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US-12618755-B2 - Methods and systems for assessing bioelectric patterns

US12618755B2US 12618755 B2US12618755 B2US 12618755B2US-12618755-B2

Abstract

Methods and systems for assessing membrane potential are provided. In some embodiments, the methods and systems, described herein, may allow spatial patterns of membrane potential to be facilely obtained. For instance, a method may comprise transferring a population of cells from a tissue to a substrate. The transfer process may substantially maintain the viability of and/or the spatial relationship between the cells. The cells on the membrane may be exposed to a voltage sensitive dye. The dye may allow the membrane potential of individual cells on the substrate to be imaged or otherwise detected. The individual cell membrane potentials when imaged together on the substrate may form a spatial membrane potential pattern. The spatial membrane potential pattern may be used to assess one or more physiological characteristics of the cells. The methods and systems may be used for a wide variety of applications, including the assessment of biopsies.

Inventors

  • Dany Adams
  • Sandra Gaston

Assignees

  • Dany S. Adam

Dates

Publication Date
20260505
Application Date
20200320

Claims (14)

  1. 1 . A method for controlling a treatment of a subject by determining a metastatic potential of cells and/or an extent of a potential pathological disorder in a tissue from the subject, the method comprising the steps of: (1) contacting a surface of a tissue from the subject with a substrate for a sufficient period of time, wherein the surface of the tissue comprises cells and/or extracellular matrix, and wherein the sufficient period of time allows for a binding comprising associations, non-covalent bonds, and/or covalent bonds to form between the substrate and the cells and/or extracellular matrix, whereby allowing at least a portion of the cells and/or extracellular matrix to bind to the substrate; transferring the at least a portion of the cells from the surface of the tissue to the substrate via the binding to the substrate, wherein a cell viability of the at least a portion of the cells after the transferring step is greater than or equal to about 75% to provide living cells thereon and removing the substrate from the surface of the tissue, (2) exposing the cells and/or extracellular matrix on the substrate to a voltage sensitive dye (VSD), obtaining a 1 st visualization of a resting membrane voltage (Vmem) in a whole layer of cells and/or extracellular matrix on the substrate using a microscope with the VSD, obtaining as a 2 nd visualization by visualizing a resting membrane voltage (Vmem) in only the living cells on the substrate with the VSD, and using a computer with software to combine the 1 st and 2 nd visualizations with a computer aided image analysis that includes a dividing up of both the visualizations each into a plurality of regions for each the 1 st visualization and the 2 nd visualization, to produce a comparison of a pattern entropy or a measure of an information versus a mean intensity for each cell type, whereby cells from a tumor with a higher metastatic potential have both higher overall entropy and greater variation across 1 st visualization; (3) using the computer with software to compare a mean intensity and/or brightness to a variance of Vmem, to obtain a measurement of variance of Vmem including a largest variance and a lowest variance, for each region from each of the plurality of regions; whereby regions that have a largest variance of Vmem contain a highest metastatic potential of cells and/or a highest amount of abnormal cells, and regions that have a lowest variance of Vmem contain a lowest metastatic potential of cells and/or a lowest amount of abnormal cells; whereby cells that are derived from a tumor with a higher metastatic potential include a charge that varies more, whereby cells that are derived from a tumor of low metastatic potential vary less in charge; and (4) executing a diagnosis and/or a pathological assessment on the tissue of the subject based upon the largest variance and the lowest variance in step (3) and the cells that have the higher metastatic potential and the cells that have the lowest metastatic potential, and controlling a treatment of the subject using the diagnosis and/or the pathological assessment.
  2. 2 . The method of claim 1 , wherein the at least a portion of the cells form a layer on the substrate, wherein the layer has a shape having a perimeter and the perimeter is within about 20% of a perimeter of the surface of the tissue.
  3. 3 . The method of claim 2 , wherein an area of the layer is within about 20% of an area of the surface of the tissue.
  4. 4 . The method of claim 1 , wherein the surface of the tissue comprises a first cell and a second cell that are separated by a first distance, wherein the first cell and the second cell are transferred to the substrate, and wherein a second distance between the first cell and the second cell on the substrate is within about 20% of the first distance.
  5. 5 . The method of claim 1 , wherein the at least a portion of the cells comprises at least about 70% of the cells on the surface of the tissue.
  6. 6 . The method of claim 1 , further comprising exposing the at least a portion of the cells on the substrate to a fluorescent voltage sensitive dye configured to provide a change in fluorescence in a response to a different voltage.
  7. 7 . The method of claim 1 , wherein the at least a portion of the cells comprise cancer cells.
  8. 8 . The method of claim 1 , wherein the at least a portion of the cells comprise one or more cells having an abnormality.
  9. 9 . The method of claim 1 , further comprising obtaining a two-dimensional fluorescence image of the at least a portion of the cells on the substrate and determining a fluorescence intensity of one or more cells on the substrate based at least in part on the two-dimensional fluorescence image.
  10. 10 . The method of claim 9 , further comprising determining a variance and/or pattern in the fluorescence intensity across at least a portion of the substrate based at least in part on the two-dimensional fluorescence image.
  11. 11 . The method of claim 9 , further comprising determining whether the subject has a pathological disorder based at least in part on the two-dimensional fluorescence image.
  12. 12 . The method of claim 1 , wherein the sufficient period of time is in a range from 1-60 seconds.
  13. 13 . The method of claim 1 , further comprising contacting the cells on the substrate with a culture medium configured to keep cells alive before and/or during a visualization.
  14. 14 . The method of claim 1 , further comprising the computer with software are accessed by an uploading of a visualization to a web-based image analysis software which is configured to return an image analysis.

Description

RELATED APPLICATIONS This application is a national stage filing under 35 U.S.C. 371 of International Patent Application Serial No. PCT/US2020/023998, filed Mar. 20, 2020, entitled “METHODS AND SYSTEMS FOR ASSESSING BIOELECTRIC PATTERNS”, which claims the benefit of U.S. Application Ser. No. 62/821,063, filed Mar. 20, 2019, entitled “METHODS AND SYSTEMS FOR ASSESSING BIOELECTRIC PATTERNS,” each of which is incorporated herein by reference in its entirety. GOVERNMENT SUPPORT This invention was made with Government support under Grant Nos. D14PC00119 (Phase I SBIR) and D15PC00054 (Phase II SBIR) awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention. TECHNICAL FIELD Methods and systems for assessing membrane potential are generally described. BACKGROUND For many disorders, biopsy remains the gold standard for diagnosis. Typically, a biopsy contains both normal and abnormal tissue. Therefore, accurate diagnosis hinges on the ability to locate abnormal tissue within the biopsy. In many cases, the biopsy is divided into a plethora of thin sections that are evaluated by a trained professional or a complex analytical technique. The evaluation of tissue sections is often time-intensive, labor-intensive, and/or expensive. Accordingly, improved compositions and methods are needed. SUMMARY Methods for assessing membrane potential and related kits, systems, methods, and components associated therewith are provided. The subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles. In one set of embodiments, kits are provided. In one embodiment, a kit comprises a voltage sensitive dye and a substrate configured to bind cells. In another set of embodiments, methods are provided. In one embodiment, a method comprises contacting a surface of a tissue with a substrate, wherein the surface of the tissue comprises cells; allowing at least a portion of the cells to bind to the substrate; and transferring the at least a portion of the cells from the surface of the tissue to the substrate, wherein a cell viability of the at least a portion of the cells after the transferring step is greater than or equal to about 75%. In another embodiment, a method comprises exposing cells on a membrane to a voltage sensitive dye, wherein the cells are living cells. In one set of embodiments, systems are provided. In one embodiment, a system comprises a layer on a membrane, wherein the layer comprises living cells; and a voltage sensitive dye, wherein the voltage sensitive dye is associated with at least a portion of the living cells. Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the invention shown where illustration is not necessary to allow those of ordinary skill in the art to understand the invention. In the figures: FIGS. 1A-D show a schematic of a method, according to one set of embodiments; FIG. 2 shows an exemplary method, according to one set of embodiments; FIGS. 3A-B show (A) images of cells that differ in metastatic potential and (B) a plot of pattern versus mean brightness, according to one set of embodiments; and FIG. 4 shows an exemplary method using a prostate tissue sample, according to one set of embodiments. DETAILED DESCRIPTION Methods and systems for assessing membrane potential are provided. In some embodiments, the methods and systems described herein may allow spatial patterns of membrane potential to be facilely obtained. For instance, in some embodiments, a method may comprise transferring a population of cells (e.g., living cells) from a tissue to a substrate. The transfer process may substantially maintain the viability of and/or the spatial relationship between the cells. For example, the spatial arrangement of the cells on the membrane may correspond to the spatial arrangement of the cells in the tissue prior to the transfer process. In some embodiments, the cells on the membrane may be exposed to a voltage sensitive dye. The dye may allow the membrane potential of individual cells on the substrate to