U.S. patent application number 17/122560 was filed with the patent office on 2021-04-01 for methods and devices for producing cellular suspensions from tissue samples.
The applicant listed for this patent is Becton, Dickinson and Company. Invention is credited to Mitchell FERGUSON, Ronald J. PETTIS.
Application Number | 20210096046 17/122560 |
Document ID | / |
Family ID | 1000005276001 |
Filed Date | 2021-04-01 |
United States Patent
Application |
20210096046 |
Kind Code |
A1 |
PETTIS; Ronald J. ; et
al. |
April 1, 2021 |
Methods and Devices for Producing Cellular Suspensions from Tissue
Samples
Abstract
Aspects of the present disclosure include methods of producing a
cellular suspension from a tissue sample by applying resonant
acoustic energy to a container comprising the tissue sample in a
manner sufficient to produce a cellular suspension from the tissue
sample. Resonant acoustic mixers and kits for use in producing a
cellular suspension from a tissue sample are also provided.
Inventors: |
PETTIS; Ronald J.; (Cary,
NC) ; FERGUSON; Mitchell; (Clayton, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Becton, Dickinson and Company |
Franklin Lakes |
NJ |
US |
|
|
Family ID: |
1000005276001 |
Appl. No.: |
17/122560 |
Filed: |
December 15, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16082513 |
Sep 5, 2018 |
10900876 |
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PCT/US17/21207 |
Mar 7, 2017 |
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17122560 |
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62306576 |
Mar 10, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01F 11/02 20130101;
G01N 1/38 20130101; G01N 15/1459 20130101; C12M 45/02 20130101;
G01N 2001/386 20130101; G01N 2001/2866 20130101; G01N 1/286
20130101; B01F 2215/0037 20130101; B01F 2215/0073 20130101; G01N
2015/1006 20130101 |
International
Class: |
G01N 1/38 20060101
G01N001/38; B01F 11/02 20060101 B01F011/02; C12M 1/33 20060101
C12M001/33; G01N 1/28 20060101 G01N001/28; G01N 15/14 20060101
G01N015/14 |
Claims
1.-11. (canceled)
12. A resonant acoustic mixer comprising: a mixing container; an
resonant acoustic energy source operatively coupled to the mixing
container; and a tissue sample present in the mixing container.
13. The resonant acoustic mixer according to claim 12, wherein the
resonant acoustic mixer is operatively coupled to a flow
cytometer.
14. The resonant acoustic mixer according to claim 12, wherein the
container further comprises at least one of an enzymatic
dissolution agent and a milling agent.
15. A kit for use in producing a cellular suspension from a tissue
sample, the kit comprising: an enzymatic dissolution agent; and a
milling agent.
16. The resonant acoustic mixer according to claim 12, wherein the
resonant acoustic energy source is configured to apply acoustic
energy having a frequency ranging from 10 to 100 Hz to the mixing
container.
17. The resonant acoustic mixer according to claim 12, wherein the
mixing container is a sealed container.
18. The resonant acoustic mixer according to claim 12, wherein the
container has a volume ranging from 10 to 500 ml.
19. The resonant acoustic mixer according to claim 12, wherein the
tissue sample is a biopsy sample.
20. The resonant acoustic mixer according to claim 13, wherein the
flow cytometer is a cell sorter.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] Pursuant to 35 U.S.C. .sctn. 119(e), this application claims
priority to the filing date of U.S. Provisional Patent Application
No. 62/306,576, filed Mar. 10, 2016; the disclosure of which
application is herein incorporated by reference.
INTRODUCTION
[0002] The current clinical gold standard for characterizing solid
tumors and stratifying patients relies partially on analyzing
formalin-fixed paraffin-embedded (FFPE) tumor slices via
immunohistochemistry (IHC), but the thin 5-7 micron (.mu.) sections
represent less than 0.01% of the total cell population in a typical
tumor biopsy and therefore not a thorough representation of a
tumor's composition. More specific diagnoses, which will ultimately
lead to better patient outcomes, will require clinicians to adopt
new, more comprehensive methods of solid tumor analysis.
[0003] One of the newest methods on the forefront of cancer
microenvironment and solid tumor research involves dissociating
solid tumors into single cell suspensions and subsequently
characterizing subpopulations of the tumor using cell surface
markers and flow cytometric analysis. This type of "deep
phenotyping" is critical to better understand which tumor
subpopulations are deleterious, which subpopulations are benign and
the complex mechanisms through which these populations interact,
evolve and evade therapeutic intervention.
[0004] A key aspect of this workflow is rendering a heterogeneous
solid tumor biopsy into a complete single cell suspension without
significantly altering surface marker expression or discriminately
affecting cell viability. This is typically performed using a
combination of mechanical, enzymatic, and/or temperature incubation
treatments. These methods are time-intensive, inefficient, and
highly subjective as the skill and experience of the researcher can
be a factor.
SUMMARY
[0005] Aspects of the present disclosure include methods of
producing a cellular suspension from a tissue sample by applying
resonant acoustic energy to a container that includes the tissue
sample in a manner sufficient to produce a cellular suspension from
the tissue sample. Additional aspects include resonant acoustic
mixers that have a mixing container, a resonant acoustic energy
source operatively coupled to the mixing container, and a tissue
sample present in the mixing container. Kits for use in producing a
cellular suspension from a tissue sample are also provided, where
such kits include an enzymatic dissolution agent and a milling
agent.
BRIEF DESCRIPTION OF THE FIGURES
[0006] The invention may be best understood from the following
detailed description when read in conjunction with the accompanying
drawings. Included in the drawings are the following figures:
[0007] FIG. 1 shows the LabRAM I acoustic mixer employed in the
Examples described herein. The LabRAM I (top left photograph) uses
low frequency, high intensity acoustic energy to create a uniform
shear field throughout the mixing vessel. A cartoon showing the
mixing vessel (top right) indicates the many intense mixing zones
(red circular arrows) produce with the application of the acoustic
energy that are approximately 50.mu. in diameter. The temperature
of the vessel and the precise frequency and duration of the
acoustic energy applied can be controlled as desired by a user. The
diagram at the bottom of FIG. 1 shows a spectrum of acoustic
frequencies and indicates where on this spectrum the LabRAM I
acoustic mixing device operates (i.e., at approximately 60 Hertz
(Hz)).
[0008] FIG. 2 is a graph showing the viability (Y-axis) of kidney
and liver tissue (X-axis) subjected to low intensity (black
circles) vs. high intensity (red squares) LabRAM I protocols (see
Example 2). Green diamonds represent Standard Protocol from Table 1
(positive control).
[0009] FIG. 3 is a graph showing total viable cells per milligram
of input tissue (Y-axis) processed as described in Example 2 with
the data separated with respect to enzyme treatment (X-axis). The
"no enzyme" and "enzyme" samples were processed by the LabRAM I
acoustic mixer while the Standard Protocol was not. Kidney samples
are shown in black circles and liver samples are shown in red
squares.
[0010] FIG. 4 is a graph showing the viability (Y-axis) of kidney
and liver tissue subjected to low intensity (black circles) vs.
high intensity (red squares) LabRAM I protocols in Example 3. Green
diamonds represent Standard Protocol from Table 1 (positive
control). This graph does not differentiate with respect to the
mincing and enzyme treatment.
[0011] FIG. 5 is a graph showing total viable cells per milligram
of input tissue (Y-axis) comparing LabRAM I protocols in Example 3
with respect to mincing (with and without) as well as the vessel
fill volume. Results using the Standard Protocol are also
shown.
[0012] FIG. 6 is a graph showing the viability (Y-axis) of kidney
and liver tissue (X-axis) subjected to the low intensity (black
circles) vs. high intensity (red squares) LabRAM I protocols in
Example 4. Green diamonds represent Standard Protocol from Table I.
This graph does not differentiate mincing and enzyme treatment.
[0013] FIG. 7 is a graph showing total viable cells per milligram
of input tissue (Y-axis) comparing LabRAM I protocols in Example 4
with respect to vessel fill volume and intensity of LabRAM I
treatment (see legend in FIG. 7). Total viable cells from the
Standard Protocol processing of liver (left-pointing triangles) and
kidney (green diamond) are also shown.
[0014] FIG. 8 is a graph showing total viable cells per milligram
of liver tissue (Y-axis) under different Standard and LabRAM I
protocols (X-axis) as described in Example 4. The last four
protocols were performed in duplicate. This graph shows clear and
reproducible evidence that acoustic energy-based mixing can be used
to dissociate liver tissue more thoroughly than the standard
protocol.
DETAILED DESCRIPTION
[0015] Aspects of the present disclosure include methods of
producing a cellular suspension from a tissue sample by applying
resonant acoustic energy to a container that includes the tissue
sample in a manner sufficient to produce a cellular suspension from
the tissue sample. Additional aspects include resonant acoustic
mixers that have a mixing container, a resonant acoustic energy
source operatively coupled to the mixing container, and a tissue
sample present in the mixing container. Kits for use in producing a
cellular suspension from a tissue sample are also provided, where
such kits include an enzymatic dissolution agent and a milling
agent.
[0016] Before the present invention is described in greater detail,
it is to be understood that this invention is not limited to
particular embodiments described, as such may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
be limiting, since the scope of the present invention will be
limited only by the appended claims.
[0017] Where a range of values is provided, it is understood that
each intervening value, to the tenth of the unit of the lower limit
unless the context clearly dictates otherwise, between the upper
and lower limit of that range and any other stated or intervening
value in that stated range, is encompassed within the invention.
The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding either or both of those
included limits are also included in the invention.
[0018] 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 this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can also be used in the practice or testing of the present
invention, representative illustrative methods and materials are
now described.
[0019] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference and are incorporated herein by reference
to disclose and describe the methods and/or materials in connection
with which the publications are cited. The citation of any
publication is for its disclosure prior to the filing date and
should not be construed as an admission that the present invention
is not entitled to antedate such publication by virtue of prior
invention. Further, the dates of publication provided may be
different from the actual publication dates which may need to be
independently confirmed.
[0020] It is noted that, as used herein and in the appended claims,
the singular forms "a", "an", and "the" include plural referents
unless the context clearly dictates otherwise. It is further noted
that the claims may be drafted to exclude any optional element. As
such, this statement is intended to serve as antecedent basis for
use of such exclusive terminology as "solely," "only" and the like
in connection with the recitation of claim elements, or use of a
"negative" limitation.
[0021] As will be apparent to those of skill in the art upon
reading this disclosure, each of the individual embodiments
described and illustrated herein has discrete components and
features which may be readily separated from or combined with the
features of any of the other several embodiments without departing
from the scope or spirit of the present invention. Any recited
method can be carried out in the order of events recited or in any
other order which is logically possible.
Methods
[0022] Methods of the present disclosure are based on the finding
that applying resonant acoustic energy to a container with a solid
tissue sample therein can produce a cellular suspension containing
viable cells. As described below, these viable cell suspensions
find use in numerous downstream assays and analyses.
[0023] Resonant acoustic energy can be supplied to the container,
with the tissue sample therein, using any convenient protocol. In
some instances, resonant acoustic energy may be supplied by cyclic
linear displacement of the container. In systems where fluid motion
is driven by resonant acoustic energy, motion may be imparted
substantially uniformly throughout all volume elements of the
container providing lower velocity gradients within the tissue
sample in the container. Although it will be appreciated that
numerous mechanical or electronic transducer arrangements can be
utilized to supply the cyclic linear displacement, one example of
commercially available equipment suitable for supplying the
necessary acoustic energy is the Resodyn.TM. LabRAM I acoustic
mixer (Resodyn Acoustic Mixers, Inc.; see FIG. 1). This technology
has been described, for example, in U.S. Pat. No. 7,188,993 to Howe
et al., and employs linear displacement to introduce a standing
linear acoustic wave into a medium, for example, gas, liquid or
solid, residing within a container affixed to the device. The
disclosure of U.S. Pat. No. 7,188,993 is incorporated herein by
reference.
[0024] In certain embodiments, the method includes placing a solid
tissue sample into a container, e.g., in an aqueous buffer or other
suitable medium, and subjecting the tissue to resonant acoustic
energy of sufficient frequency and amplitude, and for a
sufficiently sustained period of time, to produce a cellular
suspension containing viable cells derived from the solid tissue.
As the term is used herein, resonant acoustic energy (or simply
acoustic energy) is linear or spherical energy propagation through
a tangible medium which is within the frequency range of 10 hertz
to 20,000 Hertz (Hz). It will be appreciated that in methods of the
present disclosure the exact frequency may be selected by a user to
provide a standing wave in the solid tissue sample from which a
cell suspension is being produced. The frequency required to
achieve a standing wave may vary depending upon the nature of the
solid tissue, the liquid (e.g., buffer or media) in which the solid
tissue is suspended, the amount of buffer employed, the dimensions
of the container in which the tissue sample is held (and to which
the resonant acoustic energy is applied), etc. In embodiments of
the methods of the present invention, resonant acoustic energy at a
frequency of from 10 Hz up to 100 Hz is employed, including from 30
to 90 Hz, from 50 to 70 Hz, e.g., 60 Hz. In certain embodiments,
the acoustic energy is applied to the tissue sample in the
container from 50 to 150 g of acceleration (where "g" is the force
of gravity), including from 70 to 120 g, e.g., 100 g. The amplitude
of the applied resonant acoustic energy and duration that the
resonant acoustic energy is applied may also vary as desired.
[0025] The solid tissue being dissociated in the methods disclosed
herein can be any solid tissue of interest to a user, including
normal tissue and/or diseased tissue from a subject, e.g., a tumor
biopsy sample, tumor tissue, inflamed or infected tissue, cadaveric
tissue, etc. In certain embodiments, the subject from which the
tissue is derived is a mammal, e.g., a human. In some embodiments,
the method includes obtaining the tissue directly from a subject
whereas in other embodiments the tissue is received from a third
party. Examples of tissues/biopsies include gastrointestinal tract
tissue (e.g., esophagus, stomach, duodenum, colon and terminal
ileum), lung, liver, vascular (e.g., veins, arteries, lymph
vessels, lymph node), spleen, thymus, muscle, integumentary tissue
(e.g. skin, subcutis, exocrine glands), endocrine (e.g., pancreas,
pituitary, hypothalamus, thyroid, parathyroid, adrenal, thymus,
gonads) nervous system (e.g., brain, nerve, and meningeal tissue),
prostate (e.g., transrectal biopsy and/or transurethral biopsy),
urogenital system tissue (e.g., renal biopsy, endometrial biopsy
and cervical conization), breast tissue, etc. Such tissue/biopsy
tissue can be harvested according to any convenient method,
including punch biopsy, surgical methods, endoscopy-enabled
methods, needle core biopsy methods, etc. In certain embodiments,
the tissue is a normal tissue or diseased tissue grown in vitro,
e.g., an embryoid body or other differentiated tissue (e.g., muscle
tissue). No limitation in this regard is intended. The initial mass
of the tissue sample may vary as desired.
[0026] In certain embodiments, the tissue being dissociated is not
subjected to any additional tissue dissociating treatment or agent
prior to or during application of the resonant acoustic energy. In
other embodiments, the tissue being dissociated is subjected to an
additional tissue dissociating treatment or agent prior to or
during, or after application of the resonant acoustic energy, e.g.,
physical processing (e.g., mincing, crushing, and the like), the
inclusion of milling agents (e.g., beads), the enzymatic treatment
and/or chemical treatment (e.g., hyaluronidases, collagenases,
DNAses, proteases, chelating agents (e.g.,
ethylenediaminetetraacetic acid, or EDTA)). Thus, in certain
embodiments the tissue sample is contacted with an enzyme/chemical
prior to and/or during applying acoustic resonant energy. For
example, the container in which the tissue sample is placed can
include an enzymatic dissolution agent. In other embodiments, the
container includes a milling agent or milling media, e.g.,
polymeric beads.
[0027] For carrying out the method of the present disclosure, any
convenient, sealable container may be employed. Examples of
containers of interest include those that can be fixed to a
carriage of the acoustic mixing equipment utilized to prepare the
cell suspension. Examples of suitable containers include, but are
not limited to, a sealable bottle, tube (e.g., a conical tube) or
flask of any material (e.g., glass, plastic or metal), a sealable
plastic bag, and a sealable micro-titer multiple well plate. In
certain embodiments, the container has a volume ranging from 1004
to 500 mL, including from 1 mL to 250 mL, from 5 mL to 100 mL, and
anywhere in between.
[0028] Once the tissue in the container has been subjected to the
acoustic resonant energy, the acoustic energy-processed sample can
be subjected to any of a variety of clean-up or separation steps to
obtain a sample comprising live dissociated cells in a cellular
suspension. The clean-up/separation steps employed are generally
based on the specific method used to dissociate the tissue and the
desired form of the cellular suspension. For example, where a
milling agent is used, the method can further include separating
the milling agent from the acoustic energy-processed sample (e.g.,
via sieving or filtration, magnetic separation, flotation, density
centrifugation). Where enzyme and/or chemical dissociation agents
are employed, the acoustic energy-processed sample can be washed
with buffer or media to remove the dissociation agent, or the agent
can otherwise be inactivated in solution. In addition, non-cellular
debris and/or non-viable cells can be removed from the acoustic
energy-processed sample. Any convenient separation technique may be
employed in these clean-up/separation steps, including decantation,
filtering/size exclusion separation, centrifugation (e.g., density
gradient centrifugation), or combinations thereof (e.g.,
centrifuging through a properly sized sieve/mesh).
[0029] The above described steps result in the production of a
cellular suspension from an initial tissue sample. Cellular
suspensions produced by methods of the invention may be
characterized as being liquid compositions in which cells are
dissociated from each other, such that they freely move relative to
each other in the liquid composition. In cellular suspensions
produced by embodiments of the invention, the number % of cells
that are present as single cells in the suspension (i.e., not
stably associated with one or more other cells) may vary, and in
certain instances is 60% or more, such as 70% or more, including
80% or more, such as 90% or more, 95% or more, up to 100%. In some
cases the interstitial cellular fluids isolated from these cellular
suspensions may also be of interest for further manipulation or
analysis. In such instances, methods may include isolating such
fluids and, optionally manipulating or analyzing such fluids, as
desired.
[0030] Once a cellular suspension has been obtained, the cellular
suspension can be manipulated and/or analyzed as desired by the
user. Non-limiting examples include manipulations and/or analyses
related to: protein analysis (e.g., western blotting, ELISAs, cell
surface marker analysis/sorting, intracellular protein expression,
etc.), nucleic acid analysis (e.g., gene expression level
detection, genome and exome sequence analyses, etc.), determining
cellular growth/differentiation properties in vitro and/or in vivo
(e.g., in a transplantation setting, in an in vitro differentiation
assay, etc.), quantifying cellular types and subtypes and their
relative percentages within a sample, identifying therapeutically
useful cells (e.g., tumor infiltrating lymphocytes (TILs)),
identifying and/or purifying stem/progenitor cell populations,
diagnosing a disease, staging disease progression, determining the
best course of therapy for a disease/condition in a subject, etc.
Similar analyses can be utilized with interstitial fluids obtained
from the separated cell-free solution.
[0031] In certain embodiments, the cells in the cell suspension are
manipulated and/or analyzed using antibodies (or other affinity
reagents) that are specific for expression products of interest.
For example, antibodies that bind to a specific cell surface marker
can be used to purify, isolate or enrich for cells that express the
marker (or multiple different markers). In one embodiment, one or
more marker-specific antibody can be coupled to a solid support,
e.g., polymeric or magnetic beads, a tissue culture plate, a slide,
etc., to form an affinity surface to which only cells recognized by
the antibodies can adhere. After non-adherent cells are washed
away, the bound cells can then recovered by any convenient method,
e.g., by gentle shaking, by treatment with protease, by flushing
with an excess of the peptide containing the epitope for the
antibody, etc.
[0032] In certain embodiments, the cells can be separated using
antibodies coupled to a detectable label (e.g., a fluorescent dye)
by fluorescence activated cells sorting (FACS). Thus, in certain
embodiments, manipulating the cell suspension includes sorting one
or more subset(s) of cells from the cellular suspension by FACS.
While flow cytometers find use in sorting cells from cell
suspensions, flow cytometry also can be used as a purely analytical
tool (and thus the cells analyzed are not sorted for further
processing). Therefore, in certain embodiments, the cells are
analyzed for the presence or absence of one or more gene expression
products by flow cytometry. Gene expression products include both
extracellular and intracellular gene expression products and
include peptides, proteins, glycoproteins, ribonucleic acids (RNAs,
e.g., mRNA, microRNA, etc.), and the like. Where nucleic acids are
analyzed by flow cytometry, a label, such as a dye, specific for
nucleic acids can be employed to determine the total nucleic acid
content or an affinity reagent suitable to label specific cells
containing nucleic acids of a particular sequence. The affinity
reagent employed can be a detectably labeled nucleic acid probe
that include a sequence that can bind specifically to its desired
target sequence under moderate to high stringency hybridization
conditions. For example, the nucleic acid probe can include a
nucleic acid sequence that is complementary (or substantially
complementary) to a target nucleic acid sequence that is at least
15 nucleotides in length, e.g., at least 20, at least 25, at least
30, at least 50, at least 100, etc., nucleotides in length. The
design and use of nucleic acid probes that are specific for a
target of interest can be done using any convenient protocol.
[0033] In certain embodiments, non-viable cells in the cell
suspension can be identified and/or selected against, either in
sorting or purely analytical assays, by employing dyes associated
with dead cells (e.g., propidium iodide, 7-AAD, trypan blue, etc.).
Any technique or vital may be employed which is not unduly
detrimental to the viability of the selected cells may be
employed.
Compositions and Kits
[0034] Aspects of the present disclosure include a resonant
acoustic mixer that finds use in the tissue dissociation methods
described above, where the mixer includes: a mixing container; a
resonant acoustic energy source operatively coupled to the mixing
container; and a tissue sample present in the mixing container.
[0035] Resonant acoustic mixers that find use in the present
composition include an energy source that can supply cyclic linear
displacement to a mixing container with a tissue sample present
therein. The resonant acoustic energy supplied to the mixing
container in such resonant acoustic mixers is of sufficient
frequency and amplitude, and can be provided for a sufficiently
sustained period of time, to produce a cellular suspension
containing viable cells derived from the solid tissue in the
container. The resonant acoustic mixer is capable of providing
resonant acoustic energy to the mixing container within a frequency
range of from 10 Hz to 100 Hz, including from 30 to 90 Hz, from 50
to 70 Hz, e.g., 60 Hz, and at an acceleration of from 50 to 150 g,
including from 70 to 120 g, e.g., 100 g.
[0036] As noted above, one example of a commercially available
equipment suitable for supplying the necessary acoustic energy is
the Resodyn.TM. LabRAM I acoustic mixer (Resodyn Acoustic Mixers,
Inc.; see FIG. 1). This technology has been described, for example,
in U.S. Pat. No. 7,188,993 to Howe et al., (the disclosure of which
is herein incorporated by reference) and employs linear
displacement to introduce a standing linear acoustic wave into a
medium, for example, gas, liquid or solid, residing within a
container affixed to the device.
[0037] The tissue sample in the mixing container can be any solid
tissue of interest to a user, including normal tissue and/or
diseased tissue from a subject, e.g., a tumor biopsy sample, tumor
tissue, inflamed or infected tissue, cadaveric tissue, etc. In
certain embodiments, the subject from which the tissue is derived
is a mammal, e.g., a human. In some embodiments, the method
includes obtaining the tissue directly from a subject whereas in
other embodiments the tissue is received from a third party.
Examples of tissues/biopsies include gastrointestinal tract tissue
(e.g., esophagus, stomach, duodenum, colon and terminal ileum),
lung, liver, vascular (e.g., veins, arteries, lymph vessels, lymph
node), spleen, thymus, muscle, integumentary tissue (e.g. skin,
subcutis, exocrine glands), endocrine (e.g., pancreas, pituitary,
hypothalamus, thyroid, parathyroid, adrenal, thymus, gonads)
nervous system (e.g., brain, nerve, and meningeal tissue), prostate
(e.g., transrectal biopsy and/or transurethral biopsy), urogenital
system tissue (e.g., renal biopsy, endometrial biopsy and cervical
conization), breast tissue, etc. Such tissue/biopsy tissue can be
harvested according to any convenient method, including punch
biopsy, surgical methods, endoscopy-enabled methods, needle core
biopsy methods, etc.
[0038] In certain embodiments, the tissue is a normal tissue or
diseased tissue grown in vitro, e.g., an embryoid body or other
differentiated tissue (e.g., muscle tissue). No limitation in this
regard is intended.
[0039] In certain embodiments, the mixing container further
includes an agent or agents that aid in the tissue dissolution
process, e.g., an enzymatic or chemical dissolution agent(s) and/or
a milling agent. Such agents include, but are not limited to:
hyaluronidases, collagenases, DNAses, proteases, chelating agents
(e.g., ethylenediaminetetraacetic acid, or EDTA), and beads.
[0040] The mixing container can be any convenient sealable
container which can be fixed to the carriage of the resonant
acoustic mixing device. Examples of suitable containers include,
but are not limited to, a sealable bottle, tube (e.g., a conical
tube) or flask of any material (e.g., glass, plastic or metal), a
sealable plastic bag, and a sealable micro-titer multiple well
plate. In certain embodiments, the container has a volume ranging
from 1004 to 500 mL, including from 1 mL to 250 mL, from 5 mL to
100 mL, and anywhere in between.
[0041] In certain embodiments, the resonant acoustic mixer is
operatively coupled to a device or component that finds use in
downstream manipulation or analysis of the acoustic
energy-processed tissue sample. In certain embodiments, the device
or component is configured to perform any of a variety of clean-up
or separation steps to obtain a sample comprising live dissociated
cells in a cellular suspension, e.g., an automated cell
enrichment/purification system. Examples include, but are not
limited to, a magnetic activated cell sorter (e.g., MACS system,
Miltenyi Biotec), flow cytometer/cell sorters, or both.
[0042] Flow cytometer systems of interest include flow cell nozzles
and optics subsystems for detecting light emitted by a sample in a
flow stream. Suitable flow cytometer systems and methods for
analyzing samples include, but are not limited to those described
in Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford
Univ. Press (1997); Jaroszeski et al. (eds.), Flow Cytometry
Protocols, Methods in Molecular Biology No. 91, Humana Press
(1997); Practical Flow Cytometry, 3rd ed., Wiley-Liss (1995);
Virgo, et al. (2012) Ann Clin Biochem. January; 49(pt 1): 17-28;
Linden, et. al., Semin Throm Hemost. 2004 October; 30(5):502-11;
Alison, et al. J Pathol, 2010 December; 222(4):335-344; and Herbig,
et al. (2007) Crit Rev Ther Drug Carrier Syst. 24(3):203-255; the
disclosures of which are incorporated herein by reference. In
certain instances, flow cytometry systems of interest include BD
Biosciences FACSCanto.TM. flow cytometer, BD Biosciences
FACSVantage.TM., BD Biosciences FACSort.TM., BD Biosciences
FACSCount.TM., BD Biosciences FACScan.TM., and BD Biosciences
FACSCalibur.TM. systems, a BD Biosciences Influx.TM. cell sorter,
BD Biosciences JazzlM cell sorter and BD Biosciences Aria.TM. cell
sorter, BD Biosciences FACSVerse.TM., BD Accuri.TM. C6, BD
FACSCelesta.TM., BD LSRFortessa.TM., BD Biosciences FACSCanto.TM.
II, BD LSRFortessa.TM. X-20, etc. or the like.
[0043] In certain embodiments, the subject systems are flow
cytometer systems which incorporate one or more components of the
flow cytometers described in U.S. Pat. Nos. 3,960,449; 4,347,935;
4,667,830; 4,704,891; 4,770,992; 5,030,002; 5,040,890; 5,047,321;
5,245,318; 5,317,162; 5,464,581; 5,483,469; 5,602,039; 5,620,842;
5,627,040; 5,643,796; 5,700,692; 6,372,506; 6,809,804; 6,813,017;
6,821,740; 7,129,505; 7,201,875; 7,544,326; 8,140,300; 8,233,146;
8,753,573; 8,975,595; 9,092,034; 9,095,494 and 9,097,640; the
disclosures of which are herein incorporated by reference.
[0044] Also provided by the subject disclosure are kits and systems
for practicing the subject methods, as described above, e.g., for
producing a cellular suspension from a tissue sample by applying
resonant acoustic energy. In certain embodiments, the kit includes
one or more of: an enzymatic dissolution agent, a chemical
dissolution agent, and a milling agent. Enzymatic/chemical
dissolution agents include, but are not limited to hyaluronidases,
collagenases, DNAses, proteases, EDTA, and the like; and milling
agents include beads, e.g., polymeric beads.
[0045] In some embodiments, the kit includes a sealable mixing
container that is configured to hold a solid tissue sample and be
attached/fixed to the carriage of an acoustic mixing device (as
described above). Examples of suitable containers include, but are
not limited to, a sealable bottle, tube (e.g., a conical tube) or
flask of any material (e.g., glass, plastic or metal), a sealable
plastic bag, and a sealable micro-titer multiple well plate. In
certain embodiments, the container has a volume ranging from 1004
(e.g., for each well of a 96 well plate) to 500 mL, including from
1 mL to 250 mL, from 5 mL to 100 mL, and anywhere in between.
[0046] The subject systems and kits may also include one or more
other reagents for preparing or processing a tissue sample
according to the subject methods. The reagents may include one or
more sample preparation devices (e.g., scalpels, syringes, etc.),
reagents and/or buffers, sieves, filters, additional enzymatic
reagents, affinity reagents (e.g., antibodies), etc. As such, the
kits may include one or more containers such as vials or bottles,
with each container containing a separate component for carrying
out a tissue sample processing or preparing step and/or for
carrying out solid tissue dissolution using resonant acoustic
energy as described herein. It is noted that the various components
of the kits may be present in separate containers or certain
compatible components may be pre-combined into a single container,
as desired.
[0047] In addition to above-mentioned components, the subject kits
typically further include instructions for using the components of
the kit to practice the subject methods, e.g., to prepare a cell
suspension from a solid tissue sample as described herein. The
instructions for practicing the subject methods are generally
recorded on a suitable recording medium. For example, the
instructions may be printed on a substrate, such as paper or
plastic, etc. As such, the instructions may be present in the kits
as a package insert, in the labeling of the container of the kit or
components thereof (i.e., associated with the packaging or
sub-packaging) etc. In other embodiments, the instructions are
present as an electronic storage data file present on a suitable
computer readable storage medium, e.g. CD-ROM, diskette, etc. In
yet other embodiments, the actual instructions are not present in
the kit, but means for obtaining the instructions from a remote
source, e.g. via the internet, are provided. An example of this
embodiment is a kit that includes a web address where the
instructions can be viewed and/or from which the instructions can
be downloaded. As with the instructions, this means for obtaining
the instructions is recorded on a suitable substrate.
[0048] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
[0049] In the experiments described below, the use of low
frequency, high intensity acoustic energy for dissociating solid
tissues was evaluated under a series of different conditions. The
results demonstrate that acoustic energy can be used to completely
dissociate tissues, even without manual mincing or enzyme
treatments, while maintaining equivalent cell yields and
viabilities as compared to other standard protocols.
[0050] The LabRAM I from Resodyn.TM. Acoustic Mixers, Inc. (Butte,
Mont.) system was employed. The LabRAM I is a lab mixer that
thoroughly mixes gases, liquids, solids, powders and very viscous
compounds with a nominal capacity of 1 pint. A variety of mixing
vessels may be used including small vials and disposable plastic
containers and bags. The LabRAM I uses low frequency, high
intensity acoustic energy to create a uniform shear field
throughout the mixing vessel (see FIG. 1). The result is rapid
fluidization (like a fluidized bed) and dispersion of material.
ResonantAcoustic.RTM. Mixing differs from ultrasonic mixing in that
the frequency of acoustic energy is orders of magnitude lower than
ultrasonic energy (see frequency scale in FIG. 1). As a result, the
scale of mixing is larger. Unlike impeller agitation, which mixes
by inducing bulk flow, the mixing in an acoustic mixer occurs on a
microscale throughout the mixing volume. Resonant Acoustic.RTM.
Mixing introduces acoustic energy into liquids, slurries, powders
and pastes. An oscillating mechanical driver creates motion in a
mechanical system comprised of engineered plates, eccentric weights
and springs. This energy is then acoustically transferred to the
material to be mixed. The underlying technology principle is that
the system operates at resonance. In this mode, there is a nearly
complete exchange of energy between the mass elements and the
spring elements in the mechanical system. In a Resonant
Acoustic.RTM. Mixer, the only element that absorbs energy (apart
from some negligible friction losses) is the mix load itself. Thus,
the ResonantAcoustic.RTM.Mixer provides a highly efficient way of
transferring mechanical energy directly into the mixing materials.
As an example, a Resonant Acoustic.RTM. Mixer using 40 HP would
require over 770 HP in a non-resonant equipment configuration to
mix the same load.
Example 1
Experimental Design and Controls
[0051] The experiments described in this section were performed
using mouse kidney and liver tissue. Each experiment utilized a
positive control consisting of a matched piece of organ tissue
processed using a Standard Protocol (Table 1). Outputs for
evaluating the different tissue dissociation methods tested
included assessing (a) the percentage of viable (live) cells in the
dissociated sample as a proportion of the total cell output, and
(b) the total number of viable cells in the dissociated sample per
mg of input tissue. Both measurements were determined using a
commercially available Beckman Coulter Vi-Cell cell analyzer (a
trypan blue dye exclusion-based assay).
TABLE-US-00001 TABLE 1 Standard Protocol (Enzyme + Manual Mince) I.
Extraction: 1. Mice are injected i.p. with 50/5 mg/kg of
ketamine/xylazine cocktail. 2. Once sufficiently sedated, organs
are removed for subsequent dissociation. 3. The hepatic artery is
clipped to ensure euthanization. 4. All extracted tissues are
weighed and sub-divided as needed. II. Place tissues in 5 ml
chilled preservation solution DPBS. III. Mince using scissors until
all pieces are ~1-3 mm.sup.3. IV. Incubate in Collagenase IV (1500
u/ml)/(DNase I (2500 u/ml) dissociation buffer at 37 C. using a
water bath for 30 minutes, agitating every 5-10 minutes. V. Top off
Dissociation Buffer with 40 ml of RPMI 1640. VI. Centrifuge
dissociated tissue at 300x g for 5 m at RT in a 50 ml centrifuge
tube. Upon pelleting, a thick RBC band will be detected in all
samples, usually more apparent in CRC samples. VII. Resuspend the
pellet in 1 ml DPBS using a wide-orifice 1 m pipette tip, then add
5 ml of ACK Buffer, swirl the mixture, and then allow to incubate
at RT for 5-7 m. Add 45 ml 1% BSA/DPBS (no Ca+/Mg+) as a
stop-reaction and pellet the suspension at 300x g for 5 m at RT.
VIII. Resuspend in 10 ml DPBS. Filter through a 70 um sieve or
filter, and then top off filtrate with enough DPBS to bring volume
up to 10 ml. If digesting a larger piece of tumor, higher volumes
may be needed for effective filtering and to keep the cell
concentration in range of the ViCell. Change sieves or filters as
needed. IX. Mix and Transfer 600 ul of cell suspension to ViCell
tube. X. Record number and viability using ViCell Program TD 1. 50
images 2. 1 aspirate cycle 3. 3 trypan mix 4. 5 um-70 um size range
5. Cell brightness: 85 6. Cell sharpness: 100 7. Viable cell
brightness: 65 8. Viable cell spot area: 5 9. Minimum circularity:
0 10. Decluster: Med
[0052] We conducted initial experiments to optimize certain
parameters of the LabRAM I system for tissue dissociation,
including the following: [0053] 1. Exposure Time: the length of
time the tissue is exposed to the shear forces. [0054] 2.
Intensity: the LabRAM I can be tuned to deliver up to 100 g of
acceleration to the mixing vessel. [0055] 3. Vessel Geometry: the
size and shape of the vessel containing the sample can affect shear
forces. [0056] 4. Vessel Fill Volume: the ratio of liquid buffer
vs. dead space in the mixing vessel also affects shear forces.
[0057] 5. Enzyme/Mincing: some conditions were tested with and
without the presence of the enzyme cocktail from Step IV and/or
mincing of the tissue in Step III in the Standard Protocol (Table
1).
Example 2
[0058] Tissue disruption protocols were performed on mouse liver
and kidney tissues as set forth in Table 2 below.
TABLE-US-00002 TABLE 2 Sample Conditions Example 2 Sample Organ
Protocol Parameters No. Type RAM Enzyme Mincing K1 Kidney None Yes
Yes (Standard protocol) K2 Kidney Hi Yes No K3 Kidney Hi No No K4
Kidney Lo No No K5 Kidney Hi Yes Yes K6 Kidney Lo Yes Yes K7 Kidney
Hi No Yes K8 Kidney Lo No Yes L1 Liver None Yes Yes (Standard
protocol) L2 Liver Hi Yes No L3 Liver Hi No No L4 Liver Lo No No L5
Liver Hi Yes Yes L6 Liver Lo Yes Yes L7 Liver Hi No Yes L8 Liver Lo
No Yes
[0059] LabRAM I exposure of the tissue samples came after enzyme or
PBS (for "No Enzyme" conditions) incubation and prior to initial
centrifugation step (Step VI). Conditions labeled "No Mince"
skipped the scissor mincing (Step III).
[0060] Hi Ram conditions are as follows: 1 minute exposure to
LabRAM I at 90% intensity (100-110 g of acceleration) in an
inverted 50 ml conical tube with 50 ml of PBS.
[0061] Lo Ram conditions are as follows: 1 minute exposure to
LabRAM I at 50% intensity (62-70 g of acceleration) in an inverted
50 ml conical tube with 50 ml of PBS.
[0062] FIG. 2 shows the viability (as a percentage) of kidney and
liver tissue subjected to low intensity (black circles) vs. high
intensity (red squares) LabRAM I protocols. Green diamonds
represent cells dissociated using the Standard Protocol (K1 and L1
samples, positive controls). This graph does not differentiate
between mincing and enzyme treatment and is intended to simply show
that the cells can sufficiently survive the physical rigors of the
LabRAM I treatment.
[0063] FIG. 3 shows the total viable (or "Live") cells per
milligram of input tissue and separates the samples with respect to
enzyme treatment (no enzyme vs. enzyme vs. standard protocol).
Kidney samples are shown in black circles and liver samples are
shown in red squares. This Figure shows that enzyme treatment was
important in retrieving a sufficient number of viable cells from
kidney tissue, but was deleterious to obtaining the more fragile
liver cells.
Example 3
[0064] Tissue disruption protocols were performed on mouse liver
and kidney tissues as set forth in Table 3 below.
TABLE-US-00003 TABLE 3 Sample Conditions Example 3 Sample Organ
Protocol Parameters No. Type RAM Enzyme Mincing Fill (%) K1 Kidney
None (Standard Yes Yes N/A protocol) K2 Kidney Hi Long Yes No 100
K3 Kidney Hi Long Yes No 50 K4 Kidney Hi Short Yes No 100 K5 Kidney
Hi Short Yes No 50 K6 Kidney Lo Long Yes No 100 K7 Kidney Lo Long
Yes No 50 K8 Kidney Lo Short Yes No 100 K9 Kidney Lo Short Yes No
50 K10 Kidney Hi Long No Yes 100 K11 Kidney Hi Long No Yes 50 K12
Kidney Lo Long No Yes 100 K13 Kidney Lo Long No Yes 50 K14 Kidney
Lo Long Yes Yes 100 K15 Kidney Lo Long Yes Yes 50 K16 Kidney Hi
Long Yes Yes 100 L1 Liver None (Standard Yes Yes N/A protocol) L2
Liver Hi Long No No 100 L3 Liver Hi Long No No 50 L4 Liver Hi Short
No No 100 L5 Liver Hi Short No No 50 L6 Liver Lo Long No No 100 L7
Liver Lo Long No No 50 L8 Liver Lo Short No No 100 L9 Liver Lo
Short No No 50 L10 Liver Hi Long No Yes 100 L11 Liver Hi Long No
Yes 50 L12 Liver Lo Long No Yes 100 L13 Liver Lo Long No Yes 50 L14
Liver Lo Long Yes Yes 100 L15 Liver Lo Long Yes Yes 50 L16 Liver Hi
Long Yes Yes 100
[0065] Based on the results shown in Study 1 and shown in FIG. 3
(described above), the majority of kidney samples tested in this
study underwent the enzyme treatment step while the majority of
liver samples were not treated with enzyme.
[0066] In these experiments, the mixing vessel was changed from a
50 ml conical tube to a 15 ml conical tube to increase shear
forces. The vessels were either filled to 7.5 ml (50% Fill) or 15
ml (100% Fill) of media (PBS) during LabRAM I exposure.
[0067] Short LabRAM I exposure is 1 minute while Long LabRAM I
exposure is 3 minutes.
[0068] Hi LabRAM I exposure is 90% intensity (100-110 g of
acceleration) and Lo LabRAM I exposure is 50% intensity (62-70 g of
acceleration).
[0069] FIG. 4 shows the viability of kidney and liver tissue
subjected to low intensity (black circles) vs. high intensity (red
squares) LabRAM I protocols. The Green diamonds represent tissue
dissociated using the Standard Protocol from Table 1 (K1 and L1
samples, positive controls). This graph does not differentiate
between mincing and enzyme treatment and is intended to simply show
that these cells can sufficiently survive the physical rigors of
the LabRAM I treatment.
[0070] FIG. 5 shows total viable cells per milligram of input
tissue and separates the samples based on mincing (on X axis) and
vessel fill volume (see legend in FIG. 5). These data show that
liver tissue (shown in triangles) are dissociated most efficiently
without manual pre-mincing and using 50% vessel fill volume
(compare upward pointing traingles to those pointing rightward). No
significant difference in dissociation of kidney tissue was seen
between mincing/no mincing samples and between 100% fill and 50%
fill samples.
Example 4
[0071] Tissue disruption protocols were performed on mouse liver
and kidney tissues as set forth in Table 2 below.
TABLE-US-00004 TABLE 4 Sample Conditions Example 4 Sample Organ
Protocol Parameters No. Type RAM Enzyme Mincing Fill (%) K1 Kidney
None (Standard Yes Yes N/A protocol) K2 Kidney None (Standard No No
N/A protocol) K3 Kidney Hi Long Yes No 100 K4 Kidney Lo Long Yes No
100 K5 Kidney Lo Long Yes No 50 K6 Kidney Lo Long Yes No 100 K7
Kidney Lo Long Yes No 50 K8 Kidney Lo Long Yes Yes 100 K9 Kidney Lo
Long Yes Yes 50 K10 Kidney Hi Long Yes Yes 100 K11 Kidney Hi Longer
Yes No 100 K12 Kidney Lo Longer Yes No 100 K13 Kidney Lo Longer Yes
No 50 K14 Kidney Lo Longer Yes Yes 100 K15 Kidney Lo Longer Yes Yes
50 K16 Kidney Hi Longer Yes Yes 100 L1 Liver None (Standard Yes Yes
N/A protocol) L2 Liver None (Standard Yes (50%) Yes N/A protocol)
L3 Liver None No No N/A L4 Liver Hi Long No Yes 100 L5 Liver Hi
Long No No 50 L6 Liver Hi Long No Yes 50 L7 Liver Hi Short No No 50
L8 Liver Hi Short No Yes 50 L9 Liver Lo Long No No 50 L10 Liver Lo
Long No Yes 50 L11 Liver Lo Short No No 50 L12 Liver Lo Short No
Yes 50 L13 Liver Hi Long No No 50 L14 Liver Hi Short No No 50 L15
Liver Lo Long No No 50 L16 Liver Lo Short No No 50
[0072] In these experiments, all kidney samples included an enzyme
treatment step while all liver samples skipped the enzyme treatment
step. In addition, all but 4 liver samples skipped mincing step as
well. (These experimental design choices were based on results
described above.)
[0073] Vessels used were 15 ml conicals and were filled to either
7.5 ml (50%) or 15 ml (100%) (all but 1 of the liver samples were
filled to 7.5 ml based on experiments above).
[0074] Short LabRAM I exposure is 1 minute, Long LabRAM I exposure
is 3 minutes, and Longer LabRAM I exposure is 5 minutes. This
longer exposure was only tested in kidney samples as it was deemed
unnecessary for liver tissue. HI and Lo LabRAM I exposure is as
described in Example 3.
[0075] FIG. 6 shows the viability of kidney and liver tissue
subjected to low intensity (black circles) vs. high intensity (red
squares) LabRAM I protocols. Green diamonds represent tissue
dissociated using the Standard Protocol from Table 1 (K1, K2, L1
and L2 samples, positive controls)(Note that one control sample for
each tissue was dissociated using 50% of the enzyme dose). This
graph does not differentiate between mincing and enzyme treatment
and is intended to simply show that these cells can sufficiently
survive the physical rigors of the LabRAM I treatment.
[0076] FIG. 7 shows total viable cells per milligram of input
tissue and separates the data based on vessel fill volume (see
legend) and intensity of LabRAM I treatment (X axis). These data
show that liver tissue can be dissociated using only LabRAM I
treatment while achieving equivalent viability and better cell
yields than current standard protocols that are laborious and
expensive (compare right-pointing triangles and left-pointing
triangles). While viable kidney cells could be obtained in
significant numbers from the LabRAM I protocol, these protocols did
not dissociate kidney tissue as well as the standard protocol.
Regardless, it is clear from the data that sufficient live cells
can be obtained from the LAbRAM I protocol to dissociate kidney
tissue.
[0077] FIG. 8 shows total viable cells per milligram of liver
tissue dissociated with 50% fill (i.e., 7.5 mL in a 15 mL conical),
no enzyme treatment, and no mincing. The data is separated based on
the different LabRAM I protocols (X-axis). Some protocols were run
in duplicate (last 4 LabRAM I protocols in the graph, which have
two replicate experiments). From these data it is clear that for
liver tissue disruption, Short LabRAM I exposure (1 min.) is better
that Long LabRAM I exposure (3 min.), and Lo LabRAM I intensity
(50%; 62-70 g of acceleration) is better than Hi LabAM I intensity
(90%; 100-110 g of acceleration).
[0078] The data provided herein clearly shows that the LabRAM I
system can be used to dissociate liver tissue more thoroughly and
efficiently than the Standard protocol.
Discussion
[0079] The Standard Protocol employed in the experiments described
above (which is representative of protocols currently used in the
art) employs two key steps: manual mincing (e.g., using razors,
scissors, mortar and pestle, etc.) and enzyme treatment (e.g.,
collagenase). Such protocols require a researcher to cut the sample
into small pieces, the size and geometry of which is highly
variable depending on the chosen method and the individual
researcher performing the task. The enzyme treatment requires
expensive enzymes or enzyme cocktails as well as moderate to long
incubation times. In addition, such protocols require a heated
water bath or other heating element to maintain the elevated
temperature needed to ensure optimal enzymatic activity throughout
the incubation. An instrument or method of thoroughly dissociating
solid tumors into viable single-cell suspensions without the
subjectivity of manual mincing and/or the need for expensive
enzymes and/or time-consuming workflows would be highly
desirable.
[0080] The results provided above show that the low frequency, high
intensity acoustic energy, e.g., provided by the LabRAM I
instrument, can be employed to dissociate tissue into a single cell
suspension containing sufficient numbers of viable cells for
analysis and/or downstream assays.
[0081] For example, the LabRAM I was shown to be effective at
dissociating liver tissue into a highly viable single-cell
suspension in 1 minute without the need for manual mincing or
enzyme treatment. While the acoustic energy methods tested on
kidney tissue described above did not return as high a percentage
of viable kidney cells in suspension as the Standard Protocol
(calculated as viable cells/mg of input tissue), sufficient numbers
of cells were obtained from kidney samples for downstream
processing.
Embodiments
[0082] Aspects of the present disclosure include a method of
producing a cellular suspension from a tissue sample, the method
comprising: applying resonant acoustic energy to a container
comprising the tissue sample in a manner sufficient to produce a
cellular suspension from the tissue sample. In certain embodiments,
the resonant acoustic energy has a frequency ranging from 10 to 100
Hz. In any preceding embodiment, the container is a sealed
container. In any preceding embodiment, the container has a volume
ranging from 10 to 500 ml. In any preceding embodiment, the tissue
sample is a mammalian tissue sample. In certain embodiments, the
tissue sample is a biopsy sample, e.g., a tumor biopsy sample. In
any preceding embodiment, the method further comprises manipulating
the cellular suspension. In certain embodiments, the manipulating
comprises flow cytometrically processing the cellular suspension,
e.g., analyzing and/or sorting the cellular suspension. In any
preceding embodiment, the method further comprises assaying a cell
of the cellular suspension. In certain embodiments, the assaying
comprises nucleic acid analysis, e.g., nucleic acid sequencing,
expression level detection, and the like. In any preceding
embodiment, the method comprises obtaining the tissue sample. In
any preceding embodiment, the container further comprises an
enzymatic dissolution agent. In any preceding embodiment, the
container further comprises a milling agent. In certain
embodiments, the method further comprises separating the milling
agent from the cellular suspension.
[0083] Aspects of the present disclosure include a resonant
acoustic mixer comprising: a mixing container; a resonant acoustic
energy source operatively coupled to the mixing container; and a
tissue sample present in the mixing container. In certain
embodiments, the resonant acoustic energy source is configured to
apply acoustic energy having a frequency ranging from 10 to 100 Hz
to the mixing container. In any preceding embodiment, the mixing
container is a sealed container. In any preceding embodiment, the
container has a volume ranging from 10 to 500 ml. In any preceding
embodiment, the tissue sample is a mammalian tissue sample. In
certain embodiments, the tissue sample is a biopsy sample, e.g., a
tumor biopsy sample. In any preceding embodiment, the resonant
acoustic mixer is operatively coupled to a flow cytometer. In
certain embodiments, the flow cytometer is a cell sorter. In any
preceding embodiment, the container further comprises an enzymatic
dissolution agent. In any preceding embodiment, the container
further comprises a milling agent.
[0084] Aspects of the present disclosure include a kit for use in
producing a cellular suspension from a tissue sample, the kit
comprising: an enzymatic dissolution agent; and a milling
agent.
[0085] Notwithstanding the appended clauses, the disclosure set
forth herein is also defined by the following clauses:
1. A method of producing a cellular suspension from a tissue
sample, the method comprising:
[0086] applying resonant acoustic energy to a container comprising
the tissue sample in a manner sufficient to produce a cellular
suspension from the tissue sample.
2. The method according to Clause 1, wherein the resonant acoustic
energy has a frequency ranging from 10 to 100 Hz. 3. The method
according to Clause 1 or 2, wherein the container is a sealed
container. 4. The method according to any of Clauses 1 to 3,
wherein the container has a volume ranging from 10 to 500 ml. 5.
The method according to any of Clauses 1 to 4, wherein the tissue
sample is a mammalian tissue sample. 6. The method according to
Clause 5, wherein the tissue sample is a biopsy sample. 7. The
method according to Clause 6, wherein the biopsy sample is a tumor
biopsy sample. 8. The method according to any of the preceding
clauses, wherein the method further comprises manipulating the
cellular suspension. 9. The method according to Clause 8, wherein
the manipulating comprises flow cytometrically processing the
cellular suspension. 10. The method according to Clause 9, wherein
the flow cytometrically processing the cellular suspension
comprises sorting the cellular suspension. 11. The method according
to any of the preceding clauses, wherein the method further
comprises assaying a cell of the cellular suspension. 12. The
method according to Clause 11, where the assaying comprises nucleic
acid analysis. 13. The method according to Clause 12, wherein the
nucleic acid analysis comprises nucleic acid sequencing. 14. The
method according to Clause 12 or 13, wherein the nucleic acid
analysis comprises expression level detection. 15. The method
according to any of the preceding clauses, wherein the method
comprises obtaining the tissue sample. 16. The method according to
any of the preceding clauses, wherein the container further
comprises an enzymatic dissolution agent. 17. The method according
to any of the preceding clauses, wherein the container further
comprises a milling agent. 18. The method according to Clause 17,
wherein the method further comprises separating the milling agent
from the cellular suspension. 19. A resonant acoustic mixer
comprising:
[0087] a mixing container;
[0088] an resonant acoustic energy source operatively coupled to
the mixing container; and
[0089] a tissue sample present in the mixing container.
20. The resonant acoustic mixer according to Clause 19, wherein the
resonant acoustic energy source is configured to apply acoustic
energy having a frequency ranging from 10 to 100 Hz to the mixing
container. 21. The resonant acoustic mixer according to Clause 19
or 20, wherein the mixing container is a sealed container. 22. The
resonant acoustic mixer according to any of Clauses 19 to 21,
wherein the container has a volume ranging from 10 to 500 ml. 23.
The resonant acoustic mixer according to any of Clauses 19 to 22,
wherein the tissue sample is a mammalian tissue sample. 24. The
resonant acoustic mixer according to Clause 23, wherein the tissue
sample is a biopsy sample. 25. The resonant acoustic mixer
according to Clause 24, wherein the biopsy sample is a tumor biopsy
sample. 26. The resonant acoustic mixer according to any of Clauses
19 to 25, wherein the resonant acoustic mixer is operatively
coupled to a flow cytometer. 27. The resonant acoustic mixer
according to Clause 26, wherein the flow cytometer is a cell
sorter. 28. The resonant acoustic mixer according to any of Clauses
19 to 27, wherein the container further comprises an enzymatic
dissolution agent. 29. The resonant acoustic mixer according to any
of Clauses 19 to 28, wherein the container further comprises a
milling agent. 30. A kit for use in producing a cellular suspension
from a tissue sample, the kit comprising:
[0090] an enzymatic dissolution agent; and
[0091] a milling agent.
[0092] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this disclosure that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
[0093] Accordingly, the preceding merely illustrates the principles
of the invention. It will be appreciated that those skilled in the
art will be able to devise various arrangements which, although not
explicitly described or shown herein, embody the principles of the
invention and are included within its spirit and scope.
Furthermore, all examples and conditional language recited herein
are principally intended to aid the reader in understanding the
principles of the invention being without limitation to such
specifically recited examples and conditions. Moreover, all
statements herein reciting principles, aspects, and embodiments of
the invention as well as specific examples thereof, are intended to
encompass both structural and functional equivalents thereof.
Additionally, it is intended that such equivalents include both
currently known equivalents and equivalents developed in the
future, i.e., any elements developed that perform the same
function, regardless of structure. The scope of the present
invention, therefore, is not intended to be limited to the
exemplary embodiments shown and described herein. Rather, the scope
and spirit of present invention is embodied by the appended
claims.
* * * * *