Systems and methods for using a single-cell to create chromosomal spreads

Abstract

Embodiments of the present disclosure provide for methods and systems for preparing chromosomal spread for a selected cell so that chromosomal spreads and/or translocations can be correlated with the selected cell.

Claims

1. A method for assaying a plurality of nucleated cells, comprising (a) imaging a first cell at a first location in a scan area; (b) applying downward compression on the first cell in an manner sufficient to eject DNA out of the first cell into the scan area; (c) imaging the ejected DNA; (d) repeat steps (a) to (c) on a second cell at a second location in the scan area.

2. The method of claim 1, wherein the vertical downward compression is applied using a vertical rod.

3. The method of claim 1, wherein the vertical downward compression is applied using jet flow.

4. The method of claim 1, further comprising quantifying chromosomal content from the ejected DNA.

5. The method of claim 1, further comprising measuring mitochondrial activity in the cell prior to step (b).

6. The method of claim 1, wherein step (a) comprises measuring cell volume, nuclear volume, or a combination thereof.

7. The method of claim 1, wherein step (a) comprises measuring cell shape, nuclear shape, nuclear invaginations, or any combination thereof.

8. The method of claim 1, wherein step (a) comprises detecting organization of the cytoskeleton, and ER and golgi body localization.

9. The method of claim 1, wherein the cell is a cancer cell.

10. The method of claim 1, further comprising selecting a suitable therapeutic based on the chromosomal content.

11. The method of claim 1, further comprising osmotically swelling the cell prior to step (b).

12. The method of claim 1, wherein step (b) further comprises applying horizontal shear on the cell to increase spread of the ejected DNA.

Description

DESCRIPTION OF DRAWINGS

(1) FIGS. 1 to 3 are images of non-swelled cells before and after being compressed to cause chromosomal spreading.

(2) FIGS. 4 to 6 are images of swelled cells before and after being compressed to cause chromosomal spreading

DETAILED DESCRIPTION

(3) Embodiments of the present disclosure provide for methods and systems for preparing chromosomal spread for a selected cell so that chromosomal spreads and/or translocations can be correlated with the selected cell. In an embodiment, systems and methods can be used to prepare chromosomal spreads that can be correlated with the shape of the nucleus before mitosis. In an embodiment, a cell can be selected and imaged using an imaging technique (e.g., a microscope) and then a shear and/or a compression force can be applied to the selected cell and the chromosomal spread can be imaged. Once a chromosomal spread is created from the selected cell and imaged and analyzed using the imaging system, a fluidic system can be used to transfer the chromosomal spread for additional analysis. In an embodiment, shear and compression forces can be applied to mitotic, adherent cells, to obtain the chromosomal spread of the selected cell. In this regard, embodiments of the present disclosure can be used to map the chromosomal content and chromosomal translocations onto nuclear and cell shapes and cell content (e.g., pre-mitotic nuclear and cell shapes and nuclear and cell content) for a single selected cell.

(4) In an embodiment, the compression can be performed by moving a blunt vertical structure (e.g., a glass slide or a cylinder) into a substrate (e.g., a dish) with cultured cells arrested in mitosis between the structure and the substrate. The substrate can be imaged from the bottom with a 60 objective on a Nikon epifluorescence microscope, for example, before and after application of the compression and/or shear forces. The cylinder is first centered in the field of view. By compressing down with the cylinder on top of cell while observing cell simultaneously on the microscope, it is possible to image chromosomes before and after compression of the optionally osmotically swelled cell. In an embodiment, the substrate can include a fluidic or microfluidic channel that can include the selected cell or the substrate can be in fluidic communication with the chromosomal spread, so that the chromosomal spread can be flowed from the substrate and further analyzed. In an embodiment, the fluidic system can be used to flow a fluid before and/or during compression, which may further enhance chromosomal spreading by applying another shearing force. In an embodiment, the substrate can be part of a fluidic system that can be interfaced with an analysis system.

(5) The term subject refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term patient refers to a subject under the treatment of a clinician, e.g., physician.

(6) The term treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

(7) A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

EXAMPLES

Example 1

(8) FIGS. 1 to 6 illustrate the spread chromosomes in unswelled and osmotically swollen (e.g., with water or other fluid that osmotically swells) cells. In an embodiment, the method and system include both the use of hydrodynamic shear and compression to generate the chromosomal spread of a selected cell.

(9) Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

(10) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.