U.S. patent application number 17/378085 was filed with the patent office on 2021-11-04 for therapeutic cell washing, concentration, and separation utilizing acoustophoresis.
The applicant listed for this patent is FloDesign Sonics, Inc.. Invention is credited to Jason Dionne, Brian Dutra, Bart Lipkens, Walter M. Presz, JR..
Application Number | 20210340521 17/378085 |
Document ID | / |
Family ID | 1000005720441 |
Filed Date | 2021-11-04 |
United States Patent
Application |
20210340521 |
Kind Code |
A1 |
Lipkens; Bart ; et
al. |
November 4, 2021 |
THERAPEUTIC CELL WASHING, CONCENTRATION, AND SEPARATION UTILIZING
ACOUSTOPHORESIS
Abstract
Multi-stage acoustophoretic devices for continuously separating
a second fluid or a particulate from a host fluid are disclosed.
Methods of operating the multi-stage acoustophoretic devices are
also disclosed. The systems may include multiple acoustophoretic
devices fluidly connected to one another in series, each
acoustophoretic device comprising a flow chamber, an ultrasonic
transducer capable of creating a multi-dimensional acoustic
standing wave, and a reflector. The systems can further include
pumps and flowmeters.
Inventors: |
Lipkens; Bart; (Bloomfield,
CT) ; Dionne; Jason; (Simsbury, CT) ; Presz,
JR.; Walter M.; (Wilbraham, MA) ; Dutra; Brian;
(Granby, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FloDesign Sonics, Inc. |
Wilbraham |
MA |
US |
|
|
Family ID: |
1000005720441 |
Appl. No.: |
17/378085 |
Filed: |
July 16, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15942427 |
Mar 30, 2018 |
11085035 |
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17378085 |
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15586116 |
May 3, 2017 |
10640760 |
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15942427 |
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62330947 |
May 3, 2016 |
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62359182 |
Jul 6, 2016 |
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62374910 |
Aug 15, 2016 |
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62479309 |
Mar 30, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 1/02 20130101; C12M
47/02 20130101; C12M 47/04 20130101; C12N 13/00 20130101 |
International
Class: |
C12N 13/00 20060101
C12N013/00; C12N 1/02 20060101 C12N001/02; C12M 1/00 20060101
C12M001/00 |
Claims
1. A method exchanging media, the method comprising: providing an
initial mixture of a first media and particles to a chamber of an
acoustophoretic device, the acoustophoretic device including at
least one ultrasonic transducer that includes a piezoelectric
material; driving the at least one ultrasonic transducer to create
an acoustic wave an acoustic region in the chamber; providing the
initial mixture to the acoustic region, such that at least a
portion of the particles are trapped and held against fluid flow in
the acoustic region; forming clusters of the trapped particles to
grow in size in the acoustic region; and flowing a second media to
the chamber while the particles and clusters are retained in the
chamber to cause the first media to flow out of the chamber;
wherein an input rate of one or more of the initial mixture or the
second media to the chamber is in a range of from about 10 ml/min
to about 15 ml/min.
2. The method of claim 1, wherein the second media is a
biocompatible wash or a buffer solution.
3. The method of claim 1, wherein the particles are cells.
4. The method of claim 1, wherein the particles are
microcarrier/cell complexes.
5. The method of claim 1, wherein the initial mixture has a density
of about 0.5 million particles/ml to about 5 million
particles/ml.
6. The method of claim 1, further comprising concentrating the
particles in the initial mixture.
7. The method of claim 6, further comprising concentrating the
particles to a concentrate volume that is about 25 to about 50
times less than a volume of the initial mixture.
8. The method of claim 7, further comprising concentrating the
particles in the initial mixture to a concentrated particle density
of about 25 to about 50 times greater than a particle density of
the initial mixture.
9. The method of claim 1, wherein a cell density of the first media
output from the chamber is about 0.0 to about 0.5 million
cells/ml.
10. The method of claim 9, wherein the first media output is from a
concentrate process and a wash process.
11. The method of claim 1, further comprising conducting a
spectrophotometer process on the chamber to determine wash
efficacy.
12. A method of recovering cells from a cell culture, comprising:
feeding an initial mixture of the cell culture to a flow chamber of
an acoustophoretic device, the acoustophoretic device including at
least one ultrasonic transducer that includes a piezoelectric
material that is configured to be driven to generate a
multi-dimensional acoustic wave in the flow chamber; and driving
the at least one ultrasonic transducer to generate a
multi-dimensional acoustic wave in an acoustic region in the flow
chamber; providing the initial mixture to the acoustic region; and
retaining the cells from the initial mixture in the acoustic region
to form clusters of the cells to grow in size in the acoustic
region; wherein an input rate of the initial mixture to the flow
chamber is in a range of from about 10 ml/min to about 15
ml/min.
13. The method of claim 12, wherein the cell density of the
concentrated cells is about 25 to about 50 times greater than the
cell density of the initial mixture.
14. The method of claim 12, wherein a volume of the concentrated
cells is 25 to about 50 times less than a volume of the initial
mixture.
15. The method of claim 12, wherein the concentrated cells are
obtained in about 35 minutes or less.
16. The method of claim 12, further comprising washing the
concentrated cells, wherein a cell density of a wash output of the
flow chamber is about 0.0 to about 0.5 million cells/ml.
17. An acoustophoretic device, comprising: a flow chamber with a
first outlet; at least one ultrasonic transducer coupled to the
flow chamber and including a piezoelectric material that is adapted
to be driven to generate an acoustic wave in an acoustic region of
the flow chamber, such that particles are trapped to form particle
clusters in the acoustic region that can grow in size when a fluid
and particle mixture is provided to the acoustic region; a
diminished particle concentration region adjacent the acoustic
region and in fluid communication with the first outlet, the
diminished particle concentration region being interposed between
the acoustic region and the first outlet; and a fluid input region
on an opposite side of the acoustic region from the first outlet
that permits fluid flow into the acoustic region, such that an
input fluid flows through the acoustic region while the particles
and particle clusters remain trapped in the acoustic region;
wherein the dimensions of the flow chamber and the first outlet are
sized to accommodate a flow rate in a range of from about 10 ml/min
to about 15 ml/min.
18. The acoustophoretic device of claim 17, wherein the flow
chamber can contain a cell capacity of about 4 billion to about 40
billion cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/942,427, filed on Mar. 30, 2018, which is a
continuation-in-part of U.S. patent application Ser. No.
15/586,116, filed on May 3, 2017, now U.S. Pat. No. 10,640,760,
which claims priority to U.S. Provisional Patent Application Ser.
No. 62/330,947, filed on May 3, 2016, and to U.S. Provisional
Patent Application Ser. No. 62/359,182, filed on Jul. 6, 2016, and
to U.S. Provisional Patent Application Ser. No. 62/374,910, filed
on Aug. 15, 2016. U.S. patent application Ser. No. 15/942,427,
filed on Mar. 30, 2018 also claims the benefit of U.S. Provisional
Patent Application Ser. No. 62/479,309, filed on Mar. 30, 2017. The
disclosures of these applications are hereby fully incorporated
herein by reference in their entirety.
BACKGROUND
[0002] Concentrating therapeutic cells and transferring them from
one solution into another (usually referred to as washing) are two
processes involved at multiple stages of production and use of the
cells. The washing and separation of materials in cellular
processing is an important part of the overall efficacy of the cell
therapy of choice. In particular, therapeutic cells may originally
be suspended in a growth serum or in preservative materials like
dimethyl sulfoxide (DMSO). Separating the cells from these fluids
so the cells can be further processed is important in the overall
therapeutic process of using such cellular materials. In one
example, the cells are typically recovered from a bioreactor,
concentrated, and transferred from culture media into an
electroporation buffer prior to transduction, such as in
manufacturing CAR-T cells. After expansion of cells at the final
manufacturing step, they are concentrated and transferred into an
appropriate solvent depending on the desired application.
[0003] Therapeutic cells are stored in specialized media to prolong
the viability of these cells either through refrigeration and or
freezing processes. Such specialized media may not be compatible
when the therapeutic cells are introduced into a patient. It may
thus be helpful to both wash and concentrate the therapeutic cells
in a buffer or wash media that is biocompatible with both the
therapeutic cells and with the patient. These washing and
concentration processes conventionally involve the use of
centrifugation and physical filtration. The washing step may be
repeated a number of times. For example, the specialized media
(which can be pyrogenic or otherwise harmful) may be fully removed
with multiple wash steps, and the cells may be suspended in a new
buffer or wash solution. During this washing process, many of the
cells are degraded or destroyed through the centrifugation and
physical filtration processes. Moreover, the filtration process can
be rather inefficient and may entail a non-sterile intrusion into
the environment for batch processing, whereby the cell culture is
exposed to possible pathogens or outside cellular influences that
would be harmful to the target cell culture. Further yet, with
these physical filtration processes, biological waste is generated
through the use of multiple physical filters which may incur
additional steps for proper disposal. The cost and timeliness of
this process is also not conducive to a fast or low-cost process of
preparing the cells for introduction to the patient.
BRIEF SUMMARY
[0004] The present disclosure provides methods and systems for
replacing or augmenting conventional centrifugation and physical
filtration processes along with the multiple washing steps with a
simpler, lower cost, and more friendly process for particles such
as therapeutic cells. The methods/processes can be performed in a
sterile environment and in a continuous form.
[0005] Disclosed herein are methods of washing particles, which may
be cells. In some example methods, an initial mixture of a first
media and the particles is fed to a flow chamber of an
acoustophoretic device. The first media may contain preservatives
such as dimethyl sulfoxide (DMSO) which are undesirable for future
applications/uses of the particles. The acoustophoretic device has
at least one ultrasonic transducer that includes a piezoelectric
material and is configured to be driven to create a
multi-dimensional acoustic standing wave in the flow chamber. At
least a portion of the particles are trapped in the
multi-dimensional acoustic standing wave. A second media is flowed
through the flow chamber to wash out the first media while the
particles are retained in the multidimensional acoustic standing
wave. The particles may thus experience a media exchange, where the
first media is exchanged for the second media.
[0006] In some examples, the volume of the second media used to
perform the wash process may be equivalent to a volume of the flow
chamber. In some examples, the volume of the second media used to
perform the wash process may be multiples of or portions of the
volume of the flow chamber. The second media can be a biocompatible
wash or a buffer solution.
[0007] The particles may be cells. The cells may be Chinese hamster
ovary (CHO) cells, NS0 hybridoma cells, baby hamster kidney (BHK)
cells, human cells, regulatory T-cells, Jurkat T-cells, CAR-T
cells, B cells, or NK cells, peripheral blood mononuclear cells
(PBMCs), algae, plant cells, bacteria, or viruses. The cells may be
attached to microcarriers.
[0008] Sometimes, the piezoelectric material of the at least one
ultrasonic transducer is in the form of a piezoelectric array
formed from a plurality of piezoelectric elements. Each
piezoelectric element can be physically separated from surrounding
piezoelectric elements by a potting material. The piezoelectric
array can be present on a single crystal, with one or more channels
separating the piezoelectric elements from each other. Each
piezoelectric element can be individually connected to its own pair
of electrodes. The piezoelectric elements can be operated in phase
with each other, or operated out of phase with each other. The
acoustophoretic device may further comprise a cooling unit for
cooling the at least one ultrasonic transducer.
[0009] In various embodiments, the initial mixture may have a
density of about 0.5 million particles/mL to about 5 million
particles/mL. The concentrated volume can be 25 to about 50 times
less than a volume of the initial mixture. The concentrated volume
may have a particle density of 25 to about 50 times greater than a
particle density of the initial mixture.
[0010] Also disclosed in various embodiments are methods of
recovering greater than 90% of cells from a cell culture. An
initial mixture of a first media and the cell culture is fed
through a flow chamber of an acoustophoretic device, the
acoustophoretic device comprising at least one ultrasonic
transducer including a piezoelectric material that is configured to
be driven to create a multi-dimensional acoustic standing wave in
the flow chamber. The at least one ultrasonic transducer is driven
to create a multi-dimensional acoustic standing wave in the flow
chamber, and thus to concentrate the cell culture within the
acoustic standing wave. The initial mixture has an initial cell
density of about 0.5 million cells/mL to about 5 million cells/mL,
and the concentrated cell culture has a cell density at least 25
times greater than the initial cell density.
[0011] In some embodiments, the concentrated cell culture has a
cell density of 25 to about 50 times greater than the initial cell
density. In other embodiments, a volume of the concentrated cell
culture is 25 to about 50 times less than a volume of the initial
mixture. The concentrated cell culture can be obtained in about 35
minutes or less.
[0012] Also disclosed are acoustophoretic devices, comprising: a
flow chamber having a fluid inlet, a first outlet, and a second
outlet; at least one ultrasonic transducer proximate a first wall
of the flow chamber, at least one ultrasonic transducer including a
piezoelectric material that is adapted to be driven to create a
multi-dimensional acoustic standing wave; a reflector on a second
wall of the flow chamber opposite the at least one ultrasonic
transducer; and a thermoelectric generator located between the at
least one ultrasonic transducer and the first wall of the flow
chamber.
[0013] The acoustophoretic device may have a concentrated volume of
about 25 mL to about 75 mL. The acoustophoretic device may have a
cell capacity of about 4 billion to about 40 billion cells. Various
lines can connect the acoustophoretic device to containers that
provide or receive various materials to/from the acoustophoretic
device.
[0014] These and other non-limiting characteristics are more
particularly described below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0016] The following is a brief description of the drawings, which
are presented for the purposes of illustrating the example
embodiments disclosed herein and not for the purposes of limiting
the same.
[0017] FIG. 1 illustrates an example acoustophoresis process using
a transducer and reflector to create an acoustic standing wave for
trapping particles and separating them from a fluid by enhanced
gravitational settling.
[0018] FIG. 2 illustrates an example cell concentration and washing
process ("diafiltration") according to the present disclosure using
acoustophoresis.
[0019] FIG. 3 illustrates another example cell concentration and
washing process (push through) according to the present disclosure
using acoustophoresis.
[0020] FIG. 4 shows six photographs that, from left to right and
top to bottom, show the progression of cells being trapped in an
acoustophoretic device before a second media mixture (dyed blue) is
flowed into the device and gradually replaces the first media (dyed
red).
[0021] FIG. 5 is a perspective view of an example acoustophoretic
device according to the present disclosure.
[0022] FIG. 6 is a cross-sectional illustration of the
acoustophoretic device of FIG. 5.
[0023] FIG. 7 is a graph showing the performance of the
acoustophoretic device of FIG. 5. The x-axis is elapsed time
(minutes) and runs from 0 to 40 in increments of 5. The left-side
y-axis is permeate density reduction (%) and runs from 0 to 100 in
increments of 10. The right-side y-axis is permeate cell density
(.times.10.sup.6 cells/m L) and runs from 0.00 to 2.00 in
increments of 0.20. The uppermost solid line represents permeate
reduction density (%). The lowermost solid line represents permeate
cell density. The middle line running substantially horizontally
across the page represents feed cell density for reference
purposes.
[0024] FIG. 8 is a conventional single-piece monolithic
piezoelectric material used in an ultrasonic transducer.
[0025] FIG. 9 is an example rectangular piezoelectric array having
16 piezoelectric elements used in the transducers of the present
disclosure.
[0026] FIG. 10 is another example rectangular piezoelectric array
having 25 piezoelectric elements used in the transducers of the
present disclosure.
[0027] FIG. 11 is a diagram illustrating a piezoelectric material
having 16 piezoelectric elements operated in out-of-phase modes.
Dark elements indicate a 0.degree. phase angle and light elements
indicate a 180.degree. phase angle.
[0028] FIG. 12 illustrates a kerfed piezoelectric material (top)
versus a transducer array that has piezoelectric elements joined
together by a potting material (bottom).
[0029] FIG. 13 is a graph showing the performance of an
acoustophoretic device according to the present disclosure having a
16-element piezoelectric array, with the elements operated in-phase
with one another. The x-axis is elapsed time (minutes) and runs
from 0 to 60 in increments of 10. The left-side y-axis is permeate
density reduction (%) and runs from 0 to 100 in increments of 10.
The right-side y-axis is permeate cell density (.times.10.sup.6
cells/mL) and runs from 0.00 to 2.50 in increments of 0.50. The
uppermost solid line represents permeate reduction density (%). The
lowermost solid line represents permeate cell density. The middle
line running substantially horizontally across the page represents
feed cell density for reference purposes.
[0030] FIG. 14 is a graph showing the T-cell concentration
performance of an acoustophoretic process according to the present
disclosure with a low cell density culture. The x-axis is elapsed
time (minutes) and runs from 0 to 25 in increments of 5. The
left-side y-axis is percent reduction (%) and runs from 0 to 100 in
increments of 10. The right-side y-axis is cell density
(.times.10.sup.6 cells/mL) and runs from 0.00 to 1.60 in increments
of 0.20. The upper solid line represents permeate reduction (%).
The lower solid line represents permeate cell density. The dashed
line represents feed cell density for reference purposes.
[0031] FIG. 15 is a graph showing the percent density reduction
(PDR) dependency on concentration and flow rate for an
acoustophoretic process according to the present disclosure. The
x-axis is time (minutes) and runs from 0 to 40 in increments of 5.
The y-axis is permeate density reduction (%) and runs from 0 to 100
in increments of 10. The line having circle-shaped data points
represents a mixture having an initial cell concentration of
5.times.10.sup.6 cells/mL. The line having x-shaped data points
represents a mixture having an initial cell concentration of
3.times.10.sup.6 cells/mL. The line having triangle-shaped data
points represents a mixture having an initial cell concentration of
1.times.10.sup.6 cells/mL at a flow rate of 20 mL/minute. The line
having diamond-shaped data points represents a mixture having an
initial cell concentration of 1.times.10.sup.6 cells/mL at a flow
rate of 10 mL/minute.
[0032] FIG. 16 is a graph showing the T-cell performance for an
acoustophoretic process according to the present disclosure with a
high cell density culture. The x-axis is elapsed time (minutes) and
runs from 0 to 25 in increments of 5. The left-side y-axis is
percent reduction (%) and runs from 0 to 100 in increments of 10.
The right-side y-axis is cell density (.times.10.sup.6 cells/m L)
and runs from 0.00 to 3.00 in increments of 0.50. The upper solid
line represents permeate density reduction (%). The lower solid
line represents permeate cell density. The dashed line represents
feed cell density for reference purposes.
[0033] FIG. 17A is a perspective view of an example acoustophoretic
device according to the present disclosure including a cooling unit
for cooling the transducer. FIG. 17B is an exploded view of the
device of FIG. 17A.
[0034] FIG. 18 is a graph showing the temperature profile of an
acoustophoretic device without active cooling. The x-axis is
elapsed time (minutes) and runs from 0.00 to 20.00 in increments of
2.00. The y-axis is temperature (.degree. C.) and runs from 17.00
to 33.00 in increments of 2.00. The lowermost line along the right
side of the graph represents the feed temperature (.degree. C.).
The uppermost line along the right side of the graph represents the
core temperature (.degree. C.). The middle line along the right
side of the graph represents the permeate temperature (.degree.
C.).
[0035] FIG. 19 is a graph showing the temperature profile of an
acoustophoretic device with active cooling of the transducer. The
x-axis is elapsed time (minutes) and runs from 0.00 to 20.00 in
increments of 2.00. The y-axis is temperature (.degree. C.) and
runs from 17.00 to 33.00 in increments of 2.00. The lowermost line
along the right side of the graph represents the feed temperature
(.degree. C.). The uppermost line along the right side of the graph
represents the core temperature (.degree. C.). The middle line
along the right side of the graph represents the permeate
temperature (.degree. C.).
[0036] FIG. 20 illustrates a process for concentrating, washing,
and/or separating microcarriers and cells according to the present
disclosure. The leftmost portion represents a first step of
receiving complexes of microcarriers and cells surrounded by a
bioreactor serum from a bioreactor and concentrating the
microcarrier/cell complexes in an acoustophoretic device according
to the present disclosure. The middle portion represents a second
step of washing the concentrated microcarriers with attached cells
to remove the bioreactor serum. The rightmost portion represents a
third step of trypsinizing, or disassociating, the microcarriers
and cells and a fourth step of separating the microcarriers from
the cells. The bottom portion represents a final wash and
concentrate step that can be employed as desired.
[0037] FIG. 21 shows media exchange in an acoustophoretic device
according to the present disclosure. The "Concentrate" photograph
shows the concentrate (e.g., concentrated microcarriers with
attached T cells) surrounded by a first media (dyed red). The "Wash
Pass 1" photograph shows the microcarriers with attached T cells
after a first wash pass using a second media (dyed blue). The "Wash
Pass 2" photograph shows the microcarriers with attached T cells
after a second wash pass. The rightmost "Wash Pass 3" photograph
shows the microcarriers with attached T cells after a third wash
pass, and is almost completely blue.
[0038] FIG. 22 shows microscopic images of the media exchange shown
in FIG. 21.
[0039] FIG. 22 shows a microscopic image of the microcarriers with
T attached cells in the feed and during the three wash passes, and
the concentration of separated microcarriers and T cells in the
permeate.
[0040] FIG. 23 shows the concentration of T cells in the
acoustophoretic device before acoustophoresis (top row of
photographs) and after one acoustophoresis pass (bottom row of
photographs).
[0041] FIG. 24 shows the concentration of microcarriers with
attached T cells in the feed into the acoustophoretic device (top
row of photographs) and the concentration of separated
microcarriers and T cells in the permeate drawn out of the
acoustophoretic device (bottom row of photographs). The dark
circular items indicate microcarriers, and the lighter area
indicates T cells.
[0042] FIG. 25 shows microscopic images of the concentration of
microcarriers with attached T cells in the feed and the
concentration of separated microcarriers and T cells in the
permeate.
[0043] FIG. 26 is a schematic of an example acoustophoretic system
according to the present disclosure showing the flow path of the
feed material through the system.
[0044] FIG. 27 is a schematic of the example acoustophoretic system
of FIG. 28 showing the flow path of the wash material through the
system.
[0045] FIG. 28 is a schematic of the example acoustophoretic system
of FIG. 28 showing draining of the system.
[0046] FIG. 29 is a two-axis graph showing the results of trial A.
The left-hand y-axis is the percent reduction of cells in the
permeate, and runs from 0 to 100% at intervals of 20%. The
right-hand y-axis is the cell density of the permeate in units of
million cells/m L, and runs from 0 to 1.00 at intervals of 0.20.
The x-axis is elapsed time in minutes, and runs from 0 to 33
minutes at intervals of 3. The dotted line indicates the initial
cell density, which was 0.98 million cells/mL.
[0047] FIG. 30 is a two-axis graph showing the results of trial B.
The left-hand y-axis is the percent reduction of cells in the
permeate, and runs from 0 to 100% at intervals of 20%. The
right-hand y-axis is the cell density of the permeate in units of
million cells/m L, and runs from 0 to 1.00 at intervals of 0.20.
The x-axis is elapsed time in minutes, and runs from 0 to 33
minutes at intervals of 3. The dotted line indicates the initial
cell density, which was 0.85 million cells/mL.
[0048] FIG. 31 is a two-axis graph showing the results of trial C.
The left-hand y-axis is the percent reduction of cells, and runs
from 0 to 100% at intervals of 20%. The right-hand y-axis is the
cell density in units of million cells/mL, and runs from 0 to 4.00
at intervals of 1.00. The x-axis is elapsed time in minutes, and
runs from 0 to 30 minutes at intervals of 3. The dotted line
indicates the initial cell density, which was 4.08 million
cells/mL.
[0049] FIG. 32 is a graph showing the absorbance at different
wavelengths for six different samples. Those samples are: 100% wash
media (100 W-0 G), 50% wash media and 50% growth media (50 W-50 G),
100% growth media (0 W-100 G), first volume of the wash (1 Volume),
second volume of the wash (2 Volume), and third volume of the wash
(3 Volume). The y-axis is absorbance, and runs from 0 to 1 at
intervals of 0.1. The x-axis is wavelength, and runs from 540 nm to
640 nm at intervals of 50 nm.
DETAILED DESCRIPTION
[0050] The present disclosure may be understood more readily by
reference to the following detailed description of desired
embodiments and the examples included therein. In the following
specification and the claims which follow, reference will be made
to a number of terms which shall be defined to have the following
meanings.
[0051] Although specific terms are used in the following
description for the sake of clarity, these terms are intended to
refer only to the particular structure of the embodiments selected
for illustration in the drawings, and are not intended to define or
limit the scope of the disclosure. In the drawings and the
following description below, it is to be understood that like
numeric designations refer to components of like function.
Furthermore, it should be understood that the drawings are not to
scale.
[0052] The singular forms "a," "an," and "the" include plural
referents unless the context clearly dictates otherwise.
[0053] As used in the specification and in the claims, the term
"comprising" may include the embodiments "consisting of" and
"consisting essentially of." The terms "comprise(s)," "include(s),"
"having," "has," "can," "contain(s)," and variants thereof, as used
herein, are intended to be open-ended transitional phrases, terms,
or words that require the presence of the named components/steps
and permit the presence of other components/steps. However, such
description should be construed as also describing compositions or
processes as "consisting of" and "consisting essentially of" the
enumerated components/steps, which allows the presence of only the
named components/steps, along with any impurities that might result
therefrom, and excludes other components/steps.
[0054] Numerical values should be understood to include numerical
values which are the same when reduced to the same number of
significant figures and numerical values which differ from the
stated value by less than the experimental error of conventional
measurement technique of the type described in the present
application to determine the value.
[0055] All ranges disclosed herein are inclusive of the recited
endpoint and independently combinable (for example, the range of
"from 2 grams to 10 grams" is inclusive of the endpoints, 2 grams
and 10 grams, and all the intermediate values).
[0056] A value modified by a term or terms, such as "about" and
"substantially," may not be limited to the precise value specified.
The approximating language may correspond to the precision of an
instrument for measuring the value. The modifier "about" should
also be considered as disclosing the range defined by the absolute
values of the two endpoints. For example, the expression "from
about 2 to about 4" also discloses the range "from 2 to 4."
[0057] It should be noted that many of the terms used herein are
relative terms. For example, the terms "upper" and "lower" are
relative to each other in location, e.g. an upper component is
located at a higher elevation than a lower component in a given
orientation, but these terms can change if the device is flipped.
The terms "inlet" and "outlet" are relative to a fluid flowing
through them with respect to a given structure, e.g. a fluid flows
through the inlet into the structure and flows through the outlet
out of the structure. The terms "upstream" and "downstream" are
relative to the direction in which a fluid flows through various
components, e.g. the flow fluids through an upstream component
prior to flowing through the downstream component. It should be
noted that in a loop, a first component can be described as being
both upstream of and downstream of a second component.
[0058] The terms "horizontal" and "vertical" are used to indicate
direction relative to an absolute reference, e.g. ground level. The
terms "upwards" and "downwards" are also relative to an absolute
reference; an upwards flow is always against the gravity of the
earth.
[0059] The present application refers to "the same order of
magnitude." Two numbers are of the same order of magnitude if the
quotient of the larger number divided by the smaller number is a
value of at least 1 and less than 10.
[0060] The acoustophoretic technology of the present disclosure
employs acoustic standing waves to concentrate, wash, and/or
separate materials (such as particles or a secondary fluid) in a
primary or host fluid. In particular, as shown in the upper left
image (A) of FIG. 1, an ultrasonic transducer T creates an acoustic
wave in the fluid, which interacts with a reflector R positioned
across from the ultrasonic transducer to create an acoustic
standing wave. Although a reflector R is illustrated in FIG. 1,
another transducer may be used to reflect and/or generate acoustic
energy to form the acoustic standing wave.
[0061] As shown in the upper right image (B) of FIG. 1, as the host
fluid and material entrained in the host fluid flows upwards
through the acoustic standing wave, the acoustic standing wave(s)
traps (retains or holds) the material (e.g., secondary phase
materials, including fluids and/or particles). The scattering of
the acoustic field off the material results in a three-dimensional
acoustic radiation force, which acts as a three-dimensional
trapping field.
[0062] The three-dimensional acoustic radiation force generated in
conjunction with an ultrasonic standing wave is referred to in the
present disclosure as a three-dimensional or multi-dimensional
standing wave. The acoustic radiation force is proportional to the
particle volume (e.g. the cube of the radius) of the material when
the particle is small relative to the wavelength. The acoustic
radiation force is proportional to frequency and the acoustic
contrast factor. The acoustic radiation force scales with acoustic
energy (e.g. the square of the acoustic pressure amplitude). For
harmonic excitation, the sinusoidal spatial variation of the force
drives the particles to the stable positions within the standing
waves. When the acoustic radiation force exerted on the particles
is stronger than the combined effect of fluid drag force and
buoyancy and gravitational force, the particle can be trapped
within the acoustic standing wave field, as shown in the upper
right image (B) of FIG. 1.
[0063] As can be seen in the lower left image (C) of FIG. 1, this
trapping results in coalescing, clumping, aggregating,
agglomerating, and/or clustering of the trapped particles.
Additionally, secondary inter-particle forces, such as Bjerkness
forces, aid in particle agglomeration.
[0064] As the particles continue to coalesce, clump, aggregate,
agglomerate, and/or cluster the particles can grow to a certain
size at which gravitational forces on the particle cluster overcome
the acoustic radiation force. At such size, the particle cluster
can fall out of the acoustic standing wave, as shown in the lower
right image (D) of FIG. 1.
[0065] Desirably, the ultrasonic transducer(s) generate a
three-dimensional or multi-dimensional acoustic standing wave in
the fluid that exerts a lateral force on the suspended particles to
accompany the axial force so as to increase the particle trapping
capabilities of the standing wave. A planar or one-dimensional
acoustic standing wave may provide acoustic forces in the axial or
wave propagation direction. The lateral force in planar or
one-dimensional acoustic wave generation may be two orders of
magnitude smaller than the axial force. The multi-dimensional
acoustic standing wave may provide a lateral force that is
significantly greater than that of the planar acoustic standing
wave. For example, the lateral force may be of the same order of
magnitude as the axial force in the multi-dimensional acoustic
standing wave.
[0066] The acoustic standing waves of the present disclosure can be
used to trap particles (e.g. therapeutic cells such as T cells, B
cells, NK cells) suspended in a first media in the standing wave.
The first media can then be replaced with a second media (e.g., a
biocompatible wash or buffer solution). Put another way, the host
fluid of the particles can be replaced. Prior to replacing the
first media with the second media, acoustophoresis can be used to
perform a diafiltration process, as shown in FIG. 2.
[0067] In FIG. 2, starting with an initial mixture that has a low
cell density of, for example, less than 1.times.10.sup.6 cells/mL,
acoustophoresis can be used to reduce the volume of the initial
mixture, for example by at least 10.times., including 20.times. and
up to 100.times. or more. The cell concentration may be increased
by at least 10.times., including 20.times. and up to 100.times. or
more. This initial reduction process is the first volume reduction
step (A). Next, the second media (e.g., a biocompatible wash or
buffer solution) can be introduced to at least partially displace
the first media, as indicated in step (B). Next, the new mixture of
the cells and second media can be subjected to an acoustophoretic
volume reduction step (C). This series of operations is referred to
as a "diafiltration" process.
[0068] FIG. 3 illustrates a single-step, push-through process in
which particles/cells are trapped in the acoustic standing wave and
held in the acoustophoretic device. The second media (e.g., a
biocompatible wash or buffer solution) is then flowed into the
acoustophoretic device to effectively "wash out" the first media.
With the push-through process, more than 90%, including up to 99%
or more, of the first media can be removed from the
particles/cells. The push-through process can be employed as a
continuous, single-use process that uses less buffer solution and
less time than the diafiltration process of FIG. 2.
[0069] FIG. 4 shows six photographs that, from left to right and
top to bottom, show the progression of cells being trapped in an
acoustophoretic device before a second media mixture (dyed blue) is
flowed into the device and gradually replaces the first media (dyed
red). In FIG. 4, a 150 mL feed volume was used with 80 mL of
electroporation media wash for the second media. The concentrate
was drawn off at a flow rate of 10 mL/minute. As can be seen in
these pictures, over time the first media is replaced with the
second media.
[0070] With reference now to FIG. 5 and FIG. 6, a first example
embodiment of an acoustophoretic device 100 for separation of
particles/cells from fluid is depicted. The acoustophoretic device
100 includes a flow chamber 110 having at least one inlet and at
least one outlet. In this embodiment, the flow chamber 110 includes
inlet 112, permeate outlet 114, concentrate outlet 116, an
ultrasonic transducer 120, and a reflector 130. The inlet 112 can,
in certain embodiments, serve the dual function of introducing the
cells surrounded by the first media into the flow chamber 110 in
addition to introducing the second media into the flow chamber 110.
Alternatively, separate inlets can be used for introducing the
first and second media.
[0071] The flow chamber 110 is the region of the device 100 through
which is flowed the cells surrounded by the first media. In this
embodiment, the flow chamber 110 is defined by inlet 112, permeate
outlet 114, and concentrate outlet 116. The flow chamber 110 is
further defined by a sidewall 115 to which the ultrasonic
transducer 120 and the reflector 130 are coupled. As seen here, the
sidewall is shaped so that the ultrasonic transducer and reflector
are located on opposite sides thereof.
[0072] Inlet 112 is located at a first end 106 of the flow chamber
110. In particular embodiments, the ingress of material through the
inlet 112 can be configured to occur toward the bottom end of the
inlet 112, such that the inflow of fluid into the flow chamber 110
occurs closer to the bottom end of the flow chamber 110 than the
top end thereof.
[0073] As depicted in FIG. 5 and FIG. 6, the inlet 112 is located
along a first side 107 of the device 100. The first side 107 of the
device also houses the reflector 130, while a second side 109 of
the device, opposite the first side thereof, houses the ultrasonic
transducer 120. The inlet 112 could alternatively be located along
the second side 109 of the device (e.g., on the same side as the
ultrasonic transducer) or on another side of the device.
[0074] In the embodiment depicted in FIG. 5, the permeate outlet
114 is located at a second end 108 of the flow chamber 100. The
permeate outlet 114 is generally used to recover the first media
and residual cells from the flow chamber 110. In comparison, the
concentrate outlet 116 is located between the inlet 112 and the
permeate outlet 114, below the ultrasonic transducer 120 and the
reflector 130. The concentrate outlet 116 is generally configured
to recover the cells from the flow chamber 110. In certain
embodiments, however, it may be desired to recover other material
(e.g., microcarriers) from the device, in which case the
microcarriers can be recovered by the concentrate outlet and the
cells can be recovered via the permeate outlet along with the
media). As seen here, the permeate outlet 114 is generally located
above the ultrasonic transducer 120 and the reflector 130, while
and the concentrate outlet 116 is generally located below the
ultrasonic transducer 120 and the reflector 130.
[0075] In the embodiment depicted in FIG. 5 and FIG. 6, the device
100 is vertically oriented, such that the first end 106 of the
device is the bottom end thereof and the second end 108 of the
device is the top end thereof. In this way, the cells surrounded by
the first media is introduced at the bottom end of the device 100
and flows vertically upwards through the flow chamber from the
inlet 112 toward the permeate outlet 114.
[0076] As can be best seen in FIG. 6, the device 100 also includes
a collector 140. The collector 140 is located in the flow chamber
110 between the inlet 112 and the ultrasonic transducer 120 and the
reflector 130. The collector 140 is located above the concentrate
outlet 116 and, in particular, is defined by angled walls 142 that
lead to the concentrate outlet 116. Put another way, the collector
140 leads into a common well defined by angled walls 142 that taper
downwards in cross-sectional area (i.e. larger area to smaller
area) to a vertex at the bottom of the collector, which is
fluidically connected to and drains off one side into the
concentrate outlet 116 via flowpath 119. In this way, the
multi-dimensional acoustic standing wave can direct the
concentrated cells to the collector 140 for collection and removal
from the flow chamber 110 via the concentrate outlet 116. An
annular plenum 117 surrounds the collector 140, permitting the
mixture of host fluid/cells to flow from the inlet 112 around the
collector 140 into the flow chamber 110.
[0077] In some embodiments, the collector leads to a collection
container that is filled with the second media. In this way, the
second media is not flowed through the flow chamber of the device.
Instead, as the cells are trapped in the acoustic standing wave and
form clusters that grow to a critical size and subsequently fall
out of the multi-dimensional acoustic standing wave, the cell
clusters fall into the collector and are led to the collection
container. The collection container can be separable from the rest
of the device.
[0078] As seen here, preferably, fluid flows through the device
upwards. The cells surrounded by the first media enters the device
through inlet 112 at a bottom end of the device and then makes a
sharp turn to flow upwards. This change in direction desirably
reduces turbulence, producing near plug flow upwards through the
device. Flow continues upwards through the annular plenum 117 and
up into the flow chamber 110. There, the cells encounter the
multi-dimensional acoustic standing wave(s), which traps the cells,
as explained herein. Concentration of the cells occurs within the
acoustic standing wave(s), which can also cause coalescence,
clumping, aggregation, agglomeration and/or clustering of the
cells.
[0079] As the cells are concentrated, they eventually overcome the
combined effect of the fluid flow drag forces and acoustic
radiation force, and they fall downwards into collector 140. They
can then be flowed through flowpath 119 and collected at
concentrate outlet 116. A much higher number of cells is obtained
in a smaller volume (i.e., the target cells are concentrated).
[0080] FIG. 7 is a graph showing the performance of the
acoustophoretic device of FIG. 5. The device was operated at a
fixed frequency of 2.234 MHz for a mixture having a feed cell
density of about 1.5.times.10.sup.6 cells/mL. As can be seen, the
device achieved a permeate density reduction (PDR) of greater than
95% over about 35 minutes and a permeate cell density of less than
0.10.times.10.sup.6 cells/mL over the same time period.
[0081] The piezoelectric transducer(s) of the acoustophoretic
devices and systems of the present disclosure can be single
monolithic piezoelectric materials or can be made from an array of
piezoelectric materials. The piezoelectric material can be a
ceramic material, a crystal or a polycrystal, such as PZT-8 (lead
zirconate titanate). FIG. 8 shows a monolithic, one-piece, single
electrode piezoelectric crystal 200. The piezoelectric crystal has
a substantially square shape, with a length 203 and a width 205
that are substantially equal to each other (e.g. about one inch).
The crystal 200 has an inner surface 202, and the crystal also has
an outer surface 204 on an opposite side of the crystal which is
usually exposed to fluid flowing through the acoustophoretic
device. The outer surface and the inner surface are relatively
large in area, and the crystal is relatively thin (e.g. about 0.040
inches for a 2 MHz crystal).
[0082] FIG. 9 shows a piezoelectric crystal 200' made from an array
of piezoelectric materials. The inner surface 202 of this
piezoelectric crystal 200' is divided into a piezoelectric array
206 with a plurality of (i.e. at least two) piezoelectric elements
208. However, the array is still a single crystal. The
piezoelectric elements 208 are separated from each other by one or
more channels or kerfs 210 in the inner surface 202. The width of
the channel (i.e. between piezoelectric elements) may be on the
order of from about 0.001 inches to about 0.02 inches. The depth of
the channel can be from about 0.001 inches to about 0.02 inches. In
some instances, a potting material 212 (e.g., epoxy, Sil-Gel, and
the like) can be inserted into the channels 210 between the
piezoelectric elements. The potting material 212 is non-conducting,
acts as an insulator between adjacent piezoelectric elements 208,
and also acts to hold the separate piezoelectric elements 208
together. Here, the array 206 contains sixteen piezoelectric
elements 208 (although any number of piezoelectric elements is
possible), arranged in a rectangular 4.times.4 configuration
(square is a subset of rectangular). Each of the piezoelectric
elements 208 has substantially the same dimensions as each other.
The overall array 200' has the same length 203 and width 205 as the
single crystal illustrated in FIG. 8.
[0083] FIG. 10 shows another embodiment of a transducer 200''. The
transducer 200'' is substantially similar to the transducer 200' of
FIG. 9, except that the array 206 is formed from twenty-five
piezoelectric elements 208 in a 5.times.5 configuration. Again, the
overall array 200'' has the same length 203 and width 205 as the
single crystal illustrated in FIG. 8.
[0084] Each piezoelectric element in the piezoelectric array of the
present disclosure may have individual electrical attachments (e.g.
electrodes), so that each piezoelectric element can be individually
controlled for frequency and power. These elements can share a
common ground electrode. This configuration allows for not only the
generation of a multi-dimensional acoustic standing wave, but also
improved control of the acoustic standing wave. In this way, it is
possible to drive individual piezoelectric elements (or multiple,
separate ultrasonic transducers) with arbitrary phasing and/or
different or variable frequencies and/or in various out-of-phase
modes. For example, FIG. 11 illustrates an exemplary 0-180-0-180
mode, though additional modes can be employed as desired, such as a
0-180-180-0 mode. For example, for a 5.times.5 array, additional
modes can be employed as desired, such as a 0-180-0-180-0 mode, a
0-0-180-0-0 mode, a 0-180-180-180-0 mode, or a 0-90-180-90-0 mode.
The array could also be driven, for example, such that a
checkerboard pattern of phases is employed, such as is shown in
FIG. 11. In summary, a single ultrasonic transducer that has been
divided into an ordered array can be operated such that some
components of the array are out of phase with other components of
the array.
[0085] The piezoelectric array can be formed from a monolithic
piezoelectric crystal by making cuts across one surface so as to
divide the surface of the piezoelectric crystal into separate
elements. The cutting of the surface may be performed through the
use of a saw, an end mill, or other means to remove material from
the surface and leave discrete elements of the piezoelectric
crystal between the channels/grooves that are thus formed.
[0086] As explained above, a potting material may be incorporated
into the channels/grooves between the elements to form a composite
material. For example, the potting material can be a polymer, such
as epoxy. In particular embodiments, the piezoelectric elements 208
are individually physically isolated from each other. This
structure can be obtained by filling the channels 210 with the
potting material, then cutting, sanding or grinding the outer
surface 204 down to the channels. As a result, the piezoelectric
elements are joined to each other through the potting material, and
each element is an individual component of the array. Put another
way, each piezoelectric element is physically separated from
surrounding piezoelectric elements by the potting material. FIG. 12
is a cross-sectional view comparing these two embodiments. On top,
a crystal as illustrated in FIG. 9 is shown. The crystal is kerfed
into four separate piezoelectric elements 208 on the inner surface
202, but the four elements share a common outer surface 204. On the
bottom, the four piezoelectric elements 208 are physically isolated
from each other by potting material 212. No common surface is
shared between the four elements.
[0087] FIG. 13 is a graph showing the performance of an
acoustophoretic device according to the present disclosure having a
16-element piezoelectric array. The piezoelectric array was
operated at a fixed frequency of 2.244 MHz for a mixture having a
feed cell density of about 2.00.times.10.sup.6 cells/mL. As can be
seen, the device achieved a permeate density reduction (PDR) of
about 95% over about 60 minutes and a permeate cell density of
about 0.10.times.10.sup.6 cells/mL over the same time period.
[0088] The concentration efficiency of the acoustophoretic device
was tested. First, a T-cell suspension having a cell density of
1.times.10.sup.6 cells/mL was used. A feed volume of between about
500 and 1000 mL was used at a flow rate of 10-15 mL/minute. The
results are graphically depicted in FIG. 14. The device exhibited a
concentration factor of between 10.times. and 20.times., a 90% cell
recovery, and a 77% washout efficiency (i.e., the amount of the
first media that was displaced by the second media) over ten
minutes of testing. A 10.degree. C. temperature increase was
observed.
[0089] A yeast mixture was then used to test the dependency of the
percent density reduction (PDR) on concentration and flow rate. The
results are graphically depicted in FIG. 15. As seen here, the
higher initial cell concentrations generally resulted in a greater
PDR. Additionally, the varied flow rate (from 20 mL/min to 10
mL/min) did not have an observed effect on the PDR.
[0090] The concentration efficiency of the acoustophoretic device
was again tested with a higher cell density. A T-cell suspension
having a cell density of 5.times.106 cells/mL was used. A feed
volume of 1000 mL was used at a flow rate of 10-15 mL/minute. The
results are graphically depicted in FIG. 16. The device exhibited a
concentration factor of better than 10.times., a 90% cell recovery,
and a 77% washout efficiency over one hour of testing. A 10.degree.
C. temperature increase was again observed.
[0091] During testing, it was also discovered that active cooling
of the ultrasonic transducer led to greater throughput and
efficiency and more power. As such, a cooling unit was developed
for actively cooling the transducer. FIG. 17A illustrates an
acoustophoretic device 7000 containing a cooling unit, in a fully
assembled condition. FIG. 17B illustrates the device 7000, with the
various components in a partially exploded view. Referring now to
FIG. 17B, the device includes an ultrasonic transducer 7020 and a
reflector 7050 on opposite walls of a flow chamber 7010. It is
noted that the reflector 7050 may be made of a transparent
material, such that the interior of the flow chamber 7010 can be
seen. The ultrasonic transducer is proximate a first wall of the
flow chamber. The reflector is proximate a second wall of the flow
chamber or can make up the second wall of the flow chamber. A
cooling unit 7060 is located between the ultrasonic transducer 7020
and the flow chamber 7010. The cooling unit 7060 is thermally
coupled to the ultrasonic transducer 7020. In this figure, the
cooling unit is in the form of a thermoelectric generator, which
converts heat flux (i.e. temperature differences) into electrical
energy using the Seebeck effect, thus removing heat from the flow
chamber. Put another way, electricity can be generated from
undesired waste heat while operating the acoustophoretic
device.
[0092] It is noted that the various inlets and outlets (e.g. fluid
inlet, concentrate outlet, permeate outlet, recirculation outlet,
bleed/harvest outlet) of the flow chamber are not shown here. The
cooling unit can be used to cool the ultrasonic transducer, which
can be particularly advantageous when the device is to be run
continuously with repeated processing and recirculation for an
extended period of time (e.g., perfusion).
[0093] Alternatively, the cooling unit can also be used to cool the
fluid running through the flow chamber 7010. For desired
applications, the cell culture should be maintained around room
temperature (-20.degree. C.), and at most around 28.degree. C. This
is because when cells experience higher temperatures, their
metabolic rates increase. Without a cooling unit, however, the
temperature of the cell culture can rise as high as 34.degree.
C.
[0094] These components are modular and can be changed or switched
out separately from each other. Thus, when new revisions or
modifications are made to a given component, the component can be
replaced while the remainder of the system stays the same.
[0095] The goal is to begin with a culture bag having a volume of
about 1 liter (L) to about 2 L, with a density of about 1 million
cells/m L, and concentrate this bag to a volume of about 25 mL to
about 30 mL, and then to wash the growth media or exchange the
media within a time of about one hour (or less). Desirably, the
system can be made of materials that are stable when irradiated
with gamma radiation.
[0096] The advantages of providing a cooling unit for the
transducer can be seen in FIG. 18 and FIG. 19. FIG. 18 graphically
shows the temperature profile of the acoustophoretic device without
any active cooling (e.g., without a cooling unit for the
transducer). As seen in FIG. 18, the temperature difference between
the feed and the core (e.g., the transducer) was 8.6.degree. C.
FIG. 19 graphically shows the temperature profile of the
acoustophoretic device with active cooling (e.g., with a cooling
unit for the transducer). As seen in FIG. 19, through the use of
active cooling the temperature difference between the feed and the
core was reduced to 6.1.degree. C.
[0097] FIG. 20 illustrates a four-step process (with an optional
fifth step) for concentrating, washing, and separating
microcarriers from cells. The first step in the process involves
concentrating the microcarriers with attached cells in an
acoustophoretic device, such as those described herein. The
microcarriers and attached cells can be introduced to the
acoustophoretic device by receiving the microcarriers with attached
cells from a bioreactor. In the bioreactor, the microcarriers and
cells are suspended in a first media (e.g., growth serum or
preservative material used to keep the cells viable in the
bioreactor). The microcarriers with attached cells surrounded by
the first media are concentrated by the acoustic standing wave(s)
generated in the acoustophoretic device. In a second step, the
concentrated microcarriers with attached cells are then washed with
a second media to remove the first media (e.g., bioreactor growth
serum or preservative material). The third step is to then
introduce a third media containing an enzyme into the
acoustophoretic device to detach the cells from the microcarriers
through enzymatic action of the second media. In particular
embodiments, trypsin is the enzyme used to enzymatically detach the
cells from the microcarriers. The multi-dimensional acoustic
standing wave can then be used to separate the cells from the
microcarriers. Usually, this is done by trapping the microcarriers
in the multi-dimensional acoustic standing wave, while the detached
cells pass through with the third media. However, the cells can be
trapped instead, if desired. Finally, the separated cells may
optionally be concentrated and washed again, as desired.
[0098] After being concentrated and trapped/held in the
multi-dimensional acoustic standing wave, the microcarriers can
coalesce, clump, aggregate, agglomerate, and/or cluster to a
critical size at which point the microcarriers fall out of the
acoustic standing wave due to enhanced gravitational settling. The
microcarriers can fall into a collector of the acoustophoretic
device located below the acoustic standing wave, to be removed from
the flow chamber.
[0099] During testing, steps one and two (i.e., concentration and
washing) were performed using red and blue food dye to make colored
fluid. The concentration mixture included SoloHill microcarriers in
red fluid. The wash mixture included blue fluid and was passed
through the device three times. The concentrate was observed under
a microscope, as shown in the leftmost image of FIG. 21. The
concentration step was shown to have a 99% efficiency. The
remaining three images in FIG. 21 show microscopic images after the
first, second, and third wash passes, respectively. As seen from
left to right in FIG. 21, the first media (dyed red) is
progressively washed out by a second media (dyed blue) over a
series of wash passes. The light absorbance data is shown in the
table below.
TABLE-US-00001 Light Absorbance Sample Red (510 nm) Blue (630 nm)
Feed 0.138 0.041 Wash Pass 1 0.080 0.066 Wash Pass 2 0.063 0.080
Wash Pass 3 0.054 0.084
[0100] The decrease in red light absorbance and increase in blue
light absorbance evidences the feasibility of the washing
steps.
[0101] FIG. 22 shows microscopic images of the microcarriers and
attached cells during the concentration and washing steps. In
particular, the leftmost image in the top row shows the
microcarriers and attached cells in the feed, prior to introduction
into the acoustophoretic device. The rightmost image in the top row
shows the microcarriers and attached cells in the permeate, after
concentration in the acoustophoretic device. The bottom row of
images show the microcarriers and attached cells in the device
during the washing step, namely during the first, second, and third
wash passes, from left to right.
[0102] FIG. 23 shows the concentration of T-cells after being
separated in the acoustophoretic device. The top row of images show
the T-cells before acoustophoresis with a concentration of
1.14.+-.0.03.times.10.sup.6 cells/mL. The bottom row of images show
the T-cells after acoustophoresis with a concentration of
1.13.+-.0.02.times.10.sup.6 cells/mL. The comparable concentrations
evidence that substantially all of the cells pass through the
acoustophoretic device, as the concentration was substantially
unchanged by acoustophoresis.
[0103] FIG. 24 shows the presence of SoloHill microcarriers and
T-Cells in the acoustophoretic device under 4.times. magnification.
The top row of images show the microcarriers and cells in the feed
before acoustophoresis. The bottom row of images show the
microcarriers and cells in the permeate after the cells have been
separated out by acoustophoresis. The difference in the number of
microcarriers with the application of acoustophoresis evidences the
feasibility of using the device for trapping the microcarriers in
the device and separating the cells therefrom. The feasibility of
this technique and the results are further evidenced by the images
in FIG. 25, which show microscopic images of the microcarriers and
cells in the feed (top row of images) and permeate (bottom row of
images) after concentration and the first, second, and third
washes, from left to right.
[0104] The testing of the acoustophoretic concentrating, washing,
and separating process showed that the process is appropriate for
cell therapy and microcarrier applications. The concentrate and
wash steps were performed with a resulting efficiency of greater
than 99%, and the separating step e.g., separating the cells from
the microcarriers, was performed with greater than 98%
efficiency.
[0105] FIGS. 26-28 illustrate another example embodiment of an
acoustophoretic system/process 2800 including a disposable
acoustophoretic device 2810 with solenoid pinch valves that control
the flow of fluid therethrough. Starting from the left-hand side of
FIG. 26, the system includes a feed tank 2820, a wash tank 2830,
and an air intake 2805. The air intake 2805 runs through an air
intake valve 2804. Feed line 2821 runs from the feed tank 2820. The
air intake and the feed line 2821 are joined together by a
Y-connector into common feed line 2811, which runs into feed
selector valve 2801. A wash line 2831 runs from the wash tank 2830,
and also runs into feed selector valve 2801. Feed selector valve
2801 permits only one line to be open at a given time (valves 2802,
2803 also operate in this manner). Wash line 2831 and feed line
2811 are joined together by a Y-connector downstream of the feed
selector valve 2801 into input line 2812. Input line 2812 passes
through pump 2806 to inflow selector valve 2802, which is
downstream of the feed selector valve 2801 and upstream of the
acoustophoretic device 2810. The inflow selector valve 2802
selectively controls the inflow of feed or wash into the
acoustophoretic device 2810 through either feed port 2602 or
wash/drain port 2604. A feed line 2813 runs from the inflow
selector valve 2802 to feed port 2602. A wash line 2814 runs from
the inflow selector valve 2802 to common line 2815 and into
wash/drain port 2604.
[0106] On the right-hand side of FIG. 26, an outflow selector valve
2803 is located downstream of the acoustophoretic device 2810 and
controls the outflow of fluid therefrom. A waste line 2816 runs
from waste port 2608 through outflow selector valve 2803 and
subsequently to waste tank 2850. The common line 2815 runs into
drain line 2817, which then passes through outflow selector valve
2803 and subsequently to concentrate tank 2840. These tanks 2840,
2850 can be, for example, collection bags. The outflow selector
2803 thereby selectively controls the flow of fluid to the
concentrate tank and waste tank.
[0107] The use of collection bags at the ends of the concentrate
and waste lines advantageously creates an enclosed primary
environment within which concentration, washing, and/or separation
of cells and cellular materials can occur, which helps to prevent
the cells/cell culture/cellular material from being exposed to
possible intrusions, pathogens, or outside cellular influences that
would be harmful.
[0108] FIG. 26 also illustrates the flow path of the feed material
through the system. In this example embodiment, feed selector valve
2801 is operated with the bottom open (and top closed), so that the
feed from feed tank 2820 flows through. Inflow selector valve 2802
is operated with the top open (and bottom closed), so that the feed
material enters the acoustophoretic device 2810 via feed port 2602.
The outflow selector valve 2803 is also operated with the top open
(and bottom closed) so that the fluid/first media of the feed
material flows through to waste tank 2850. The targeted particles
in the feed material (e.g., microcarriers or cells) are trapped in
the acoustophoretic device 2810 by action of a multi-dimensional
acoustic standing wave(s), as explained in detail herein.
[0109] FIG. 27 illustrates the flow path of the wash material
through the system. Feed selector valve 2801 is operated with the
top open (and bottom closed), so that the wash material from wash
tank 2830 flows through. The inflow selector valve 2802 is operated
with the bottom open (and top closed) and the outflow selector
valve 2803 is operated with the top open (and bottom closed). As a
result, the wash material enters the acoustophoretic device 2810
via wash/drain port 2604, which operates as a wash inlet. Note that
the closed outflow selector valve 2803 prevents the wash material
from entering concentrate tank 2840. The wash material can then
pass through the acoustophoretic device 2810 and remove the first
media (e.g., bioreactor serum or preservative material). The wash
material then exits via waste port 2608 and flows to waste tank
2850. The target particles remain trapped in the acoustophoretic
device 2810.
[0110] FIG. 28 illustrates the draining of the system (e.g., the
collection of the target particles). Air intake valve 2804 is
opened. The feed selector valve 2801 is operated with the bottom
open (and top closed), and the inflow selector valve 2802 is
operated with the top open (and bottom closed), so that air enters
the acoustophoretic device 2810 via feed port 2602. The air
generally aids in dislodging the clusters of target particles from
the acoustophoretic device 2810. The outflow selector valve 2803 is
operated with the bottom open (and top closed). The target
particles flow out of wash/drain port 2604 through common line
2815, through drain line 2817 and subsequently to concentrate tank
2840.
[0111] Concentrating and washing cell culture is useful for
producing biological products for industrial use. The systems of
the present disclosure can be continuously improved and scaled up
for handling of larger volumes.
[0112] In some examples, the acoustophoretic devices of the present
disclosure may have a concentrated volume ranging from about 25 mL
to about 75 mL. The devices may have a total cell capacity of about
4 billion to about 40 billion cells, or from about 4 billion to
about 8 billion cells, or from about 20 billion to about 40 billion
cells, or from about 16 billion to about 35 billion cells. The
fluids entering and exiting the acoustophoretic devices may have
cell densities from about 160 million cells/mL to about 670 million
cells/mL, or from about 160 million cells/mL to about 320 million
cells/mL, or from about 260 million cells/mL to about 535 million
cells/mL, or from about 305 million cells/mL to about 670 million
cells/mL, or from about 0.5 million cells/mL to about 5 million
cells/m L.
[0113] The following examples are provided to illustrate the
devices and processes of the present disclosure. The examples are
merely illustrative and are not intended to limit the disclosure to
the materials, conditions, or process parameters set forth
therein.
EXAMPLES
[0114] The ability of an acoustophoretic system of the present
disclosure to concentrate Jurkat T-cells was tested. Jurkat T-cells
have a diameter of 11 micrometers (.mu.m) to 14 .mu.m. An
acoustophoretic device was used, and a Beckman Coulter Vi-CELL X
was used at various test conditions to measure the cell density
reduction.
[0115] In the first trial A, the T-cells were concentrated, and the
cell density of the permeate was measured. The dotted line
indicates the feed cell density. Desirably, the cell density in the
permeate is as low as possible, indicating that the cells are
retained in the concentrate. The graph in FIG. 29 shows the results
of trial A over time. The results show very low cell densities in
the permeate, between 0.0 and 0.2 million cells/mL, showing that
most of the cells are in the concentrate. The results also show a
high permeate reduction percentage, between 80% and 99%.
[0116] In the second trial B, the T-cells were concentrated, and
the cell density of the permeate was measured. The dotted line
indicates the feed cell density. FIG. 30 shows the results over
time. The results show good performance, with the permeate cell
density being below 0.1 million cells/mL after minute 1, and
greater than 95% permeate reduction after minute 2.
[0117] In the third trial C, the T-cells were concentrated and
washed. The concentrating occurred for the first 18 minutes, and
washing was subsequently performed. FIG. 31 shows the results over
time. The dotted line indicates the feed cell density. The solid
vertical lines indicate when concentrated system volumes were
processed (three total volumes were processed). Note that this
graph includes data on the concentrate and the permeate (not just
the permeate). All of the cells obtained from concentration were
maintained through washing, e.g., concentrated cells were not lost
due to the addition of the washing process. The table below
provides additional information on these three trials. Retention
and recovery rates of greater than 90% were obtainable for Jurkat
T-cells.
TABLE-US-00002 Feed Feed Concen- Process Volume Density Concentrate
Cell tration Time Trial (mL) (cells/mL) Volume Recovery Factor
(min) A 997 0.98 .times. 10.sup.6 21 mL 91% 47X 33 B 1004 0.85
.times. 10.sup.6 21 mL 95% 48X 33 C 555 4.08 .times. 10.sup.6 20 mL
92% 28X 31
[0118] The liquid volumes used to completely wash the concentrated
cells were tracked. Tracking the liquid volumes can be useful in
applications such as, for example, removing electroporation buffer
from a cell culture prior to transduction or transfection of the
cell culture.
[0119] A blue wash media and a red growth media were used. A
Molecular Devices SpectraMax spectrophotometer was used to measure
the two different wavelengths of these two media to identify a
complete flush/washing out of the old growth media from the system.
Three samples were measured: 100% wash media (100 W-0 G), 50% wash
media and 50% growth media (50 W-50 G), and 100% growth media (0
W-100 G). Three samples of the actual process were then tested (1
Volume, 2 Volume, 3 Volume). As seen in the spectrophotometer
results shown in FIG. 32, the second and third volumes fall on top
of the 100% wash media curve (100 W-0 G), indicating that all of
the growth media has been washed from the concentrated cells after
2 or 3 volumes have been used for washing.
[0120] The present disclosure has been described with reference to
exemplary embodiments. Modifications and alterations may occur to
others upon reading and understanding the preceding detailed
description. It is intended that the present disclosure be
construed as including all such modifications and alterations
insofar as they come within the scope of the appended claims or the
equivalents thereof.
* * * * *