U.S. patent application number 11/508286 was filed with the patent office on 2007-02-15 for continuous culture apparatus with mobile vessel, allowing selection of fitter cell variants and producing a culture in a continuous manner.
Invention is credited to Eudes Francois Marie De Crecy.
Application Number | 20070037276 11/508286 |
Document ID | / |
Family ID | 38738923 |
Filed Date | 2007-02-15 |
United States Patent
Application |
20070037276 |
Kind Code |
A1 |
De Crecy; Eudes Francois
Marie |
February 15, 2007 |
Continuous culture apparatus with mobile vessel, allowing selection
of fitter cell variants and producing a culture in a continuous
manner
Abstract
A method and device for growing plant, animal or stem cells in a
continuous manner.
Inventors: |
De Crecy; Eudes Francois Marie;
(Gainesville, FL) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET
2ND FLOOR
ARLINGTON
VA
22202
US
|
Family ID: |
38738923 |
Appl. No.: |
11/508286 |
Filed: |
August 23, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US05/05616 |
Feb 23, 2005 |
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11508286 |
Aug 23, 2006 |
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60833821 |
Jul 28, 2006 |
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60547379 |
Feb 23, 2004 |
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Current U.S.
Class: |
435/293.1 ;
435/304.1 |
Current CPC
Class: |
C12N 5/04 20130101; C12N
5/0606 20130101; C12N 5/0622 20130101; C12N 7/00 20130101; C12M
23/34 20130101; C12N 5/0605 20130101; C12M 23/26 20130101; C12N
5/0669 20130101; C12M 41/26 20130101; C12N 5/0647 20130101; C12M
23/06 20130101; C12M 41/36 20130101; C12M 41/40 20130101; C12N 1/16
20130101; C12N 1/20 20130101 |
Class at
Publication: |
435/293.1 ;
435/304.1 |
International
Class: |
C12M 1/00 20060101
C12M001/00 |
Claims
1. A device for growing living cells in a continuous manner,
comprising: a flexible tubing containing culture medium and a
surface in which the living cells can grow on; and a system of
clamps, each capable of open and closed positions, the clamps being
positioned so as to be able to divide the tubing into: i) an
upstream region containing unused culture medium; ii) a downstream
region containing spent culture medium; and iii) a growth chamber
region for growing said cells disposed between the upstream and
downstream regions; wherein the system of clamps is constructed and
arranged to open and close so as to clamp off and define the growth
chamber region of the tubing between the upstream and downstream
regions of the tubing, and to cyclically redefine the growth
chamber region of the tubing so that a first portion of the
previously defined growth chamber region becomes a portion of the
downstream region of the tubing, and a portion of the previously
defined upstream region of the tubing becomes a portion of the
growth chamber region of the tubing.
2. The device according to claim 1, wherein the system of clamps is
structured and arranged so that each of the clamps does not move
with respect to the tubing when said clamp is in the closed
position.
3. The device according to claim 1, wherein the surface is the
interior surface of the tubing.
4. The device according to claim 3, wherein the surface is a
continuous support inserted inside the tubing.
5. The device according to claim 4, wherein the means for providing
a surface is a continuous fiber.
6. The device according to claim 1, wherein the tubing is gas
permeable
7. The device according to claim 1, wherein the tubing is gas
impermeable.
8. The device according to claim 1, wherein the tubing is one of
transparent and translucent to permit a turbidimeter to determine
the density of the culture.
9. The device according to claim 1, wherein the device further
comprises a pressure regulator constructed to change a pressure of
the growth chamber portion of the tubing relative to ambient
pressure.
10. The device according to claim 1, wherein the tubing comprises a
pH indicator.
11. The device according to claim 1, further comprising a
temperature regulator constructed to allow control of a temperature
of the growth chamber region of the tubing.
12. The device according to claim 1, wherein the device further
comprises an agitator constructed to allow agitation of the growth
chamber portion of the tubing.
13. The device according to claim 12, wherein the agitator
comprises at least one stirring bar.
14. The device according to claim 1, further comprising an emitter
constructed to subject the growth culture chamber region to at
least one of radio waves, light waves, x-rays, sound waves, an
electro magnetic field, and a radioactive field.
15. The device according to claim 1, further comprising a means for
subjecting the growth chamber region to a different gravitational
force.
16. The device according to claim 1, wherein said growth chamber
region comprises one or more growth chambers containing culture
medium.
17. A method for growing cells, comprising: a) providing flexible
tubing containing culture medium, and a surface in which the living
cells can grow on in said tubing, and a system of clamps, each of
the clamps being capable of open and closed positions, the clamps
being positioned so as to be able to divide the tubing into: i) an
upstream region containing unused culture medium; ii) a downstream
region containing spent culture medium; and iii) a growth chamber
region for growing said cells disposed between the upstream and
downstream regions; and b) closing selected ones of the clamps on
the tubing to define the growth chamber region of the tubing
between the upstream and downstream regions of the tubing, and
introducing viable cells into the growth chamber region; c)
cyclically closing and opening selected ones of the clamps to
redefine the growth chamber region of the tubing so that a first
portion of the previously defined growth chamber region becomes a
portion of the downstream region of the tubing, and a portion of
the previously defined upstream region of the tubing becomes a
portion of the growth chamber region of the tubing; and d)
repeating step c) until a sufficient amount of cells have been
grown.
18. The method according to claim 17, comprising the further step
of withdrawing a sample of living cells from said culture medium
from the downstream region.
19. The method according to claim 17, further comprising isolating
said living cells from the downstream region.
20. The method according to claim 17, wherein the living cells are
selected from the group consisting of yeast cells, animal cells,
plant cells, and stem cells.
21. The method according to claim 17, wherein the living cells are
stem cells.
22. The method according to claim 21, wherein the living cells are
selected from the group consisting of hematopoietic stem cells,
bone marrow stem cells, stromal cells, astrocytes,
oligidendrocytes, embryonic stem cells, fetal stem cells, umbilical
cord stem cells, placenta derived stem cells, and adult stem
cells.
23. The method according to claim 22, wherein the living cells are
selected from the group consisting of hematopoietic stem cells,
bone marrow stem cells, stromal cells, astrocytes and
oligidendrocytes.
24. The method according to claim 17, wherein the surface in which
the cells are grown is the interior surface of the tubing.
25. The method according to claim 17, wherein the surface in which
the cells are grown is a continuous fiber.
26. The method according to claim 17, further comprising growing
the cells in one or more growth chambers that are present in the
growth chamber region.
27. The method according to claim 17, further comprising growing
one or more types of cells in the growth chamber region.
28. The method according to claim 17, wherein the sufficient amount
of cells of step d) is defined as a pre-determined density level of
the cells.
29. The method according to claim 17, wherein the tubing is gas
permeable
30. The method according to claim 17, wherein the tubing is gas
impermeable.
31. The method according to claim 17, wherein the tubing is one of
transparent and translucent, a turbidimeter being used to determine
the density level of the cells.
32. The method according to claim 17, further comprising regulating
the pressure of the growth chamber portion of the tubing relative
to ambient pressure.
33. The method according to claim 17, further comprising measuring
a pH of the culture medium in the growth chamber region.
34. The method according to claim 17, further comprising regulating
the temperature of the growth chamber region with a temperature
regulator constructed to control the temperature of the growth
chamber region of the tubing.
35. The method according to claim 17, further comprising agitating
the culture medium in the growth chamber region with an
agitator.
36. The method according to claim 35, wherein the agitator
comprises at least one stirring bar.
37. The method according to claim 17, further comprising subjecting
the growth culture chamber region to at least one of radio waves,
light waves, x-rays, sound waves, an electro magnetic field, and
radioactive field.
38. The method according to claim 17, further comprising subjecting
the growth chamber region to a different gravitational force.
Description
FIELD OF THE INVENTION
[0001] The described invention provides a method and a device that
allow selection of living cells, with increased rates of
reproduction and specific metabolic properties, in a liquid or
semi-solid medium. For the process of selection (adaptive
evolution), genetically variant organisms (mutants) arise in a
population and compete with other variants of the same origin.
Those with the fastest rate of reproduction increase in relative
proportion over time, leading to a population (and individual
organisms) with increased reproductive rate. This process can
improve the performance of organisms used in industrial processes
or academic purpose. The present invention utilizes a continuous
culture apparatus to achieve the viable production of living cells,
for example, yeast, plant cells, animal cells or stem cells. The
present invention may be used to produce an active ingredient or
biologic that is produced by the living cells. The active
ingredient or biologic may in turn be used as a diagnostic,
preventive, or therapeutic agent.
BACKGROUND OF THE INVENTION
[0002] Selection for increased reproductive rate (fitness) requires
sustained growth, which is achieved through regular dilution of a
growing culture. In the prior art this has been accomplished two
ways: serial dilution and continuous culture, which differ
primarily in the degree of dilution.
[0003] Serial culture involves repetitive transfer of a small
volume of grown culture to a much larger vessel containing fresh
growth medium. When the cultured cells have grown to saturation in
the new vessel, the process is repeated. This method has been used
to achieve the longest demonstrations of sustained culture in the
literature (Lenski & Travisano: Dynamics of adaptation and
diversification: a 10,000-generation experiment with bacterial
populations. 1994. Proc Natl Acad Sci USA. 15:6808-14), in
experiments which clearly demonstrated consistent improvement in
reproductive rate over period of years. This process is usually
done manually, with considerable labor investment, and is subject
to contamination through exposure to the outside environment.
Serial culture is also inefficient, as described in the following
paragraph.
[0004] The rate of selection, or the rate of improvement in
reproductive rate, is dependant on population size (Fisher: The
Genetical Theory of Natural Selection.1930. Oxford University
Press, London, UK). Furthermore, in a situation like serial
transfer where population size fluctuates rapidly, selection is
proportional to the harmonic mean (N) of the population (Wright:
Size of population and breeding structure in relation to evolution.
1938. Science 87: 430-431), and hence can be approximated by the
lowest population during the cycle.
[0005] Population size can be sustained, and selection therefore
made more efficient, through continuous culture. Continuous
culture, as distinguished from serial dilution, involves smaller
relative volume such that a small portion of a growing culture is
regularly replaced by an equal volume of fresh growth medium. This
process maximizes the effective population size by increasing its
minimum size during cyclical dilution. Devices allowing continuous
culture are termed "chemostats" if dilutions occur at specified
time intervals, and "turbidostats" if dilution occur automatically
when the culture grows to a specific density.
[0006] For the sake of simplicity, both types of devices will
hereafter be grouped under the term "chemostat". Chemostats were
invented simultaneously by two groups in the 1950's (Novick &
Szilard: Description of the chemostat. 1950. Science 112: 715-716)
and (Monod: La technique de la culture continue--Theorie et
applications.1950. Ann. Inst. Pasteur 79:390-410). Chemostats have
been used to demonstrate short periods of rapid improvement in
reproductive rate (Dykhuizen DE. Chemostats used for studying
natural selection and adaptive evolution.1993. Methods Enzymol.
224:613-31).
[0007] Traditional chemostats are unable to sustain long periods of
selection for increased reproduction rate, due to the unintended
selection of dilution-resistant (static) variants. These variants
are able to resist dilution by adhering to the surface of the
chemostat, and by doing so, outcompete less sticky individuals
including those that have higher reproductive rates, thus obviating
the intended purpose of the device (Chao & Ramsdell: The
effects of wall populations on coexistence of bacteria in the
liquid phase of chemostat cultures,. 1985. J. Gen. Microbiol. 131:
1229-36).
[0008] One method and chemostatic device (the Genetic Engine) has
been invented to avoid dilution resistance in continuous culture
(patent U.S. Pat. No. 6,686,194-B1 filed by PASTEUR INSTITUT [FR]
& MUTZEL RUPERT [DE]) . This method uses valve controlled fluid
transfer to periodically move the growing culture between two
chemostats, allowing each to be sterilized and rinsed between
periods of active culture growth. The regular sterilization cycles
prevent selection of dilution-resistant variants by destroying
them. This method and device achieves the goal, but requires
independent complex manipulations of several fluids within a
sterile (sealed) environment, including one (NaOH) which is both
very caustic and potentially very reactive, quickly damaging
valves, and posing containment and waste-disposal problems. The
chemostatic device is also limited in that no provisions are made
to provide a support for cells to grow on
[0009] There are some types of cells that are difficult to culture
in large amounts due to the conditions the cells require to survive
and grow. It is believed that these cells could grow in conditions
provided by a continuous culture approach. This is particularly the
case for stem cells.
[0010] For example, human embryonic stem cells are typically grown
by isolating and transferring a stem cell mass into a plastic
laboratory culture dish that contains a nutrient broth known as
culture medium. The cells divide and spread over the surface of the
dish. The inner surface of the culture dish is typically coated
with mouse embryonic skin cells that have been treated so they will
not divide. This coating layer of cells is called a feeder layer.
The reason for having the feeder layer in the bottom of the culture
dish is to give the human embryonic stem cells a sticky surface to
which they can attach. Also, the feeder cells release nutrients
into the culture medium. Recently, scientists have begun to devise
ways of growing embryonic stem cells without the mouse feeder
cells. This is a significant scientific advancement because it
avoids the risk that viruses or other macromolecules in the mouse
cells may be transmitted to the human cells.
[0011] Over the course of several days, the cells of the inner cell
mass proliferate and begin to crowd the culture dish. When this
occurs, they are removed gently and plated into several fresh
culture dishes. The process of replating the cells is repeated many
times and for many months, and is called subculturing. Each cycle
of subculturing the cells is referred to as a passage. After six
months or more, the original cells of the cell mass yield millions
of embryonic stem cells. Embryonic stem cells that have
proliferated in cell culture for six or more months without
differentiating, are pluripotent, and appear genetically normal are
referred to as an embryonic stem cell line.
[0012] Once cell lines are established, or even before that stage,
batches of them can be frozen and shipped to other laboratories for
further culture and experimentation. However, continuous culture
grants the advantage of suppressing a maximum of manipulations that
stress the living cells and create a potential source of
contamination. When a culture is started, continuous culture
conditions allow the skilled artisan to take advantage of a
continuous production of cells. Once stem cells are being produced,
the production of stems cells could continue without interruption
to produce substantially more stem cells than methods that are
typically used today.
SUMMARY OF THE INVENTION
[0013] It is therefore an object of the present invention to
provide an improved (and completely independent) method and device
for continuous culture of cells (including bacteria, archaea,
eukaryotes and viruses) without interference from
dilution-resistant variants. Like other chemostats, the device
provides a means for regular dilution of a grown culture with fresh
growth medium, a means for gas exchange between the culture and the
outside environment, sterility, and automatic operation as either a
chemostat or a turbidostat.
[0014] Additionally, it is an object of the present invention to
provide an improved and distinct method and device for continuous
culture of cells such as plant cells, animal cells or stem cells.
Stem cells that may be cultured with the present invention include
but are not limited to embryonic stem cells, fetal stem cells,
umbilical cord stem cells, placenta derived stem cells, and adult
stem cells. The adult stem cells that may be cultured with the
present invention include but are not limited to hematopoietic stem
cells, bone marrow stem cells, stromal cells, astrocytes and
oligidendrrocytes (e.g, Hematopoietic Stem Cell Protocols by C.
Klug and C. Jordan, Humana Press, Totowa, N.J., 2002, incorporated
by reference herein).
[0015] The present invention is designed to achieve these goals
without any fluid transfer, including sterilization or rinsing
functions. This represents a specific advantage of the present
invention with respect to prior art in so far as it avoids the
hazards and difficulties associated with sterilization and rinsing,
including containment and complex fluid transfers involving caustic
solvents.
[0016] Continuous culture is achieved inside a flexible sterile
tube filled with growth medium. The medium and the chamber surface
are static with respect to each other, and both are regularly and
simultaneously replaced by peristaltic movement of the tubing
through "gates", or points at which the tube is sterilely
subdivided by clamps that prevent the cultured cells from moving
between regions of the tube. UV gates can also (optionally) be
added upstream and downstream of the culture vessel for additional
security.
[0017] The present method and device are also an improvement over
prior art insofar as they continually, rather than periodically,
select against adherence of dilution-resistant variants to the
chemostat surfaces, as replacement of the affected surfaces occurs
in tandem with the process of dilution.
[0018] The tube is subdivided in a transient way such that there
are regions that contain saturated (fully grown) culture, regions
that contain fresh medium, and a region between these two, wherein
one or more chambers referred to as growth or culture chambers are
present to form a growth chamber region in which grown culture is
mixed with fresh medium to achieve dilution. The gates are
periodically released from one point on the tube and replaced at
another point, such that grown culture along with its associated
growth chamber surface and attached static cells, is removed by
isolation from the growth chamber and replaced by both fresh medium
and fresh chamber surface.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Without being exhaustive and limiting, one possible general
configuration will include several components as described
hereafter. In the following the present invention is exemplarily
explained on the basis of a preferred embodiment, thereby referring
to the drawings in which:
[0020] FIG. 1 displays an overall view of a possible configuration
of the device in which:
[0021] (1) represents the flexible tubing containing the different
regions of the device which are: upstream fresh medium (7), growth
chamber (10), sampling chamber (11) and disposed grown culture
region (15)
[0022] (2) represents the thermostatically controlled box allowing
regulation of temperature according to conditions determined by
user, and in which may be located: [0023] a. said growth chamber
(10), [0024] b. said sampling chamber (11), [0025] c. upstream gate
(3) defining the beginning of said growth chamber (10), [0026] d.
downstream gate (4) defining the end of said growth chamber (10)
and the beginning of said sampling chamber (11) [0027] e. second
downstream gate (5) defining the end of said sampling chamber (11),
[0028] f. turbidimeter (6) allowing the user or automated control
system to monitor optical density of growing culture and to operate
a feedback control system (13), allowing controlled movement of the
tubing (1) on the basis of culture density (turbidostat function),
[0029] g. one or several agitators (9).
[0030] It should be noted that the device elements listed in a-g
may also be located outside
[0031] of, or in the absence of, a thermostatically controlled
box.
[0032] (7) represents the fresh medium in unused flexible
tubing,
[0033] (8) represents a barrel loaded with fresh medium filled
tubing, in order to dispense said fresh medium and tubing during
operations.
[0034] (12) represents optional ultra-violet radiation gates,
[0035] (13) represents the control system that can consist of a
computer connected with means of communication to different
monitoring or operating interfaces, like optical density
turbidimeters, temperature measurement and regulation devices,
agitators and tilting motors, etc, that allow automation and
control of operations,
[0036] (14) represents the optional disposal barrel on which to
wind up tubing containing disposed grown culture filled tubing,
[0037] (15) represents disposed grown culture located downstream of
said sampling chamber.
[0038] FIG. 2 displays two possible positions of the device,
exemplifying the fact that said thermostatically controlled box (2)
and other pieces of said device associated with said culture
chamber can be tilted to various degrees for agitation purposes,
gas circulation and removal purposes, and purposes of guaranteeing
the removal of granulated (aggregated) cells that might escape
dilution by settling to the bottom.
[0039] FIGS. 3 to 9 represents said flexible tubing (1) in place in
said thermostatically controlled box (2) and introduced through
gates (3), (4) and (5) through which said tubing will stay during
all steps of process and through which said tubing will move
according to its peristaltic movement.
[0040] FIG. 3 symbolizes status T0 of the device in which all
regions of said flexible tubing are filled with fresh medium before
injection of the cell intended for continuous culture.
[0041] FIG. 4 symbolizes status T1 of said flexible tubing just
after injection of cell strain.
[0042] FIG. 5 symbolizes status T2 of the device which is a growing
period during which the culture grows in the region defined as the
growth chamber (10) limited by said gates (3) and (4).
[0043] FIG. 6 symbolizes status T3 of device, just after the first
peristaltic movement of tubing and associated medium, which
determines the beginning of the second growing cycle, introducing
fresh tubing and medium through movement of gate 3 simultaneous
with a transfer of equivalent volume of tubing, medium, and grown
culture out of the growth chamber region (10) and into the sampling
chamber region (11) by movement of gate 4. It is critical to
recognize that the tubing, the medium that is within the tubing,
and any culture that has grown in that medium, all move together.
Fluid transfer only occurs insofar as fresh medium and grown
culture mix together through agitation within the growth chamber
region.
[0044] FIG. 7 symbolizes status T4 of the device which is the
second growing cycle; during this cycle cells that remain in the
growth chamber after peristaltic movement of the tubing can now
grow using nutrients provided in the fresh medium that is mixed
with the remaining culture during this step.
[0045] FIG. 8 symbolizes status T5 of device, just after the second
peristaltic movement of the tubing and the contained medium, which
determines the beginning of the third growing cycle, introducing
fresh tubing and medium through movement of gate 3 simultaneous
with a transfer of equivalent volume of tubing, medium, and grown
culture out of the growth chamber region (10) and into the sampling
chamber region (11) by movement of gate 4.
[0046] FIG. 9 symbolizes status T6 of device which is the third
growing cycle; this step is equivalent to status T4 and indicates
the repetitive nature of further operations. Samples of selected
cells may be removed at any time from the sampling chamber region
(11) using a syringe or other retrieval device.
[0047] FIG. 10 displays a possible profile of teeth determining a
gate in the configuration which consists of two stacking teeth
pinching flexible tubing. Gates could also be determined by single
teeth pressing against a moveable belt, removable clamps, or other
mechanisms that prevent movement of cells through the gate and
which can be alternately placed and removed in variable positions
along the tubing.
DETAILED DESCRIPTION OF INVENTION
[0048] The basic operation of the device is depicted in FIGS. 3
through 9.
[0049] One potential configuration for the present device is shown
in FIG. 1, as it appears after having been loaded with a fresh tube
of sterile medium (shown divided into regions A-H by said gates
(3), (4) and (5)).
[0050] Inoculation of the device with the chosen cell could be
achieved by introduction of the cell into the growth chamber (FIG.
3), through injection (FIG. 4, region B). The culture would then be
allowed to grow to the desired density and continuous culture would
begin (FIG. 5).
[0051] Continuous culture would proceed by repetitive movements of
the gated regions of tubing. This involves simultaneous movements
of the gates, the tubing, the medium, and any culture within the
tubing. The tubing will always move in the same direction; unused
tubing containing fresh medium (and hereafter said to be `upstream`
of the growth chamber (7)) will move into the growth chamber and
mix with the culture remaining there, providing the substrate for
further growth of the cells contained therein. Before introduction
into the growth chamber region, this medium and its associated
tubing will be maintained in a sterile condition by separation from
the growth chamber by the upstream gates (3). Used tubing
containing grown culture will simultaneously be moved `downstream`
and separated from the growth chamber by the downstream gates
(4).
[0052] When one or more growth chambers are present, the growth
chambers may be used for the same or different purpose. For
example, living cells could be grown in a first growth chamber and
a second growth chamber with the same or different conditions. In
one embodiment, a first growth chamber can be used to grow cells
and a second growth chamber may then be used to treat the living
cells under different conditions. For example, the cells may be
treated to induce the expression of a desired product. Components
or additives of the culture medium itself may be added prior to or
after the culture begins. For example, all components or additives
could be included in the media before beginning the culture, or
components can be injected into one or more of the growth chambers
after the culture has been initiated.
[0053] Gate configuration is not a specific point of the present
patent application. For example, in a given configuration, gates
can be designed through one chain of multiple teeth simultaneously
moved or in another configuration separated in distinct
synchronized chains as depicted in FIG. 1. Gates can consist of a
system made of two teeth pinching the tubing in a stacking manner
as described in FIG. 10, avoiding contamination between regions G
and H of the tubing through the precision of the interface between
the teeth. In another configuration, sterile gates can be obtained
by pressing one tooth against one side of the tubing and thereby
pressing the tubing tightly against a fixed chassis along which
tubing is slid during its peristaltic movement, as sketched in FIG.
3 to 9, marks 3, 4 and 5.
[0054] Said thermostatically controlled box (2) is obtained by
already known means such as a thermometer coupled with a heating
and cooling device.
[0055] Aeration (gas exchange), when required for growth of the
cultured cell or by the design of the experiment, is achieved
directly and without mechanical assistance by the use of gas
permeable tubing. For example and without being limiting, flexible
gas permeable tubing can be made of silicone. Aeration could be
achieved through exchange with the ambient atmosphere or through
exchange with an artificially defined atmosphere (liquid or gas)
that contacts the growth chamber or the entire chemostat. When an
experiment demands anaerobiosis the flexible tubing can be gas
impermeable. For example and without being limiting, flexible gas
impermeable tubing can be made of coated or treated silicone.
[0056] For anaerobic evolution conditions, regions of the tubing
can also be confined in a specific and controlled atmospheric area
to control gas exchange dynamics. This can be achieved either by
making said thermostatically controlled box gastight and then
injecting neutral gas into it or by placing the complete device in
an atmosphere controlled room.
[0057] Counter-selection of static variants is achieved by
replacement of the growth chamber surface along with growth
medium.
[0058] The device is further designed to be operable in a variety
of orientations with respect to gravity, that is, to be tilted as
shown by FIG. 2, along a range of up to 360.degree..
[0059] Dilution-resistant variants may avoid dilution by sticking
to one another, rather than to the chamber wall if aggregated cells
can fall upstream and thereby avoid removal from the chamber. Hence
it is desirable that the tubing generally be tilted downward, such
that aggregated cells will fall toward the region that will be
removed from the growth chamber during a cycle of tube movement.
This configuration involves tilting the device so that the
downstream gates are below the upstream gates with respect to
gravity.
[0060] The growing chamber can be depressurized or over pressurized
according to conditions chosen by the experimenter. Different ways
of adjusting pressure can be used, for instance applying vacuum or
pressurized air to the fresh medium and tubing through its upstream
extremity and across the growth chamber; another way of
depressurizing or overpressurizing tubing can be done by alternate
pinching and locking tubing upstream of or inside the growth
chamber.
[0061] When the medium is contained in gas permeable tubing, air
bubbles may form within the medium. These will rise to the top of a
sealed region of tubing and become trapped there until the movement
of the region (and the gates defining it) releases the region into
either the growth chamber, the sampling chamber or the endpoint of
the chemostat (FIGS. 6, regions D-C, B or A, respectively). If the
device is tilted downward such bubbles will accumulate in the
growth chamber or sampling chamber and displace the culture. The
device is designed to periodically tilt upward for a cycle of the
tube movement, allowing for the removal of accumulated gas from
said chambers.
[0062] Tilting movements of the device, and/or shaking of the
growth chamber by an external device (9) can be used to decrease
aggregation of cells within the growth chamber. Alternatively, one
or several stirring bars can be included in the tubing filled with
fresh medium before sterilization and magnetically agitated during
culture operations.
[0063] The proportional length of the regions of fresh medium
defined by the upstream gates as compared to the length of the
culture chamber will define the degree of dilution achieved during
a cycle.
[0064] The frequency of dilution can be determined either by timing
(chemostat function) or by feedback regulation whereby the density
of the culture in the growth chamber is measured by a turbidimeter
(FIG. 1--mark 6) and the dilution cycle occurs when the turbidity
reaches a threshold value (turbidostat function).
[0065] The sampling chamber allows withdrawing grown culture in
order to analyze the outcome of an experiment, collect cells with
improved growth rate for further culture, storage, or functional
implementation, or other purposes such as counting the population,
checking the chemical composition of the medium, or testing the pH
of grown culture. In order to achieve permanent monitoring of pH
inside growth chamber, tubing can include by construction a pH
indicator line embedded/encrusted in the wall of the tubing.
[0066] Any form of liquid or semi-solid material can be used as a
growth medium in the present device. The ability to utilize
semi-solid growth substrates is a notable advancement over prior
art. The growth medium, which will define the metabolic processes
improved by the selection process, can be chosen and defined by the
user.
[0067] If needed, this device can contain multiple growth chambers,
such that the downstream gates of one growth chamber become the
upstream gates of another. This could, for example, allow one cell
to grow alone in the first chamber, and then act as the source of
nutrition for a second cell (or virus) in the second chamber.
[0068] The invention may be used to produce a preparation, such as
a biologic for a drug, a vaccine, or an antitoxin, that is
synthesized from cells grown by the invention or their products.
The biologic may be used as a diagnostic, preventive, or
therapeutic agent. For example, the present invention may be used
to produce therapeutic proteins such as insulin.
[0069] In a preferred embodiment, the device and/or method may be
cycled in a manner to continually collect stem cells in their
undifferentiated state. Furthermore, the culture conditions may be
modified to inhibit the differentiation of the stem cells. For
example, stem cell differentiation inhibitors (e.g., inhibitors of
aldehyde dehydrogenase, inhibitors of phosphoinositide 3-kinase,
TGF Receptor Kinase inhibitors, TGF-B Receptor Kinase Inhibitor
etc. . . . ) may be added to the culture medium. Alternatively,
process conditions such as the amount of oxygen delivered to the
culture medium may be increased or decreased to improve the growth
of certain stem cells and/or slow down or improve differentiation
of the stem cells.
[0070] As some cells require a substrate to grow, a physical
support or structure can be added to the vessel culture chamber. In
a preferred embodiment, a continuous support could be added inside
the tubing like a continuous fiber bed, constituted by a thin
continuous fiber like support structure, can be added to the vessel
culture chamber which could let cells grow in 3 dimensions. For
example, the support could be a fiber bed, which provides support
for the growth of cells such as stem cells, plants cells and other
types of cells that prefer such a support structure, and in some
specific conditions or change of conditions, to process the natural
selection for targeted mutations.
[0071] In a preferred embodiment, a fibrous material as described
in Huang et al., Continuous Production of butanol by Clostridium
acetobutylicum immobilized in a fibrous bed reactor, Appl Biochem
Biotechnol. 2004 Spring; 113-116:887-98, incorporated by reference
herein. The structure and size of the tubing may also be varied to
avoid the need for incorporating a support structure into the
mobile vessel culture chamber. In a preferred embodiment, tubing
with a smaller diameter is used so the cells may adhere in a more
natural manner.
[0072] This device and method allows researchers and product
developers to evolve any strain of culturable living cells in
suspension or any strain of culturable living cells which are not
in suspension which grow on the wall of the tubing or on a support
which could be a fiber bed in the tubing through sustained growth
(continuous culture); the resulting improved cell can constitute a
new strain or species. These new cells can be identified by
mutations acquired during the course of culture, and these
mutations may allow the new cells to be distinguished from their
ancestors' genotype characteristics. This device and method allow
the researcher to select new strains of any living cell by
segregating individuals with improved rates of reproduction through
the process of natural selection. The invention also provides an
improved and completely distinct method and device for continuous
culture of cells such as yeast, plant cells, animal cells or stem
cells.
[0073] In a further embodiment, an emitter can be used to subject
the cells, permanently or temporarily, to at least one of radio
waves, light waves, x-rays, sound waves, an electro magnetic field,
a radioactive field, radioactive media, or combinations thereof.
The following publications are incorporated by reference:
Biofizika. 2005 July-August, 50(4):689-92; Bioelectromagnetics.
2005 September, 26(6):431-9; Chem Commun (Camb). Jan. 14, 2005,
(2):174-6; Biophys J. 2005 February, 88(2):1496-9;
Bioelectromagnetics. 1981, 2(3):285-9; Sb Lek. 1998, 99(4):455-64;
Antimicrob Agents Chemother. 2004 December, 48(12):4662-4; J Food
Prot. 2003 September, 66(9):1712-5; Astrobiology. 2006 April,
6(2):332-47; Life Sci Space Res. 1970, 8:33-8; Adv Space Res. 1995
March, 15(3):211-4; Radiat Res. 2006 May, 165(5):532-7;
Mutagenesis. 2004 September, 19(5):349-54; Cancer Sci. 2006 June,
97(6):535-9; Appl Environ Microbiol. 2006 May, 72(5):3608-14; and
Pol J Microbiol. 2005, 54 Suppl:7-11.
[0074] In another embodiment, the growth chamber region of the
device could be subjected to, permanently or temporarily,
subjecting the cells to a different gravitational force. For
example, the cells can be grown in a microgravity environment. The
following publications are incorporated by reference: J Gravit
Physiol. 2004 March;11(1):75-80; Immunol Rev. 2005 Dec;208:267-80;
and J Gravit Physiol. 2004 July;11(2):P181-3.
[0075] Modifications and variations of the present invention
relating to the apparatus and method will be obvious to those
skilled in the art from the foregoing detailed description of the
invention. Such modifications and variations are intended to come
within the scope of the appended claims.
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