U.S. patent application number 15/789414 was filed with the patent office on 2019-03-07 for systems and methods for using a single-cell to create chromosomal spreads.
The applicant listed for this patent is University of Florida Research Foundation, Inc.. Invention is credited to Tanmay P. Lele, Wallace Gregory Sawyer.
Application Number | 20190073766 15/789414 |
Document ID | / |
Family ID | 61970256 |
Filed Date | 2019-03-07 |
United States Patent
Application |
20190073766 |
Kind Code |
A2 |
Lele; Tanmay P. ; et
al. |
March 7, 2019 |
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.
Inventors: |
Lele; Tanmay P.; (Alachua,
FL) ; Sawyer; Wallace Gregory; (Gainesville,
FL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Florida Research Foundation, Inc. |
Gainesville |
FL |
US |
|
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20180114316 A1 |
April 26, 2018 |
|
|
Family ID: |
61970256 |
Appl. No.: |
15/789414 |
Filed: |
October 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62410553 |
Oct 20, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6841 20130101;
B01L 2400/0481 20130101; C12N 15/1003 20130101; C12Q 2527/109
20130101; C12Q 2523/303 20130101; C12Q 1/6841 20130101; C40B 40/06
20130101; B01L 3/50273 20130101; G06T 7/0012 20130101; C12Q 1/6851
20130101; C12N 15/1003 20130101; G01N 33/50 20130101; G06T
2207/30024 20130101 |
International
Class: |
G06T 7/00 20060101
G06T007/00; B01L 3/00 20060101 B01L003/00; C40B 40/06 20060101
C40B040/06; C12Q 1/68 20060101 C12Q001/68; G01N 33/50 20060101
G01N033/50 |
Claims
1. A method for assaying a nucleated cell, comprising (a) imaging
the cell; (b) applying downward compression on the cell in an
manner sufficient to eject DNA out of the cell; and (c) imaging the
ejected DNA.
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.
13. A device for processing cells, comprising a stage configured to
hold a container or slide comprising a cell; an imaging apparatus
configured to acquire an image at a plurality of locations in a
scan area of the container or slide; and a compression apparatus
configured to apply vertical pressure on the cell in a manner
sufficient to eject DNA from the cell.
14. The device of claim 13, wherein the compression apparatus
comprises a vertical rod positioned over the scan area.
15. The device of claim 13, further comprises a fluidic apparatus
configured to apply horizontal shear flow on the cell while
vertical pressure is applied sufficient to increase spread of the
ejected DNA.
16. The device of claim 13, further comprising a computer program
on computer readable medium with instructions to cause the device
to carry out a method comprising (a) imaging a first cell at a
first location in the scan area; (b) applying downward compression
using the compression apparatus in an manner sufficient to eject
DNA out of the cell; and (c) imaging the ejected DNA.
17. The device of claim 15, wherein the instructions further cause
the device to repeat steps (a) to (c) on a second cell at a second
location in the scan area.
18. The device of claim 15, wherein the imaging apparatus comprises
an image processor operable to process the image to identify and
select a cell in a scan area for processing by the method.
19. The device of claim 15, wherein the computer readable medium
further comprises instructions to process the image of the cell to
measure one or more of cell volume, nuclear volume, cell shape,
nuclear shape, and nuclear invaginations.
20. The device of claim 15, wherein the computer readable medium
further comprises instructions to process the image of the ejected
DNA to detect chromosomal content.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 62/410,553, filed Oct. 20, 2016, which is hereby
incorporated herein by reference in its entirety.
BACKGROUND
[0002] The shape and size of the nucleus is an important prognostic
marker for diseases. In particular, the shape and size of the
nucleus in a cell can be used to identify a number of different
types of cancers. However, the mechanisms by which the cancer
nucleus becomes abnormal in shape are poorly understood. One
potential mechanism is that altered number of chromosomes and/or
chromosomal translocations contribute to abnormal cancer nuclear
shapes.
[0003] Currently, methods that can be used to prepare chromosomal
spreads rely on colliding a drop containing many cells (e.g.,
millions of mitotic cells (cells lacking a nucleus)) against a
surface to spread out the DNA. The disadvantage of this method is
that it is not possible to map a given chromosomal spread to an
image of the nucleus that housed it, nor the cell. This makes it
difficult to correlate chromosomal spreads and/or translocations
with cell and nuclear phenotype.
SUMMARY
[0004] Disclosed herein is a method for assaying chromosomal
content in a cell containing a nucleus. The method involves imaging
the cell first, then applying downward compression on the cell in a
manner sufficient to eject DNA out of the cell; and then imaging
the ejected DNA. In some embodiments, the cells are adherent and
mitotic, and could include any of the cell types in the human body.
In other embodiments, cells could be in suspension, such as cancer
stem cells or part of tissue such as myotubes, endothelium or
cardiovascular tissue. In particular embodiments, the cell is a
cancer cell. Alternatively, it could be a cell from patients with
progeria, cell from muscular dystrophy, laminopathy or
lipodystrophy patients, or it could be a cell from aging
humans.
[0005] Vertical downward compression can be applied using any means
suitable to compress and rupture the cell in a controlled manner.
For example, the compression can be applied using a glass slide or
slide of other material, cantilever, sphere, or cylinder (rod) made
of glass or other material. In particular embodiments, the cell is
compressed with a rod using downward/vertical pressure. In some
cases, the rod is also actuated in a horizontal plane to apply
shear in addition to compression. Compression and/or shear can also
be applied using fluid flow, such as jet flow or hydrostatic
pressure. Alternatively, cells may be extruded under pressure
through narrow pores to burst the nucleus and remove chromosomal
contents.
[0006] In some embodiments, the compression involves a single
downward force at a speed and force sufficient to eject the DNA. In
other embodiments, the compression involves oscillating vertical
and tangential forces at controlled frequencies, with or without
fluid flow.
[0007] In order for ejected DNA to adequately spread, compression
is preferably accompanied by a horizontal force. In some
embodiments, this horizontal force is achieved by the force of
ejection. This force can be increased by, for example, increasing
intracellular/intranuclear pressure prior to compression. In
particular embodiments, the method involves osmotically swelling
the cell prior to compression to increase DNA ejection. For
example, in some embodiments, the cells are arrested (e.g. the
combined treatment of thymidine and nocodazole) and fixed at the
pro-metaphase/metaphase. The culture media can then be replaced,
for example, with a hypotonic solution (e.g. 0.56% KCl solution) to
induce osmotic imbalance for cell/nucleus swelling. The
consequentially increased hydrostatic pressure inside these
inflated cell, which is balanced by the cellular/nuclear membrane
tension, can enhance the horizontal DNA ejection once the membranes
are disrupted either by the vertical compression of microprobe
[0008] In some embodiments, the method involves applying horizontal
flow during compression to increase spread of the ejected DNA. This
flow can also be used to transfer the ejected DNA after imaging for
further analysis.
[0009] The disclosed method can further involve quantifying
chromosomal content from the ejected DNA. For example, images of
the DNA can be used to identify chromosomal duplications,
deletions, or rearrangements. DNA can be collected, and labeled
with chromosome specific probes that allow identification of
chromosomes. Specific genes could be labeled to determine their
position on chromosomes, and this information correlated with
nuclear and cell phenotype before compression.
[0010] The disclosed method involves imaging the cell prior to
compression. For example, the nucleus becomes abnormally shaped in
a large number of cancers, and its appearance can be a diagnostic
metric. Imaging can be done after fluorescently labeling specific
targets in the cell using immuno-labeling, expressing proteins
conjugated with fluorescent dyes or green fluorescent protein, or
using quantum dots to recognize proteins, confocal fluorescence or
other fluorescence imaging methods (epifluorescence,
super-resolution microscopy) can be used to image cells. Images
collected can be used, for example, to measure cell volume, nuclear
volume, cell shape, nuclear shape, nuclear invaginations, or any
combination thereof. Images can also be used to detect organization
of the cytoskeleton and/or to detect localization of the
mitochondria, ER and golgi body and similar such cell biological
organelles. Images can be used to measure mitochondrial activity in
the cell prior to compression. Images can be used to quantify
localization of chosen proteins like transcription factors in
specific locations of the cell and or fluorescence methods like
photobleaching, FRET or other methods can be used to quantify
protein interactions or protein dynamics.
[0011] One advantage of the disclosed methods is the ability to
correlate observations of these types with chromosomal content.
Quantifying chromosomal abnormalities simultaneously with cellular
parameters like nuclear volume and shape, protein dynamics, protein
interactions, localization of proteins, size and localization of
other organelles like ER, golgi or mitochondria, and cellular
geometry may improve diagnostic outcomes. For example, the nucleus
can be abnormally shaped in a certain cancer, without changes to
chromosomal content. While chromosomal duplications, deletions or
rearrangements might occur in other cancers and cause abnormal
cellular parameters. Collecting such information about cancers can
improve diagnose and treatment of cancers. Nuclear abnormalities
also occur in human aging, and therefore combining chromosomal
spreads with nuclear shape measurements in aging populations can
help understand how to `normalize` aged cell populations. Such
abnormalities also occur in a host of other diseases like progeria,
laminopathies, lipodystrophies, muscular dystrophies and
cardiomyopathies.
[0012] In some embodiments, the disclosed method can be used to
select a suitable therapeutic, e.g. based on the chromosomal
content and/or its relationship to other observations of the cell
prior to compression. For example, if chromosomal abnormalities are
determined to be not responsible for nuclear volume and shape
changes in a pathological state, this may suggest a different
target for nuclear abnormalities and a different treatment modality
as compared to if chromosomal abnormalities are observed.
Similarly, if chromosomal abnormalities are found to correlate with
mitochondrial abnormalities or abnormal localization of proteins to
the ER or golgi, or with changes in cell volume, this information
can help develop better target for therapies.
[0013] Also disclosed is a device for processing cells that
includes a stage configured to hold a container or slide comprising
a cell, an imaging apparatus configured to acquire an image at a
plurality of locations in a scan area of the container or slide,
and a compression apparatus configured to apply vertical pressure
on the cell in a manner sufficient to eject DNA from the cell.
[0014] In some embodiments, the compression apparatus involves a
rod (e.g. glass rod) positioned over the scan area.
[0015] In some embodiments, the compression apparatus involves a
fluidic apparatus configured to apply compression flow on the cell,
configured to apply horizontal shear flow on the cell in a manner
sufficient to increase spread of the ejected DNA, or a combination
thereof.
[0016] In some embodiments, the device also includes a computer
program on computer readable medium with instructions to cause the
device to carry out a method that involves imaging a first cell at
a first location in the scan area, applying downward compression
using the compression apparatus in an manner sufficient to eject
DNA out of the cell, and imaging the ejected DNA. In some cases,
the instructions further cause the device to repeat these steps on
a second cell at a second location in the scan area.
[0017] The imaging apparatus can also contain an image processor
operable to process the image to identify and select a cell in a
scan area for processing by the method. The computer readable
medium can also include instructions to process the image of the
cell to measure one or more of cell volume, nuclear volume, cell
shape, nuclear shape, and nuclear invaginations. The computer
readable medium can also comprises instructions to process the
image of the ejected DNA to detect chromosomal content.
[0018] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0019] FIGS. 1 to 3 are images of non-swelled cells before and
after being compressed to cause chromosomal spreading.
[0020] FIGS. 4 to 6 are images of swelled cells before and after
being compressed to cause chromosomal spreading
DETAILED DESCRIPTION
[0021] 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.
[0022] 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.times. 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.
[0023] 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.
[0024] 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.
[0025] 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
[0026] 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.
[0027] 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.
[0028] 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.
* * * * *