, e.g., a one-tenth serially diluted concentrations of 1 M, 0.1 M, 0.01 M, etc.

[0091] Suitable for cell attachment or growth” is used herein to refer to a surface, such as an electrode and / or non-electrode area, that has appropriate physical, chemical or biological properties such that cells of interest can viably attach on the surface and new cells can continue to attach, while the cell culture grows, on the surface of the apparatus. However, it is not necessary that the device, or the surface thereof, contain substances necessary for cell viability or growth. These necessary substances, e.g., nutrients or growth factors, can be supplied in a medium. Preferably, when a suspension of viable, unimpaired, excitable cells is added to the “surface suitable for cell attachment” at least 50% of the cells adhere to the surface within twelve hours. More preferably, a surface that is suitable for cell attachment has surface properties so that at least 70% of the cells are adhering to the surface within twelve hours of plating (i.e., adding cells to the chamber or well that comprises the said device). Even more preferably, the surface properties of a surface that is suitable for cell attachment results in at least 90% of the cells adhering to the surface within twelve hours of plating. Most preferably, the surface properties of a surface that is suitablefor cell attachment results in at least 90% of the cells adhering to the surface within eight, six, four, two hours of plating.

[0092] As used herein, various chemical compounds are referred to by associated element abbreviations set by the International Union of Pure and Applied Chemistry (IUPAC), which one of ordinary skill in the relevant art will be familiar with. Similarly, various units of measure may be used herein, which are referred to by associated short forms as set by the International System of Units (SI), which one of ordinary skill in the relevant art will be familiar with.

[0093] As used herein, various terms provided with reference to the body of a biological subject are to be understood with reference to the standard anatomical position of that biological subject using anatomical terms of location e.g., as set by the International Federation of Associations of Anatomists or the World Associate of Veterinary Anatomists that will be understood by the person on ordinary skill in the relevant art without further explanation.

[0094] As used herein, “about,” “approximately” and “substantially” arc understood to refer to numbers in a range of the referenced number, for example the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1 % to +1% of the referenced number, most preferably -0.1% to +0.1% of the referenced number.

[0095] Furthermore, all numerical ranges herein should be understood to include all integers, whole numbers, or fractions, within the range. Moreover, these numerical ranges should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of a range from 1 to 10 should be construed as supporting ranges of any two numbers that fall into the initial range of from 1 to 10 (e.g., from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from X to Y where X > 1 and Y < 10).

[0096] As used in the present disclosure, a phrase referring to “at least one of’ a list of items refers to any set of those items, including sets with a single member, and every potential combination thereof. For example, when referencing “at least one of A, B, or C” or “at least one of A, B, and C”, the phrase is intended to cover the sets of: A, B, C, A-B, B-C, and A-B-C, where the sets may include one or multiple instances of a given member (e.g., A-A, A-A-A, A- A-B, A- A-B-B-C-C-C, etc.) and any ordering thereof. For avoidance of doubt, the phrase “at least one of A, B, and C” shall not be interpreted to mean “at least one of A, at least one of B, and at least one of C”.

[0097] As used in the present disclosure, the term “determining” encompasses a variety of actions that may include calculating, computing, processing, deriving, investigating, looking up (e.g., via a table, database, or other data structure), ascertaining, receiving (e.g., receiving information), accessing (e.g., accessing data in a memory), retrieving, resolving, selecting, choosing, establishing, and the like.Operational Setup and Practice

[0098] Figure 1 illustrates an example titer setup 100, according to embodiments of the present disclosure. Sample cells 110 are extracted from a biological subject and loaded into bioreactor 140, where the sample cells 110 are encouraged to multiply in a cultivation process to produce cultivated cells 130. In various embodiments, the cultivation process may include various gene editing or gene expression / activation operations to affect the ability of the sample cells 110 to treat, prevent, or mitigate various medical conditions when the cultivated cells 130 are introduced to a biological subject (which may be the same or a different biological subject from which the cells 110 were initially extracted). In various embodiments, the cultivation process yields an increased number of target cells for analysis relative to various therapeutic agents and / or viruses. Additionally or alternatively, the sample cells 110 may be received from an established cell line, which may be commercially available, developed via a proprietary cell line, and may be unrelated to biological subject currently undergoing analysis or treatment for a medical condition.

[0099] To avoid confusion, as cells arc understood to grow, divide, and die off during cultivation, the cells placed in the bioreactor are referred to as reactor cells 120, which may display the properties of the sample cells 110 (as input to the bioreactor 140), the cultivated cells 130 (as extracted from the bioreactor 140), or properties intermediate thereto.

[0100] In various embodiments, during the cultivation process, the bioreactors 140 may control for one or more of: nutrient and gas exchange, oxygen (O2) control, pH control, a feeding regime of media exchange, media addition during cultivation, mixing or shear force applied to the reactor cells 120, O2 permeability of the bioreactor 140 or wells therein, a vessel size (e.g., of a well in a wellplate used in the bioreactor 140), and whether activation beads are removed during cultivation, automated cell population selection or enrichment during cultivation process.

[0101] After cultivation of the cultivated cells 130, the cells, if not already held within the wells of a wellplate during cultivation, are added to individual wells of a plurality of wells that arecapable of measuring cellular impedance of a cell sample 160 held therein. In various aspects, the number of wells on an individual wellplate may vary (e.g., 6 wells, 8 wells, 16 wells, 32 wells, 96 wells, 384 wells, or any number), but each individual well includes at least a first electrode and a second electrode across which an cellular impedance for a cell sample held therein may be measured. Example circuit layouts for the electrodes are discussed with respect to Figures 2A-2C. [001021 A plurality of vims preparations 150 having serially diluted volumes are prepared and used to inoculate at least one of the cell samples 160 in individual wells in the wellplate. For example, each row or column of the wellplate may be inoculated with a shared concentration of the vims, such that a first virus preparation 150 of a first virus concentration is applied to the cell samples 160 in a first row of the wellplate, a second vims preparation 150 of a second vims concentration is applied to the cell samples 160 in a second row of the wellplate, a third virus preparation 150 of a third virus concentration is applied to the cell samples 160 in a third row of the wcllplatc, etc. In some embodiments, at least one row / column / wcll is left uninoculatcd, having a virus preparation 150 of zero or no vims applied thereto.

[0103] In various embodiments, the various concentrations of the virus in the virus preparations 150 can include dilution factors of 1:2, 1:5, and 1:10. In some embodiments, the concentrations include one or more of about IxlO'2, IxlO'3, IxlO'4, IxlO'5, IxlO'6, IxlO'7, IxlO'8, IxlO'9, and IxlO’10

[0104] The wellplate including the cell samples 160 and vims preparations 150 are provided to an impedance analysis instrument 170, such as the RCTA system (offered by Agilent of Santa Clara, California), which may also offer imaging functionality. The impedance analysis instrument 170 measures the CI for each well at various time intervals (e.g., once every 5, 10 15, 30, 60 minutes) for the duration of the assay. In various embodiments, the impedance analysis instrument 170 included or is connected to an output device 180, which may store, analyze, or display the measured CI values and various values derived therefrom.

[0105] Figures 2A-2C illustrate example circuit layouts 200 in an impedance sensing well, according to embodiments of the present disclosure. In some aspects, the first electrode and the second electrode are disposed on a base or bottom of the well, such that an imaging window is formed between the electrodes.

[0106] In Figure 2A, the first electrode structure 230a (generally or collectively, electrode stmeture 230) has electrode elements 210a-c (generally or collectively, electrode elements 210)connected to one another via a first bus 220a (generally or collectively, bus 220) and interdigitated with the electrode elements 210d-f of the second electrode structure 230b, which are connected to one another via a second bus 220b. The electrode elements 210 and the buses 220 are made of electrically conductive material (e.g., gold film, platinum film, gold film over a chromium or titanium film). In some embodiments, the buses 220 may have an insulating coating. Connection pads 250a-b (generally or collectively, connection pad 250) are included in the layout, which can be connected to an external impedance measurement circuit. Electrical connection traces 240a-b (generally or collectively, connection trace 240) connect the connection pad 250 to the electrode structures 230. Such connection traces 240 can extend in any direction in the plane of the electrodes.

[0107] As illustrated in Figure 2A, the electrode elements 210 comprise electrode lines with connected circles added on the line, but other shapes are contemplated. The overall area of electrode elements 210 and gaps 260a-c (generally or collectively, gaps 260) between electrode elements 210 may correspond to, or may be slightly larger than, or may be slightly smaller than, the bottom of a well (e.g., a cylinder shaped well, a conical shaped well, or a cubic shaped well), for example, a 24-well wellplate, a 96-well wellplate, or a 384-well wellplate that are commonly used, or any number of wells in another size of wellplate.

[0108] In some embodiments, the whole surfaces of the wells may be covered with electrodes to ensure that the cell attachment at nearly any locations of the bottom surface of the well can contribute to the impedance change. This arrangement has an advantage that non-uniform landing and attachment of cells on the bottom surface of the different wells would result in only a small variation in the impedance measured between electrode elements 210.

[0109] In some embodiments, portions of the well (e.g., the gaps 260) are substantially free of electrode elements 210, to thereby define an imaging window through which an imaging device or light source can penetrate through the cell sample and collect images of the cell sample in a particular well. In some embodiments, an imaging device used for capturing the image of an individual or a plurality of cell samples is located below the wells and a light source is located above the wells, such that light from the light source passes first through the cell samples and then through the imaging window of the respective well. In some embodiments, an imaging device used for capturing the image of an individual or a plurality of cell samples is located above the wellsand a light source is located below the wells, such that light from the light source passes first through the imaging window of the respective well and then through the cell samples.

[0110] Figure 2B is a schematic representation of a device with two electrode structures 230a- b of similar areas deposited on a substrate. The electrode structures 230a-b each comprise multiple interconnected electrode elements 210. As illustrated, the electrode elements 210 are rectangular lines and together form an interdigitated electrode structure unit. Similarly to Figure 2A, the electrode elements 210 within each electrode structure 230 are connected through arc-shaped, buses 220. Connection pads 250 are connected to the electrode structures 230 through the electrical connection traces 240.

[0111] Figure 2C is a schematic representation of a device with two electrodes of similar areas deposited on a substrate in which each electrode include two electrode structures separately connected to a respective pad 250 (or trace 240). The electrode structures 230a-d each comprise multiple interconnected electrode elements 210a-h. As illustrated, the electrode elements 210 arc rectangular lines and together form an interdigitated electrode structure unit. In contrast to the layouts shown in Figure 2A and Figure 2B, the electrode structures 230 of Figure 2C have electrode elements 210 that are connected to connection pads 250.

[0112] Figure 3 is a flowchart of an example operating procedure 300 for impedance-based determination of Tissue Culture Infections Dose (TCID) values, according to embodiments of the present disclosure. Any of the described operations performed by an operator may be performed directly by the operator, via software controlled by the operator, or via robotic assistance. Additionally, although describe with an example for determining a TCID value, other CPE-based values may be determined according to procedure 300 in addition to or alternatively from a TCID value.

[0113] At block 310, where an operator prepares the cell samples for performing the titer and assay. In various embodiments, the operator can include an initial cell sample in each well of wellplate as a sample container, thereby allowing the incubating cells to expand within the well that includes the electrodes that will measure the impedances during the IMP assay, or the operator may incubate the cells in a separate sample container and then transfer the cell samples to the wells. Accordingly, the operator places each cell sample prepared in a corresponding well of a wellplate (which may include any number of individual wells with corresponding electrodes forimpedance measurement), and allows the cells to grow / incubate for a predetermined amount of time (e.g., 4, 8, 12, 24, 36, 48, 60, or 72 hours, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days).

[0114] The incubation may be performed within a bioreactor or other similar device, and may control for one or more parameters for the duration of incubation. The parameters may include one or more of nutrient and gas exchange, oxygen control, pH control, a feeding regime of media exchange versus media addition, mixing or shear force, device oxygen (O2) permeability, vessel size, removal of activation beads, addition of additional growth media, enrichment / selection of a particular subpopulation, etc.

[0115] In various embodiments, the operator adds the host cells and provides growth media for the cells, which may be periodically refreshed, and may optionally add various candidate therapeutic agents or gene expression / alteration media to the cells.

[0116] At block 320, the operator determines baseline CI values for each of the cell samples. In various embodiments, determining the baseline CI values includes obtaining a base CI reading for each cell sample of the plurality of cell samples when the predefined time period for incubation has concluded. In some embodiments, the operator may also obtain readings for CI values during the incubation process to determine when to extend the predetermined period (e.g., to allow for additional growth time) or to terminate the assay early if insufficient growth or overgrowth has occurred relative to a target growth values for the cell samples or when uneven growth among the cell samples is identified by the CI values collected by during the incubation time period.

[0117] At block 330, the operator prepares a plurality of virus dilutions. These virus dilutions may be prepared to have various serially different concentrations of the virus to be assayed. For example, the plurality of different dilutions may include virus concentrations of 1:2, 1:5, and 1:10 in a standardized volume of media to add to the wells in which the cell samples are located. In another example, the plurality of different dilutions may include virus concentrations in standardized volumes of about IxlO'2, IxlO'3, IxlO’4, IxlO'5, IxlO'6, IxlO'7, IxlO’8, IxlO'9, and IxlO'10.

[0118] At block 340, the operator inoculates the cell samples with the virus dilutions prepared per block 330. These virus dilutions are used to inoculate different populations of the cell samples with different amounts of the virus as part of the assay, and may be added via different procedures. In a first procedure, inoculating the plurality of cell samples with the plurality of different dilutions of the virus includes aspirating culture media from the plurality of cell samples, adding the pluralityof different dilutions of the virus to the cell samples according to a predefined layout for tracking in the wellplate, adding vims absorption media (e.g., approximately 150 microliters (pL) ) for a predefined absorption time after adding the plurality of different dilutions of the vims to the cell samples, and aliquoting fresh growth medium (e.g., approximately 50 microliters pL) into each of the plurality of cell samples. In a second procedure, inoculating the plurality of cell samples with the plurality of different dilutions of the vims includes: aspirating culture media from the plurality of cell samples; adding the plurality of different dilutions of the virus to the cell samples according to a predefined layout, and adding fresh cell growth medium (e.g., approximately 200 pL) into each of the plurality of cell samples.

[0119] In various embodiments, the plurality of cell samples include one or more control samples in which a given concentration of the vims to which the control samples are inoculated is no virus. In such embodiments, the operator may add fresh growth medium (e.g., approximately 200 pL) to the wells that docs not include the virus. Similarly, in embodiments in which one or more wells are empty or otherwise do not include a viable cell sample, the operator may omit adding vims and associated media to that well.

[0120] As will be noted, the addition of the virus and fresh growth media to the various wells in block 340 to inoculate the cell samples is a last time an operator physically interacts with the plurality of cells samples until the IMP assay concludes.

[0121] At block 350, the operator determines subsequent CI values for each of the cells samples in the well. In various embodiments, the operator may perform impedance readings at a predefined interval (e.g., every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 minutes, every 0.5, 1, 1.5. 2 hours, etc.). These CI values may be collected every interval starting with the inoculation and terminating at the end of the assay such that a plurality of CI a plurality of CI measurements corresponding to the plurality of cell samples at different times are determined. This plurality includes the first / initial plurality of CIs and the second / final plurality of CIs, such that the CPE status experienced by the plurality of cell samples can be determined (per block 360) based on any two sets of pluralities of CI measurements taken at different times from the plurality of Cl measurements.

[0122] In various embodiments, when the impedance analysis instrument includes optical imaging functionality (e.g., such as the RCTA system available from Agilent Technologies, Inc. of Santa Clara, California), some or all of impedance measurements may be accompanied by visualimages captures for the cell samples in the wells. For example, an imaging device may operate in conjunction with a light source to capture images of the cell samples through a window in the well (e.g., a gap between the electrodes).

[0123] In various embodiments, the plurality of CI measurements are output in real-time, but may additionally or alternatively be stored for historical review. In various embodiments, the historical data for the control samples is stored so that the growth patterns of the control samples in a present assay may be compared against the growth patterns previously collected from one or more quality control samples from one or more previously conducted assays in which no virus was inoculated in the quality control samples. Using these historical growth patterns, an operator may reject any TCID, TCID / ml, other CPE-derived metrics from a present assay in response to the cell growth of the one or more control samples falling outside of quality threshold relative to the historic cell growth patterns. Because the quality control samples are not inoculated with the vims under test, the growth patterns from the quality control samples from an assay of a first virus may be used in assay of any other virus.

[0124] At block 360, the operator determines CPE status based on the CI values. The operator may select any two (or more) CI values collected at different times to calculate the CPE status of a given cell sample, and may provide various definitions for how to define the CPE status of the given cells sample. In various embodiments, the user-defined CPE definition may be selected from among: a change in impedance between a first time and a second time greater that is greater than a threshold for that difference in time (e.g., a CPE timed threshold), a trend in impedance change between the first time and the second time above a threshold for that difference in time (e.g., a trending CPE threshold), and an impedance value outside of an expected CPE window.

[0125] At block 370, the operator calculates the TCID and other measures of interest based on the CPE status, and when the various different populations change CPE statuses. In various embodiments, these other measures may include various values for TCID (e.g., TCID50, TCIDn), TCID / ml, and a virus reduction neutralization test (VRNT) value. In various embodiments, the calculation for these metrics may be based on various user-defined formulas. For example, for the TCID, the user may select a formula provided by the software including the Reed-Muench method, Spearman- Karber method, and improved-Karber method.

[0126] In various embodiments, the TCID and other measures of interest may be calculated at various times during the assay or at the conclusion of the assay. The assay may run for apredetermined duration between determining the first plurality of Cis (per block 320) and determining the CPE status (per block 360) of at least one of: 15, 30, 60, 75, 90, 105, 120, 135, 150, 165, 180, or 195 minutes; 4, 8, 12, 24, 36, 48, 60, or 72 hours; and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.

[0127] Optionally, at block 380, the operator performs an optical TCID assay, which may be used to supplement or confirm the results produced in the IMP assay (e.g., from block 320-370). In various embodiments, the operator adds one or more optical reagents to the cell samples and then performs an optical TCID assay on the plurality of cell samples as will be understood by the operator.

[0128] Figure 4 is a flowchart of an example method 400 for impedance-based determination of Tissue Culture Infections Dose (TCID) values, according to embodiments of the present disclosure. Additionally, although describe with an example for determining a TCID value, other CPE-bascd values may be determined according to method 400 in addition to or alternatively from a TCID value.

[0129] Method 400 begins at block 410, where the system receives a command to perform analysis in a virology mode. In various embodiments, the software operating with the system may operate in various modes, and the selection of a virology mode offers analyses a plurality of cell samples held in corresponding wells of a wellplate with respect to an impedance analysis device capable of also operating in a basic operation mode, an immunotherapy operation mode, an imaging operation mode, and combinations thereof.

[0130] At block 420, the system receives dilution or concentration information for a vims under test. The dilution or concentration information identifies various populations of the wells on the wellplate and which dilutions / concentrations are applied to those wells. In various embodiments, the populations may include excluded (e.g., untested) populations that may omit cell samples and applied viruses and control populations in which the dilution / concentration indicates that no vims is applied to the associated cell samples.

[0131] At block 430, the system monitors the impedances of the cell samples in the various wells. In various embodiments, the system may monitor the impedances during an incubation period (e.g., before the viruses inoculate the cell samples) and may periodically monitor the impedances at a predefined interval throughout the duration of incubation. In various embodiments the system may identify a first or initial plurality of CI values for the plurality of cell samples afterthe cells samples are inoculated with the virus and a second or final plurality of CI values for the plurality of cell samples after duration of the assay concludes. In some embodiments, the system periodically determines the CI values for the plurality of cell samples at a predefined interval throughout the course of the assay (e.g., once every n minutes). These intermediate CI values may be used to monitor the growth conditions of the cell samples during the assay and to identify CPE statues for the cell samples at various times during the assay.

[0132] At block 440, the system determines the CPE status of the cells samples. In various embodiments, the CPE status may be determined at the end of the assay (e.g., once the duration has expired) or at any time during the assay. In various embodiments, the system may select any two or more impedance readings from different times (as monitored per block 430) to determine the CPE status of the cell samples. An operator may identify CPE-positive wells of the cell samples, which may be selected from CI or NCI; % CPE; and an area under the curve of CI or NCI for the cell samples treated with different dilutions of virus.

[0133] At block 450, the system calculates the TCID, and other measures derived from the CI values determined per block 440. In various embodiments, the operator selects the calculation formulas provided by the software for calculating the TCID, which may be selected from the Reed- Muench method, Spearman-Karber method, and improved- Karber method. Although generally discussed in relation to determining a TCID50, the operator may also specify other percentage of tissue infectious dose (e.g., TCID25, TCID90, TCIDn). When the system has received dilution or concentration information for a virus used to inoculate the plurality of cell samples and has received volume information for the virus and the cell samples, the system may calculate a TCID / ml value based on the TCID and the volume information. Other metrics or measures that may be calculated include a virus reduction neutralization test (VRNT) value (e.g.,VRNT25, VRNT50, VRNT90, VRNT / z). which is also based on the CPE status and a specified dose percentage, and a time dependent TCID based on the CPE determined from various points in time over the duration of the assay.

[0134] At block 460, the system determines whether any quality control samples in the assay are within a window of historically viable growth patterns. The system compares a series of Cl values for a cell sample of the plurality of cell samples indicated to be a control sample in which the vims was not inoculated against a historical series of CI values for a historical control sample in which no vims was inoculated. In response to the series of CI values for the control sampledeviating more than an allowed amount from the historical series of CI values (e.g., falling outside of the window), the system marks the calculation results (e.g., the TCID or other measures calculated per block 450) as compromised as the control is not behaving as expected, and the inoculated cell samples under test may also be behaving abnormally (e.g., due to contamination, alterations to environmental conditions, faulty cell lines, equipment malfunction, etc.). In response to the series of CI values for the control sample deviating no more than the allowed amount from the historical series of CI values (e.g., falling within the window), the system marks the calculation results (e.g., the TCID or other measures calculated per block 450) as acceptable, as the control is behaving as expected, and the inoculated cell samples under test are also likely to be behaving as expected.

[0135] Figure 5 illustrates a computing device 500, as may be used for impedance-based determination of CPE-derived values, according to embodiments of the present disclosure. The computing device 500 may include at least one processor 510, a memory 520, and a communication interface 530. In various embodiments, the computing device 500 may be configured for processing method used in procedure 300 (described in greater detail in regard to Figure 3) and method 400 (described in greater detail in regard to Figure 4).

[0136] The processor 510 may be any processing unit capable of performing the operations and procedures described in the present disclosure. In various embodiments, the processor 510 can represent a single processor, multiple processors, a processor with multiple cores, and combinations thereof.

[0137] The memory 520 is an apparatus that may be either volatile or non-volatile memory and may include RAM, flash, cache, disk drives, and other computer readable memory storage devices. Although shown as a single entity, the memory 520 may be divided into different memory storage elements such as RAM and one or more hard disk drives. As used herein, the memory 520 is an example of a device that includes computer-readable storage media, and is not to be interpreted as transmission media or signals per se.

[0138] As shown, the memory 520 includes various instructions that are executable by the processor 510 to provide an operating system 522 to manage various features of the computing device 500 and one or more programs 524 to provide various functionalities to users of the computing device 500, which include one or more of the features and functionalities described in the present disclosure. One of ordinary skill in the relevant art will recognize that differentapproaches can be taken in selecting or designing a program 524 to perform the operations described herein, including choice of programming language, the operating system 522 used by the computing device 500, and the architecture of the processor 510 and memory 520. In various embodiments, the program 524 may include or make use of a machine learning model 526 that is trained to make determinations as set forth in the present disclosure, and may be retrained or updated based on data collected as set forth in the present disclosure. Accordingly, the person of ordinary skill in the relevant art will be able to select or design an appropriate program 524 based on the details provided in the present disclosure.

[0139] The communication interface 530 facilitates communications between the computing device 500 and other devices, which may also be computing devices as described in relation to Figure 5. In various embodiments, the communication interface 530 includes antennas for wireless communications and various wired communication ports. The computing device 500 may also include or be in communication, via the communication interface 530, one or more input devices (e.g., a keyboard, mouse, pen, touch input device, etc.) and one or more output devices (e.g., a display, speakers, a printer, etc.).

[0140] Although not explicitly shown in Figure 5, it should be recognized that the computing device 500 may be connected to one or more public and / or private networks via appropriate network connections via the communication interface 530. It will also be recognized that software instructions may also be loaded into a non-transitory computer readable medium, such as the memory 520, from an appropriate storage medium or via wired or wireless means.

[0141] Accordingly, the computing device 500 is an example of a system that includes a processor 510 and a memory 520 that includes instructions that (when executed by the processor 510) perform various embodiments of the present disclosure. Similarly, the memory 520 is an apparatus that includes instructions that, when executed by a processor 510, perform various embodiments of the present disclosure.EXAMPLESExperimental Results

[0142] The concepts described in the present disclosure are generally counter-intuitive to the current understanding for performing a TCID assay. Accordingly, the present disclosure supportsthe position that traditional optical-based assay methodology may be replaced by or supplemented with impedance-based measurements of equal or greater precision, with lower setup time and inputs. Outputs from the analysis may be provided to end users via software programs that are integrated in the hardware collecting and measuring the impedance data (e.g., the impedance measuring device 180) or in communication with the hardware collecting and measuring the impedance data, via several example graphical user interfaces such as those shown in Figures 6- 11 or in various data files (e.g., spreadsheets, comma separate value files, etc.).Example 1:

[0143] Figure 6 is a chart 600 displaying experimental results collected from a plurality of cell samples monitored via the IMP assay, according to embodiments of the present disclosure. The normalized CI for each of the cell samples is plotted on the Y axis, while time of the assay is plotted. The data from different ranges of the wells, which correspond to the various different concentrations of the virus inoculated with the cell samples therein may be shown with different colors, data marker shapes, or combinations thereof. Additionally, some wells in a wellplate may remain unfilled (e.g., include no cell samples), which may be unplotted with the rest of the data.

[0144] In various embodiments, these data are plotted in real-time as the data are collected, or may include historical plots of data. Additionally, in some embodiments, historical data may be plotted with live-collected data to show comparisons between the currently collected data and historical averages, ranges, bounds, or the like. For example, data from control wells (e.g., wells in which a cell sample is not exposed to a virus) from a first assay may be plotted on the chart for comparison to data collected from a second assay as a quality control metric, to identify that the regular growth of the cell samples in the second assay matches expected growth patterns, as conveyed by the CI values collected from the wells. Similarly, CI data from the various inoculation concentrations of the virus may be compared against one another from different assays to confinn repeatability of the results or to compare the effects of different growth conditions, therapeutic substances, host cells, virus strains, or the like.

[0145] As illustrated in Figure 6, the time period from hour-0 to hour-24 corresponds to the initial cell growth period for the cell samples, during which time each of the cell samples exhibit substantially similar CI values over time, which should be expected. At the 24-hour mark in these data, the cell samples are inoculated with several different concentrations of a virus. The present disclosure contemplates that various other incubation times may be used other than 24-hours.

[0146] Several different well populations 610a-h (generally or collectively, populations 610) are shown having effects on the CI values at different times; causing the CI in the respective wells of the inoculated cell samples to deviate from the behavior. The populations 610 each describe a subset of the wells on a wellplate in which a shared virus concentration is was applied to the respective cell samples held therein. For example, the first population 610a may describe those cells.

[0147] The software may calculate an average (e.g., mean) values from the plurality of wells sharing one population 610, and may output this average value with or without error or range bars to represent all of the data collected from the individual wells. For example, with the illustrated eight populations 610a-h, each population 610 may represent all of the n wells to which a given concentration of the virus (including a null-concentration for control wells) has been applied. For example, the concentrations may be of substantially equal sizes such that each population 610 may represent ten wells in an eighty-four well wcllplatc (leaving four wells unfilled), nine wells in an eighty-four well wellplate (leaving twelve wells unfilled), to twelve wells in a ninety-six well wellplate (leaving no wells unfilled), to forty-eight wells in a 384-well wellplate (leaving no wells unfilled), etc., according to the experimental setup. In some embodiments, one or more populations 610 may be of different sizes than other populations (e.g., to fill all available wells, when a given concentration is more important, when a given population is susceptible to outlier effects, etc.). For example, a first population may include In wells, while other populations include n wells or a first population may include n+x wells, while other populations include n wells when the number of wells w is not evenly divisible by n (but w-x is evenly divisible by ri).

[0148] In various embodiments, individual wells of a population 610 may be removed as outliers from the calculations, and may be removed from display in an associated chart 600. For example, when a given well in a population does not output a CI within a given range of a quality control standard or within a given range of other members of the population 610, the CI values collected from that well may be marked as outlier values, and be quarantined or excluded from the calculations.

[0149] As illustrated in Figure 6, beginning after hour-24 and inoculation, the different populations 610a-h begin to display different normalized CI values as the corresponding different concentrations of the virus begin to invoke CPE on the cell samples. These data may be used to calculate various metrics related to CPE, such as TCID50, as described in the present disclosure.Example Graphical User Interfaces[001501 The present disclosure contemplates that an operator may control the collection and calculation of the IMP data using software integrated with, or in communication with the software used to manage the operations of the impedance analysis instrument. Figures 7-11 illustrate several example GUIs, according to embodiments of the present disclosure that may be used to control and analyze the results of the IMP assay. The present disclosure contemplates that various artistic, stylistic, and hierarchical changes may be made to the non-limiting example GUIs without departing from the spirit and scope of the present disclosure.

[0151] Figure 7 illustrates an example selection GUI, where an operator is enabled to select an operation and analysis mode for the impedance analysis system. In various embodiments, the operator is able to select from modes for basic operation, immunotherapy operation, and virology operation, which may cause other elements of the GUI (e.g., menus, ribbons, plots / windows) to update to expose elements that are more relevant and hide elements that are less relevant for the selected mode of operation.

[0152] Figure 8A and Figure 8B illustrates example representations of wellplates and populations thereof for an assay. As illustrated, the representations are provided in a grid that corresponds to the various physical wells in a wellplate and the properties applied thereto and coordinate markers for those wells. The illustrated grid in Figures 8A-8B is of a ninety-six wellplate, having an eight-by-twelve arrangement. Figure 8A illustrates that applied test program for each well, in which test “293A” is applied to seventy-two wells, and no test applied to twenty- four wells. Figure 8B illustrates the serial virus concentrations applied to each of the wells, with each population corresponding to a column of the wells. Other data may be displayed according to the example grid layout via displayed numerical representations, color / shading, size, etc., such as the currently measured Cl value, a time at which CPE effects are identified above a CPE threshold, etc.

[0153] Figure 9 illustrates an example Y-axis selection GUI for plotting the data from the wells, according to embodiments of the present disclosure. In various embodiments, the Y-axis may include plots for CI, normalized CI, delta CI (e.g., the change in CI value from a designated baseline), scaled CI, baseline normalized CI, % positive control, % negative control, slope, % CPE, and % neutralization.

[0154] Figures 10 A- 10C illustrate example parameter selection GUIs for TCID assays, according to embodiments of the present disclosure. Figure 10A illustrates that an operator may choose between representing the data for display as a bar chart of a time dependent TCID representation (such as is shown in Figure 6). Figure 10B illustrates that an operator may choose various formulas and various criteria for determining the CPE-positive status of the various wells. Figure 10C illustrates that an operator can choose for the parameters for display in the assay analysis, which may include: % neutralization, slope, area under the curve (AUC), highest dilution, and TCID (e.g., TCID50).

[0155] Figure 11 illustrates an example parameter selection GUI with respect to a virus reduction neutralization test display. The operator can choose for various parameters for display in the assay analysis, which may include: % neutralization, slope, area under the curve (AUC), highest dilution, and TCID (e.g., TCID50). The operator may also choose various output types, which may include: bar chart; time dependent VRNT; and DRC with Y at time point vs. concentration, maximum Y in a time period vs. concentration, minimum Y in a time period vs. concentration, max-min Y in a time period vs. concentration, and AUC in a time period vs. concentration. The operator may also choose from various formulas and various constraints for determining the VRNT values.GENERAL APPLICABILITY OF THE TEACHINGS PROVIDED HEREIN

[0156] Certain terms are used throughout the description and claims to refer to particular features or components. As one skilled in the art will appreciate, different persons may refer to the same feature or component by different names. This document does not intend to distinguish between components or features that differ in name but not function.

[0157] Without further elaboration, it is believed that one skilled in the art can use the preceding description to use the claimed inventions to their fullest extent. The examples and aspects disclosed herein are to be construed as merely illustrative and not a limitation of the scope of the present disclosure in any way. It will be apparent to those having skill in the art that changes may be made to the details of the above-described examples without departing from the underlying principles discussed. In other words, various modifications and improvements of the examplesspecifically disclosed in the description above are within the scope of the appended claims. For instance, any suitable combination of features of the various examples described is contemplated.

[0158] Within the claims, reference to an element in the singular is not intended to mean “one and only one” unless specifically stated as such, but rather as “one or more” or “at least one”. Unless specifically stated otherwise, the term “some” refers to one or more. No claim element is to be construed under the provision of 35 U.S.C. § 112(f) unless the element is expressly recited using the phrase “means for” or “step for”. All structural and functional equivalents to the elements of the various embodiments described in the present disclosure that are known or come later to be known to those of ordinary skill in the relevant art are expressly incorporated herein by reference and are intended to be encompassed by the claims. Moreover, nothing disclosed in the present disclosure is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

Claims

CLAIMSWhat is claimed is:

1. A method for performing an impedance assay, the method comprising: preparing a plurality of cell samples; determining a first plurality of cellular impedance (CI) values corresponding to the plurality of cell samples while performing the impedance assay; preparing a plurality of different dilutions of a virus; inoculating, after determining the first plurality of CI values, the plurality of cell samples with the plurality of different dilutions of the virus such that a first subset of the plurality of cell samples is inoculated with a first dilution of the plurality of different dilutions of the vims and a second subset of the plurality of cell samples is inoculated with a second dilution of the plurality of different dilutions of the vims; determining, after inoculating the plurality of cell samples, a second plurality of CI values corresponding to the plurality of cell samples, wherein each CI value of the second plurality of CI values is associated with one CI value of the first plurality of CI values; determining, from the first plurality of CI values and the second plurality of CI values, a plurality of cytopathic effect (CPE) statuses experienced by the plurality of cell samples; and calculating, based on the plurality of CPE statuses, one or more of a TCID for a predefined percentage of the samples experiencing the CPE statuses and a TCID / ml (milliliter).

2. The method of claim 1, wherein preparing the plurality of cell samples includes adding a candidate therapeutic agent to the cell samples.

3. The method of claim 1, wherein preparing the plurality of cell samples includes: adding host cells with growth medium to a plurality of separate containers for each cell sample of the plurality of cell samples; incubating the host cells for a predefined incubation time period; and obtaining a base CI reading for each cell sample of the plurality of cell samples when the predefined time period concluded.

4. The method of claim 1, wherein inoculating the plurality of cell samples with the plurality of different dilutions of the virus is a last time an operator physically interacts with the plurality of cells samples until the impedance assay concludes.

5. The method of claim 1, further comprising: determining, at a predefined interval, a plurality of CI measurements corresponding to the plurality of cell samples at different times between determining the first plurality of CI values and the second plurality of CI values, which are included as an initial set of the plurality of CI measurements and a final set of the plurality of CI measurements, respectively.

6. The method of claim 5, wherein determining the plurality of CPE statuses experienced by the plurality of cell samples is based on any two sets of pluralities of CI measurements taken at different times from the plurality of CI measurements.

7. The method of claim 1, wherein the plurality of cell samples include one or more control samples in which a given concentration of the vims to which the control samples are inoculated is no virus, further comprising: comparing cell growth of the one or more control samples against historic cell growth patterns previously collected from a quality control sample in which no virus was inoculated; and in response to the cell growth of the one or more control samples falling outside of quality threshold relative to the historic cell growth patterns, rejecting the TCID and the TCID / ml milliliter calculated.

8. The method of claim 7, wherein the control sample was collected from a previous impedance assay of a different vims from respective control samples in the impedance assay.

9. The method of claim 1, wherein each individual cell sample of the plurality of cell samples is included in an individual well of a plurality of wells, each well of the plurality of wells including a first electrode and a second electrode across which each CI value of the first plurality of CI values and the second plurality of CI values is calculated for a corresponding cell sample of the plurality of cell samples.

10. The method of claim 9, wherein the first electrode and the second electrode in each well of the plurality of wells are spaced apart to define an imaging window in a base of the well, the method further comprising: capturing an image of the individual cell sample, wherein an imaging device used for capturing the image of the individual cell sample is located on a first one of above or below the individual well and a light source is located on a second one of above or below the individual well, such that light from the light source passes first through the individual cell sample and then through the imaging window.

11. The method of claim 1, further comprising, receiving a user-defined CPE definition, selected from among: a change in impedance between a first time and a second time greater than a timed CPE threshold; a trend in impedance change between the first time and the second time above a trending CPE threshold; and an impedance value outside of an expected CPE window.

12. The method of claim 1, further comprising, after calculating the TCID and the TCID / ml: adding an optical reagent to the plurality of cells; and performing an optical TCID assay on the plurality of cell samples.

13. A method, comprising: receiving selection of a virology mode for analyzing a plurality of cell samples held in corresponding wells of a wellplate for use by an impedance analysis device; receiving dilution or concentration information for a virus used to inoculate the plurality of cell samples; monitoring impedances of the cell samples over a duration of an assay; determining cytopathic effect (CPE) status experienced by the plurality of cell samples based on the impedances of the cell samples over the duration of the assay; andcalculating, based on the CPE status, a TCID for a predefined percentage of the samples experiencing the CPE status and a TCID / ml (milliliter).

14. The method of claim 13, further comprising: receiving volume information for the virus and the cell samples; and calculating a TCID / ml value based on the TCID and the volume information.

15. The method of claim 13, further comprising: comparing a series of CI values for a cell sample of the plurality of cell samples indicated to be a control sample in which the virus was not inoculated against a historical series of CI values for a historical control sample; and in response to the series of CI values for the control sample deviating more than an allowed amount from the historical scries of CI values, marking the TCID as compromised.

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