U.S. patent application number 17/569901 was filed with the patent office on 2022-04-28 for culture container, method for culturing lymphocytes, culture-container production method, and solid-phasing apparatus.
This patent application is currently assigned to TOYO SEIKAN GROUP HOLDINGS, LTD.. The applicant listed for this patent is TOYO SEIKAN GROUP HOLDINGS, LTD.. Invention is credited to Takeshi Aihara, Yoichi Ishizaki, Satoshi Tanaka, Takahiko Totani.
Application Number | 20220127554 17/569901 |
Document ID | / |
Family ID | |
Filed Date | 2022-04-28 |
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United States Patent
Application |
20220127554 |
Kind Code |
A1 |
Totani; Takahiko ; et
al. |
April 28, 2022 |
CULTURE CONTAINER, METHOD FOR CULTURING LYMPHOCYTES,
CULTURE-CONTAINER PRODUCTION METHOD, AND SOLID-PHASING
APPARATUS
Abstract
A culture container for culturing lymphocytes includes an
immobilized surface and a non-immobilized surface, wherein the
culture container is formed of a gas permeable film, the
immobilized surface and the non-immobilized surface are container
inner surfaces facing each other, and anti-CD3 antibodies are
immobilized in the immobilized surface at a concentration of 10 to
300 ng/cm.sup.2.
Inventors: |
Totani; Takahiko; (Kanagawa,
JP) ; Tanaka; Satoshi; (Kanagawa, JP) ;
Aihara; Takeshi; (Kanagawa, JP) ; Ishizaki;
Yoichi; (Kanagawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TOYO SEIKAN GROUP HOLDINGS, LTD. |
Tokyo |
|
JP |
|
|
Assignee: |
TOYO SEIKAN GROUP HOLDINGS,
LTD.
Tokyo
JP
|
Appl. No.: |
17/569901 |
Filed: |
January 6, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15184649 |
Jun 16, 2016 |
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17569901 |
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PCT/JP2014/006252 |
Dec 16, 2014 |
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15184649 |
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International
Class: |
C12M 1/12 20060101
C12M001/12; C12N 5/0783 20060101 C12N005/0783; C12M 1/00 20060101
C12M001/00; C12M 1/04 20060101 C12M001/04 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 18, 2013 |
JP |
2013-261181 |
Dec 25, 2013 |
JP |
2013-267082 |
Claims
1.-12. (canceled)
13. An immobilizing apparatus for allowing proteins to be
immobilized on the inner surface of a culture container,
comprising: a mounting table on which the liquid droplets
containing the proteins being dissolved therein and a culture
container are mounted, and a driving means that move the mounting
table to allow the liquid droplets in the culture container to move
on the inner surface of the culture container.
14. The immobilizing apparatus according to claim 13, comprising:
as the driving means, a driving means for moving in the
longitudinal direction that allows the mounting table to rotate
with a lateral direction being a central axis, thereby to allow the
liquid droplets to move in the longitudinal direction on the inner
surface of the culture container, and a driving means for moving in
the lateral direction that allows the mounting table to rotate with
a longitudinal direction being a central axis, thereby to allow the
liquid droplets to move in the lateral direction on the inner
surface of the culture container.
15. The immobilizing apparatus according to claim 13, wherein it
comprises a retention member having a semi-cylindrical recess part
for retaining the culture container such that the bottom surface of
the culture container forms a semi-cylindrical shape and the
retention member is fixed to the mounting table or is formed
integrally with the mounting table.
16. The immobilizing apparatus according to claim 15, wherein the
retention member is fixed to the mounting table or the retention
member is formed integrally with the mounting table such that the
direction of the central axis of the semi-cylindrical shape of the
retention member is identical with the longitudinal direction of
the mounting table.
17. The immobilizing apparatus according to claim 15, wherein the
retention member is provided with a pressing part for pressing the
culture container such that the bottom surface of the culture
container forms a semi-cylindrical shape.
Description
TECHNICAL FIELD
[0001] The present invention relates to a cell culture technology.
In particular, the present invention relates to a culture
container, a method for culturing lymphocytes, a method for
producing a culture container in which proteins are immobilized and
an immobilizing apparatus.
BACKGROUND ART
[0002] In recent years, in the fields of pharmaceuticals
production, gene therapy, regenerative therapy, immune therapy or
the like, there has been a demand for culturing a large amount of
cells, tissues, microorganisms efficiently in an artificial
environment.
[0003] Under such circumstances, culture of a large amount of cells
in a closed system by sealing cells and a culture liquid in a
culture bag formed of a gas-permeable film has been conducted.
[0004] In the meantime, in order to proliferate lymphocytes, it is
required to activate lymphocytes at first. Therefore, lymphocytes
were activated on a base member to which anti-CD3 antibodies had
been immobilized, and then the activated lymphocytes were sealed in
a culture bag and proliferated.
[0005] At this time, as shown in FIG. 8, in general, a flask in
which anti-CD3 antibodies are immobilized to the bottom surface
thereof was used for activation of lymphocytes and, after
activation of the lymphocytes, the activated lymphocytes were
transferred to a culture bag to conduct cultivation of the
lymphocytes. The reason therefor is as follows: lymphocytes have
properties that, while they are required to be irritated by
anti-CD3 antibodies in order to be proliferated, they become hardly
be proliferated when irritated continuously by anti-CD3 antibodies.
Therefore, after activation of lymphocytes, the activated
lymphocytes are required to be cultured in a container where
anti-CD3 antibodies are not immobilized.
[0006] As a culture container for activating lymphocytes, a closed
cell culture container (disclosed in Patent Document 1) or the like
can be given. In this closed cell culture container, anti-CD3
antibodies are immobilized on the entire surface of the container
(paragraphs 0048 and 0057), whereby lymphocytes can be activated
efficiently.
[0007] As mentioned above, activation of lymphocytes is generally
conducted by anti-CD3 antibodies. That is, lymphocytes are
activated in a container in which anti-CD3 antibodies are
immobilized, and thereafter, the activated lymphocytes are sealed
in a culture bag in which anti-CD3 antibodies are not immobilized,
whereby lymphocytes are proliferated.
[0008] Therefore, prior to activation of lymphocytes, it was
required to immobilize (coat) anti-CD3 antibodies in a container
used for activation of lymphocytes.
[0009] As the conventional method for immobilizing, in general,
after immersing the bottom surface of a flask in a solution
containing anti-CD3 antibodies and allowing the flask to stand, the
solution is then removed, whereby anti-CD3 antibodies are
immobilized.
[0010] Specifically, for example, Patent Document 2 discloses
preparation of a flask in which antibodies are immobilized, in
which anti-CD3 antibodies are immersed homogenously in the bottom
surface of a flask, and stored in a refrigerator overnight, and
then, the anti-CD3 antibodies are withdrawn, thereby to prepare a
flask in which antibodies are immobilized (paragraphs 0035 to 0036,
and FIG. 1).
[0011] Further, Patent Document 1 states that a solution obtained
by dissolving anti-CD3 antibodies is sealed in an accommodating
part of a container, and the container is allowed to stand for a
prescribed period of time, whereby the anti-CD3 antibodies are
immobilized on the film surface of the container (paragraph 0015
and FIG. 1).
Patent Document 1
[0012] JP-A-2007-175028
Patent Document 2
[0013] Japan Patent No. 4399710
SUMMARY OF THE INVENTION
[0014] However, if lymphocytes are cultured by using only the
closed cell culture container disclosed in Patent Document 1, since
the lymphocytes are continued to be irritated by anti-CD3
antibodies after the activation thereof, the lymphocytes are
excessively irritated, and as a result, proliferation thereof is
suppressed. Accordingly, in order to culture a large amount of
lymphocytes efficiently, it is desirable to use this closed cell
culture container as a container dedicated for activation, and to
conduct proliferation of the cells in a separate culture
container.
[0015] Therefore, in this conventional technology, if a large
amount of lymphocytes is efficiently cultured, it may be difficult
to transfer activated cells or there may be a risk of contamination
occurs.
[0016] The inventors of the present invention made intensive
studies, and have developed a culture container for culturing
lymphocytes, wherein one or more embodiments of the culture
container are formed of a gas-permeable film, anti-CD3 antibodies
are immobilized on only one of the container inner surfaces facing
each other, thereby to provide an immobilizing surface and a
non-immobilizing surface. The culture container is arranged such
that the immobilized surface becomes a bottom surface, thereby to
activate lymphocytes, and thereafter, the culture container is
arranged such that the non-immobilized surface becomes a bottom
surface, thereby to proliferate lymphocytes, whereby the inventors
successfully enabled activation and proliferation of lymphocytes
efficiently and simultaneously in a single container.
[0017] That is, one or more embodiments of the present invention
provide a culture container capable of efficiently activating and
proliferating lymphocytes in a single culture container, as well as
a method for culturing lymphocytes.
[0018] Further, the conventional immobilizing methods described in
Patent Documents 1 and 2 may require a long resting time to allow
antibodies to be sufficiently immobilized.
[0019] Further, since most of antibodies remain in a solution
without being adsorbed to the inside of the container, the
conventional methods may require antibodies to be used in an amount
larger than the amount immobilized. For example, as mentioned
later, when a container in which antibodies are sealed by a
conventional method is allowed to stand for 1 hour, only 10% of the
antibodies were adsorbed to the inside of the container, and the
remaining 90% of the antibodies were discarded without being
immobilized in the container.
[0020] Meanwhile, proteins such as antibodies are easily affected
by heat, and the functions thereof disappear when adsorbed at high
temperatures. Therefore, antibodies are materials that are
difficult to be immobilized in a container. On the other hand, it
is desired that immobilizing be conducted in a required amount for
a short period of time by using a small amount of proteins.
Further, since antibodies are generally expensive, it is desired
that the amount of antibodies being discarded wastefully be
reduced.
[0021] The inventors of the present invention made intensive
studies, and found that, by sealing liquid droplets of a protein
solution in a container together with a gas and by moving these
liquid droplets on the inner surface of the container, protein
molecules concentrated in the gas-liquid interface of the liquid
droplets are efficiently adsorbed in the container. One or more
embodiments of the present invention have been completed based on
this finding.
[0022] That is, one or more embodiments of the present invention is
aimed at providing, when immobilizing proteins to the inner surface
of a container, a method for efficiently immobilizing proteins in a
container by sealing liquid droplets of a protein solution in the
container together with a gas, and by moving the liquid droplets on
the inner surface of the container, thereby allowing the proteins
to be immobilized in the container efficiently, as well as a
immobilizing apparatus for implementing this method.
[0023] According to one or more embodiments of the present
invention, the culture container is a culture container for
culturing lymphocytes, wherein the culture container is formed of a
gas-permeable film, antibodies are immobilized on only one of the
container inner surfaces facing each other, thereby to provide a
immobilizing surface and a non-immobilizing surface, and in the
immobilized surface, anti-CD3 antibodies are immobilized at a
concentration of 10 to 300 ng/cm.sup.2.
[0024] According to one or more embodiments of the present
invention, the method for culturing lymphocytes is a method for
culturing lymphocytes using the above-mentioned culture container,
comprising the steps of: an activation step in which lymphocytes
and a culture liquid are sealed in the culture container, and the
culture container is arranged such that the immobilized surface
becomes a bottom surface, thereby to activate the lymphocytes, and
a proliferation step in which the culture container is inverted
upside down and arranged such that the non-immobilized surface
becomes a bottom surface, thereby to proliferate the
lymphocytes.
[0025] According to one or more embodiments of the present
invention, the method for producing a culture container is a method
for producing a culture container in which proteins are
immobilized, wherein liquid droplets in which the proteins are
dissolved are injected into the culture container and the liquid
droplets are moved to part or the whole of the inner surface of the
culture container.
[0026] Further, according to one or more embodiments of the present
invention, the immobilizing apparatus is a immobilizing apparatus
for allowing proteins to be immobilized on the inner surface of a
culture container, comprising: a mounting table on which the liquid
droplets containing the proteins being dissolved therein and a
culture container are mounted, and a driving means that move the
mounting table to allow the liquid droplets in the culture
container to move on the inner surface of the culture
container.
Advantageous Effects of the Invention
[0027] According to one or more embodiments of the present
invention, it is possible to conduct activation and proliferation
of lymphocytes efficiently in a single culture container without
using a dedicated culture apparatus or the like. Therefore, it
becomes possible to eliminate troublesomeness in transfer conducted
in order to prevent excessive irritation exerted by antibodies on
lymphocytes and risk of contamination. According to one or more
embodiments of the present invention, it is possible to provide a
method for producing a culture container that efficiently
immobilized proteins and an immobilizing apparatus for conducting
this method.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1A is a view showing the culture container according to
one or more embodiments of the present invention;
[0029] FIG. 1B is a view showing the culture container according to
one or more embodiments of the present invention;
[0030] FIG. 2 is a graph showing the results of move of antibodies
when storing the culture container in a state where the inner
surfaces of the container are laminated;
[0031] FIG. 3A is a view showing the state of storage of the
culture container according to one or more embodiments of the
present invention;
[0032] FIG. 3B is a view showing the state of storage of the
culture container according to one or more embodiments of the
present invention;
[0033] FIG. 4 is a view showing the method for culturing
lymphocytes according to a first embodiment of the present
invention;
[0034] FIG. 5 is a view showing the method for culturing
lymphocytes according to a second embodiment of the present
invention;
[0035] FIG. 6A is a schematic view showing Example 1 of the culture
container and the method for culturing lymphocytes according to one
or more embodiments of the present invention;
[0036] FIG. 6B is a schematic view showing Example 2 of the culture
container and the method for culturing lymphocytes according to one
or more embodiments of the present invention;
[0037] FIG. 6C is a schematic view showing Comparative Example 1 of
the culture container and the method for culturing lymphocytes
according to one or more embodiments of the present invention;
[0038] FIG. 6D is a schematic view showing Comparative Example 2 of
the culture container and the method for culturing lymphocytes
according to one or more embodiments of the present invention;
[0039] FIG. 7 is a graph showing the results of an experiment
conducted on Examples and Comparative Examples of the culture
container and the method for culturing lymphocytes according to one
or more embodiments of the present invention;
[0040] FIG. 8 is a view showing a conventional method for culturing
lymphocytes;
[0041] FIG. 9 is a view for explaining the principle of
immobilizing in the method for producing a culture container
according to one or more embodiments of the present invention;
[0042] FIG. 10A is a view showing a culture bag in which antibodies
are immobilized obtained by the method for producing a culture
container according to one or more embodiments of the present
invention;
[0043] FIG. 10B is a view showing a culture bag in which antibodies
are immobilized obtained by the method for producing a culture
container according to one or more embodiments of the present
invention;
[0044] FIG. 11 is a view (plan view) showing a immobilizing
apparatus according to one or more embodiments of the present
invention;
[0045] FIG. 12 is a view (front view) showing an immobilizing
apparatus according to one or more embodiments of the present
invention;
[0046] FIG. 13 is a view showing the manner of immobilizing by the
immobilizing apparatus according to one or more embodiments of the
present invention;
[0047] FIG. 14A is a front view showing retention means in the
immobilizing apparatus according to one or more embodiments of the
present invention;
[0048] FIG. 14B is a side view showing retention means in the
immobilizing apparatus according to one or more embodiments of the
present invention;
[0049] FIG. 15A is a view showing the state of usage of the
retention means in the immobilizing apparatus according to one or
more embodiments of the present invention;
[0050] FIG. 15B is a view showing the state of usage of the
retention means in the immobilizing apparatus according to one or
more embodiments of the present invention;
[0051] FIG. 15C is a view showing the state of usage of the
retention means in the immobilizing apparatus according to one or
more embodiments of the present invention;
[0052] FIG. 15D is a view showing the state of usage of the
retention means in the immobilizing apparatus according to one or
more embodiments of the present invention;
[0053] FIG. 16A is a schematic view showing Example 3 of the
culture container (culture bag in which antibodies are immobilized)
according to one or more embodiments of the present invention;
[0054] FIG. 16B is a schematic view showing Example 4 of the
culture container (culture bag in which antibodies are immobilized)
according to one or more embodiments of the present invention;
[0055] FIG. 16C is a schematic view showing Comparative Example 3
of the culture container (culture bag in which antibodies are
immobilized) according to one or more embodiments of the present
invention;
[0056] FIG. 16D is a schematic view showing Comparative Example 4
of the culture container (culture bag in which antibodies are
immobilized) according to one or more embodiments of the present
invention;
[0057] FIG. 17 is a graph showing the results of experiments
conducted in Examples and Comparative Examples in the method for
producing a culture container (culture bag in which antibodies are
immobilized) according to one or more embodiments of the present
invention;
[0058] FIG. 18A is a schematic view showing Example 5 of the method
for producing a culture container (culture flask in which
antibodies are immobilized) according to one or more embodiments of
the present invention;
[0059] FIG. 18B is a schematic view showing Comparative Example 5
of the method for producing a culture container (culture flask in
which antibodies are immobilized) according to one or more
embodiments of the present invention;
[0060] FIG. 18C is a schematic view showing Comparative Example 6
of the method for producing a culture container (culture flask in
which antibodies are immobilized) according to one or more
embodiments of the present invention;
[0061] FIG. 19 is a graph showing the results of an experiment
conducted in the Examples and the Comparative Examples of the
method for producing a culture container (culture flask in which
antibodies are immobilized) according to one or more embodiments of
the present invention;
[0062] FIG. 20A is a schematic view showing Example 6 of the method
for producing a culture container (culture bag in which fibronectin
is immobilized) according to one or more embodiments of the present
invention;
[0063] FIG. 20B is a schematic view showing Comparative Example 7
of the method for producing a culture container (culture bag in
which fibronectin is immobilized) according to one or more
embodiments of the present invention; and
[0064] FIG. 21 is a graph showing the results of an experiment in
the Examples and the Comparative Examples of the method for
producing a culture container (culture bag in which fibronectin is
immobilized) according to one or more embodiments of the present
invention.
MODE FOR CARRYING OUT THE INVENTION
[0065] Hereinbelow, a detailed explanation will be made on one or
more embodiments of the culture container and the method for
culturing lymphocytes according to one or more embodiments of the
present invention. First, one or more embodiments of the culture
container according to the present invention will be explained with
reference to FIG. 1. FIG. 1 shows a schematic view as viewed from
the above and a schematic view as viewed from the side of the
culture container being mounted on a mounting table (not
shown).
[Culture Container]
[0066] As shown in FIG. 1, the culture container 10 according to
one or more embodiments of the present invention has container
walls facing with each other in a vertical direction. One of the
container inner surfaces is a immobilized surface 11 (bottom
surface of the container shown in FIG. 1) in which antibodies 20
are immobilized. The other of the container inner surfaces is a
non-immobilized surface 12 (upper surface of the container shown in
FIG. 1) in which no antibodies 20 are immobilized. The culture
container 10 is provided with a tube 13. By this tube 13, sealing
of lymphocytes and a culture liquid in the culture container 10 and
recovery of the lymphocytes cultured and the culture liquid are
conducted. In the example shown in the figure, only one tube 13 is
attached to the culture container 10, but two or more tubes 13 may
be attached.
[0067] The culture container 10 is formed in the shape of a bag by
using a film having gas permeability required for cell culture. As
the material for the culture container 10, a polyolefin resin such
as polyethylene and polypropylene can be preferably used.
[0068] As the antibodies 20 to be immobilized to the immobilized
surface 11, it is preferable to use anti-CD3 antibodies.
Lymphocytes can be activated by anti-CD3 antibodies and
proliferated.
[0069] The concentration of the antibodies 20 immobilized on the
immobilized surface 11 is preferably 10 to 300 ng/cm.sup.2, more
preferably 10 to 40 ng/cm.sup.2.
[0070] By allowing the concentration of the immobilized antibodies
20 to be 10 ng/cm.sup.2 or more, lymphocytes can be effectively
activated, and thereafter, the activated lymphocytes can be
efficiently proliferated. If the concentration of the immobilized
antibodies 20 is allowed to be larger than 40 ng/cm.sup.2, no
differences arise in proliferation efficiency of lymphocytes, and
on the contrary, the cost of the container increases due to
excessive use of antibodies.
[0071] The closed packing concentration of anti-CD3 antibodies in
the immobilized surface 11 is 300 ng/cm.sup.2. Within this range,
it is possible to sufficiently activate lymphocytes and then
proliferate the lymphocytes efficiently. On the other hand, the
concentration of anti-CD3 antibodies exceeding 300 ng/cm.sup.2 is
not preferable, since the anti-CD3 antibodies may float in the
culture liquid in the culture container 10, causing excessive
irritation on the lymphocytes.
[0072] The culture container 10 according to one or more
embodiments of the present invention can be produced as follows,
for example.
[0073] First, low-density polyethylene is extruded by means of a
plastic extrusion molding apparatus to form a film. Then, by using
an impulse sealer, a bag-shaped culture container 10 is produced
from this film. As shown in FIG. 1, the culture container 10 is
produced such that it is provided with the tube 13.
[0074] Subsequently, the culture container 10 is mounted on a
mounting table, and a prescribed amount of a gas is sealed. At that
time, a buffer solution in which the antibodies 20 are dissolved is
continuously sealed. By swinging or the like of the culture
container 10, the liquid droplets of the buffer solution are moved
on the bottom surface of the culture container 10, whereby the
antibodies 20 contained in the buffer solution are adhered to the
bottom surface of the culture container 10. As a result, the
antibodies 20 are adhered only to the bottom surface in the culture
container 10, whereby the immobilized surface 11 is formed. At this
time, the upper surface of the culture container 10 is formed as a
non-immobilized surface 12 on which no antibodies 20 are
adhered.
[0075] Subsequently, the state of storage of the culture container
10 according to one or more embodiments of the present invention
will be explained.
[0076] It is preferred that the culture container 10 be stored in a
state in which the immobilized surface 11 and the non-immobilized
surface 12 are not in contact with each other.
[0077] That is, when the culture container 10 is stored at
37.degree. C. for 2 hours under a load of 1.6 kg in a state where
the immobilized surface 11 and the non-immobilized surface 12 are
in contact with each other, as shown in FIG. 2, about 20% of the
antibodies 20 are moved from the immobilized surface 11 to the
non-immobilized surface 12. The antibodies 20 that have been moved
to the non-immobilized surface 12 in this way may irritate
lymphocytes excessively to lower the rate of proliferation thereof
when proliferating is conducted after activation of lymphocytes.
Therefore, it is preferable to suppress move of the antibodies 20
from the immobilized surface 11 to the non-immobilized surface 12
as much as possible.
[0078] When storing the culture container 10 according to one or
more embodiments of the present invention, it is preferred that a
prescribed amount of a gas be sealed in the culture container 10
and that a state in which the immobilized surface 11 and the
non-immobilized surface 12 are not in contact with each other be
maintained.
[0079] Specifically, it is preferred that the amount of a gas to be
sealed be 0.01 to 4 ml per cm.sup.2 of the bottom surface of the
culture container 10. By allowing the amount of a gas to be sealed
to be in this range, it is possible to prevent contact of the
immobilized surface 11 and the non-immobilized surface 12.
[0080] Although no specific restrictions are imposed on the gas to
be sealed, an inert gas is preferable, and air, nitrogen gas or the
like can be used. Such gas can be injected into the culture
container 10 through the tube 13 by a gas supply apparatus.
[0081] As the specific manner of storage of the culture container
10 according to one or more embodiments of the present invention,
as shown in FIG. 3A, it is preferred that the shape of the culture
container 10 be retained by an outer covering container 60 having
rigidity. When the culture container 10 is stored by using such
outer covering container 60, by sealing only a small amount of a
gas in the culture container 10, the culture container 10 can be
stored without causing movement of the antibodies 20.
[0082] Further, as shown in FIG. 3B, it is preferred that the
culture container 10 be stored in a state swollen with a gas and
wrapped with a packaging container 70. Due to such a configuration,
it is possible to package a plurality of culture containers 10
simply and store them without causing movement of the antibodies
20.
[0083] As explained hereinabove, the culture container 10 according
to one or more embodiments of the present invention is made of a
gas-permeable film, and the antibodies 20 are immobilized on only
one surface of container inner walls facing each other, whereby it
is provided with the immobilized surface 11 and the non-immobilized
surface 12. The lymphocytes sealed within the culture container 10
are gathered at the bottom of the container.
[0084] Therefore, by using the culture container 10 such that the
immobilized surface 11 becomes a bottom surface when activating
lymphocytes and such that the non-immobilized surface 12 becomes a
bottom surface when proliferating lymphocytes, excessive irritation
of lymphocytes exerted by the antibodies 20 can be prevented,
whereby activation and proliferation of lymphocytes can be
efficiently conducted in a single container. As a result, it
becomes possible to eliminate troublesomeness in transfer or risk
of contamination. Further, since moving from activation to
proliferation is a simple operation (i.e. only inverting the
container), it can be conducted simply without using a dedicated
apparatus or the like.
[0085] Further, by sealing a prescribed amount of a gas in the
culture container 10 according to one or more embodiments of the
present invention and retaining a state where the immobilized
surface 11 and the non-immobilized surface 12 are not in contact
with each other, it is possible to store the culture container 10
without lowering the effect of preventing excessive irritation on
lymphocytes.
[0086] In the meantime, in a conventional flask that is used for
activating lymphocytes, anti-CD3 antibodies are immobilized only on
the bottom surface, and lymphocytes can be activated by this
configuration. However, proliferation of lymphocytes after
activation cannot be conducted by using a single flask. The reason
is that, in a flask, the number of cells per area cannot be
maintained at an appropriate density. Namely, lymphocytes are
proliferated after activation, it is required to enlarge the
culture area. In one or more embodiments, it is possible to enlarge
the culture area by partitioning part of the culture container by
means of a clip or a roller, and by moving, removing or the like of
the clip or the roller in accordance with the proliferation of
cells. However, such enlargement of the culture area cannot be
conducted in a flask. A flask can be used for activating
lymphocytes, but is not suited to proliferation.
Method for Culturing Lymphocytes (First Embodiment)
[0087] Subsequently, a first embodiment of the method for culturing
lymphocytes according to one or more embodiments of the present
invention will be explained with reference to FIG. 4. As shown in
FIG. 4, the method for culturing lymphocytes according to one or
more embodiments of the present invention comprises the step of an
activation step (1) and a proliferation step (2).
(1) Activation Step
[0088] The activation step in the method for culturing lymphocytes
according to one or more embodiments of the present invention is a
step in which lymphocytes 30 and a culture liquid 40 are sealed in
the culture container 10, and the culture container 10 is arranged
such that the immobilized surface 11 becomes a bottom surface,
thereby to activate the lymphocytes 30.
[0089] As mentioned above, the antibodies 20 are immobilized on the
immobilized surface 11. As such antibodies 20, anti-CD3 antibodies
can be preferably used.
[0090] The type of the lymphocytes 30 is not particularly
restricted, and NK cells, B cells, T cells and mononuclear cells or
the like can be objects to be cultured.
[0091] As the culture liquid 40, one commonly used for culturing
the lymphocytes 30 can be used. For example, a culture liquid to
which Interleukin-2 has been added can be preferably used.
[0092] In this activation step, the lymphocytes 30 in the culture
container 10 are activated since the CD3 as the receptor molecule
is irritated by the anti-CD3 antibodies that have been immobilized
on the immobilized surface 11.
(2) Proliferation Step
[0093] The proliferation step in the method for culturing
lymphocytes according to one or more embodiments of the present
invention is a step in which the culture container 10 is inverted
upside down to arrange the culture container 10 such that the
non-immobilized surface 12 becomes a bottom surface, thereby to
proliferate the lymphocytes 30.
[0094] By arranging the culture container 10 such that the
non-immobilized surface 12 becomes a bottom surface, it becomes
possible to culture the lymphocytes 30 on the side of the
non-immobilized surface 12 in the culture container 10.
[0095] As a result, the lymphocytes 30 can be proliferated without
being irritated by anti-CD3 antibodies that have been immobilized
on the immobilized surface 11. Therefore, lowering in proliferation
efficiency that occurs when the lymphocytes 30 are excessively
irritated by the anti-CD3 antibodies can be prevented.
Method for Culturing Lymphocytes (Second Embodiment)
[0096] Subsequently, a second embodiment of the method for
culturing lymphocytes according to one or more embodiments of the
present invention will be explained with reference to FIG. 5. The
method for culturing lymphocytes according to one or more
embodiments of the present invention has, as shown in FIG. 5, steps
of activation step (1), first proliferation step (2),
volume-enlarging step (3) and second proliferation step (4).
[0097] That is, the method for culturing lymphocytes according to
one or more embodiments of the present invention has a
volume-enlarging step in the midst of the proliferation step. As a
result, proliferation efficiency of lymphocytes can be further
improved as compared with that in the first embodiment. In one or
more embodiments, the proliferation step will be explained by
subdividing it into three steps of (2) to (4) mentioned above.
Other points are the same as those in the first embodiment.
(1) Activation Step
[0098] In the activation step in the method for culturing
lymphocytes according to one or more embodiments of the present
invention, by partitioning the culture container 10 by means of a
partitioning member 50, the culture container 10 is divided into a
culture part 10-1 and an enlargeable part 10-2.
[0099] The culture part 10-1 is a chamber where lymphocytes 30 and
the culture liquid 40 are sealed to activate the lymphocytes
30.
[0100] The enlargeable part 10-2 is a chamber where no lymphocytes
30 and the culture liquid 40 are sealed, and used as a space that
enlarges the culture part 10-1 in accordance with proliferation of
the lymphocytes 30.
[0101] The lymphocytes 30 and the culture liquid 40 cannot pass
between the culture part 10-1 and the enlargeable part 10-2.
[0102] Here, in general, cells have properties that they hardly
proliferate unless they have a prescribed level or more of cell
density at the initial stage of culture. Therefore, it is preferred
that the volume of the culture part 10-1 be adjusted such that it
is small at the initial stage of culture and then is increased in
size later.
[0103] Further, FIG. 5 shows a step in which the culture container
10 is partitioned by using a clip as a partitioning member 50 and
then the clip is removed later to allow the whole of the culture
container 10 to be the culture part 10-1. The manner of
partitioning the culture container 10 is not limited thereto. For
example, the culture part 10-1 may be continuously changed by using
a roller as a partitioning member 50. It is also possible to change
the culture part 10-1 multiple times into an arbitrary size.
[0104] Subsequently, the lymphocytes 30 and the culture liquid 40
are sealed in the culture part 10-1 in the culture container 10,
and then, the culture container 10 is arranged such that the
immobilized surface 11 in the culture part 10-1 becomes a bottom
surface, whereby the lymphocytes 30 are activated.
[0105] During this activation step, the lymphocytes 30 in the
culture part 10-1 are activated by the anti-CD3 antibodies that
have been immobilized on the immobilized surface 11.
(2) First Proliferation Step
[0106] Subsequently, the culture container 10 is inverted upside
down and the culture container 10 is arranged such that the
non-immobilized surface 12 becomes a bottom surface, whereby the
lymphocytes 30 are proliferated.
[0107] That is, by arranging the culture container 10 such that the
non-immobilized surface 12 becomes a bottom surface, it becomes
possible to culture the lymphocytes 30 on the non-immobilized
surface side 12 in the culture part 10-1. As a result, the
lymphocytes 30 can be proliferated without being irritated by the
anti-CD3 antibodies that have been immobilized on the immobilized
surface 11. Therefore, it becomes possible to proliferate
lymphocytes efficiently in a single container.
(3) Volume-Enlarging Step
[0108] A volume-enlarging step is a step of enlarging the volume of
the culture part 10-1 by moving or removing the partitioning member
50.
[0109] By this step, it becomes possible to adjust the volume of
the culture part 10-1 in accordance with the number of the
lymphocytes 30 that have been proliferated. As a result, the
efficiency of proliferation of the lymphocytes 30 can further be
improved.
(4) Second Proliferation Step
[0110] A second proliferation step is a step of continuously
conducting culturing of the lymphocytes 30 in a state where the
culture part 10-1 in the culture container 10 is enlarged. At this
time, the culture container 10 is in the state where the
non-immobilized surface 12 is arranged such that the
non-immobilized surface 12 becomes a bottom surface.
[0111] As a result, the lymphocytes 30 can be proliferated without
being irritated by the anti-CD3 antibodies that have been
immobilized on the immobilized surface 11, and it becomes possible
to prevent lowering in rate of proliferation of the lymphocytes 30
due to an excessive density of the lymphocytes 30 in the culture
part 10-1.
[0112] In the meantime, by using plural clips or rollers as the
partitioning member 50, the steps (3) and (4) are repeated plural
times, whereby enlargement of the volume of the culture part 10-1
in a stepwise manner.
[0113] As explained hereinabove, according to the method for
culturing lymphocytes according to one or more embodiments of the
present invention, by using the culture container 10 according to
one or more embodiments of the present invention, arranging the
culture container 10 such that the immobilized surface 11 thereof
becomes a bottom surface to activate the lymphocytes 30, and
thereafter, arranging the culture container 10 such that the
non-immobilized surface 12 thereof becomes a bottom surface to
proliferate the lymphocytes 30.
[0114] Therefore, it is possible to prevent the lymphocytes 30 from
being excessively irritated by the antibodies 20, whereby
activation and proliferation of the lymphocytes 30 can be conducted
efficiently in a single container.
[0115] Further, it is also possible to adjust the volume of the
culture part 10-1 in the culture container 10 in accordance with
the number of cells of proliferated lymphocytes 30, whereby the
efficiency of proliferation of the lymphocytes 30 can be further
improved.
[0116] Subsequently, a detailed explanation will be given on one or
more embodiments of the method for producing a culture container
and the immobilizing apparatus of the present invention.
[Method for Producing Culture Container]
[0117] The method for producing a culture container according to
one or more embodiments of the present invention is a method for
producing a culture container in which proteins are immobilized,
characterized in that liquid droplets containing proteins being
dissolved therein are injected and the liquid droplets are moved to
part or the whole of an inner surface of the culture container.
[0118] First, a principle for enabling proteins to be immobilized
efficiently on the inner surface of the culture container in the
method for producing a culture container of one or more embodiments
of the present invention will be explained with reference to FIG.
9.
[0119] When a solution containing proteins being dissolved therein
is allowed to stand, the proteins exhibit their properties that
they are adsorbed to the gas-liquid interface of this solution
(BUNSEKI KAGAKU Vol. 59, No. 6. pp. 437-445 (2010)). Therefore, in
the method for producing a culture container according to one or
more embodiments of the present invention, liquid droplets
containing proteins being dissolved therein are moved on the base
member, whereby protein molecules concentrated in the gas-liquid
interface are allowed to be in contact with the base member
actively.
[0120] As mentioned above, by allowing liquid droplets to roll in
the container to allow the gas-liquid interface to move in the
container, proteins are adsorbed to the base member on the
borderline of the gas, the liquid and the base member. When
proteins are adsorbed to the base member, proteins in the liquid
droplets are adsorbed to a newly generated gas-liquid interface.
Then, if the liquid droplets are moved on the inner surface of the
container, due to the contact of the gas-liquid interface having a
high protein concentration and the inner surface of the container,
proteins are adsorbed to the container inner surface at a high
probability.
[0121] As a result, according to the method for producing a culture
container of one or more embodiments of the present invention, it
is possible to allow proteins to be immobilized efficiently on the
inner surface of the container.
[0122] For example, when proteins are immobilized only on the
bottom surface of the container, it becomes possible to prevent
loss of proteins caused by adsorption of proteins to the upper
surface of the container.
[0123] In one or more embodiments, the culture container means all
of containers used in culturing cells, and includes containers used
for activation and/or proliferation of cells.
[0124] No specific restrictions are imposed on the shape of the
culture container in one or more embodiments of the present
invention, and a bag-shaped culture bag made of a soft packaging
material or a flask made of glass or polystyrene can be preferably
used.
[0125] For example, a culture bag having opposing walls on the
container inner surface can be preferably used, and proteins are
immobilized on part or the whole of one or both of the inner
surface of the culture bag, whereby the culture bag according to
one or more embodiments of the present invention can be
produced.
[0126] Specifically, as shown in FIG. 10, one of the opposing inner
surfaces of a culture bag 100 can be a immobilized surface 110 in
which proteins 200 are immobilized (bottom surface of the container
in FIG. 10), and the other one of the inner surfaces of the culture
bag can be a non-immobilized surface 120 in which no proteins 200
are immobilized (upper surface of the container in FIG. 10).
[0127] The culture bag 100 is provided with the tube 130, and
through this tube 130, the liquid droplets containing proteins
dissolved therein and a gas can be introduced into or removed from
the culture bag 100. In the example shown in this figure, the
culture bag 100 is provided with two tubes 130. However, one or
three or more tubes 130 may be provided.
[0128] It is preferred that the culture bag 100 be formed by using
a film having permeability for a gas required for culturing cells.
As the material for such culture bag 100, a polyolefin-based resin
such as polyethylene and polypropylene can be preferably used.
[0129] In one or more embodiments of the present invention, the
proteins 200 to be immobilized on the culture bag 100 are not
particularly restricted. Antibodies such as anti-CD3 antibodies and
cell adhesive proteins such as fibronectin, collagen and laminin
can be used. Anti-CD3 antibodies are preferably used for activating
lymphocytes. Cell adhesive proteins are preferably used in order to
allow adhesive cells efficiently on the culture base member.
[0130] When anti-CD3 antibodies are immobilized as the proteins
200, it is preferred that the anti-CD3 antibodies be immobilized at
a concentration of 10 to 300 ng/cm.sup.2. If the anti-CD3
antibodies are immobilized at a concentration of 10 ng/cm.sup.2 or
more, it is possible to effectively activate lymphocytes, and
thereafter, proliferate the lymphocytes efficiently. On the other
hand, immobilizing at a concentration of 300 ng/cm.sup.2 or higher
is not preferable, since anti-CD3 antibodies float in a culture
liquid in the culture bag 100 and excessively irritate
lymphocytes.
[0131] The liquid droplets for dissolving proteins in one or more
embodiments of the present invention are not particularly
restricted. However, a phosphate buffer solution can be preferably
used.
[0132] Further, it is preferred that the size of the liquid
droplets (amount of liquid droplets) containing proteins dissolved
therein be 1 cc to 20 cc. Since a friction force acts between the
liquid droplets and the inner surface when the liquid droplets move
on the inner surface of the culture bag 100, if the droplets are
too small, they cannot move appropriately. Further, if the size of
the liquid droplets is too large, the concentration of proteins in
the liquid droplets is lowered, and the area of the gas-liquid
interface per volume of the liquid droplet is reduced, whereby the
adhesion efficiency is lowered. In this respect, the size of the
liquid droplet is preferably 1 cc to 10 cc, further preferably 1 cc
to 5 cc, and further more preferably around 2 cc.
[0133] In one or more embodiments of the present invention,
although the gas to be sealed in the culture bag 100 is not
particularly restricted, an inert gas is preferable. Air, nitrogen
gas or the like can be used. Such a gas can be injected into the
culture bag 100 through the tube 130 by a gas supply apparatus, for
example.
[0134] The amount of a gas to be sealed in the culture bag 100 is
preferably 0.1 to 4 ml, further preferably 1 to 3 ml, per cm.sup.2
of the bottom surface of the culture bag 100. If the amount of the
gas to be sealed is allowed to be in this range, it is possible to
prevent the opposing walls on the inner surface of the culture bag
100 from contacting each other, whereby the liquid droplets
containing the proteins 200 being dissolved therein can be
efficiently moved in the culture bag 100.
[0135] The culture bag 100 can be produced as follows, for
example.
[0136] First, low-density polyethylene is extruded by using a
plastic extrusion molding apparatus to form a film. By using an
impulse sealer, the culture bag 100 is produced from this film. As
shown in FIG. 10, the culture bag 100 is produced with the tube 130
being attached thereto.
[0137] Subsequently, the culture bag 100 is mounted on a mounting
table, and a prescribed amount of a gas is sealed. At that time, a
buffer solution in which the proteins 200 are dissolved is
continuously sealed. Then, by swinging or the like of the culture
bag 100, the liquid droplets of the buffer solution are moved on
the bottom surface of the culture bag 100. As a result, the
proteins 200 contained in the buffer solution are adhered to the
bottom surface of the culture bag 100, whereby the culture bag 100
in which the proteins 200 are immobilized can be obtained.
[Immobilizing Apparatus]
[0138] Subsequently, an immobilizing apparatus of one or more
embodiments of the present invention that can be preferably used in
the method for producing a culture container of one or more
embodiments of the present invention and a retention means used in
this immobilizing apparatus will be explained with reference to
FIGS. 11 to 15. FIG. 11 shows a plan view of the immobilizing
apparatus and FIG. 12 is a front view of the immobilizing
apparatus. FIG. 13 shows a manner of immobilizing by means of the
immobilizing apparatus. FIG. 14 shows a front view and a lateral
view of the retention means. FIG. 15 shows how the retention means
is used.
[0139] In FIG. 11, the lower side shows the front side of the
immobilizing apparatus 300, the upper side shows the back side of
the immobilizing apparatus 300, the left side shows the left side
of the immobilizing apparatus 300 and the right side shows the
right side of the immobilizing apparatus 300, respectively. In this
figure, the lateral direction (the longitudinal direction of the
mounting table mentioned later) is taken as the X-axis direction
and the vertical direction (the lateral direction of the mounting
table) is taken as the Y-axis direction.
[0140] The immobilizing apparatus 300 is provided with a supporting
table 310 for moving in the X-axis direction and a supporting table
360 for moving in the Y-axis direction. The culture bag 100 seals
liquid droplets containing proteins being dissolved therein and a
prescribed amount of a gas, and then is mounted on the supporting
table 360 for moving in the Y-axis direction. The supporting table
310 for moving in the X-axis direction and the supporting table 360
for moving in the Y-axis direction are moved integrally.
Hereinafter, the supporting table 310 for moving in the X-axis
direction and the supporting table 360 for moving in the Y-axis
direction may comprehensively be referred to as the "mounting
table".
[0141] The supporting table 310 for moving in the X-axis direction
is supported at the both sides (front and back sides) in the middle
of the longitudinal direction by two vertically provided supporting
columns 320 that are fixed to a base stand 400.
[0142] The supporting table 310 for moving in the X-axis direction
is connected to a servo motor 330 for moving in the X-axis
direction (driving means for moving in the longitudinal direction)
through a gear box 340 for moving in the X-axis direction.
[0143] By driving this servo motor 330 for moving in the X-axis
direction, the supporting table 310 for moving in the X-axis
direction can move alternatively in the left-handed direction and
the right-handed direction around the Y-axis direction. As a
result, the supporting table 310 for moving in the X-axis direction
can conduct seesaw movement laterally.
[0144] Accordingly, the liquid droplets in the culture bag 100
mounted on the supporting table containing proteins being dissolved
therein can move laterally and reciprocally from the left end to
the right end in the culture bag 100.
[0145] At this time, the angle of the rotational movement of the
supporting table 310 for moving in the X-axis direction may be in a
range of -10.degree. to +10.degree., for example. In FIGS. 11 and
12, a case where the left side becomes high is minus (-) and a case
where the right side becomes high is plus (+).
[0146] The speed of the rotational movement of the supporting table
310 for moving in the X-axis direction can be a speed at which the
droplets containing proteins being dissolved therein move in the
culture bag 100 at a speed of 5 m/min to 15 m/min, for example.
[0147] An origin limit detection sensor for the Y-axis 350 is used
in order to detect the origin and the limit of the Y-axis.
[0148] The supporting table 360 for moving in the Y-axis direction
is supported by the both sides (the left and right sides) in the
middle of the lateral direction by two supporting parts that are
vertically provided on the left and right end parts of the
supporting table 310 for moving in the X-axis direction.
[0149] Further, the supporting table 360 for moving in the Y-axis
direction is connected to a servo motor 370 (driving means for
moving in the lateral direction) through a gear box 380 for moving
in the Y-axis direction.
[0150] By driving this servo motor 370 for moving in the Y-axis
direction, the supporting table 360 for moving in the Y-axis
direction can conduct a rotational movement together with the
supporting table 310 for moving in the X-direction with the X-axis
direction being as a central axis alternatively in the left-handed
direction and the right-handed direction. As a result, the mounting
table can move laterally; i.e. conduct seesaw movement (move up and
down in FIG. 11).
[0151] Therefore, the liquid droplets containing proteins being
dissolved therein in the culture bag 100 mounted on the mounting
table can move in the culture bag 100 from the lower end to the
upper end.
[0152] At this time, the angle of the rotational movement of the
supporting table 360 for moving in the Y-axis direction may be in a
range of -50.degree. to +50.degree., for example. In FIGS. 11 and
12, a case where the front side becomes high is minus (-) and a
case where the back side becomes high is plus (+).
[0153] It is preferred that movement of liquid droplets in the
Y-axis direction be conducted for a distance that is equal to or
smaller than the size of the liquid droplets. By allowing the
liquid droplets to move in the Y-axis direction and by allowing the
liquid droplets to move in the X-axis direction from the left end
to the right end in the culture bag 100, it is possible to allow
the liquid droplets to move over the entire bottom surface of the
culture bag 100, as well as to efficiently adsorb proteins
contained in the liquid droplets in the culture bag 100.
[0154] An X-axis origin limit detection sensor 390 is used to
detect the origin and limit of the X-axis.
[0155] Subsequently, an explanation will be made on the method for
producing the culture bag 100 by using the immobilizing apparatus
300 of one or more embodiments of the present invention.
[0156] First, the culture bag 100 is mounted on the mounting table,
and a prescribed amount of air is sealed. Then, liquid droplets
containing proteins being dissolved therein are injected.
Subsequently, by driving a servo motor 370 for moving in the Y-axis
direction, as shown in FIG. 13, the mounting table is rotationally
moved to the front side with the X-axis direction being the central
axis. As a result, the liquid droplets are allowed to move to the
end part at the front side of the culture bag 100.
[0157] Subsequently, by driving the servo motor 330 for moving in
the X-axis direction, the mounting table is allowed to move
rotationally in the lateral direction with the Y-axis direction
being a central axis. As a result, the liquid droplets are allowed
to move in the culture bag 100 laterally from the left end and the
right end.
[0158] Subsequently, by driving the servo motor 370 for moving in
the Y-axis direction, the mounting table is slightly moved
rotationally to the backside, whereby the liquid droplets are
slightly moved towards the backside. Then, by driving again the
servo motor 330 for moving in the X-axis direction to allow the
mounting table to move rotationally in a lateral direction, the
liquid droplets in the culture bag 100 are allowed to move
laterally from the left end to the right end. The above-mentioned
operation was repeated until the liquid droplets are moved to the
end part on the backside of the culture bag 100 and moved laterally
from the left end to the right end.
[0159] As mentioned above, by allowing proteins to be adsorbed to
the culture bag 100 by using the immobilizing apparatus 300,
immobilizing efficiency of proteins can be further improved.
[0160] The immobilizing apparatus 300 of one or more embodiments of
the present invention can be preferably used when proteins are
immobilized not only to the culture bag 100 but also to a flask or
other containers.
[0161] Next, an explanation will be made on a retention member used
in the immobilizing apparatus of one or more embodiments of the
present invention.
[0162] The retention member is a member for supporting the culture
container such that the bottom surface thereof forms a
semi-cylindrical shape, and is fixed on the mounting table or
formed integrally with the mounting table.
[0163] As shown in FIG. 14, the retention member 500 is provided
with a main body 510, an upper lid part 520 and a pressuring part
530.
[0164] The main body part 510 is provided with a semi-cylindrical
recess part 510-1 for retaining the culture bag 100 in a curved
state. The upper lid part 520 is attached to the main body part 510
by covering this recess part 510-1. The method for attaching the
upper lid part 520 to the main body part 510 is not particularly
restricted. For example, attaching by screwing can be given.
[0165] The upper lid part 520 is provided with a pressing part 530
for pressing the culture bag 100 from the outside. By allowing the
pressing part 530 to move downwardly, the culture bag 100 arranged
in the recess part 510-1 of the main body part 510 is pressed,
whereby the bottom surface of the culture bag 100 can be stabilized
in a semi-cylindrical shape.
[0166] The method for attaching the pressing part 530 to the upper
lid part 520 is not particularly restricted as long as the pressing
part 530 can be moved downwardly and the culture bag 100 can be
pressed. For example, the upper lid part 520 and the pressing part
530 are engaged by a screw.
[0167] The shape of the pressing part 530 is not limited to a rod
as shown in FIG. 14. The pressing part 530 may be in other shapes.
For example, it is preferable to keep the bottom surface of the
culture bag 100 in a semi-cylindrical shape by allowing the lower
end of the pressing part 530 to be branched or by allowing the
pressing part 530 to be semi-sphere more stably with the lower part
thereof to be curved.
[0168] FIG. 14 shows a state in which three pressing parts 530 are
attached to the upper lid part 520. The number of the pressing
parts 530 to be attached is not particularly limited. It may be
one, two or four or more.
[0169] FIG. 15 shows a state where the retention member 500 is
used, and shows how the culture bag 100 is retained by the
retention member 500.
[0170] At first, in the recess part 510-1 of the main body part 510
of the retention member 500, the culture bag 100 is arranged. At
this time, the culture bag is arranged such that the bottom surface
of the culture bag 100 becomes curved along the recess part
510-1.
[0171] Subsequently, the upper lid part 520 is attached to the main
body part 510, and the pressing part 530 is moved downward. As a
result, the bottom surface of the culture bag 100 can be kept in a
semi-cylindrical shape.
[0172] The retention member 500 is fixed to the mounting table of
the immobilizing apparatus 300 such that the direction of the
central axis of the semi-cylindrical shape of the recess part 510-1
becomes identical with the longitudinal direction of the mounting
table. Further, the retention member 500 can be formed integrally
with the mounting table with this positional relationship.
[0173] If the culture bag 100 is retained by such retention member
500 such that the bottom surface of the culture bag 100 to be kept
in a semi-cylindrical shape, and the liquid droplets in the culture
bag 100 are moved in the Y-axis direction, since the liquid
droplets are always positioned on the lowermost part of the recess
part 510-1 of the retention member 500, the movement of the liquid
droplets in the Y-axis direction can be precisely controlled.
[0174] As explained hereinabove, according to the method for
producing a culture container of one or more embodiments of the
present invention, it is possible to allow proteins to be
immobilized efficiently on the inner surface of the culture
container. As a result, the time required for immobilizing can be
shortened, and at the same time, adsorption efficiency to the
culture container can be improved, whereby a required amount of
immobilizing can be conducted with a small amount of proteins.
Accordingly, the amount of proteins discarded without being
immobilized can be reduced. Further, by using the immobilizing
apparatus of one or more embodiments of the present invention, it
is possible to further improve the efficiency of immobilizing of
proteins. In addition, by using the retention member, movement of
liquid droplets can be controlled more precisely, whereby
efficiency of immobilizing of proteins can be further improved.
EXAMPLES
[0175] Hereinbelow, the Examples and Comparative Examples of the
culture container and the method for culturing lymphocytes will be
explained with reference to FIGS. 6 and 7. FIG. 6 is a schematic
view showing the Examples and the Comparative Examples, and FIG. 7
is a graph showing the results of experiments conducted in the
Examples and the Comparative Examples. FIG. 6 shows a difference
between the Examples and the Comparative Examples, and the
enlargement of the volume of the culture part is omitted.
Experiment 1: Production of Culture Container
Example 1
[0176] By using Labo Plastmill (manufactured by Toyoseiki
Seisakusho, Co., Ltd.) as a plastic extrusion molding apparatus,
low-density polyethylene was extruded to form a 100 .mu.m-thick
film. Subsequently, by using an impulse sealer, a bag of 11
cm.times.22.5 cm (about 225 cm.sup.2) was prepared from this film.
This bag was sterilized with .gamma. rays to be used for an
experiment.
[0177] Subsequently, about 600 ml of air was sealed in this bag,
followed by sealing of 10 ml of a phosphate buffer solution
(manufactured by Lifetechnologies, Japan) containing 50 .mu.g of
anti-CD3 antibodies (manufactured by Miltenyi Biotec K.K.) being
dissolved therein. At this time, sealing was conducted such that
the liquid droplets of the phosphate buffer solution were brought
into contact with only one surface (immobilizing surface) of the
inner surfaces of the bag.
[0178] Then, the bag was swung by hands for 1 minute at 26.degree.
C., thereby to move the liquid droplets of the phosphate buffer
solution on the bottom surface in the bag at a speed of 10 m/min,
whereby a immobilized surface was formed in the bag to produce a
culture container.
[0179] In the meantime, the culture containers were produced in a
quantity of two. One of the culture containers was used for
measuring the concentration of antibodies that were immobilized and
the other was used for a test for culturing lymphocytes. The same
is applied to other Examples and Comparative Examples.
[0180] Measurement of the concentration of antibodies that were
immobilized was conducted as follows.
[0181] First, the liquid in the culture container was removed, and
500 .mu.l of a phosphate buffer solution (same as above) comprising
1% sodium dodecyl sulfate (manufactured by Sigma-Aldrich Japan) was
brought into contact with the immobilized surface, and the
container was allowed to stand for 30 minutes. Then, strong
vibration was applied by means of a PresentMixer (manufactured by
TAITEC Co., Ltd.), whereby adsorbed antibodies were peeled off. The
amount of the antibodies in the peeling liquid was measured by
means of Micro BCATM Protein Assay Kit (manufactured by
ThermoFisher Scientific K.K.), and adsorption concentration was
calculated by dividing with an immobilizing area.
[0182] As for the culture container in Example 1, anti-CD3
antibodies were immobilized on only one of the inner surfaces
thereof.
[0183] As a result of measurement of the concentration of
antibodies that were immobilized in the culture container in
Example 1, the concentration of the anti-CD3 antibodies in the
immobilized surface was 40 ng/cm.sup.2.
Example 2
[0184] A bag of 11 cm.times.22.5 cm (about 225 cm.sup.2) was
prepared in the same manner as in Example 1.
[0185] Subsequently, about 600 ml of air was sealed in this bag,
followed by sealing of 10 ml of a phosphate buffer solution
(manufactured by Lifetechnologies, Japan) containing 5 .mu.g of
anti-CD3 antibodies (manufactured by Miltenyi Biotec K.K.) being
dissolved therein. As for other points, the same procedures as
those in Example 1 were conducted to produce the culture container
in Example 2.
[0186] In the culture container in Example 2, anti-CD3 antibodies
were immobilized on only one of the inner surfaces thereof.
[0187] As the result of measurement of the concentration of the
immobilized antibodies in the culture container in Example 2, the
concentration of the anti-CD3 antibodies in the immobilized surface
was 11 ng/cm.sup.2.
Comparative Example 1
[0188] A bag of 11 cm.times.22.5 cm (about 225 cm.sup.2) was
prepared in the same manner as in Example 1.
[0189] Subsequently, in this bag, 10 ml of a phosphate buffer
solution (manufactured by Lifetechnologies, Japan) containing 50
.mu.g of anti-CD3 antibodies (manufactured by Miltenyi Biotec K.K.)
being dissolved therein was sealed. The liquid droplets of the
phosphate buffer solution were brought into contact with the upper
and lower surfaces of the bag, and the bag was allowed to stand at
26.degree. C. for 60 minutes. Then, the inside of the bag was
washed with 40 ml of the phosphate buffer solution three times,
whereby the culture container of Comparative Example 1 was
produced.
[0190] In the culture container in Comparative Example 1, anti-CD3
antibodies were immobilized on the both inner surfaces thereof.
[0191] As a result of measurement of the concentration of the
immobilized antibodies in the culture container in Comparative
Example 1, the concentration of the anti-CD3 antibodies in the
immobilized surface was 25 ng/cm.sup.2.
Comparative Example 2
[0192] A bag of 11 cm.times.22.5 cm (about 225 cm.sup.2) was
prepared in the same manner as in Example 1.
[0193] Subsequently, about 600 ml of air was sealed in this bag,
followed by sealing of 10 ml of a phosphate buffer solution
(manufactured by Lifetechnologies, Japan) containing 5 .mu.g of
anti-CD3 antibodies (manufactured by Miltenyi Biotec K.K.) being
dissolved therein. At this time, sealing was conducted such that
the liquid droplets of the phosphate buffer solution were brought
into contact with only one surface (immobilized surface) of the
inner surface of the bag.
[0194] Then, the bag was swung by hands for 1 minute at 26.degree.
C. to move the liquid droplets of the phosphate buffer solution on
the bottom surface in the bag at a speed of 10 m/min, whereby a
immobilized surface was formed in the bag. Further, the inside of
the bag was washed three times with 10 ml of the phosphate buffer
solution, thereby to produce the culture container of Comparative
Example 2.
[0195] In the culture container in Comparative Example 2, anti-CD3
antibodies were immobilized on only one inner surface thereof.
[0196] As a result of measurement of the concentration of the
immobilized antibodies in the culture container in Comparative
Example 2, the concentration of the anti-CD3 antibodies in the
immobilized surface was 8 ng/cm.sup.2.
Experiment 2: Culture of Lymphocytes
(Activation Step)
[0197] Each of the culture containers obtained in Examples 1 and 2
and Comparative Examples 1 and 2 of Experiment 1 were partitioned
by means of a clip such that the culture part became 5 cm.times.11
cm.
[0198] Subsequently, 5.8.times.10.sup.4 human mononuclear cells
(manufactured by Cell Applications, Inc.) were suspended in an
ALyS505N-7 culture medium containing 10% fetal bovine serum
(manufactured by Cell Science & Technology, Inc.) and 4 ml of
the suspension was sealed in the culture container. The culture
container was allowed to stand at 37.degree. C. for 75 hours,
thereby to activate lymphocytes.
[0199] In Examples 1 and 2 and Comparative Example 2, activation
was conducted with the immobilized surface of the culture container
facing downward. As for the culture container in Comparative
Example 1, as mentioned above, the same amount of the anti-CD3
antibodies were immobilized on the both upper and lower surfaces of
the inside thereof. Therefore, there is no distinction between the
upper surface and the lower surface; that is, there is no
distinction between the immobilized surface and the non-immobilized
surface.
(Proliferation Step)
[0200] To the culture container that had completed the activation
step, 4 ml of the above-mentioned culture medium was added. The
culture container was inverted upside down, and in that state, the
culture container was allowed to stand at 37.degree. C. for 26
hours, thereby to proliferate the lymphocytes (first proliferation
step in the second embodiment).
[0201] Subsequently, the clip was removed to enlarge the volume of
the culture part of the culture container (volume enlargement step
in the second embodiment). Then, 20 ml of the above-mentioned
culture medium was added, and the container was allowed to stand at
37.degree. C. for 62 hours to continue the proliferation of the
lymphocytes (second proliferation step in the second
embodiment).
[0202] At the timing of each operation after the start of the
culture (after 75 hours, 75+26 (=101) hours, 75+26+62 (=163) hours,
and 18 hours after the removal of the clip (75+26+18 (=119)), the
number of the lymphocytes was counted. The results are shown in
FIG. 7.
[0203] As shown in FIG. 7, in Examples 1 and 2 where 40 ng/cm.sup.2
and 11 ng/cm.sup.2 of anti-CD3 antibodies were immobilized only on
one surface of the culture container, it became possible to
significantly proliferate lymphocytes during the proliferation
step. At this time, no significant difference in proliferation
efficiency was observed between the immobilizing concentration of
40 ng/cm.sup.2 (Example 1) and 11 ng/cm.sup.2 (Example 2).
[0204] In Comparative Example 1 in which anti-CD3 antibodies were
immobilized to the both surfaces of the culture container, it could
be understood that the proliferation efficiency of lymphocytes in
proliferation step was significantly lowered as compared with
Examples 1 and 2.
[0205] Further, in Comparative Example 2 in which the concentration
of the anti-CD3 antibodies to be immobilized was slightly reduced
than that in Example 2, most of lymphocytes could not be
proliferated during the proliferation step. From this result, it
can be understood that it is preferable to allow the concentration
of the anti-CD3 antibodies to be immobilized on the immobilizing
surface to be about 10 ng/cm.sup.2 or more.
[0206] Subsequently, the Examples and the Comparative Examples of
the method for producing a culture container according to one or
more embodiments of the present invention will be explained with
reference to FIGS. 16 to 21. FIGS. 16, 18 and 20 are schematic
views showing the Examples and the Comparative Examples, and FIGS.
17, 19 and 21 are graphs showing the results of the experiments in
the Examples and the Comparative Examples.
Experiment 3: Production of Culture Bag in which Antibodies are
Immobilized
Example 3
[0207] By using a Labo Plastmill (manufactured by Toyo Seiki Kogyo
Co., Ltd.) as a plastic extrusion molding apparatus, low-density
polyethylene was extruded to form a 100 .mu.m-thick film.
Subsequently, by using an impulse sealer, a bag of 11 cm.times.22.5
cm (about 225 cm.sup.2) was prepared from this film. This bag was
sterilized with .gamma. rays to be used for an experiment.
[0208] Subsequently, this bag was mounted on the supporting table,
and about 600 ml of air was sealed in this bag, followed by sealing
of 10 ml of a phosphate buffer solution (manufactured by
Lifetechnologies, Japan) containing 50 .mu.g of anti-CD3 antibodies
(manufactured by Miltenyi Biotec K.K.) being dissolved therein. At
this time, sealing was conducted such that the liquid droplets of
the phosphate buffer solution were brought into contact with only
the bottom surface of the inner surface of the bag.
[0209] Then, the bag was swung by hands for 1 minute at 26.degree.
C., thereby to move the liquid droplets of the phosphate buffer
solution on the bottom surface in the bag at a speed of 10 m/min.
As a result, antibodies were adsorbed to the bottom surface in the
bag, whereby a culture bag in which the antibodies were immobilized
was produced.
[0210] The measurement of the antibodies that were immobilized was
conducted as follows:
[0211] First, the liquid in the culture container was removed, and
500 .mu.l of a phosphate buffer (same as above) comprising 1%
sodium dodecyl phosphate (manufactured by Sigma-Aldrich Japan) was
brought into contact with the immobilized surface, and the
container was allowed to stand for 30 minutes. Then, strong
vibration was applied by means of a PresentMixer (manufactured by
TAITEC Co., Ltd.), whereby adsorbed antibodies were peeled off. The
amount of the antibodies in the peeling liquid was measured by
means of Micro BCATM Protein Assay Kit (manufactured by
ThermoFisher Scientific K.K.). The amount of proteins adsorbed per
unit area (hereinafter referred to as the adsorption concentration)
was calculated by dividing the amount of antibodies by the area
where the antibodies were immobilized.
[0212] In the culture container of Example 3, anti-CD3 antibodies
were immobilized only on one surface (bottom surface) of the inside
thereof. As a result of measuring the concentration of antibodies
that had been immobilized in the culture container in Example 3,
the concentration of the anti-CD3 antibodies adsorbed was found to
be 41.5 ng/cm.sup.2.
Example 4
[0213] A bag of 11 cm.times.22.5 cm (about 225 cm.sup.2) was
prepared in the same manner as in Example 3.
[0214] Subsequently, about 600 ml of air was sealed in this bag,
followed by sealing of 10 ml of a phosphate buffer solution
(manufactured by Lifetechnologies, Japan) containing 50 .mu.g of
anti-CD3 antibodies (manufactured by Miltenyi Biotec K.K.) being
dissolved therein.
[0215] Then, the bag was swung by hands for 2.5 minutes at
26.degree. C., thereby to move the liquid droplets of the phosphate
buffer solution on the bottom surface in the bag at a speed of 10
m/min, thereby to allow antibodies to be adsorbed to the bottom
surface in the bag. Subsequently, the bag was turned upside down,
and then again swung by hand for 2.5 minutes, thereby to allow the
liquid droplets to move on the bottom surface in the bag (upper
surface before turning upside down) at a speed of 10 m/min. As a
result, the antibodies were adsorbed on the both surfaces of the
inner wall of the bag, whereby a culture bag in which the
antibodies were immobilized was produced.
[0216] In the culture container of Example 4, the anti-CD3 bodies
were immobilized on the both surfaces of the inside thereof. As a
result of measuring the concentration of the antibodies that were
immobilized in the culture container of Example 4, the
concentration of anti-CD3 antibodies that were adsorbed was 31.2
ng/cm.sup.2.
Comparative Example 3
[0217] A bag of 11 cm.times.22.5 cm (about 225 cm.sup.2) was
prepared in the same manner as in Example 3.
[0218] Subsequently, without sealing air in this bag, 10 ml of a
phosphate buffer solution (manufactured by Lifetechnologies, Japan)
containing 50 .mu.g of anti-CD3 antibodies (manufactured by
Miltenyi Biotec K.K.) being dissolved therein was sealed, and this
phosphate buffer solution was allowed to be in contact with both
the upper and lower surfaces of the bag, and the bag was allowed to
stand at 26.degree. C. for 60 minutes. The inside of the bag was
washed three times with 40 ml of the phosphate buffer solution,
whereby a culture bag in which the antibodies were immobilized was
produced.
[0219] In the culture container of Comparative Example 3, the
anti-CD3 antibodies were immobilized on the both surfaces of the
inside thereof. As a result of measuring the concentration of the
antibodies that were immobilized in the culture container in
Comparative Example 3, it was found that the concentration of the
anti-CD3 antibodies that were adsorbed was 26.1 ng/cm.sup.2.
Comparative Example 4
[0220] A bag of 11 cm.times.22.5 cm (about 225 cm.sup.2) was
prepared in the same manner as in Example 3.
[0221] Subsequently, without sealing air in this bag, 10 ml of a
phosphate buffer solution (manufactured by Lifetechnologies, Japan)
containing 50 .mu.g of anti-CD3 antibodies (manufactured by
Miltenyi Biotec K.K.) being dissolved therein was sealed. Then, the
bag was swung by hands at 26.degree. C. for 5 minutes, whereby the
liquid droplets of the phosphate buffer solution were allowed to
move in the bag at a speed of 10 m/min. As a result, the antibodies
were adsorbed to the inner surface of the bag. Further, the inside
of the bag was washed three times with 10 ml of the phosphate
buffer solution, whereby a bag in which the antibodies were
immobilized was produced.
[0222] In the culture container of Comparative Example 4, the
anti-CD3 antibodies were immobilized on the both surfaces of the
inside thereof. As a result of measuring the concentration of the
antibodies that were immobilized in the culture container in
Comparative Example 4, it was found that the concentration of the
anti-CD3 antibodies that were adsorbed was 23.8 ng/cm.sup.2.
[0223] As shown in FIG. 17, efficiencies of adsorption (adsorption
concentration.times.225 cm.sup.2/50000 ng.times.100) of the
antibodies on the culture container bottom surface per area of the
antibodies in Example 3, Example 4, Comparative Example 3 and
Comparative Example 4 were 19%, 14%, 12% and 11%, respectively.
[0224] That is, according to the method in Example 3, although the
immobilizing time was 1 minute, the efficiency of adsorption was
increased by about 60% as compared with the results of Comparative
Example 3 in which the immobilizing was conducted for 60
minutes.
[0225] On the other hand, in Comparative Example 4 in which the
antibodies were immobilized by moving the liquid droplets without
sealing a gas in the culture container, the adsorption efficiency
was small as compared with Comparative Example 3.
[0226] The reason for the fact that the adsorption efficiency in
Example 4 was smaller than that in Example 3 is thought that the
absorption area in Example 4 was twice as large as the absorption
area of Example 3.
[0227] Further, when the results of Example 4 are compared with the
results of Comparative Example 4, it can be understood that the
adsorption efficiency could be improved by about 30% by allowing
antibodies to be immobilized after incorporating a gas in the
culture container while moving liquid droplets.
[0228] As mentioned above, according to the method for producing a
culture container of one or more embodiments of the present
invention, it is possible to allow a larger amount of antibodies to
be immobilized in the culture container for a shorter period of
time. In addition, it is possible to allow a larger amount of
antibodies to be immobilized on a culture container by using
antibodies in an amount smaller than that used in the conventional
method.
Experiment 4: Production of Flask in which Antibodies are
Immobilized
Example 5
[0229] In a suspension culture flask 800 (manufactured by Sumitomo
Bakelite Co., Ltd., made of polystyrene having a bottom surface
area of 225 cm.sup.2), 10 ml of a phosphate buffer solution
(manufactured by Lifetechnologies, Japan) containing 50 .mu.g of
anti-CD3 antibodies (manufactured by Miltenyi Biotec K.K.) being
dissolved therein was sealed in the state of liquid droplets. The
flask was swung by hands at 26.degree. C. for 5 minutes, the liquid
droplets of the phosphate buffer solution were allowed to move on
the bottom surface in the bag at a speed of 10 m/min, and were
adsorbed on the bottom surface of the flask, whereby a flask in
which antibodies were immobilized was produced. The concentration
of the anti-CD3 antibodies in the flask in which the antibodies
were immobilized in Example 5 was 27.9 ng/cm.sup.2.
Comparative Example 5
[0230] In a suspension culture flask 800 (manufactured by Sumitomo
Bakelite Co., Ltd., made of polystyrene having a bottom surface
area of 225 cm.sup.2), 10 ml of a phosphate buffer solution
(manufactured by Lifetechnologies, Japan) containing 50 .mu.g of
anti-CD3 antibodies (manufactured by Miltenyi Biotec K.K.) being
dissolved therein was put, and the solution of the antibodies was
spread over the entire surface. The flask was allowed to stand at
26.degree. C. for 60 hours. As a result, the antibodies were
adsorbed on the bottom surface of the flask, whereby a flask in
which the antibodies were immobilized was produced. The
concentration of the anti-CD3 antibodies in the flask in which the
antibodies were immobilized in Comparative Example 5 was 27.6
ng/cm.sup.2.
Comparative Example 6
[0231] In a suspension culture flask 800 (manufactured by Sumitomo
Bakelite Co., Ltd., made of polystyrene having a bottom surface
area of 225 cm.sup.2), 10 ml of a phosphate buffer solution
containing (manufactured by Lifetechnologies, Japan) 50 .mu.g of
anti-CD3 antibodies (manufactured by Miltenyi Biotec K.K.) being
dissolved therein was put, and the solution of the antibodies was
spread over the entire surface. The flask was allowed to stand at
26.degree. C. for 5 minutes. As a result, the antibodies were
adsorbed on the bottom surface of the flask, whereby a flask in
which the antibodies were immobilized was produced. In the flask of
Comparative Example 6 in which the anti-CD3 antibodies were
immobilized, the concentration of the antibodies was 21.8
ng/cm.sup.2.
[0232] As shown in FIG. 19, efficiencies of adsorption of the
antibodies per area of the flask in Example 5, Comparative Example
5 and Comparative Example 6 was 13%, 12% and 10%, respectively.
[0233] Namely, as shown in Comparative Example 5, in the
conventional method for immobilizing antibodies in which the
antibodies are allowed to stand, the efficiency of absorption was
12% after the lapse of 60 minutes from the start of immobilizing.
On the other hand, as shown in Example 5, in the method for
producing a culture container according to one or more embodiments
of the present invention, the efficiency of absorption could be 13%
after the lapse of 5 minutes from the start of immobilizing.
[0234] Accordingly, it has been revealed that, according to the
method for producing a culture container of one or more embodiments
of the present invention, even if proteins are immobilized in a
flask, an equivalent amount of proteins can be immobilized for a
shorter period of time as compared with the conventional
methods.
Experiment 5: Production of Culture Bag in which Fibronectin is
Immobilized
Example 6
[0235] A bag of 11 cm.times.22.5 cm (about 225 cm.sup.2) was
prepared in the same manner as in Example 3.
[0236] Subsequently, about 600 ml of air was sealed in this bag,
and then 10 ml of a phosphate buffer solution (manufactured by
Lifetechnologies, Japan) containing 50 .mu.g of human plasma
fibronectin (manufactured by Sigma-Aldrich Japan) being dissolved
therein was continuously sealed in the liquid droplet state.
[0237] The bag was swung by hands for 1 minute at 26.degree. C.,
whereby the liquid droplets of the phosphate buffer solution were
allowed to move on the bottom surface in the bag at a speed of 10
m/min. As a result, the fibronectin was adsorbed to the bottom
surface of the bag, whereby a bag in which fibronectin was
immobilized was produced.
[0238] In the culture container of Example 6, fibronectin was
immobilized only on one side (bottom surface) in the inside
thereof. As a result of measuring the concentration of immobilized
fibronectin for the culture container of Example 6, it was found
that the concentration of fibronectin adsorbed was 53.6
ng/cm.sup.2.
Comparative Example 7
[0239] A bag of 11 cm.times.22.5 cm (about 225 cm.sup.2) was
prepared in the same manner as in Example 3.
[0240] Subsequently, without sealing air in this bag, 10 ml of a
phosphate buffer solution (manufactured by Lifetechnologies, Japan)
containing 50 .mu.g of human plasma fibronectin (manufactured by
Sigma-Aldrich Japan) being dissolved therein was sealed. The
droplets of the phosphate buffer solution were brought into contact
with the both upper and lower surfaces in the bag, and the bag was
allowed to stand at 26.degree. C. for 60 minutes. The inside of the
bag was washed three times with 40 ml of the phosphate buffer
solution, whereby a culture bag in which fibronectin was
immobilized was produced.
[0241] In the culture container of Comparative Example 7,
fibronectin was immobilized on both surfaces in the inside thereof.
As a result of measuring the concentration of immobilized
fibronectin for the culture container of Comparative Example 7, it
was found that the concentration of fibronectin adsorbed was 30.7
ng/cm.sup.2.
[0242] As shown in FIG. 21, efficiencies of adsorption of the
fibronectin per area of the culture container in Example 6 and
Comparative Example 7 were 24% and 14%, respectively.
[0243] Namely, when fibronectin is used as protein, according to
the method for producing a culture container of one or more
embodiments of the present invention, it is possible to allow
fibronectin to be immobilized in an absorption efficiency that is
higher by 70% than that of the conventional methods.
[0244] Therefore, according to the method for producing a culture
container of one or more embodiments of the present invention, it
has been revealed that proteins other than antibodies can be
immobilized efficiently for a short period of time.
Experiment 6: Production of a Culture Bag in which Antibodies are
Immobilized by Using an Immobilizing Apparatus
Example 7
[0245] A bag of 8 cm.times.20 cm (about 160 cm.sup.2) was prepared
in the same manner as in Example 3.
[0246] Subsequently, this bag was mounted on the retention member
in the immobilizing apparatus. By using the pressing part, the bag
was fixed such that the bottom surface thereof became
semi-cylindrical. Subsequently, about 280 ml of air was sealed, and
2 ml of a phosphate buffer solution (manufactured by
Lifetechnologies, Japan) containing 10 .mu.g of anti-CD3 antibodies
(manufactured by Miltenyi Biotec K.K.) being dissolved therein was
continuously sealed in the state of liquid droplets.
[0247] As shown in FIG. 13, the mounting table in the immobilizing
apparatus was inclined by about -50.degree. in the Y-axis direction
(rotational movement with the X-axis direction being a central
axis) to allow the liquid droplets to move towards the end part of
the culture bag. Subsequently, the mounting table was rotated in
the X-axis direction by -10.degree. to 10.degree. (rotational
movement with the Y-axis direction being a central axis) to allow
the liquid droplets to move towards the X-axis direction, whereby
the liquid droplets were moved laterally in the culture bag
reciprocally from the left end to the right end.
[0248] Subsequently, the mounting table was rotated by 10.degree.
in the Y-axis direction to allow the liquid droplets to move
slightly towards the backside. Subsequently, the mounting table was
again rotated in the X-axis direction by -10.degree. to 10.degree.
to allow the liquid droplets to move in the X-axis direction,
whereby the liquid droplets were laterally moved in the culture bag
reciprocally from the left end to the right end.
[0249] The operations mentioned above were repeated until the
rotation angle in the Y-axis direction (X-axis rotation angle)
became 50.degree., whereby the liquid droplets were moved over the
entire bottom surface of the culture bag. Taking this operation as
one cycle, four cycles were conducted (immobilizing time: 3.5
minutes, temperature: 26.degree. C.), and the amount of the
antibodies that were immobilized was measured.
[0250] In the culture container of Example 7, the anti-CD3
antibodies were immobilized on only one surface in the inside
thereof. As a result of measuring the concentration of immobilized
antibodies for the culture container in Example 7, it was found
that the concentration of the anti-CD3 antibodies adsorbed was 30.1
ng/cm.sup.2.
[0251] Accordingly, the efficiency of adsorption of the antibodies
per area of the culture container in Example 7 (adsorption
concentration.times.160 cm.sup.2/10000 ng.times.100) was 48%. This
adsorption efficiency was 4 times as large as the adsorption
efficiency of the culture container in Comparative Example 3
(12%).
[0252] As mentioned above, by producing a culture container by
using the immobilizing apparatus of one or more embodiments of the
present invention, a culture container that exhibits high
adsorption efficiency that cannot be realized by conventional
culture containers can be obtained.
[0253] The present invention is not limited to the above-mentioned
embodiments or examples, and it is needless to say that various
modifications are possible within the scope of the present
invention.
[0254] For example, the size of a culture container, the type of
lymphocytes, the type of a culture medium, the type of proteins, or
the like can be appropriately changed such that they are different
from those in the Examples.
INDUSTRIAL APPLICABILITY
[0255] One or more embodiments of the present invention can be
preferably utilized for culturing a large amount of lymphocytes by
using a single culture container while eliminating troublesomeness
in transfer of activated cells or risk of contamination. In
addition, one or more embodiments of the present invention can be
preferably utilized for producing a culture container in which
proteins are immobilized on the inner surface thereof efficiently
for a short period of time.
EXPLANATION OF REFERENTIAL NUMERALS
[0256] 10. Culture container [0257] 11. Immobilized surface [0258]
12. Non-immobilized surface [0259] 13. Tube [0260] 20. Antibodies
[0261] 30. Lymphocytes [0262] 40. Culture liquid [0263] 50.
Partitioning member [0264] 60. Outer covering container [0265] 70.
Packaging container [0266] 100. Culture bag [0267] 110. Immobilized
surface [0268] 120. Non-immobilized surface [0269] 130. Tube [0270]
200. Proteins [0271] 300. Immobilizing apparatus [0272] 310.
Supporting table for moving in the X-axis direction [0273] 320.
Supporting column [0274] 330. Servo motor for moving in the X-axis
direction [0275] 340. Gear box for moving in the X-axis direction
[0276] 350. Origin limit detection senor for the Y axis [0277] 360.
Supporting table for moving in the Y-axis direction [0278] 370.
Servo motor for moving in the Y-axis direction [0279] 380. Gear box
for moving in the Y-axis direction [0280] 390. Origin limit
detection sensor for the X axis [0281] 400. Base stand [0282] 500.
Retention member [0283] 510. Main body part [0284] 510-1. Recess
part [0285] 520. Upper lid part [0286] 530. Pressing part
* * * * *