Note: Descriptions are shown in the official language in which they were submitted.
1
HYPERBARIC INCUBATION SYSTEM AND METHOD
TECHNICAL FIELD
[0001] The technical field generally relates to an incubation system and
method for incubating cell cultures, and more particularly to a multizone
incubation
system operable at hyperbaric conditions.
BACKGROUND
[0002] Cell culture incubators are units configured to control a desired
temperature, humidity, or other parameters within the unit to allow cell
cultures
placed therein to grow. However, there are various challenges to providing
hyperbaric conditions for cell culture incubation, even though such hyperbaric
conditions could be of interest. There is a need for a technology that
facilitates cell
culture growth in hyperbaric conditions.
SUMMARY
[0003] Various aspects of the systems and methods for the incubation of
cell
cultures are described herein. Hyperbaric incubation can leverage certain
features
such as providing chambers as well as air displacement components such that
air
is fed through the chambers establishing hyperbaric pressure conditions while
removing CO2 generated by the cell culture.
[0004] In accordance with one aspect, there is provided a hyperbaric
incubation system comprising a compressor for providing pressurized air; a
hyperbaric chamber comprising a hyperbaric chamber wall defining a hyperbaric
chamber cavity; a hyperbaric chamber inlet defined in the wall for receiving
the
pressurized air from the compressor; a hyperbaric chamber pressure relief open
port defined in the wall, wherein closing the hyperbaric chamber pressure
relief
open port allows hyperbaric chamber pressurization and opening the hyperbaric
chamber pressure relief open port allows hyperbaric chamber depressurization;
and a hyperbaric chamber door provided in the hyperbaric chamber wall and
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having an open position for allowing access to the hyperbaric chamber cavity
and
a closed position for providing hyperbaric conditions; a reservoir located
within the
hyperbaric chamber cavity and comprising a reservoir wall defining a reservoir
cavity; a reservoir inlet defined in the reservoir wall and being in fluid
communication with the hyperbaric chamber cavity for receiving the pressurized
air therefrom; and a reservoir outlet defined in the reservoir wall; an
incubator
located within the hyperbaric chamber cavity and comprising an incubator wall
defining an incubator cavity, and configured to receive at least one cell
culture
container therein, wherein the at least one cell culture container is
configured to
contain a cell culture which produces CO2 that becomes part of the pressurized
air
to create CO2 enriched pressurized air; an incubator inlet defined in the
incubator
wall and being in fluid communication with the reservoir outlet for receiving
the
pressurized air therefrom; an incubator outlet defined in the incubator wall;
and an
incubator door provided in the incubator wall and having an open position for
allowing access to the incubator cavity and a closed position for providing
hyperbaric conditions; an exhaust container located within the hyperbaric
chamber
cavity and comprising an exhaust container wall defining an exhaust container
cavity; an exhaust container inlet defined in the exhaust container wall and
being
in fluid communication with the reservoir outlet for receiving the pressurized
air
therefrom; and an exhaust container outlet defined in the exhaust container
wall
and being in fluid communication with the hyperbaric chamber cavity; and an
exhaust pump in fluid communication with the exhaust container inlet for
drawing
the CO2 enriched pressurized air out of the incubator to maintain a stable pH
for
the cell culture.
[0005] In
accordance with another aspect, there is provided an incubation
system comprising a compressor for providing airflow; a reservoir comprising a
reservoir wall defining a reservoir cavity; a reservoir inlet defined in the
reservoir
wall and being in fluid communication with the entry flow displacement device
for
receiving the airflow therefrom; and a reservoir outlet defined in the
reservoir wall;
an incubator comprising an incubator wall defining an incubator cavity, and
configured to receive at least one cell culture container therein, wherein the
at least
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one cell culture container is configured to contain a cell culture which
produces
CO2 that becomes part of the airflow to create a CO2 enriched airflow; an
incubator
inlet defined in the incubator wall and being in fluid communication with the
reservoir outlet for receiving the airflow therefrom; an incubator outlet
defined in
the incubator wall; and an incubator door provided in the incubator wall and
having
an open position for allowing access to the incubator cavity and a closed
position
for providing hyperbaric conditions; an exhaust container comprising an
exhaust
container wall defining an exhaust container cavity; an exhaust container
inlet
defined in the exhaust container wall and being in fluid communication with
the
reservoir outlet for receiving the airflow therefrom; and an exhaust container
outlet
defined in the exhaust container wall; and an exhaust pump in fluid
communication
with the exhaust container inlet for drawing the CO2 enriched airflow out of
the
incubator to maintain a stable pH level for the cell culture.
[0006] In accordance with yet another aspect, there is provided a method
of
providing cell incubation comprising providing a reservoir, an incubator, and
an
exhaust container; supplying air at atmospheric pressure to the reservoir;
supplying the air from the reservoir to at least one cell culture container
located in
the incubator for incubation of a cell culture which produces CO2 that forms a
CO2
enriched air; supplying the CO2 enriched air from the incubator into the
exhaust
container to maintain a stable pH for the cell culture; and providing isobaric
pressure conditions in the reservoir, the incubator, and the exhaust
container.
[0007] In accordance with yet another aspect, there is provided a method
of
providing cell incubation in a hyperbaric environment comprising supplying
pressurized air to a hyperbaric chamber in which a reservoir, an incubator,
and an
exhaust container are located; supplying the pressurized air from the
hyperbaric
chamber into the reservoir; supplying the pressurized air from the reservoir
to at
least one cell culture container located in the incubator for incubation of a
cell
culture which produces CO2 that forms a CO2 enriched pressurized air;
supplying
the CO2 enriched pressurized air from the incubator into the exhaust container
to
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maintain a stable pH for the cell culture; and providing isobaric pressure
conditions
in the hyperbaric chamber, the reservoir, the incubator, and the exhaust
container.
[0008] In accordance with yet another aspect, there is provided a method
of
providing cell incubation in a hyperbaric environment comprising providing a
reservoir, an incubator, and an exhaust container; supplying pressurized air
into
the reservoir; supplying the pressurized air from the reservoir to at least
one cell
culture container located in the incubator for incubation of a cell culture
which
produces CO2 that forms a CO2 enriched pressurized air; supplying the CO2
enriched pressurized air from the incubator into the exhaust container to
maintain
a stable pH for the cell culture; and providing isobaric pressure conditions
in the
hyperbaric chamber, the reservoir, the incubator, the at least one cell
culture
container and the exhaust container.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is a diagram of an implementation of a hyperbaric
incubation
system, which could also be operated at atmospheric conditions.
[0010] FIG. 2 is a diagram of another implementation of a hyperbaric
incubation system, which could also be operated at atmospheric conditions.
DETAILED DESCRIPTION
[0011] Various aspects of the incubation system and associated methods
and
its use for incubating cell cultures will be described in further detail
below.
[0012] The present disclosure relates to systems and methods that
facilitate
incubation of cell cultures contained within an incubator, such as a
hyperbaric
incubator, while mitigating issues related to the release and accumulation of
CO2
in a cell culture medium that may increase acidity and kill a culture.
[0013] There are various contexts in which hyperbaric conditions may be
of
interest for incubating cell cultures. Hyperbaric conditions, such as those
provided
during oxygen therapy, have been found to be beneficial for improved recovery
in
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patients. Providing hyperbaric conditions in cell cultures may provide
benefits to
the cell cultures as well, such as improved growth rates, and/or experimental
conditions that mimic certain real-life conditions of interest. For example,
it is
envisaged that skin grafts for burn patients may be more efficiently grown
using
methods and systems of the present disclosure. It may also be advantageous to
incubate certain cell cultures in hyperbaric conditions that would mimic real-
life
conditions for certain organisms and organs such as the lungs.
[0014] It may additionally be advantageous to incubate cell cultures at
normal
atmospheric conditions according to the present disclosure, mimicking real-
life
conditions for certain organisms or organs such as the lungs, and facilitating
CO2
extraction as well as controlling the concentration of CO2 and the associated
pH
level in cell cultures.
[0015] Referring to the drawings, and more particularly to FIG. 1, an
incubation
system 100 is disclosed. In one implementation, the incubation system 100
comprises a compressor 102, particulate filters 104, 106 mounted upstream and
downstream of the compressor 102, a first heat exchanger 108 mounted
downstream of the compressor 102, an activated carbon filter 110, a second
heat
exchanger 114, and a High Efficiency Particulate Air (HEPA) filter 116, which
together make up an input assembly. The input assembly can include various
components for supplying a pressurized gas, such as air, to the rest of the
system.
The input assembly is connected to a hyperbaric chamber 120 having therein a
reservoir 140, an incubator 170 and an exhaust container 200. As shown by the
flow arrows in FIG. 1, air is supplied by the compressor 102 through the input
assembly and into the hyperbaric chamber 120. In some implementations, the
order of the components may be modified compared to what is shown in FIG. 1
and certain components, such as one or more of the filters and/or heat
exchangers,
can be absent from the input assembly.
[0016] The compressor 102 is operable to increase the pressure in the
hyperbaric chamber 120 above atmospheric pressure. In one implementation, the
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hyperbaric chamber 120 is pressurized to comprise pressurized air at a
pressure
between 1.2 and 1.6 atm, such as a pressure of about 1.4 atm. In one
implementation, the hyperbaric chamber 120 is pressurized to comprise
pressurized air at a pressure between 2 and 5 atm.
[0017] The compressor 102 is configured to receive air under ambient
conditions, for example ambient conditions in a laboratory, through a
compressor
inlet and to pressurize the air so that the air exits through a compressor
outlet
under hyperbaric conditions. If components presenting air resistance, such as
filters, are mounted downstream of the compressor 102, the compressor 102 may
be configured to provide air pressure to a level to compensate for these
pressure
losses. In one implementation, one or more filters, such as illustrated
filters 104,
106, carbon filter 110 and FIEPA filter 116, may be placed upstream and/or
downstream of the compressor 102. This may help, for example, to filter out
dust
or other particles that may reduce the performance of the incubation
environment
or impair or influence cell cultures. In one implementation, the one or more
filters
are configured to additionally filter out microorganisms such as bacteria,
mold, and
viruses that may be present in the air. In one implementation, one or more of
the
filters are FIEPA rated filters. It is noted that other units could be
provided for
removing and/or deactivating microorganisms via mechanisms other than
filtering.
The input assembly is thus configured such that the air supplied to the
incubation
environment carries no bacteria or molds and is appropriate for the cell
culture of
interest.
[0018] Still referring to FIG. 1, the first heat exchanger 108 is
positioned
downstream of the compressor 102 and is configured to receive pressurized air
from the compressor 102. Air temperature increases as its pressure increases
in
the compressor 102, and the first heat exchanger 108 is configured to cool
this
pressurized air. In one implementation, the first heat exchanger 108 may be
configured to cool the pressurized air to ambient conditions. In some
implementations, the first heat exchanger 108 may be configured to cool or
heat
Date Recue/Date Received 2022-06-01
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the pressurized air to a given target temperature, which can depend on the
conditions desired for a particular incubation run.
[0019] In some implementations, filters 104, 106 may be positioned both
upstream and downstream of the compressor 102. Although the downstream filter
106 has been illustrated as being directly upstream of the first heat
exchanger 108,
the filter may instead be positioned differently, for example downstream of
the first
heat exchanger 108. In addition to the filters 104, 106 positioned upstream
and
downstream of the compressor 102, additional filters may be provided as part
of
the input assembly. For example, as illustrated in FIG. 1, the carbon filter
110 may
be added and configured to filter out volatile organic compounds (VOC's). As
illustrated in FIG. 1, FIEPA filter 116 may also be positioned in the input
assembly.
In some implementations, the positioning and type of filters may be changed.
[0020] In some implementations, the second heat exchanger 114 may be
positioned downstream of the first heat exchanger 108 for further adjusting or
regulating the temperature of the pressurized air. In one implementation, the
second heat exchanger 114 is a Peltier heat exchanger. A Peltier heat
exchanger
may be configured to either cool or heat the fluid passing therethrough.
Accordingly, a Peltier heat exchanger may allow the temperature of the
pressurized air to be increased or decreased, providing greater control and
stability
over the incubation process. In one implementation, the first heat exchanger
108
may be removed entirely, leaving only the second heat exchanger 114 to
regulate
the temperature of the pressurized air. In one implementation, the second heat
exchanger 114 may be removed entirely, leaving only the first heat exchanger
108.
In one implementation, there are no heat exchangers or more than two heat
exchangers. In one implementation, as shown in FIG. 1, an additional filter
may be
positioned downstream of the second heat exchanger 114, further removing
contaminants from the pressurized air. In the illustrated implementation, the
additional filter is FIEPA filter 116.
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[0021] Temperature regulated pressurized air exits the second heat
exchanger
114 and the NEPA filter 116, where applicable, and is supplied to the
hyperbaric
chamber 120. The hyperbaric chamber 120 comprises a wall 122 defining a cavity
124. The hyperbaric chamber wall 122 has an inlet 126 for receiving the
filtered
and temperature regulated pressurized air. In one implementation, the
hyperbaric
chamber wall 122 may be a flexible wall. The hyperbaric chamber 120 further
comprises an access opening (not shown in FIG. 1) in the wall 122, which can
take
the form of a door. The door has an open position to allow a user access
inside
the hyperbaric chamber 120, and a closed airtight position to allow
pressurizing of
the hyperbaric chamber 120 to hyperbaric conditions. The door can be
configured
in various ways. In one implementation, the hyperbaric chamber wall 122 is a
flexible wall and the chamber door is a slit with an airtight zipper which
extends
across part of the wall 122 of the chamber 120. In one implementation, the
hyperbaric chamber 120 is a flexible wall hyperbaric chamber such as those
used
in oxygen therapy, e.g., a Yada TM hyperbaric chamber.
[0022] In one implementation, the hyperbaric chamber 120 may comprise an
open port 128 which allows for setting the working the pressure of the
hyperbaric
chamber 120. In one implementation, the open port 128 is a valve which may be
closed after the door is closed to allow the hyperbaric chamber pressure to
increase to the desired level. In some implementations, and as illustrated in
FIG.
1, the open port 128 may be a pressure relief valve configured to open and
release
pressurized air from inside the hyperbaric chamber 120 to ambient conditions
once
a certain pressure is reached in order to release the pressurized air from
inside the
hyperbaric chamber 120 back to ambient conditions. A user may then be able to
open the access door to access the inside of the hyperbaric chamber 120.
Alternatively, the open port 128 may be an electrically actuated port,
configured to
open or close in response to a pressure configured by a user and detected by a
pressure sensor coupled to the open port 128.
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[0023] In one implementation, this open port 128 is kept open during
incubation, preventing pressurization to hyperbaric conditions, but permitting
CO2
extraction under atmospheric conditions.
[0024] The hyperbaric chamber 120 may comprise an additional open port
129,
the open port 129 being coupled to a CO2 sensor. The additional open port 129
may be configured to open when the CO2 sensor detects an excess amount of CO2
in the hyperbaric chamber 120. In this configuration, the hyperbaric chamber
may
be configured to be open or closed to the ambient environment in response to
one
or both of air pressure and CO2 concentration.
[0025] In one implementation, the hyperbaric chamber 120 may comprise an
ultraviolet (UV) light 130 which can be operable, for example, by an external
controller, to disinfect the interior of the hyperbaric chamber 120 when a
culture is
removed and before a new culture is placed therein. The hyperbaric chamber 120
is sized and dimensioned to accommodate the reservoir 140, the incubator 170,
and the exhaust container 200 within the cavity. In one implementation, the UV
light emits light at a wavelength of 250-280nm, such as 253nm.
[0026] The reservoir 140 can have a form of a container that receives the
pressurized air from the hyperbaric chamber 120. The reservoir 140 comprises a
reservoir wall 142 defining a cavity 144 for receiving the pressurized air.
The
reservoir wall 142 comprises an inlet 146 defined therein and in fluid
communication with the hyperbaric chamber 120 for receiving the pressurized
air
therefrom. The inlet 146 may have a filter 148 mounted thereon. In one
implementation, the filter 148 is a NEPA filter. The reservoir 140 also
comprises
an outlet 150 defined in the wall 142, shown mounted opposite to the inlet 146
in
FIG. 1, and in fluid communication with cell culture containers 180 for
supplying
the pressurized air to the cell culture containers 180. In one implementation,
the
reservoir 140 additionally comprises a UV light 154 which can be operable, for
example, by an external controller, to disinfect the interior of the reservoir
140. The
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reservoir 140 may additionally have an access opening having a door to allow,
for
example, to change the UV light 154, where applicable.
[0027] In one implementation, an oxygen concentrator 158 may additionally
be
coupled to be in fluid communication with the reservoir cavity 144. An oxygen
concentrator is a device that removes nitrogen from the air it receives. Given
that
ambient air is mostly composed of nitrogen and oxygen, an oxygen concentrator
removes the nitrogen to provide an oxygen rich output airflow. In one
implementation, the oxygen concentrator 158 is mounted outside the hyperbaric
chamber 120 with air entering through inlet 160 from outside the hyperbaric
chamber 120 and entering the reservoir 140 via outlet tubes 162 connected to
the
reservoir 140. In one implementation, the outlet tubes 162 are tygon tubes.
The
tubes 162 may have a diameter between 7-10 mm. Alternatively, they may have
any other desired diameter. The oxygen concentrator draws air from the outside
and discharges oxygen-enriched air to the reservoir 140. In one
implementation,
filters 164 are positioned on an outlet and an inlet of the oxygen
concentrator. In
one implementation, filters 164 are NEPA filters. Alternatively, if oxygen
rich air is
not required, the oxygen concentrator may be removed entirely.
[0028] The reservoir 140 further comprises an open port 152 provided on
the
reservoir wall. Open ports are mounted on the reservoir 140, incubator 170 and
exhaust compartment 200 and configured to maintain the reservoir 140,
incubator
170 and exhaust compartment 200 in fluid communication with one another. Open
ports allow pressure equalization between said compartments, so that isobaric
conditions may be maintained between the reservoir 140, incubator 170 and
exhaust compartment 200. In one implementation, the calibrated port is an
electrically actuated port. The open ports are calibrated to open or close
depending
on the desired pressure in the reservoir 140, incubator 170 and exhaust
compartment 200. For example, the open ports may be configured to receive
pressure readings from pressure gauges and to open or close in response. The
open ports thus permit pressure regulation across the reservoir 140, incubator
170
and exhaust compartment 200 by selectively opening or closing. The open port
Date Recue/Date Received 2022-06-01
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152 may have a filter 166 mounted thereon. In one implementation, the filter
166
is a NEPA filter.
[0029] The incubator 170 can have a form of a cabinet that receives the
pressurized air from the reservoir 140. The incubator 170 comprises an
incubator
wall 172 defining an incubator cavity 174. Cell culture containers 180 may be
received in the incubator cavity 174 for allowing cell cultures to metabolize
and
reproduce in the cell culture containers 180. The incubator 170 is a container
configured to maintain temperature and humidity to encourage growth of the
cell
cultures. In one implementation, the incubator 170 is a so-called CO2
incubator,
such as those sold by Thermo Fisher ScientificTM. In one implementation, the
CO2
incubator resembles a small refrigerator with a metal housing having heating
elements, at least one shelf for receiving cell culture containers 180
thereon, and
an access opening comprising a door for allowing a user access to the cell
culture
containers 180 (for example, for removing and replacing the cell culture
containers
180). A water container configured to receive water may additionally be
included
or added to the incubator 170 to ensure that a desired level of humidity is
maintained in the incubator 170. In one implementation, the incubator 170 may
be
a water-jacketed incubator. A water-jacketed incubator may comprise a double
wall housing with water flowing between the walls. A water-jacketed incubator
may
be more temperature stable than a CO2 incubator without a water jacket due to
the
higher specific heat capacity of water.
[0030] The incubator 170 comprises an incubator inlet 176 defined in the
incubator wall 172 and in fluid communication with the reservoir outlet 150.
Accordingly, pressurized air from the reservoir 140 is supplied to the
incubator 170
through the incubator inlet 176. The incubator 170 additionally comprises an
incubator outlet 178 defined in the incubator wall 172 opposite to the
incubator inlet
176 and in fluid communication with the exhaust container 200. The incubator
170
additionally comprises an access opening in the incubator wall 172 having a
door.
The door has an open position to allow access to the incubator cavity 174 and
a
closed position for sealing the incubator cavity 174. Typically, a user may
open the
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door to monitor, place, replace or remove cell culture containers 180 from
within
the incubator 170. Once the user has manipulated the cell culture containers
180,
the door is closed so that the conditions in the incubator 170 may be
regulated as
desired, for example to a specific pressure, temperature, or humidity. As the
user
opens and closes the incubator 170, the incubator 170 may be prone to
contaminants entering. In one implementation, the reservoir 140 additionally
comprises a UV light 190 which can be operable, for example by an external
controller, to disinfect the interior of the incubator 170 between cell
cultures.
[0031] The cell culture containers 180 received in the incubator 170 can
each
be configured to comprise an inlet 182 and an outlet 184. In one
implementation,
the cell culture container inlet 182 may be connected to the incubator inlet
176
through an inlet tube 186 and the cell culture container outlet 184 may be
connected to the incubator outlet 178 through an outlet tube 188. In one
implementation, the tubes are Tygon tubes having a diameter between 7-10 mm.
The tubes 186, 188 can provide the benefit of providing fresh and sterilized
air from
the reservoir 140, preventing potentially contaminated air from inside of the
incubator 170 as a result of opening the incubator door to manipulate the cell
culture containers 180. In the implementation illustrated in FIG. 1, the tubes
186,
188 may have a main artery which branches out to inlets 182 and outlets 184 of
the cell culture containers 180, where there is a plurality of cell culture
containers.
[0032] In one implementation, the inlet 182 and outlet 184 of each cell
culture
container 180 may comprise a disposable syringe-type FIEPA filter sized and
dimensioned to be mounted onto the inlet 182 and outlet 184 of the cell
culture
containers 180.
[0033] In one implementation, the cell culture container 180 is composed
of a
light polymer material. In one implementation, the cell culture container 180
has a
filter at the cell culture container inlet 182 and another filter at the cell
culture
container outlet 184. In one implementation, the air/cell culture medium ratio
is 1:1
or higher, that is to say, the volume of air is the same or greater than the
volume
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of the cell culture medium, such as a liquid. A 1:1 ratio or greater ratio
between air
and the cell culture medium may improve passive diffusion by providing a
greater
amount of air.
[0034] The cell cultures in the cell culture containers 180 are
configured to
receive the pressurized (and filtered) air from the reservoir 140 through the
incubator inlet 176, to metabolize and reproduce, and in so doing can produce
CO2. The cell culture containers 180 are also configured to be in isobar with
the air
inside the incubator 170. The produced CO2 becomes part of the pressurized air
to create CO2 enriched pressurized air, or polluted air. The CO2 enriched
pressurized air is subsequently removed through the incubator outlet 178.
[0035] In one implementation, the incubator 170 is an insulated incubator
(including a water-jacketed incubator) to reduce temperature fluctuations in
the
incubator cavity. The incubator 170 further comprises a first open port 192
and a
second open port 194 provided on the incubator wall 172.
[0036] The exhaust container 200 is configured to be in isobar with the
incubator 170, and to receive the CO2 enriched pressurized air from the cell
culture
containers 180 in the incubator 170. The exhaust container 200 comprises an
exhaust container wall 202 defining an exhaust container cavity 204. The
exhaust
container wall 202 comprises an inlet 206 defined thereon and in fluid
communication with the cell culture containers 180 in the incubator 170 for
receiving the CO2 enriched pressurized air therefrom.
[0037] The exhaust container 200 also comprises an outlet 208 defined in
the
wall 202 and in fluid communication with the hyperbaric chamber 120 for
supplying
the CO2 enriched pressurized air thereto. The outlet 208 may be coupled to a
pressure sensor to open or close in response to pressure inside the exhaust
container 200. The exhaust container 200 may also comprise an open port 209
coupled to a CO2 sensor. The open port 209 may be configured to open when the
CO2 sensor detects an excess amount of CO2 in the exhaust container 200. In
this
configuration, the exhaust container 200 may be configured to be open or
closed
Date Recue/Date Received 2022-06-01
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to the ambient environment in response to one or both of air pressure and CO2
concentration.
[0038] In one implementation, the exhaust container 200 additionally
comprises a UV light 210 which can be operable, for example, by an external
controller, to disinfect the interior of the exhaust container 200. In one
implementation, the UV lights 130, 154, 190, 210 of the hyperbaric chamber
120,
the reservoir 140, the incubator 170, and the exhaust container 200 can be run
for
a total of five minutes to disinfect their respective interior surfaces prior
to starting
a new culture. They may additionally be run throughout the incubation. The
exhaust container 200 may additionally comprise an access opening having a
door
thereon for providing access to the interior components.
[0039] The exhaust container 200 further comprises an open port 212
provided
on the exhaust container wall 202. The open port 212 is in fluid communication
with the second open port 194 on the incubator wall 172, allowing fluid
communication between the exhaust container 200 and the incubator 170. The
open ports 152, 192, 194, 212 on the reservoir 140, the incubator 170 and the
exhaust container 200 may be operable to open and close in response to
pressure
fluctuation across the reservoir 140, incubator 170, and exhaust container
200.
That is to say, the open ports 152, 192, 194, 212 may be configured to ensure
isobaric conditions across the reservoir 140, the incubator 170, and the
exhaust
container 200 and to mitigate pressure fluctuations across said compartments.
[0040] The hyperbaric chamber 120 is also isobaric with the reservoir
140, the
incubator 170, and the exhaust container 200, such that the pressure Ric of
the
hyperbaric chamber 120 is equal to pressure Pr of the reservoir 140, pressure
Pi
of the incubator 170, and pressure Pe of the exhaust container 200. At least
one of
the hyperbaric chamber 120, reservoir 140, incubator 170, and exhaust
container
200 may have a pressure gauge to allow a user to determine the pressure in the
respective compartment. In one implementation, the compressor 102 may be
computer controlled and configured to increase or reduce flow to regulate the
Date Recue/Date Received 2022-06-01
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pressure in response to pressure readings from the hyperbaric chamber 120,
reservoir 140, incubator 170 and/or exhaust container 200.
[0041] In some implementations, an exhaust pump 220 is positioned in the
exhaust container 200. The exhaust pump 220 is in fluid communication with the
inlet 206 of the exhaust container 200 for drawing air from the cell culture
containers 180 in the incubator 170. The exhaust pump 220 is configured to
draw
air from the cell culture containers 180, in effect mimicking exhalation, to
regulate
the CO2 present in the cell culture containers 180. In one implementation, the
exhaust pump 220 is a centrifugal pump. The exhaust pump 220 is configured to
exhaust out the CO2 enriched pressurized air into the exhaust container 200.
The
exhausted CO2 enriched pressurized air is supplied into the hyperbaric chamber
120 through the exhaust container outlet 208, which may additionally have a
filter
mounted thereon. The exhausted CO2 enriched pressurized air may then circulate
in the hyperbaric chamber 120 or exit the hyperbaric chamber 120 through the
hyperbaric chamber valve 128. The hyperbaric chamber valve 128 may therefore
release pressure prior to opening the door to the hyperbaric chamber 120.
[0042] The combined effect of the open ports 152, 192, 194, 212 across
the
reservoir 140, incubator 170 and exhaust container 200 is to maintain an
isobaric
condition between the said containers and with the cell culture containers
180, thus
preventing any pressure differential that could cause violent rupture of the
cell
culture containers 180. It is accordingly possible to maintain a sterile and
constant
flow of air through the hyperbaric incubation system 100 as described.
Although
three separate compartments (reservoir 140, incubator 170, exhaust container
200) have been illustrated, it is envisaged that in some implementations the
user
may instead use two compartments. For example, the exhaust container 200 may
be removed, and the exhaust pump may instead be positioned in the hyperbaric
chamber 120. Alternatively, both the exhaust container 200 and the reservoir
200
may be removed, so that only the incubator 170 is positioned inside the
hyperbaric
chamber 120. Additionally, although the hyperbaric chamber 120 is configured
to
receive the reservoir 140, incubator 170 and the exhaust container 200 therein
to
Date Recue/Date Received 2022-06-01
16
effectively maintain a null pressure differential between the interior and
exterior of
each of these compartments, it will also be possible to run the incubation
system
100 under normal atmospheric conditions. The closed-circuit fresh air
circulation
would then make it possible to control cell culture pH with less pH balancing
compounds, such as buffer salts.
[0043] In
accordance with the above, a method of starting up the hyperbaric
incubation will now be described. Once the hyperbaric incubation system 100 is
prepared, and prior to placing the cell cultures in the incubator 170 as
illustrated
for example in FIG. 1, the user may operate the UV lights 130, 154, 190, 210
of
each of the compartments 120, 140, 170, 200 to sterilize the interior of each
compartment. In one implementation, the UV lights 130, 154, 190, 210 are
operated for five minutes each to provide adequate sterilization. Cell culture
containers 180 are then placed within the incubator 170. Tubes such as tubes
186,
188 may be used to connect the cell culture container inlets 182 and outlets
184
to the incubator inlet 176 (connected to the cavity 144 of the reservoir 140)
and
outlet 178 (connected to the cavity 204 of the exhaust container 200). The
incubator door is then closed and the incubator 170 allowed to regulate the
temperature and humidity therein. The valve 128 of the hyperbaric chamber 120
and the door are closed to seal the hyperbaric chamber 120. The compressor 102
is then started to pressurize the hyperbaric chamber 120 (and the compartments
140, 170, 200 therein). The pressurized air passes by any filters and/or heat
exchangers to provide temperature controlled filtered air to the hyperbaric
chamber
120. Once the interior pressure of the hyperbaric chamber 120 reaches the
desired
pressure, the flow of the compressor 102 may be reduced to a level needed to
maintain the pressure while reducing air movement and potential associated
disturbances. In one implementation, the pressure in the hyperbaric chamber
120
may be reached in about ten minutes. In one implementation, the compressor 102
may begin pressurizing at hundreds of liters per minute, and ramp down to tens
of
liters per minute as the pressure increases. The valve 128 on the hyperbaric
chamber 120 may additionally allow any excess air to escape into the ambient.
Once the isobaric condition has been satisfied between the compartments, the
Date Recue/Date Received 2022-06-01
17
exhaust pump 220 may be activated to start drawing CO2 enriched pressurized
air
from the cell culture containers 180. The flowrate of the exhaust pump 220 may
be
adjusted as a function of the amount of CO2 enriched pressurized air that
needs to
be removed to maintain the pH level of the cell culture containers 180 within
the
desired range.
[0044] If access is required to the cell culture containers 180 or the
interior of
the hyperbaric chamber 120, the compressor 102 may be stopped. The valve 128
on the hyperbaric chamber 120 may be opened to allow the hyperbaric chamber
120 to depressurize to atmospheric condition. In one implementation, the
depressurization process takes approximately ten minutes. Once the incubation
system 100 is depressurized, the user may access the interior of the
hyperbaric
chamber 120 or the cell culture containers within the incubator 170.
[0045] In accordance with another aspect and as illustrated in FIG. 2,
there is
provided an incubation system 300 operating at hyperbaric conditions while
placed
in normal atmospheric conditions. The atmospheric incubation system 300 of
FIG.
2 is similar to the hyperbaric incubation system 100 of FIG. 1, with the
exception
that there is no hyperbaric chamber 120 since the cabinets are strong enough
to
maintain the air pressure inside. For example, the body of each of the
reservoir
140, incubator 170 and exhaust compartment 200 may be reinforced. In this
configuration, the hyperbaric chamber may be omitted as each of the reservoir
140, incubator 170 and exhaust compartment 200 is a standalone hyperbaric
component. In the illustrated implementation of FIG. 2, there is a compressor
302
which is configured to supply air from the ambient to the reservoir 140.
Similarly to
previously described incubation system 100, airflow emanating from the
compressor 302 is supplied to an input assembly comprising at least one
filter,
such as particulate, charcoal and/or HEPA filters. The input assembly may
further
comprise a heat exchanger 314 configured to either cool or heat the fluid
passing
therethrough. The heat exchanger 314 may, in one implementation, be a Peltier
heat exchanger.
Date Recue/Date Received 2022-06-01
18
[0046] The incubation system 300 further comprises an exhaust pump 320
positioned in the exhaust container 200 and configured to draw CO2 enriched
airflow out of the cell culture containers 180 in the incubator 170 to
maintain a
stable pH level for the cell culture. Similar to the exhaust pump 220, the
exhaust
pump 320 helps to mimic exhalation to regulate the CO2 present in (and
therefore
the acidity of) the cell culture containers 180. The CO2 enriched airflow is
then
exhausted to the exhaust container 200, which allows the CO2 enriched airflow
to
exit the incubation system 300 via the outlet 208 into the ambient. In one
implementation the outlet 208 may be an electrically actuated port, configured
to
open or close in response to a pressure configured by a user and detected by a
pressure sensor coupled to the open port 128.
[0047] In one implementation, a valve 211 may be mounted to the outlet
208
to allow a user to manually release pressure when the exhaust container 200
has
a pressure different to the exterior of the exhaust container 200, for example
when
the exhaust container 200 is hyperbaric while the surrounding environment is
under atmospheric pressure. The valve 211 therefore allows the user to release
pressure, allowing the user to open the access door of the exhaust container
200.
[0048] The incubation system 300 provides stronger cabinets for the
reservoir
140, incubator 170, and exhaust container 200 without the need for a
hyperbaric
chamber, allowing each of the reservoir 140, incubator 170, and exhaust
container
200 to maintain the hyperbaric inside without rupture of their structures. The
reservoir 140, incubator 170 and exhaust container 200 are configured to
withstand a pressure differential between their respective interior and
exterior.
They may additionally resist fluid leakage at a given pressure differential.
There is
accordingly a need fora pressure release valve on each of the containers to
permit
safely opening the door to the respective container, as well as the open ports
152,
192, 194, 212 to maintain an isobar between the reservoir 140, incubator 170
and
exhaust container 200. It is envisaged that the incubation system 300 may also
be
run at atmospheric conditions.
Date Recue/Date Received 2022-06-01
19
[0049] The embodiments described in the present disclosure provide
multiple
benefits. In a gas mixture, the total pressure of the mixture is equal to the
sum of
the partial pressures of its constituent components. Accordingly, an increase
in
pressure from atmospheric to hyperbaric conditions will lead to an increase in
the
partial pressure of CO2 in the gas mixture. The pH level of a cell culture is
related
to the partial pressure of CO2 in the cell culture environment. Accordingly,
providing
hyperbaric conditions to a cell culture without regulating the amount of CO2
in the
gas mixture will lead to a reduction in pH level, creating greater acidity in
the cell
culture environment. If the pH level of the cell culture is not precisely
controlled,
the cell culture may die. For example, the pH level in a cell culture may need
to be
controlled within a range of 7.3-7.6 to maintain the cell culture in ideal
conditions.
[0050] The human body regulates the pH level of its blood by regular
breathing.
It is, possible, however, to vary the pH of blood by changing our breathing
pattern.
As an example, hyperventilating wherein a person rapidly inhales air,
increases
the pH level of blood making it more alkaline. By contrast, inhaling air from
a small,
closed container, such as a paper bag, will result in the person inhaling CO2
that
had previously been exhaled. This increases the amount of CO2 in the blood,
reducing its pH level making it more acidic. It has been noted that this
additional
acidity may ultimately to death of the cell culture.
[0051] It is therefore noted that a person changing their rate of
respiration may
change their blood pH level by changing the concentration of CO2 in their
blood. It
is an object of the present disclosure as set out above to artificially
replicate this
process in an incubation system to regulate the level of CO2 in cell cultures
within
the incubation system by use of a CO2 extraction and incubation method or an
incubator as described herein.
[0052] While the above description provides examples of the embodiments,
it
will be appreciated that some features and/or functions of the described
embodiments are susceptible to modification without departing from the spirit
and
principles of operation of the described embodiments. Accordingly, what has
been
Date Recue/Date Received 2022-06-01
20
described above has been intended to be illustrative and non-limiting and it
will be
understood by persons skilled in the art that other variants and modifications
may
be made without departing from the scope of the invention as defined in the
claims
appended hereto.
Date Recue/Date Received 2022-06-01
