U.S. patent application number 16/485409 was filed with the patent office on 2020-07-30 for cell processing system and cell processing device.
The applicant listed for this patent is Koji I PEACE, INC. TANABE. Invention is credited to Ryoji HIRAIDE, Koji TANABE.
Application Number | 20200239823 16/485409 |
Document ID | 20200239823 / US20200239823 |
Family ID | 1000004795867 |
Filed Date | 2020-07-30 |
Patent Application | download [pdf] |
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United States Patent
Application |
20200239823 |
Kind Code |
A1 |
TANABE; Koji ; et
al. |
July 30, 2020 |
CELL PROCESSING SYSTEM AND CELL PROCESSING DEVICE
Abstract
A cell processing system comprising an enclosure 601, an outer
enclosure 701 that envelops the enclosure 601, an intake air
purification filter 602 provided in the enclosure 601, that
purifies gas that has been drawn in from outside the enclosure 601,
a circulating apparatus, inside the outer enclosure 701, that
circulates gas inside and outside the enclosure 601 in such a
manner that gas in the outer enclosure 701 is drawn into the
enclosure 601 through the intake air purification filter 602 and
gas inside the enclosure 601 is discharged into the outer enclosure
701, and a cell processing apparatus for processing of cells,
disposed inside the enclosure 601.
Inventors: |
TANABE; Koji; (Palo Alto,
CA) ; HIRAIDE; Ryoji; (Kyoto-shi, Kyoto, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
TANABE; Koji
I PEACE, INC. |
Palo Alto
Palo Alto |
CA
CA |
US
US |
|
|
Family ID: |
1000004795867 |
Appl. No.: |
16/485409 |
Filed: |
February 27, 2017 |
PCT Filed: |
February 27, 2017 |
PCT NO: |
PCT/JP2017/007577 |
371 Date: |
August 12, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 25/00 20130101;
C12M 33/14 20130101; C12M 23/34 20130101; C12M 29/04 20130101 |
International
Class: |
C12M 1/00 20060101
C12M001/00; C12M 1/26 20060101 C12M001/26; C12M 1/12 20060101
C12M001/12 |
Claims
1-116. (canceled)
117. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises a mononuclear cell separating unit, disposed inside the
embedding member, that separates mononuclear cells from blood, the
cell processing apparatus further comprises a separating agent
storing unit, disposed outside the embedding member, that stores a
separating agent for separation of mononuclear cells, and the
communicating solution-feeding channel comprises a separating agent
solution-feeding channel that allows communication between the
separating agent storing unit and the mononuclear cell separating
unit.
118. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises: a mononuclear cell separating unit, disposed inside the
embedding member, that separates mononuclear cells from blood, a
factor introducing device, disposed inside the embedding member,
that introduces an inducing factor into cells to prepare inducing
factor-introduced cells, and a preintroduction cell
solution-feeding channel, disposed inside the embedding member,
that allows communication between the mononuclear cell separating
unit and the factor introducing device.
119. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises: a mononuclear cell purifying filter, disposed inside the
embedding member, a factor introducing device, disposed inside the
embedding member, that introduces an inducing factor into cells to
prepare inducing factor-introduced cells, and a preintroduction
cell solution-feeding channel, disposed inside the embedding
member, that allows communication between the mononuclear cell
purifying filter and the factor introducing device.
120. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises an initializing culturing vessel, disposed inside the
embedding member, that cultures inducing factor-introduced cells
into which an inducing factor has been introduced, the cell
processing apparatus further comprises a blood cell culture medium
storing unit, disposed outside the embedding member, that stores
blood cell culture medium, and the communicating solution-feeding
channel comprises a culture medium solution-feeding channel that
allows communication between the blood cell culture medium storing
unit and the initializing culturing vessel.
121. The cell processing apparatus according to claim 120, wherein
the blood cell culture medium is supplied to the initializing
culturing vessel at a prescribed timing.
122. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises an initializing culturing vessel, disposed inside the
embedding member, that cultures inducing factor-introduced cells
into which an inducing factor has been introduced, the cell
processing apparatus further comprises a stem cell culture medium
storing unit, disposed outside the embedding member, that stores
stem cell culture medium, and the communicating solution-feeding
channel comprises a culture medium solution-feeding channel that
allows communication between the stem cell culture medium storing
unit and the initializing culturing vessel.
123. The cell processing apparatus according to claim 122, wherein
the stem cell culture medium is supplied to the initializing
culturing vessel at a prescribed timing.
124. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises an initializing culturing vessel, disposed inside the
embedding member, that cultures inducing factor-introduced cells
into which an inducing factor has been introduced, the cell
processing apparatus further comprises a waste liquid storage
section, disposed outside the embedding member, that stores waste
liquid, and the communicating solution-feeding channel comprises a
waste liquid solution-feeding channel that allows communication
between the initializing culturing vessel and the waste liquid
storage section.
125. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises an initializing culturing vessel, disposed inside the
embedding member, that cultures inducing factor-introduced cells
into which an inducing factor has been introduced, and the
initializing culturing vessel comprises: a suspension culture
vessel, which comprises: a dialysis tube in which the inducing
factor-introduced cells and culture medium are to be accommodated,
and a vessel in which the dialysis tube is to be placed, with the
culture medium accommodable around the periphery of the dialysis
tube.
126. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises: a factor introducing device, disposed inside the
embedding member, that introduces an inducing factor into cells to
prepare inducing factor-introduced cells, an initializing culturing
vessel, disposed inside the embedding member, that cultures the
inducing factor-introduced cells, and an introduced cell
solution-feeding channel, disposed inside the embedding member,
that allows communication between the factor introducing device and
the initializing culturing vessel.
127. The cell processing apparatus according to claim 126, which
further comprises a driving unit disposed outside the embedding
member, for feeding of a solution in the introduced cell
solution-feeding channel.
128. The cell processing apparatus according to claim 127, wherein
the driving unit is connected to an outer wall of the embedding
member.
129. The cell processing apparatus according to claim 127, which is
provided with a slave unit in the embedding member, to which
driving force from the driving unit is transmitted, with the slave
unit being connected to the introduced cell solution-feeding
channel.
130. The cell processing apparatus according to claim 129, wherein
the driving unit and the slave unit are connected by magnetic
force.
131. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises an amplifying culturing vessel, disposed inside the
embedding member, that carries out amplifying culturing of a
plurality of cell masses comprising established stem cells, the
cell processing apparatus further comprises a stem cell culture
medium storing unit, disposed outside the embedding member, that
stores stem cell culture medium, and the communicating
solution-feeding channel comprises a culture medium
solution-feeding channel that allows communication between the stem
cell culture medium storing unit and the amplifying culturing
vessel.
132. The cell processing apparatus according to claim 131, wherein
the stem cell culture medium is supplied to the amplifying
culturing vessel at a prescribed timing.
133. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises: an initializing culturing vessel, disposed inside the
embedding member, that cultures cells, an amplifying culturing
vessel, disposed inside the embedding member, that carries out
amplifying culturing of a plurality of cell masses comprising
established stem cells, and an introduced cell solution-feeding
channel, disposed inside the embedding member, that allows
communication between the initializing culturing vessel and the
amplifying culturing vessel.
134. The cell processing apparatus according to claim 133, which
further comprises a driving unit disposed outside the embedding
member, for feeding of a solution in the introduced cell
solution-feeding channel.
135. The cell processing apparatus according to claim 134, wherein
the driving unit is connected to an outer wall of the embedding
member.
136. The cell processing apparatus according to claim 134, which is
provided with a slave unit in the embedding member, to which
driving force from the driving unit is transmitted, with the slave
unit being connected to the introduced cell solution-feeding
channel.
137. The cell processing apparatus according to claim 133, wherein
the cell processing instrument further comprises a cell
dissociater, disposed inside the embedding member and provided to
the introduced cell solution-feeding channel, for dissociation of
the cell masses.
138. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises: an amplifying culturing vessel, disposed inside the
embedding member, that carries out amplifying culturing of a
plurality of cell masses comprising established stem cells, a
solution exchanger that exchanges a solution surrounding cells, and
an introduced cell solution-feeding channel, disposed inside the
embedding member, that allows communication between the amplifying
culturing vessel and the solution exchanger.
139. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, wherein the cell processing instrument
comprises a cell mass dissociater that dissociates cell masses.
140. The cell processing apparatus according to claim 117, which
further comprises a driving unit disposed outside the embedding
member, for feeding of a solution in the communicating
solution-feeding channel.
141. The cell processing apparatus according to claim 140, wherein
the driving unit is connected to an outer wall of the embedding
member.
142. The cell processing apparatus according to claim 140, which is
provided with a slave unit in the embedding member, to which
driving force from the driving unit is transmitted, with the slave
unit being connected to the communicating solution-feeding
channel.
143. A cell processing apparatus comprising: a cell processing
instrument that processes cells, an embedding member that embeds
the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member, and a tubing pump, disposed inside the
embedding member, for feeding of a solution in the communicating
solution-feeding channel, wherein the cells are cultured by the
cell processing instrument.
Description
FIELD
[0001] The present invention relates to cell technology, and
specifically it relates to a cell processing system and a cell
processing apparatus.
BACKGROUND
[0002] Embryonic stem cells (ES cells) are stem cells established
from early embryos of human or mice. ES cells are pluripotent,
being capable of differentiating into all cells in the body. At the
current time, human ES cells are able to be used in cell
transplantation therapy for numerous diseases including Parkinson's
disease, juvenile onset diabetes and leukemia. However, certain
barriers exist against transplantation of ES cells. In particular,
transplantation of ES cells can provoke immunorejection similar to
the rejection encountered after unsuccessful organ transplantation.
Moreover, there are many ethical considerations as well as critical
and dissenting opinions against the use of ES cell lines that have
been established by destruction of human embryos.
[0003] It was against this background that Professor Shinya
Yamanaka of Kyoto University successfully established a line of
induced pluripotent stem cells (iPS cells) by transferring four
genes: Oct3/4, Klf4, c-Myc and Sox2, into somatic cells. For this,
Professor Yamanaka received the Nobel Prize in Physiology or
Medicine in 2012 (see PTLs 1 and 2, for example). iPS cells are
ideal pluripotent cells which are free of issues of rejection or
ethical problems. Therefore, iPS cells are considered promising for
use in cell transplantation therapy.
CITATION LIST
Patent Literature
[0004] PTL 1: Japanese Patent Publication No. 4183742
[0005] PTL 2: Japanese Unexamined Patent Publication No.
2014-114997
SUMMARY
Technical Problem
[0006] Induced stem cells such as iPS cells are established by
introducing inducing factors such as genes into cells which are
then subjected to amplifying culturing and cryopreservation. Stem
cells have conventionally been produced manually by technicians in
a cleanroom. Blood and inducing factors used for production of stem
cells must be prevented from coming into contact with humans.
Furthermore, because cells can potentially be infected with viruses
and other pathogens, it is essential to keep the cells contained to
prevent them from being released to the exterior. For example, when
cells infected with hepatitis virus or human immunodeficiency virus
(HIV) diffuse out they can have fatal effects on humans and
animals. It is therefore necessary to prevent release of blood,
inducing factors, preinduction cells and induced cells out of
cleanrooms. In addition, iPS cells from only a single individual
are prepared in the same cleanroom over a given period of time in
order to prevent cross-contamination with iPS cells of other
individuals.
[0007] It is therefore an object of the present invention to
provide a cell processing system and a cell processing apparatus
that allow treatment of cells without contamination of the
surroundings.
Solution to Problem
[0008] According to one aspect of the invention there is provided a
cell processing system comprising an enclosure, an outer enclosure
that envelops the enclosure, an intake air purification filter
provided in the enclosure, that purifies gas that has been drawn in
from outside the enclosure, a circulating apparatus, inside the
outer enclosure, that circulates gas inside and outside the
enclosure in such a manner that gas in the outer enclosure is drawn
into the enclosure through the intake air purification filter and
gas inside the enclosure is discharged into the outer enclosure,
and a cell processing apparatus for processing of cells, disposed
inside the enclosure.
[0009] In this cell processing system, a pressure adjustment hole
may be provided in the outer enclosure.
[0010] In this cell processing system, the circulating apparatus
may comprise a gas discharger, provided in the enclosure, that
draws in gas from the enclosure and discharges purified gas out of
the enclosure.
[0011] In this cell processing system, the gas discharger may
comprise an exhaust system that exhausts gas inside the enclosure
to the exterior of the enclosure, and an exhaust purification
filter that purifies gas that has been drawn in by the exhaust
system.
[0012] In this cell processing system, the exhaust purification
filter may be situated upstream from the exhaust system.
[0013] This cell processing system may further comprise a shielding
member that can be attached to the exhaust purification filter so
as to shield the exhaust purification filter.
[0014] In this cell processing system, the shielding member may
comprise an enclosure side shielding member that can be attached to
the exhaust purification filter so as to shield the exhaust
purification filter from the enclosure interior, and an exhaust
system side shielding member that can be attached to the exhaust
purification filter so as to shield the exhaust purification filter
from the exhaust system.
[0015] This cell processing system may further comprise a
sterilizing device that sterilizes the exhaust purification
filter.
[0016] This cell processing system may still further comprise a
second exhaust purification filter disposed downstream from the
exhaust system.
[0017] This cell processing system may still further comprise a
shielding member that can be attached to the second exhaust
purification filter so as to shield the second exhaust purification
filter.
[0018] In this cell processing system, the shielding member may
further comprise an exhaust system side shielding member that can
be attached to the second exhaust purification filter, so as to
shield the second exhaust purification filter from the exhaust
system.
[0019] This cell processing system may further comprise a
sterilizing device that sterilizes the second exhaust purification
filter.
[0020] In this cell processing system, the circulating apparatus
may comprise an injector, provided in the enclosure, that draws in
gas that has been purified by the intake air purification filter,
from out of the enclosure.
[0021] In this cell processing system, the intake air purification
filter may be situated downstream from the injector.
[0022] This cell processing system may further comprise a shielding
member that can be attached to the intake air purification filter
so as to shield the intake air purification filter.
[0023] In this cell processing system, the shielding member may
comprise an enclosure side shielding member that can be attached to
the intake air purification filter so as to shield the intake air
purification filter from the enclosure interior, and an injector
side shielding member that can be attached to the intake air
purification filter so as to shield the intake air purification
filter from the injector.
[0024] This cell processing system may further comprise a
sterilizing device that sterilizes the intake air purification
filter.
[0025] In this cell processing system, the enclosure interior may
be at negative pressure compared to the enclosure exterior.
[0026] Stem cells may be cultured in the cell processing apparatus
of this cell processing system.
[0027] This cell processing system may still further comprise a
sterilizing device that sterilizes the space in which the cell
processing apparatus is disposed inside the enclosure.
[0028] This cell processing system may still further comprise a
temperature regulating device that regulates the temperature in the
space in which the cell processing apparatus is disposed inside the
enclosure.
[0029] This cell processing system may still further comprise a
carbon dioxide concentration control device that controls the
carbon dioxide concentration of the space in which the cell
processing apparatus is disposed inside the enclosure.
[0030] In this cell processing system, the enclosure interior may
be demarcated into multiple zones.
[0031] The cell processing apparatus of this cell processing system
may also comprise a preintroduction cell solution-feeding channel
through which a cell-containing solution passes, a factor
introducing device that is connected to the preintroduction cell
solution-feeding channel and introduces a pluripotency inducing
factor into cells to prepare inducing factor-introduced cells, and
a cell mass preparation device in which the inducing
factor-introduced cells are cultured to prepare a plurality of cell
masses comprising stem cells.
[0032] This cell processing system may still further comprise an
enclosure, an intake air purification filter provided in the
enclosure, that purifies gas that has been drawn in from outside
the enclosure, a returning member to return gas discharged from the
enclosure back to the intake air purification filter, a circulating
apparatus that circulates gas between the enclosure and the
returning member, in such a manner that the gas inside the
enclosure is discharged into the returning member and the gas in
the returning member is drawn into the enclosure through the intake
air purification filter, and a cell processing apparatus for
processing of cells, disposed inside the enclosure.
[0033] The returning member in this cell processing system may have
a shape that engages with the enclosure.
[0034] In this cell processing system, the returning member may
comprise a base with a hollow interior, in contact with the bottom
of the enclosure, a first cover that allows communication between a
first opening provided in the base and a ventilation unit provided
on the first end face of the enclosure, and a second cover that
allows communication between a second opening provided in the base
and a ventilation unit provided on a second end face of the
enclosure.
[0035] The returning member in this cell processing system may be a
duct.
[0036] In this cell processing system, the circulating apparatus
may comprise a gas discharger, provided in the enclosure, that
draws in gas from the enclosure and discharges purified gas into
the returning member.
[0037] In this cell processing system, the gas discharger may
comprise an exhaust system that exhausts gas inside the enclosure
into the returning member, and an exhaust purification filter that
purifies gas that has been drawn in by the exhaust system.
[0038] In this cell processing system, the exhaust purification
filter may be situated upstream from the exhaust system.
[0039] This cell processing system may further comprise a shielding
member that can be attached to the exhaust purification filter so
as to shield the exhaust purification filter.
[0040] In this cell processing system, the shielding member may
comprise an enclosure side shielding member that can be attached to
the exhaust purification filter so as to shield the exhaust
purification filter from the enclosure interior, and an exhaust
system side shielding member that can be attached to the exhaust
purification filter so as to shield the exhaust purification filter
from the exhaust system.
[0041] This cell processing system may further comprise a
sterilizing device that sterilizes the exhaust purification
filter.
[0042] This cell processing system may still further comprise a
second exhaust purification filter disposed downstream from the
exhaust system.
[0043] This cell processing system may still further comprise a
shielding member that can be attached to the second exhaust
purification filter so as to shield the second exhaust purification
filter.
[0044] In this cell processing system, the shielding member may
further comprise an exhaust system side shielding member that can
be attached to the second exhaust purification filter, so as to
shield the second exhaust purification filter from the exhaust
system.
[0045] This cell processing system may further comprise a
sterilizing device that sterilizes the second exhaust purification
filter.
[0046] In this cell processing system, the circulating apparatus
may comprise an injector, provided in the enclosure, that draws in
gas that has been purified by the intake air purification filter,
from inside the returning member.
[0047] In this cell processing system, the intake air purification
filter may be situated downstream from the injector.
[0048] This cell processing system may further comprise a shielding
member that can be attached to the intake air purification filter
so as to shield the intake air purification filter.
[0049] In this cell processing system, the shielding member may
comprise an enclosure side shielding member that can be attached to
the intake air purification filter so as to shield the intake air
purification filter from the enclosure interior, and an injector
side shielding member that can be attached to the intake air
purification filter so as to shield the intake air purification
filter from the injector.
[0050] This cell processing system may further comprise a
sterilizing device that sterilizes the intake air purification
filter.
[0051] In this cell processing system, the enclosure interior may
be at negative pressure compared to the returning member
interior.
[0052] Stem cells may be cultured in the cell processing apparatus
of this cell processing system.
[0053] This cell processing system may still further comprise a
sterilizing device that sterilizes the space in which the cell
processing apparatus is disposed inside the enclosure.
[0054] This cell processing system may still further comprise a
temperature regulating device that regulates the temperature in the
space in which the cell processing apparatus is disposed inside the
enclosure.
[0055] This cell processing system may still further comprise a
carbon dioxide concentration control device that controls the
carbon dioxide concentration of the space in which the cell
processing apparatus is disposed inside the enclosure.
[0056] In this cell processing system, the enclosure interior may
be demarcated into multiple zones.
[0057] The cell processing apparatus of this cell processing system
may also comprise a preintroduction cell solution-feeding channel
through which a cell-containing solution passes, a factor
introducing device that is connected to the preintroduction cell
solution-feeding channel and introduces a pluripotency inducing
factor into cells to prepare inducing factor-introduced cells, and
a cell mass preparation device in which the inducing
factor-introduced cells are cultured to prepare a plurality of cell
masses comprising stem cells.
[0058] According to one aspect of the invention there is
additionally provided a cell processing apparatus comprising a cell
processing instrument that processes cells, an embedding member
that embeds the cell processing instrument, and a communicating
solution-feeding channel that allows communication between the
outside of the embedding member and the cell processing instrument
inside the embedding member.
[0059] The embedding member in the cell processing apparatus may be
made of glass or a resin.
[0060] The embedding member in the cell processing apparatus may
also be made of a metal.
[0061] The cell processing apparatus may further comprise a driving
unit disposed outside the embedding member, for feeding of a
solution in the communicating solution-feeding channel.
[0062] The driving unit in the cell processing apparatus may be
connected to an outer wall of the embedding member.
[0063] The cell processing apparatus may also be provided with a
slave unit in the embedding member, to which driving force from the
driving unit is transmitted, with the slave unit being connected to
the communicating solution-feeding channel.
[0064] The cell processing instrument in the cell processing
apparatus may also comprise a mononuclear cell separating unit
disposed inside the embedding member, that separates mononuclear
cells from blood.
[0065] The cell processing apparatus may further comprise a blood
storing unit, disposed outside the embedding member, that stores
either or both blood and blood cells, and the communicating
solution-feeding channel may also comprise a blood delivery channel
that allows communication between the blood storing unit and the
mononuclear cell separating unit.
[0066] The cell processing apparatus may still further comprise a
separating agent storing unit, disposed outside the embedding
member, that stores a separating agent for separation of
mononuclear cells, and the communicating solution-feeding channel
may further comprise a separating agent solution-feeding channel
that allows communication between the separating agent storing unit
and the mononuclear cell separating unit.
[0067] The cell processing instrument in the cell processing
apparatus may also comprise a filter disposed inside the embedding
member, that isolates cells.
[0068] The cell processing instrument in the cell processing
apparatus may also comprise a mononuclear cell purifying filter
disposed inside the embedding member.
[0069] The cell processing instrument in the cell processing
apparatus may also comprise a mononuclear cell separating unit,
disposed inside the embedding member, that separates mononuclear
cells from blood, a mononuclear cell purifying filter disposed
inside the embedding member, and a mononuclear cell
solution-feeding channel disposed inside the embedding member, that
allows communication between the mononuclear cell separating unit
and the mononuclear cell purifying filter.
[0070] The cell processing apparatus may further comprise a driving
unit disposed outside the embedding member, for feeding of a
solution in the mononuclear cell solution-feeding channel.
[0071] The driving unit in the cell processing apparatus may be
connected to an outer wall of the embedding member.
[0072] The cell processing apparatus may also be provided with a
slave unit in the embedding member, to which driving force from the
driving unit is transmitted, with the slave unit being connected to
the mononuclear cell solution-feeding channel.
[0073] The cell processing instrument in the cell processing
apparatus may also comprise a factor introducing device, disposed
inside the embedding member, that introduces a pluripotency
inducing factor into cells to create inducing factor-introduced
cells.
[0074] The cell processing apparatus may still further comprise a
factor storing unit, disposed outside the embedding member, that
stores the pluripotency inducing factor, and the communicating
solution-feeding channel may comprise a factor solution-feeding
channel that allows communication between the factor storing unit
and the factor introducing device.
[0075] In the cell processing apparatus, the pluripotency inducing
factor may be introduced into the cells by RNA lipofection at the
factor introducing device.
[0076] The pluripotency inducing factor in the cell processing
apparatus may be DNA, RNA or protein.
[0077] The pluripotency inducing factor in the cell processing
apparatus may be incorporated into a vector.
[0078] The vector in the cell processing apparatus may be Sendai
virus vector.
[0079] The cell processing instrument in the cell processing
apparatus may further comprise a mononuclear cell separating unit,
disposed inside the embedding member, that separates mononuclear
cells from blood, a factor introducing device, disposed inside the
embedding member, that introduces a pluripotency inducing factor
into cells to create inducing factor-introduced cells, and a
preintroduction cell solution-feeding channel, disposed inside the
embedding member, that allows communication between the mononuclear
cell separating unit and the factor introducing device.
[0080] The cell processing apparatus may further comprise a driving
unit disposed outside the embedding member, for feeding of a
solution in the preintroduction cell solution-feeding channel.
[0081] The driving unit in the cell processing apparatus may be
connected to an outer wall of the embedding member.
[0082] The cell processing apparatus may also be provided with a
slave unit in the embedding member, to which driving force from the
driving unit is transmitted, with the slave unit being connected to
the preintroduction cell solution-feeding channel.
[0083] The cell processing instrument in the cell processing
apparatus may further comprise a mononuclear cell purifying filter,
disposed inside the embedding member, a factor introducing device,
disposed inside the embedding member, that introduces a
pluripotency inducing factor into cells to create inducing
factor-introduced cells, and a preintroduction cell
solution-feeding channel, disposed inside the embedding member,
that allows communication between the mononuclear cell purifying
filter and the factor introducing device.
[0084] The cell processing apparatus may further comprise a driving
unit disposed outside the embedding member, for feeding of a
solution in the preintroduction cell solution-feeding channel.
[0085] The driving unit in the cell processing apparatus may be
connected to an outer wall of the embedding member.
[0086] The cell processing apparatus may also be provided with a
slave unit in the embedding member, to which driving force from the
driving unit is transmitted, with the slave unit being connected to
the preintroduction cell solution-feeding channel.
[0087] The cell processing instrument in the cell processing
apparatus may also comprise an initializing culturing vessel,
disposed inside the embedding member, that cultures the inducing
factor-introduced cells into which the pluripotency inducing factor
has been introduced.
[0088] The cell processing apparatus may still further comprise a
blood cell culture medium storing unit, disposed outside the
embedding member, that stores blood cell culture medium, and the
communicating solution-feeding channel may also comprise a culture
medium solution-feeding channel that allows communication between
the blood cell culture medium storing unit and the initializing
culturing vessel.
[0089] The blood cell culture medium in the cell processing
apparatus may be continuously supplied to the initializing
culturing vessel.
[0090] The blood cell culture medium in the cell processing
apparatus may also be supplied to the initializing culturing vessel
at a prescribed timing.
[0091] The cell processing apparatus may still further comprise a
cold storage unit, disposed outside the embedding member, that
keeps the blood cell culture medium in cold storage.
[0092] The cell processing apparatus may still further comprise a
stem cell culture medium storing unit, disposed outside the
embedding member, that stores stem cell culture medium, and the
communicating solution-feeding channel may also comprise a culture
medium solution-feeding channel that allows communication between
the stem cell culture medium storing unit and the initializing
culturing vessel.
[0093] The stem cell culture medium in the cell processing
apparatus may be continuously supplied to the initializing
culturing vessel.
[0094] The stem cell culture medium in the cell processing
apparatus may also be supplied to the initializing culturing vessel
at a prescribed timing.
[0095] The cell processing apparatus may still further comprise a
cold storage unit, disposed outside the embedding member, that
keeps the stem cell culture medium in cold storage.
[0096] The cell processing apparatus may still further comprise a
waste liquid storage section, disposed outside the embedding
member, that stores waste liquid, and the communicating
solution-feeding channel may also comprise a waste liquid
solution-feeding channel that allows communication between the
initializing culturing vessel and the waste liquid storage
section.
[0097] The initializing culturing vessel in the cell processing
apparatus may also comprise a suspension culture vessel that
comprises a dialysis tube in which the inducing factor-introduced
cells and culture medium are to be accommodated, and a vessel in
which the dialysis tube is to be placed, with the culture medium
accommodable around the periphery of the dialysis tube.
[0098] The cell processing instrument in the cell processing
apparatus may further comprise a factor introducing device,
disposed inside the embedding member, that introduces a
pluripotency inducing factor into cells to create inducing
factor-introduced cells, an initializing culturing vessel, disposed
inside the embedding member, that cultures the inducing
factor-introduced cells, and an introduced cell solution-feeding
channel, disposed inside the embedding member, that allows
communication between the factor introducing device and the
initializing culturing vessel.
[0099] The cell processing apparatus may further comprise a driving
unit disposed outside the embedding member, for feeding of a
solution in the introduced cell solution-feeding channel.
[0100] The driving unit in the cell processing apparatus may be
connected to an outer wall of the embedding member.
[0101] The cell processing apparatus may also be provided with a
slave unit in the embedding member, to which driving force from the
driving unit is transmitted, with the slave unit being connected to
the introduced cell solution-feeding channel.
[0102] The driving unit and slave unit in the cell processing
apparatus may also be connected by magnetic force.
[0103] The cell processing instrument in the cell processing
apparatus may also comprise an amplifying culturing vessel,
disposed inside the embedding member, that carries out amplifying
culturing of a plurality of cell masses comprising established stem
cells.
[0104] The cell processing apparatus may still further comprise a
stem cell culture medium storing unit, disposed outside the
embedding member, that stores stem cell culture medium, and the
communicating solution-feeding channel may also comprise a culture
medium solution-feeding channel that allows communication between
the stem cell culture medium storing unit and the amplifying
culturing vessel.
[0105] The stem cell culture medium in the cell processing
apparatus may be continuously supplied to the amplifying culturing
vessel.
[0106] The stem cell culture medium in the cell processing
apparatus may also be supplied to the amplifying culturing vessel
at a prescribed timing.
[0107] The cell processing apparatus may still further comprise a
cold storage unit, disposed outside the embedding member, that
keeps the stem cell culture medium in cold storage.
[0108] The cell processing apparatus may still further comprise a
waste liquid storage section, disposed outside the embedding
member, that stores waste liquid, and the communicating
solution-feeding channel may also comprise a waste liquid
solution-feeding channel that allows communication between the
amplifying culturing vessel and the waste liquid storage
section.
[0109] The amplifying culturing vessel in the cell processing
apparatus may also comprise a suspension culture vessel that
comprises a dialysis tube in which the inducing factor-introduced
cells and culture medium are to be accommodated, and a vessel in
which the dialysis tube is to be placed, with the culture medium
accommodable around the periphery of the dialysis tube.
[0110] The cell processing instrument in the cell processing
apparatus may also comprise an initializing culturing vessel,
disposed inside the embedding member, that cultures the inducing
factor-introduced cells, an amplifying culturing vessel, disposed
inside the embedding member, that carries out amplifying culturing
of a plurality of cell masses comprising established stem cells,
and an introduced cell solution-feeding channel, disposed inside
the embedding member, that allows communication between the
initializing culturing vessel and the amplifying culturing
vessel.
[0111] The cell processing apparatus may further comprise a driving
unit disposed outside the embedding member, for feeding of a
solution in the introduced cell solution-feeding channel.
[0112] The driving unit in the cell processing apparatus may be
connected to an outer wall of the embedding member.
[0113] The cell processing apparatus may also be provided with a
slave unit in the embedding member, to which driving force from the
driving unit is transmitted, with the slave unit being connected to
the introduced cell solution-feeding channel.
[0114] The cell processing instrument in the cell processing
apparatus may also comprise a cell dissociater, disposed inside the
embedding member, for dissociation of the cell masses provided to
the introduced cell solution-feeding channel.
[0115] The cell processing instrument in the cell processing
apparatus may also comprise a solution exchanger that exchanges the
solution surrounding the cells.
[0116] The cell processing apparatus may still further comprise a
cryopreservation liquid storing unit, disposed outside the
embedding member, that contains a cryopreservation liquid, and the
communicating solution-feeding channel may also comprise a
cryopreservation liquid-feeding channel that allows communication
between the cryopreservation liquid storing unit and the solution
exchanger.
[0117] The cell processing apparatus may still further comprise a
cryopreservation vessel, disposed outside the embedding member, for
storage of the cryopreservation liquid in which cell masses have
been dispersed, and the communicating solution-feeding channel may
also comprise a freezing cell solution-feeding channel, that allows
communication between the solution exchanger and the
cryopreservation vessel.
[0118] The cell processing instrument in the cell processing
apparatus may further comprise an amplifying culturing vessel,
disposed inside the embedding member, that carries out amplifying
culturing of a plurality of cell masses comprising established stem
cells, a solution exchanger that exchanges the solution surrounding
the cells, and an introduced cell solution-feeding channel,
disposed inside the embedding member, that allows communication
between the amplifying culturing vessel and the solution
exchanger.
[0119] The cell processing apparatus may further comprise a driving
unit disposed outside the embedding member, for feeding of a
solution in the introduced cell solution-feeding channel.
[0120] The driving unit in the cell processing apparatus may be
connected to an outer wall of the embedding member.
[0121] The cell processing apparatus may also be provided with a
slave unit in the embedding member, to which driving force from the
driving unit is transmitted, with the slave unit being connected to
the introduced cell solution-feeding channel.
[0122] The cell processing instrument in the cell processing
apparatus may also comprise a cell dissociater, disposed inside the
embedding member, for dissociation of the cell masses provided to
the introduced cell solution-feeding channel.
[0123] The cell processing instrument in the cell processing
apparatus may also comprise a cell mass dissociater that
dissociates cell masses.
Advantageous Effects of Invention
[0124] According to the invention it is possible to provide a cell
processing system and a cell processing apparatus that allow
treatment of cells without contamination of the surroundings.
BRIEF DESCRIPTION OF DRAWINGS
[0125] FIG. 1 is a schematic perspective view of a cell processing
system according to an embodiment of the invention.
[0126] FIG. 2 is a schematic perspective view of a cell processing
system according to an embodiment.
[0127] FIG. 3 is a schematic cross-sectional perspective view of a
cell processing system according to an embodiment.
[0128] FIG. 4 is a schematic cross-sectional view of a cell
processing system according to an embodiment.
[0129] FIG. 5 is a schematic cross-sectional perspective view of a
cell processing system according to an embodiment.
[0130] FIG. 6 is a schematic cross-sectional view of a cell
processing system according to an embodiment.
[0131] FIG. 7 is a schematic cross-sectional perspective view of a
cell processing system according to an embodiment.
[0132] FIG. 8 is a schematic cross-sectional view of a cell
processing system according to an embodiment.
[0133] FIG. 9 is a schematic perspective view of a cell processing
system according to an embodiment of the invention.
[0134] FIG. 10 is a schematic perspective view of a cell processing
system according to an embodiment of the invention.
[0135] FIG. 11 is a schematic cross-sectional perspective view of a
cell processing system according to an embodiment.
[0136] FIG. 12 is a schematic perspective view of a cell processing
system according to an embodiment of the invention.
[0137] FIG. 13 is a schematic perspective view of a cell processing
system according to an embodiment of the invention.
[0138] FIG. 14 is a schematic perspective view of a cell processing
system according to an embodiment of the invention.
[0139] FIG. 15 is a schematic cross-sectional view of a cell
processing system according to an embodiment.
[0140] FIG. 16 is a schematic view of a cell processing apparatus
according to an embodiment.
[0141] FIG. 17 is a schematic cross-sectional view of an example of
an introduced cell solution-feeding channel in a cell processing
apparatus according to an embodiment.
[0142] FIG. 18 is a schematic cross-sectional view of an example of
an introduced cell solution-feeding channel in a cell processing
apparatus according to an embodiment.
[0143] FIG. 19 is a schematic view of a culturing bag to be used in
a cell processing apparatus according to an embodiment.
[0144] FIG. 20 is a schematic view of a suspension culture vessel
according to an embodiment.
[0145] FIG. 21 is a schematic view of a supply culture medium
solution-feeding pump and suspension culture vessel according to an
embodiment.
[0146] FIG. 22 is a schematic view of a supply culture medium
solution-feeding pump and suspension culture vessel according to an
embodiment.
[0147] FIG. 23 is a schematic view of a suspension culture vessel
and photographing device according to an embodiment.
[0148] FIG. 24 is a schematic view of a suspension culture vessel
and photographing device according to an embodiment.
[0149] FIG. 25 is an example of an image of cells according to an
embodiment.
[0150] FIG. 26 is a schematic view of a central processing unit
according to an embodiment.
[0151] FIG. 27 is an example of an image of cell masses according
to an embodiment.
[0152] FIG. 28 is an example of a binarized image of cell masses
according to an embodiment.
[0153] FIG. 29 is an example of an image of cell masses to which a
highpass filter has been applied, according to an embodiment.
[0154] FIG. 30 is an example of images of cell masses to which a
watershed algorithm has been applied, according to an
embodiment.
[0155] FIG. 31 is an example of an image of cell masses to which a
distance transform method has been applied, according to an
embodiment.
[0156] FIG. 32 is an example of images of cell masses to which a
watershed algorithm has been applied, according to an
embodiment.
[0157] FIG. 33 is an example of an image of cell masses dissociated
into multiple regions, according to an embodiment.
[0158] FIG. 34 is an example of an image of cell masses from which
the outlines have been extracted, according to an embodiment.
[0159] FIG. 35 is an example of an image of cell masses from which
the outlines have been extracted, according to an embodiment.
[0160] FIG. 36 is an example of a size histogram for cells
according to an embodiment.
[0161] FIG. 37 is a schematic view of a suspension culture vessel
and photographing device according to an embodiment.
[0162] FIG. 38 is an example of a graph showing the relationship
between culture medium pH and culture medium hue, according to an
embodiment.
[0163] FIG. 39 is a schematic view of a cell mass dissociater
according to an embodiment.
[0164] FIG. 40 is a schematic view of a cell mass dissociater
according to an embodiment.
[0165] FIG. 41 is a schematic view of a cell mass dissociater
according to an embodiment.
[0166] FIG. 42 is a schematic view of a cell mass dissociater
according to an embodiment.
[0167] FIG. 43 is a schematic view of a cell mass dissociater
according to an embodiment.
[0168] FIG. 44 is a schematic view of a cell mass dissociater
according to an embodiment.
[0169] FIG. 45 is a schematic view of a cell mass dissociater
according to an embodiment.
[0170] FIG. 46 is a schematic view of a cell mass dissociater
according to an embodiment.
[0171] FIG. 47 is an example of an image of dissociated cell masses
according to an embodiment.
[0172] FIG. 48 is a schematic view of a solution exchanger
according to an embodiment.
[0173] FIG. 49 is a schematic view of a cell processing apparatus
according to an embodiment.
[0174] FIG. 50 is a schematic perspective view of a cell processing
apparatus according to an embodiment.
[0175] FIG. 51 is a schematic perspective view of a cell processing
apparatus according to an embodiment.
[0176] FIG. 52 is a schematic perspective view of a cell processing
apparatus according to an embodiment.
[0177] FIG. 53 is a schematic perspective view of a driving unit in
a cell processing apparatus according to an embodiment.
[0178] FIG. 54 is a schematic perspective view of a driving unit in
a cell processing apparatus according to an embodiment.
[0179] FIG. 55 is a fluorescent microscope photograph for Example
1.
[0180] FIG. 56 is a graph showing analysis results for Example 1,
using a fluorescence activated flow cytometer.
[0181] FIG. 57 is a pair of photographs of iPS cell colonies, for
Example 2.
[0182] FIG. 58 is a pair of photographs of iPS cell colonies, for
Example 2.
[0183] FIG. 59 is a pair of photographs of iPS cell colonies, for
Example 2.
[0184] FIG. 60 is a graph showing the state of differentiation of
iPS cell colonies, for Example 2.
[0185] FIG. 61 is a pair of photographs of iPS cell colonies, for
Example 3.
[0186] FIG. 62 is a graph showing the results for Example 4.
[0187] FIG. 63 is a set of photographs of iPS cell masses, for
Example 5.
[0188] FIG. 64 is a graph showing the results for Example 5.
DESCRIPTION OF EMBODIMENTS
[0189] An embodiment of the invention will now be explained. In the
accompanying drawings, identical or similar parts will be indicated
by identical or similar reference numerals. However, the drawings
are only schematic representations. The specific dimensions,
therefore, should be judged in light of the following explanation.
Furthermore, this naturally includes parts that have different
dimensional relationships and proportions between drawings.
[0190] The present disclosure includes an invention that has been
provisionally filed in the U.S. (62/356,199), and has already been
issued a foreign application permit.
[0191] As shown in FIG. 1 to FIG. 3, the cell processing system
according to this embodiment comprises an enclosure 601, an outer
enclosure 701 that envelops the enclosure 601, an intake air
purification filter 602 provided in the enclosure 601, that
purifies gas that has been drawn in from outside the enclosure 601,
a circulating apparatus, inside the outer enclosure 701, that
circulates gas inside and outside the enclosure 601 in such a
manner that gas in the outer enclosure 701 is drawn into the
enclosure 601 through the intake air purification filter 602 and
gas inside the enclosure 601 is discharged into the outer enclosure
701, and a cell processing apparatus for processing of cells,
disposed inside the enclosure 601.
[0192] The enclosure 601 has a rectangular solid shape, for
example, but this is not limitative. The enclosure 601 is made of a
material that is able to withstand heat sterilization and
ultraviolet sterilization, for example, but this is also not
limitative. At least a portion of the enclosure 601 may be
transparent so that the interior can be observed from outside. The
enclosure 601 has an openable and closeable structure to allow the
cell processing apparatus to be inserted into and removed from
it.
[0193] The circulating apparatus comprises, for example, a gas
discharger 613, provided in the enclosure 601, that draws in gas
from inside the enclosure 601 and discharges purified gas out of
the enclosure 601. The intake air purification filter 602 and gas
discharger 613 are situated opposite each other, for example.
Examples to be used for the intake air purification filter 602
include, but are not limited to, HEPA (High Efficiency Particulate
Air) filters and URPA (Ultra Low Particulate Air) filters. A MEPA
(Medium Efficiency Particulate Air) filter may also be used as the
intake air purification filter 602, depending on the usage
environment. The intake air purification filter 602 purifies gas
that is to be drawn into the enclosure 601 from outside the
enclosure 601.
[0194] As shown in FIG. 3, the gas discharger 613 comprises an
exhaust system 605 that exhausts gas inside the enclosure 601 to
the exterior of the enclosure 601, and an exhaust purification
filter 603 that purifies gas that has been drawn in by the exhaust
system 605. The exhaust system 605 comprises a fan, for example.
The exhaust purification filter 603 may be situated facing the
interior of the enclosure 601, upstream from the exhaust system
605. It is often difficult to accomplish sterilization of the
exhaust system 605, which is an electrical device. Therefore, the
exhaust purification filter 603 may be arranged upstream from the
exhaust system 605, making it possible to inhibit contamination of
the exhaust system 605. The gas discharger 613 may also comprise a
second exhaust purification filter 606 disposed downstream from the
exhaust system 605.
[0195] The materials of the exhaust purification filter 603 and the
second exhaust purification filter 606 may be the same as for the
intake air purification filter 602, for example. Even if the
enclosure 601 interior becomes contaminated by the cell processing
apparatus, the gas purified by the exhaust purification filter 603
and second exhaust purification filter 606 is exhausted to the
outside of the enclosure 601. For example, even if the gas inside
the enclosure 601 includes blood components or viruses, such
impurities are captured by the exhaust purification filter 603.
Moreover, even if the exhaust system 605 causes contamination of
dust and the like in the gas, the dust is captured by the second
exhaust purification filter 606.
[0196] As shown in FIG. 1 to FIG. 3, the outer enclosure 701 has a
rectangular solid shape, for example, although this is not
limitative. The outer enclosure 701 is made of a material that is
able to withstand heat sterilization and ultraviolet sterilization,
for example, but this is also not limitative. At least a portion of
the outer enclosure 701 may be transparent so that the interior can
be observed from outside. The outer enclosure 701 has an openable
and closeable structure to allow the enclosure 601 to be inserted
into and removed from it. However, the outer enclosure 701
preferably has an openable and closeable structure wherein the
interior is completely closed when the pressure adjustment hole 702
is in the closed state, as described below.
[0197] As shown in FIG. 3, the outer enclosure 701 may be provided
with a pressure adjustment hole 702 for adjustment of the pressure
inside the outer enclosure 701. An occluding member 703 capable of
occluding the pressure adjustment hole 702 is also preferably
attached to the outer enclosure 701. A filter is disposed in the
pressure adjustment hole 702. The material of the filter disposed
in the pressure adjustment hole 702 is the same as for the intake
air purification filter 602, for example.
[0198] Gas is able to flow through the pressure adjustment hole 702
when the gas pressure in the outer enclosure 701 is the same as
outside the outer enclosure 701, for example. Gas that has exited
out from the outer enclosure 701 through the pressure adjustment
hole 702 is purified by the filter disposed in the pressure
adjustment hole 702. Gas that has entered into the outer enclosure
701 through the pressure adjustment hole 702 is also purified by
the filter disposed in the pressure adjustment hole 702.
[0199] As shown in FIG. 4, gas in the outer enclosure 701 is drawn
into the enclosure 601 through the intake air purification filter
602. The gas inside the enclosure 601 is also discharged into the
outer enclosure 701 by the gas discharger 613. Gas is therefore
circulated inside and outside the enclosure 601 in the outer
enclosure 701. Circulation of gas inside and outside the enclosure
601 in the outer enclosure 701 purifies not only gas inside the
enclosure 601, but also gas inside the outer enclosure 701 which is
outside of the enclosure 601, by the intake air purification filter
602, the exhaust purification filter 603 and the second exhaust
purification filter 606. A cell processing system can thus be
constructed wherein the cleanliness of the air inside the enclosure
601 and inside the outer enclosure 701 conforms to, for example,
class ISO1 to ISO6 based on ISO standard 14644-1.
[0200] As shown in FIG. 5 and FIG. 6, the second exhaust
purification filter 606 may be omitted, depending on the usage
environment. In addition, the circulating apparatus may further
comprise an injector 608, provided in the enclosure 601, that draws
in gas purified by the intake air purification filter 602, to the
outside of the enclosure 601. The injector 608 comprises a fan, for
example. The intake air purification filter 602 may be situated
facing the interior of the enclosure 601, downstream from the
injector 608.
[0201] Incidentally, as shown in FIG. 7 and FIG. 8, the cell
processing system may comprise both the injector 608 and the second
exhaust purification filter 606. The circulating apparatus may
comprise both the injector 608 and the gas discharger 613, or it
may comprise only either one.
[0202] Either or both the exhaust system 605 and injector 608
stream gas so that the enclosure 601 interior is at negative
pressure compared to the enclosure 601 exterior. This can help
prevent impurities in the enclosure 601 from diffusing to the
outside of the enclosure 601. Depending on the usage environment,
however, the enclosure 601 interior may be at positive pressure
compared to the enclosure 601 exterior.
[0203] The cell processing system may also comprise a cleanliness
sensor that monitors the cleanliness of gas inside the enclosure
601.
[0204] The cell processing system may further comprise a carbon
dioxide concentration control device that controls the
concentration of carbon dioxide (CO.sub.2) inside the enclosure
601. The carbon dioxide concentration control device may control
the carbon dioxide concentration so that the carbon dioxide
concentration inside the enclosure 601 is at a prescribed value,
such as 5%, for example. The carbon dioxide concentration control
device may also comprise a carbon dioxide concentration sensor that
monitors the carbon dioxide concentration of the gas inside the
enclosure 601.
[0205] The cell processing system may further comprise an oxygen
concentration control device that controls the concentration of
oxygen (02) inside the enclosure 601. For example, the oxygen
concentration control device controls the oxygen concentration so
that the oxygen concentration inside the enclosure 601 is at a low
oxygen state of no greater than 20%. The oxygen concentration
control device may also comprise an oxygen concentration sensor
that monitors the oxygen concentration of the gas inside the
enclosure 601.
[0206] The cell processing system may further comprise a
temperature regulating device that regulates the temperature inside
the enclosure 601. The temperature regulating device may regulate
the temperature so that the temperature inside the enclosure 601 is
a prescribed value such as 37.degree. C., for example. The
temperature regulating device comprises, for example, a Peltier
element. The temperature regulating device may also comprise a
temperature sensor that monitors the temperature of the gas inside
the enclosure 601.
[0207] The cell processing system may still further comprise a
sterilizing device that performs sterilization inside the enclosure
601. The sterilizing device may be a dry heat sterilizing device.
Alternatively, the sterilizing device may atomize or release a
sterilizing gas such as ozone gas, hydrogen peroxide gas or
formalin gas or a sterilizing solution such as ethanol, into the
enclosure 601, to sterilize the interior of the enclosure 601. Also
alternatively, the sterilizing device may irradiate ultraviolet
rays (UV) or an electron beam into the enclosure 601 to sterilize
the enclosure 601 interior. Sterilization of the enclosure 601
interior allows repeated culturing of cells to be carried out
inside the enclosure 601. It can also minimize contamination of the
enclosure 601 exterior and infection of operating personnel. The
cell processing system may also comprise a sterilizing device that
performs sterilization of the outer enclosure 701.
[0208] The enclosure 601 interior may be demarcated into multiple
zones by partitions 604 or the like.
[0209] The cell processing system may also comprise a shielding
member 800 that can be attached to the exhaust purification filter
603 so as to shield the exhaust purification filter 603, as shown
in FIG. 9, FIG. 10 and FIG. 11. For example, the shielding member
800 comprises an enclosure side shielding member 801 that can be
attached to the exhaust purification filter 603 so as to shield the
exhaust purification filter 603 from the interior of the enclosure
601, and an exhaust system side shielding member 802 that can be
attached to the exhaust purification filter 603 so as to shield the
exhaust purification filter 603 from the exhaust system 605.
[0210] This cell processing system may further comprise a
sterilizing device that sterilizes the exhaust purification filter
603 that is shielded by the shielding member 800. The sterilizing
device sterilizes the exhaust purification filter 603 that is
shielded by the shielding member 800, by the same method as for
sterilization of the interior of the enclosure 601. Sterilization
of the exhaust purification filter 603 with the sterilizing device
can help prevent contamination of operating personnel during
exchange of the exhaust purification filter 603.
[0211] Sterilization of the exhaust purification filter 603 that is
shielded by the shielding member 800 can also help prevent exposure
of the exhaust system 605 to the sterilization environment that may
result in damage to the exhaust system 605. When the sterilizing
device is a dry heat sterilizing device, the shielding member 800
may be made of an insulating material, for example. The shape of
the shielding member 800 is not particularly restricted, and it may
be in the form of a plate or a film.
[0212] The enclosure side shielding member 801 may be movable by a
guide 811, as shown in FIG. 10 and FIG. 11. For example, the
enclosure side shielding member 801 may move back and forth along
the guide 811, between a location that does not shield the exhaust
purification filter 603 and a location that does shield the exhaust
purification filter 603.
[0213] The exhaust system side shielding member 802 may also be
movable by a guide 812. For example, the exhaust system side
shielding member 802 may move back and forth along the guide 812,
between a location that does not shield the exhaust purification
filter 603 and a location that does shield the exhaust purification
filter 603.
[0214] The enclosure side shielding member 801 and the exhaust
system side shielding member 802 may be movable in the lateral
direction with respect to the enclosure 601, or as shown in FIG.
12, they may be movable in the vertical direction with respect to
the enclosure 601.
[0215] This cell processing system may still further comprise a
shielding member that can be attached to the second exhaust
purification filter 606 so as to shield the second exhaust
purification filter 606. The shielding member that can be attached
to the second exhaust purification filter 606 may comprise, for
example, an exhaust system side shielding member that can be
attached to the second exhaust purification filter 606 so as to
shield the second exhaust purification filter 606 from the exhaust
system 605.
[0216] The cell processing system may further comprise a
sterilizing device that sterilizes the second exhaust purification
filter 606 that is shielded by the shielding member. The
sterilizing device sterilizes the second exhaust purification
filter 606 that is shielded by the shielding member, by the same
method as for sterilization of the interior of the enclosure 601.
Sterilization of the second exhaust purification filter 606 with
the sterilizing device can help prevent contamination of operating
personnel during exchange of the second exhaust purification filter
606.
[0217] Sterilization of the second exhaust purification filter 606
that is shielded by the shielding member can also help prevent
exposure of the exhaust system 605 to the sterilization environment
that may result in damage to the exhaust system 605. When the
sterilizing device is a dry heat sterilizing device, the shielding
member that shields the second exhaust purification filter 606 may
be made of an insulating material, for example. The shape of the
shielding member that shields the second exhaust purification
filter 606 is not particularly restricted, and it may be in the
form of a plate or a film.
[0218] The shielding member that shields the second exhaust
purification filter 606 may also be movable by a guide. For
example, the shielding member may move back and forth along the
guide, between a location that does not shield the second exhaust
purification filter 606 and a location that does shield the second
exhaust purification filter 606.
[0219] This cell processing system may still further comprise a
shielding member that can be attached to the intake air
purification filter 602 so as to shield the intake air purification
filter 602. For example, the shielding member that can be attached
to the intake air purification filter 602 comprises an enclosure
side shielding member that can be attached to the intake air
purification filter 602 so as to shield the intake air purification
filter 602 from the interior of the enclosure 601, and an injector
side shielding member that can be attached to the intake air
purification filter 602 so as to shield the intake air purification
filter 602 from the injector 608.
[0220] This cell processing system may further comprise a
sterilizing device that sterilizes the intake air purification
filter 602 that is shielded by the shielding member. The
sterilizing device sterilizes the intake air purification filter
602 that is shielded by the shielding member, by the same method as
for sterilization of the interior of the enclosure 601.
Sterilization of the intake air purification filter 602 with the
sterilizing device can help prevent contamination of operating
personnel during exchange of the intake air purification filter
602.
[0221] Sterilization of the intake air purification filter 602 that
is shielded by the shielding member can also can help prevent
exposure of the injector 608 to the sterilization environment that
may result in damage to the injector 608. When the sterilizing
device is a dry heat sterilizing device, the shielding member that
shields the intake air purification filter 602 may be made of an
insulating material, for example. The shape of the shielding member
that shields the intake air purification filter 602 is not
particularly restricted, and it may be in the form of a plate or a
film.
[0222] The shielding member that shields the intake air
purification filter 602 may also be movable by a guide. For
example, the shielding member may move back and forth along the
guide, between a location that does not shield the intake air
purification filter 602 and a location that does shield the intake
air purification filter 602.
[0223] Alternatively, as shown in FIG. 13 to FIG. 15, the cell
processing system according to this embodiment may comprise an
enclosure 601, an intake air purification filter 602 provided in
the enclosure 601, that purifies gas that has been drawn in from
outside the enclosure 601, a returning member 651 with a hollow
interior, to return gas discharged from the enclosure 601 back to
the intake air purification filter 602, a circulating apparatus
that circulates gas between the enclosure and the returning member,
in such a manner that the gas inside the enclosure 601 is
discharged into the returning member 651 and the gas in the
returning member 651 is drawn into the enclosure 601 through the
intake air purification filter 602, and a cell processing apparatus
for processing of cells, disposed inside the enclosure 601.
[0224] The enclosure 601 shown in FIG. 13 to FIG. 15 may have the
same construction as the enclosure 601 explained using FIG. 1 to
FIG. 9. The enclosure 601 therefore has a rectangular solid shape,
for example, although this is not limitative. The enclosure 601 is
made of a material that is able to withstand the sterilization
mentioned above, for example, although this is also not limitative.
At least a portion of the enclosure 601 may be transparent so that
the interior can be observed from outside. The enclosure 601 has an
openable and closeable structure to allow the cell processing
apparatus to be inserted into and removed from it.
[0225] The circulating apparatus comprises, for example, a gas
discharger 613, provided in the enclosure 601, that draws in gas
from inside the enclosure 601 and discharges purified gas out of
the enclosure 601. The intake air purification filter 602 and gas
discharger 613 are situated opposite each other, for example.
Examples to be used for the intake air purification filter 602
include, but are not limited to, HEPA (High Efficiency Particulate
Air) filters and URPA (Ultra Low Particulate Air) filters. A MEPA
(Medium Efficiency Particulate Air) filter may also be used as the
intake air purification filter 602, depending on the usage
environment. The intake air purification filter 602 purifies gas
that is to be drawn into the enclosure 601 from outside the
enclosure 601.
[0226] In the cell processing system illustrated in FIG. 13 to FIG.
15, the gas discharger 613 comprises an exhaust system 605 that
exhausts gas inside the enclosure 601 into the returning member
651, and an exhaust purification filter 603 that purifies gas that
has been drawn in by the exhaust system 605, as shown in FIG. 3.
The exhaust system 605 comprises a fan, for example. The exhaust
purification filter 603 may be situated facing the interior of the
enclosure 601, upstream from the exhaust system 605. It is often
difficult to accomplish sterilization of the exhaust system 605,
which is an electrical device. By therefore arranging the exhaust
purification filter 603 upstream from the exhaust system 605, it is
possible to inhibit contamination of the exhaust system 605. The
gas discharger 613 may also comprise a second exhaust purification
filter 606 disposed downstream from the exhaust system 605.
[0227] The materials of the exhaust purification filter 603 and the
second exhaust purification filter 606 may be the same as for the
intake air purification filter 602, for example. Even if the
enclosure 601 interior becomes contaminated by the cell processing
apparatus, the gas purified by the exhaust purification filter 603
and second exhaust purification filter 606 is exhausted into the
returning member 651. For example, even if the gas inside the
enclosure 601 includes blood components or viruses, such impurities
are captured by the exhaust purification filter 603. Moreover, even
if the exhaust system 605 causes contamination of dust and the like
in the gas, the dust is captured by the second exhaust purification
filter 606.
[0228] As shown in FIG. 13 to FIG. 15, the returning member 651 has
a shape that engages with the enclosure 601, for example, although
this is not limitative. The returning member 651 comprises, for
example, a base 652 with a hollow interior, in contact with the
bottom of the enclosure 601, a first cover 654 that allows
communication between a first opening 653 provided in the base 652
and a ventilation unit provided on the first end face of the
enclosure 601, and a second cover 656 that allows communication
between a second opening 655 provided in the base 652 and a
ventilation unit provided in the enclosure 601.
[0229] The first cover 654 covers the intake air purification
filter 602 serving as the ventilation unit provided on the first
end face. The second cover 656 covers the gas discharger 613
serving as the ventilation unit provided on the second end
face.
[0230] The returning member 651 is made of a material that is able
to withstand sterilization, similar to the enclosure 601, although
this is not limitative. The returning member in this cell
processing system may be a duct.
[0231] As shown in FIG. 15, the gas inside the enclosure 601 is
also discharged into the returning member 651 by the gas discharger
613. Also, the gas in the returning member 651 is drawn into the
enclosure 601 through the intake air purification filter 602. The
gas therefore circulates between the enclosure 601 and the
returning member 651. Circulation of gas between the enclosure 601
and the returning member 651 purifies not only gas inside the
enclosure 601, but also gas inside the returning member 651 which
is outside of the enclosure 601, by the intake air purification
filter 602, the exhaust purification filter 603 and the second
exhaust purification filter 606. A cell processing system can thus
be constructed wherein the cleanliness of the air inside the
enclosure 601 and inside the returning member 651 conforms to, for
example, class ISO1 to ISO6 based on ISO standard 14644-1.
[0232] In the cell processing system illustrated in FIG. 13 to FIG.
15 as well, the second exhaust purification filter 606 may be
omitted as was shown in FIG. 5 and FIG. 6, depending on the usage
environment. In addition, the circulating apparatus may further
comprise an injector 608, provided in the enclosure 601, that draws
in gas purified by the intake air purification filter 602, from
inside the returning member 651. The injector 608 comprises a fan,
for example. The intake air purification filter 602 may be situated
facing the interior of the enclosure 601, downstream from the
injector 608.
[0233] The cell processing system illustrated in FIG. 13 to FIG. 15
as well, may comprise both the injector 608 and the second exhaust
purification filter 606, as was shown in FIG. 7 and FIG. 8. The
circulating apparatus may comprise both the injector 608 and the
gas discharger 613, or it may comprise only either one.
[0234] Either or both the exhaust system 605 and injector 608
stream gas so that the enclosure 601 interior is at negative
pressure compared to the enclosure 601 exterior. This can help
prevent impurities in the enclosure 601 from diffusing to the
outside of the enclosure 601. Depending on the usage environment,
however, the enclosure 601 interior may be at positive pressure
compared to the enclosure 601 exterior.
[0235] Similar to the cell processing system illustrated in FIG. 1
to FIG. 9, the cell processing system shown in FIG. 13 to FIG. 15
may also comprise one or all of a cleanliness sensor that monitors
the cleanliness of gas inside the enclosure 601, a carbon dioxide
concentration control device that controls the concentration of
carbon dioxide (CO.sub.2) inside the enclosure 601, an oxygen
concentration control device that controls the concentration of
oxygen (02) inside the enclosure 601, a temperature regulating
device that regulates the temperature inside the enclosure 601, and
a sterilizing device that sterilizes the interior of the enclosure
601.
[0236] The enclosure 601 interior may be demarcated into multiple
zones by partitions 604 or the like.
[0237] The cell processing system shown in FIG. 13 to FIG. 15 as
well may comprise a shielding member 800 that can be attached to
the exhaust purification filter 603 so as to shield the exhaust
purification filter 603, as shown in FIG. 9, FIG. 10 and FIG. 11.
For example, the shielding member 800 comprises an enclosure side
shielding member 801 that can be attached to the exhaust
purification filter 603 so as to shield the exhaust purification
filter 603 from the interior of the enclosure 601, and an exhaust
system side shielding member 802 that can be attached to the
exhaust purification filter 603 so as to shield the exhaust
purification filter 603 from the exhaust system 605.
[0238] The cell processing system shown in FIG. 13 to FIG. 15 as
well may further comprise a sterilizing device that sterilizes the
exhaust purification filter 603 that is shielded by the shielding
member 800, as shown in FIG. 9, FIG. 10 and FIG. 11. The
sterilizing device sterilizes the exhaust purification filter 603
that is shielded by the shielding member 800, by the same method as
for sterilization of the interior of the enclosure 601.
Sterilization of the exhaust purification filter 603 with the
sterilizing device can help prevent contamination of operating
personnel during exchange of the exhaust purification filter
603.
[0239] Sterilization of the exhaust purification filter 603 that is
shielded by the shielding member 800 can also help prevent exposure
of the exhaust system 605 to the sterilization environment that may
result in damage to the exhaust system 605. When the sterilizing
device is a dry heat sterilizing device, the shielding member 800
may be made of an insulating material, for example. The shape of
the shielding member 800 is not particularly restricted, and it may
be in the form of a plate or a film.
[0240] In the cell processing system shown in FIG. 13 to FIG. 15 as
well, the enclosure side shielding member 801 may be movable by a
guide 811, as shown in FIG. 10 and FIG. 11. For example, the
enclosure side shielding member 801 may move back and forth along
the guide 811, between a location that does not shield the exhaust
purification filter 603 and a location that does shield the exhaust
purification filter 603.
[0241] The exhaust system side shielding member 802 may also be
movable by the guide 812. For example, the exhaust system side
shielding member 802 may move back and forth along the guide 812,
between a location that does not shield the exhaust purification
filter 603 and a location that does shield the exhaust purification
filter 603.
[0242] The enclosure side shielding member 801 and the exhaust
system side shielding member 802 may be movable in the lateral
direction with respect to the enclosure 601, or as shown in FIG.
12, they may be movable in the vertical direction with respect to
the enclosure 601.
[0243] The cell processing system shown in FIG. 13 to FIG. 15 as
well may further comprise a shielding member that can be attached
to the second exhaust purification filter 606 so as to shield the
second exhaust purification filter 606, as shown in FIG. 12. The
shielding member that can be attached to the second exhaust
purification filter 606 may comprise, for example, an exhaust
system side shielding member that can be attached to the second
exhaust purification filter 606 so as to shield the second exhaust
purification filter 606 from the exhaust system 605.
[0244] The cell processing system shown in FIG. 13 to FIG. 15 may
further comprise a sterilizing device that sterilizes the second
exhaust purification filter 606 that is shielded by the shielding
member, as shown in FIG. 12. The sterilizing device sterilizes the
second exhaust purification filter 606 that is shielded by the
shielding member, by the same method as for sterilization of the
interior of the enclosure 601. Sterilization of the second exhaust
purification filter 606 with the sterilizing device can help
prevent contamination of operating personnel during exchange of the
second exhaust purification filter 606.
[0245] Sterilization of the second exhaust purification filter 606
that is shielded by the shielding member can also help prevent
exposure of the exhaust system 605 to the sterilization environment
that may result in damage to the exhaust system 605. When the
sterilizing device is a dry heat sterilizing device, the shielding
member that shields the second exhaust purification filter 606 may
be made of an insulating material, for example. The shape of the
shielding member that shields the second exhaust purification
filter 606 is not particularly restricted, and it may be in the
form of a plate or a film.
[0246] The shielding member that shields the second exhaust
purification filter 606 may also be movable by a guide. For
example, the shielding member may move back and forth along the
guide, between a location that does not shield the second exhaust
purification filter 606 and a location that does shield the second
exhaust purification filter 606.
[0247] The cell processing system shown in FIG. 13 to FIG. 15 as
well may further comprise a shielding member that can be attached
to the intake air purification filter 602 so as to shield the
intake air purification filter 602, as shown in FIG. 12. For
example, the shielding member that can be attached to the intake
air purification filter 602 comprises an enclosure side shielding
member that can be attached to the intake air purification filter
602 so as to shield the intake air purification filter 602 from the
interior of the enclosure 601, and an injector side shielding
member that can be attached to the intake air purification filter
602 so as to shield the intake air purification filter 602 from the
injector 608.
[0248] The cell processing system shown in FIG. 13 to FIG. 15 as
well may further comprise a sterilizing device that sterilizes the
intake air purification filter 602 that is shielded by the
shielding member. The sterilizing device sterilizes the intake air
purification filter 602 that is shielded by the shielding member,
by the same method as for sterilization of the interior of the
enclosure 601. Sterilization of the intake air purification filter
602 with the sterilizing device can help prevent contamination of
operating personnel during exchange of the intake air purification
filter 602.
[0249] Sterilization of the intake air purification filter 602 that
is shielded by the shielding member can also can help prevent
exposure of the injector 608 to the sterilization environment that
may result in damage to the injector 608. When the sterilizing
device is a dry heat sterilizing device, the shielding member that
shields the intake air purification filter 602 may be made of an
insulating material, for example. The shape of the shielding member
that shields the intake air purification filter 602 is not
particularly restricted, and it may be in the form of a plate or a
film.
[0250] The shielding member that shields the intake air
purification filter 602 may also be movable by a guide. For
example, the shielding member may move back and forth along the
guide, between a location that does not shield the intake air
purification filter 602 and a location that does shield the intake
air purification filter 602.
[0251] The cell processing apparatus disposed inside the enclosure
601 comprises, as shown in FIG. 16, a separating device 10 that
separates cells from blood, a preintroduction cell solution-feeding
channel 20 through which a cell-containing solution that has been
separated by the separating device 10 passes, an inducing factor
solution-feeding mechanism 21 that introduces a pluripotency
inducing factor into the preintroduction cell solution-feeding
channel 20, a factor introducing device 30 connected to the
preintroduction cell solution-feeding channel 20, that introduces
the pluripotency inducing factor into the cells to prepare inducing
factor-introduced cells, a cell mass preparation device 40 that
cultures the inducing factor-introduced cells to prepare a
plurality of cell masses comprising stem cells, and a packaging
device 100 that packages each of the plurality of cell masses in
order.
[0252] The separating device 10 receives vials containing human
blood, for example. The separating device 10 comprises an
anticoagulant tank that stores anticoagulants such as
ethylenediaminetetraacetic acid (EDTA), heparin and biologically
standardized blood storage Solution A (ACD Solution A, product of
Terumo Corp.), for example. The separating device 10 employs a pump
or the like to add an anticoagulant to human blood from the
anticoagulant tank.
[0253] In addition, the separating device 10 comprises a separating
reagent tank that stores a mononuclear cell separating reagent such
as Ficoll-Paque PREMIUM.RTM. (product of GE Healthcare, Japan). The
separating device 10 employs a pump or the like to inject 5 mL of
mononuclear cell separating reagent from the separating reagent
tank into each of two 15 mL tubes, for example. Resin bags may also
be used instead of tubes.
[0254] The separating device 10 also comprises a buffering solution
tank that stores a buffering solution such as phosphate-buffered
saline (PBS). The separating device 10 employs a pump to add 5 mL
of buffering solution from the buffering solution tank to 5 mL of
human blood, for example, to dilute it. In addition, the separating
device 10 employs a pump or the like to add 5 mL of the diluted
human blood to each of the mononuclear cell separating reagents in
the tubes.
[0255] The separating device 10 further comprises a
temperature-adjustable centrifuge. The centrifuge may be set to
18.degree. C., for example. The separating device 10 employs a
moving apparatus or the like to place the tubes in which the
mononuclear cell separating reagent and human blood have been
placed, into holders of the centrifuge. The centrifuge performs
centrifugation of the solutions in the tubes for 30 minutes at
400.times.g, for example. Resin bags may also be centrifuged
instead of tubes.
[0256] After centrifugation, the separating device 10 collects the
intermediate layers that have become turbid and white by the
mononuclear cells in the solutions in the tubes, using a pump or
the like. The separating device 10 employs a pump or the like to
deliver the recovered mononuclear cell suspensions to the
preintroduction cell solution-feeding channel 20. Alternatively,
the separating device 10 also adds 12 mL of PBS, for example, to 2
mL of the recovered mononuclear cell solutions, and places the
tubes in holders of the centrifuge. The centrifuge performs
centrifugation of the solutions in the tubes for 10 minutes at
200.times.g, for example.
[0257] After centrifugation, the separating device 10 employs a
pump or the like to remove the supernatants of the solutions in the
tubes by suction, and adds 3 mL of mononuclear cell culture medium
such as X-VIVO 10.RTM. (Lonza, Japan) to the mononuclear cell
solutions in the tubes to prepare suspensions. The separating
device 10 employs a pump or the like to deliver the mononuclear
cell suspensions to the preintroduction cell solution-feeding
channel 20. The separating device 10 may also employ a dialysis
membrane to separate the mononuclear cells from the blood. When
using somatic cells such as fibroblasts previously separated from
skin or the like, the separating device 10 is not necessary.
[0258] The separating device 10 may also separate cells suitable
for induction by a method other than centrifugal separation. For
example, if the cells to be separated are T cells, cells that are
CD3-, CD4- or CD8-positive may be separated by panning. If the
cells to be separated are vascular endothelial precursor cells,
then cells that are CD34-positive may be separated by panning. If
the cells to be separated are B cells, then cells that are CD10-,
CD19- or CD20-positive may be separated by panning. The separation
may also be carried out by a magnetic-activated cell sorting (MACS)
method or flow cytometry, without limitation to panning. Moreover,
the cells suitable for induction are not limited to cells derived
from blood.
[0259] The inducing factor solution-feeding mechanism 21 comprises
an inducing factor introducing reagent tank that stores an inducing
factor introducing reagent solution. The inducing factor
introducing reagent solution such as a gene transfer reagent
solution includes, for example, an electroporation solution such as
Human T Cell Nucleofector.RTM. (Lonza, Japan), a supplement
solution, and a plasmid set. The plasmid set includes, for example,
0.83 .mu.g of pCXLE-hOCT3/4-shp53-F, 0.83 .mu.g of pCXLE-hSK, 0.83
.mu.g of pCE-hUL and 0.5 .mu.g of and pCXWB-EBNA1. The inducing
factor solution-feeding mechanism 21 employs a micropump or the
like to deliver the inducing factor introducing reagent solution to
the preintroduction cell solution-feeding channel 20, in such a
manner that the mononuclear cell suspension is suspended in the
inducing factor introducing reagent solution.
[0260] The inner wall of the preintroduction cell solution-feeding
channel 20 may be coated with poly-HEMA (poly 2-hydroxyethyl
methacrylate) to render it non-cell-adherent, so that the cells do
not adhere. Alternatively, a material resistant to cell adhesion
may be used as the material for the preintroduction cell
solution-feeding channel 20. Also, by using a material with good
thermal diffusivity and CO.sub.2 permeability as the material of
the preintroduction cell solution-feeding channel 20, the
conditions in the preintroduction cell solution-feeding channel 20
will be equivalent to the controlled temperature and CO.sub.2
concentration in the enclosure 601. In addition, a back-flow valve
may be provided in the preintroduction cell solution-feeding
channel 20 from the viewpoint of preventing contamination.
[0261] The factor introducing device 30 connected to the
preintroduction cell solution-feeding channel 20 is an
electroporator, for example, and it receives a liquid mixture of
the inducing factor introducing reagent solution and mononuclear
cell suspension and carries out plasmid electroporation in the
mononuclear cells. After carrying out electroporation, the factor
introducing device 30 adds mononuclear cell culture medium to the
solution containing the plasmid-electroporated mononuclear cells.
The factor introducing device 30 employs a pump or the like to
deliver the solution containing the plasmid-electroporated
mononuclear cells (hereunder referred to as "inducing
factor-introduced cells") to the introduced cell solution-feeding
channel 31.
[0262] The factor introducing device 30 is not limited to an
electroporator. The factor introducing device 30 may also introduce
RNA coding for an initializing factor into the cells by a
lipofection method. A lipofection method is a method in which a
complex of nucleic acid as a negatively charged substance with
positively charged lipids, is formed by electrical interaction, and
the complex is incorporated into cells by endocytosis or membrane
fusion. Lipofection is advantageous as it creates little damage to
cells and has excellent introduction efficiency, while operation is
convenient and less time is required. In addition, since there is
no possibility of the initializing factor being inserted into the
genome of the cells in lipofection, there is no need to confirm the
presence or absence of insertion of exogenous genes by full genome
sequencing of the obtained stem cells. Initializing factor RNA used
as a pluripotency inducing factor may include, for example, Oct3/4
mRNA, Sox2 mRNA, Klf4 mRNA, and c-Myc mRNA.
[0263] Lipofection of initializing factor RNA uses small
interfering RNA (siRNA) or a lipofection reagent, for example. An
siRNA lipofection reagent or mRNA lipofection reagent may be used
as an RNA lipofection reagent. More specifically, the RNA
lipofection reagent used may be Lipofectamine.RTM. RNAiMAX (Thermo
Fisher Scientific), Lipofectamine.RTM. MessengerMAX (Thermo Fisher
Scientific), Lipofectamin.RTM. 2000, Lipofectamin.RTM. 3000,
NeonTransfection System (Thermo Fisher scientific), Stemfect RNA
transfection reagent (Stemfect), NextFect.RTM. RNA Transfection
Reagent (BiooSientific), Amaxa.RTM. Human T cell Nucleofector.RTM.
kit (Lonza, VAPA-1002), Amaxa.RTM. Human CD34 cell
Nucleofector.RTM. kit (Lonza, VAPA-1003), or ReproRNA.RTM.
transfection reagent STEMCELL Technologies).
[0264] When the factor introducing device 30 is to introduce an
initializing factor into cells by lipofection, the initializing
factor RNA and reagents are introduced into the preintroduction
cell solution-feeding channel 20 by the inducing factor
solution-feeding mechanism 21.
[0265] The inner wall of the introduced cell solution-feeding
channel 31 may be coated with poly-HEMA to render it non-adhesive,
so that the cells do not adhere. Alternatively, a material
resistant to cell adhesion may be used as the material for the
introduced cell solution-feeding channel 31. Also, by using a
material with good thermal diffusivity and CO.sub.2 permeability as
the material for the introduced cell solution-feeding channel 31,
the conditions in the introduced cell solution-feeding channel 31
will be equivalent to the controlled temperature and CO.sub.2
concentration in the enclosure 601. In addition, a back-flow valve
may be provided in the introduced cell solution-feeding channel 31
from the viewpoint of preventing contamination. Numerous cells die
after electroporation, and cell masses of dead cells often result.
Therefore, a filter may be provided in the introduced cell
solution-feeding channel 31 to remove the dead cell masses.
Alternatively, as shown in FIG. 17, one or a plurality of folds may
be formed in the interior of the introduced cell solution-feeding
channel 31 to intermittently vary the inner diameter. As another
alternative, the inner diameter of the introduced cell
solution-feeding channel 31 may be intermittently varied, as shown
in FIG. 18.
[0266] As shown in FIG. 16, the cell mass preparation device 40
connected to the introduced cell solution-feeding channel 31
comprises an initializing culturing apparatus 50 that cultures the
inducing factor-introduced cells prepared at the factor introducing
device 30, a first dissociating mechanism 60 that dissociates the
cell masses comprising stem cells (cell colonies) established at
the initializing culturing apparatus 50 into a plurality of cell
masses, an amplifying culturing apparatus 70 that carries out
amplifying culturing of the plurality of cell masses that have been
dissociated at the first dissociating mechanism 60, a second
dissociating mechanism 80 that dissociates the cell masses
comprising stem cells that have been amplifying cultured at the
amplifying culturing apparatus 70 into a plurality of cell masses,
and a cell mass transport mechanism 90 that delivers the plurality
of cell masses in order to the packaging device 100.
[0267] The initializing culturing apparatus 50 can house a well
plate in its interior. The initializing culturing apparatus 50 also
comprises a pipetting machine. The initializing culturing apparatus
50 receives the solution containing the inducing factor-introduced
cells from the introduced cell solution-feeding channel 31, and
allocates the solution into the wells with the pipetting machine.
The initializing culturing apparatus 50 adds stem cell culture
medium such as StemFit.RTM. (Ajinomoto Co., Inc.) on the 3rd, 5th
and 7th days, for example, after allocating the inducing
factor-introduced cells to the wells. Basic fibroblast growth
factor (basic FGF) may also be added to the culture medium as a
supplement. Sustained-release beads, such as StemBeads FGF2
(Funakoshi Corp.), may also be added to the culture medium, for
continuous supply of the FGF-2 (basic FGF, bFGF, FGF-b) to the
culture medium. Also, since FGF is often unstable, a heparin-like
polymer may be conjugated with the FGF to stabilize the FGF.
Transforming growth factor beta (TGF-.beta.), activin or the like
may also be added to the culture medium. At the initializing
culturing apparatus 50, the culture medium is exchanged on the 9th
day, for example, after allocating the inducing factor-introduced
cells to the wells, and thereafter the culture medium is exchanged
every 2 days until the iPS cell masses (colonies) exceed 1 mm.
Exchange of the medium may be partial exchange of the culture
medium, or it may be replenishment.
[0268] When cell masses form, the initializing culturing apparatus
50 collects the cell masses with a pipetting machine, and adds a
trypsin-substituting recombinant enzyme such as TrypLE Select.RTM.
(Life Technologies Corp.) to the collected cell masses. In
addition, the initializing culturing apparatus 50 places a vessel
containing the collected cell masses in an incubator, and reacts
the cell masses with the trypsin-substituting recombinant enzyme
for 10 minutes at 37.degree. C., 5% CO.sub.2. When the cell masses
are to be physically disrupted, there is no need for a
trypsin-substituting recombinant enzyme. For example, the
initializing culturing apparatus 50 disrupts the cell masses by
pipetting with a pipetting machine. Alternatively, the initializing
culturing apparatus 50 may disrupt the cell masses by passing the
cell masses through a pipe provided with a filter, or a pipe that
intermittently varies the inner diameter, similar to the introduced
cell solution-feeding channel 31 shown in FIG. 17 or FIG. 18. Next,
the initializing culturing apparatus 50 adds culture medium for
pluripotent stem cells such as StemFit.RTM. (Ajinomoto Co., Inc.),
to the solution containing the disrupted cell masses.
[0269] Culturing in the initializing culturing apparatus 50 may be
carried out in a CO.sub.2-permeable bag instead of a well plate.
The culturing may be by adhesion culture or suspension culture. In
the case of suspension culture, agitation culture may be carried
out. The culture medium may also be in the form of agar. Agar
culture media include gellan gum polymers and deacylated gellan gum
polymers. When an agar culture medium is used, there is no settling
or adhesion of cells, and therefore agitation is not necessary even
though it is suspension culture, and it is possible to form a
single cell mass deriving from one cell, while the culturing in the
initializing culturing apparatus 50 can also be by hanging drop
culture.
[0270] The initializing culturing apparatus 50 may also comprise a
first culture medium supply device that supplies culture medium
including culture solution to a well plate or a CO.sub.2-permeable
bag. The first culture medium supply device collects the culture
solution in the well plate or CO.sub.2-permeable bag, and it may
use a filter or dialysis membrane to filter the culture solution,
to allow reuse of the purified culture solution. During this time,
growth factors or the like may be added to the culture solution
that is to be reused. Furthermore, the initializing culturing
apparatus 50 may also comprise a temperature regulating device that
regulates the temperature of the culture medium, and a humidity
control device that controls the humidity in the vicinity of the
culture medium.
[0271] In the initializing culturing apparatus 50, the cells may be
placed in a culture solution-permeable bag 301 such as a dialysis
membrane as shown in FIG. 19, for example, and the culture
solution-permeable bag 301 may be placed in a culture
solution-impermeable CO.sub.2-permeable bag 302, so that the
culture solution is placed in bags 301, 302. The initializing
culturing apparatus 50 may have multiple bags 302 prepared
containing fresh culture solution, and the bag 302 in which the
cell-containing bag 301 is placed may be replaced by a bag 302
containing fresh culture solution, at prescribed intervals of
time.
[0272] The method of culturing in the initializing culturing
apparatus 50 is not limited to the method described above, and a
suspension culture vessel such as shown in FIG. 20 may be used. The
suspension culture vessel shown in FIG. 20 comprises a dialysis
tube 75 in which the inducing factor-introduced cells and gel
medium are to be inserted, and a vessel 76 in which the dialysis
tube 75 is to be placed, with the gel medium accommodable around
the periphery of the dialysis tube 75. The suspension culture
vessel may also comprise a pH sensor that measures the hydrogen ion
exponent (pH) of the gel medium surrounding the dialysis tube
75.
[0273] The dialysis tube 75 is made of a semipermeable membrane,
and it allows permeation of ROCK inhibitor, for example. The
molecular cutoff of the dialysis tube 75 is .gtoreq.0.1 KDa,
.gtoreq.10 KDa, or .gtoreq.50 KDa. The dialysis tube 75 is made of,
for example, cellulose ester, ethyl cellulose, a cellulose ester
derivative, regenerated cellulose, polysulfone, polyacrylnitrile,
polymethyl methacrylate, ethylenevinyl alcohol copolymer,
polyester-based polymer alloy, polycarbonate, polyamide, cellulose
acetate, cellulose diacetate, cellulose triacetate, copper ammonium
rayon, saponified cellulose, a Hemophan membrane, a
phosphatidylcholine membrane or a vitamin E coated membrane.
[0274] The vessel 76 used may be a conical tube such as a
centrifugation tube. The vessel 76 is made of polypropylene, for
example. The vessel 76 may also be CO.sub.2-permeable. G-Rex.RTM.
(Wilson Wolf) may be used as a CO.sub.2-permeable vessel 76.
[0275] The inducing factor-introduced cells are placed in the
dialysis tube 75. The gel medium is not agitated. Also, the gel
medium does not include feeder cells. A solution-feeding channel
may be connected to the dialysis tube 75 to feed cell-containing
culture medium into the dialysis tube 75. A solution-feeding
channel may also be connected to the dialysis tube 75 to feed the
cell-containing culture medium in the dialysis tube 75 to the
outside of the vessel.
[0276] The gel medium is prepared, for example, by adding
deacylated gellan gum to the blood cell culture medium or stem cell
culture medium, to a final concentration of 0.5 wt % to 0.001 wt %,
0.1 wt % to 0.005 wt % or 0.05 wt % to 0.01 wt %. At the start of
initializing culturing, for example, gel medium prepared from the
blood cell culture medium is used, and then gel medium prepared
from stem cell culture medium is used.
[0277] The stem cell culture medium used may be human ES/iPS
culture medium such as Primate ES Cell Medium (ReproCELL), for
example.
[0278] The stem cell culture medium is not limited to this,
however, and various stem cell culture media may be used. For
example, Primate ES Cell Medium, Reprostem, ReproFF, ReproFF2,
ReproXF (Reprocell), mTeSR1, TeSR2, TeSRE8, ReproTeSR (STEMCELL
Technologies), PluriSTEM.RTM. Human ES/iPS Medium (Merck),
NutriStem.RTM. XF/FF Culture Medium for Human iPS and ES Cells,
Pluriton reprogramming medium (Stemgent), PluriSTEMR, Stemfit
AK02N, Stemfit AK03 (Ajinomoto), ESC-Sure.RTM. serum and feeder
free medium for hESC/iPS (Applied StemCell) and L7.RTM. hPSC
Culture System (LONZA) may be used.
[0279] The gel medium may include one or more high molecular
compounds selected from the group consisting of gellan gum,
hyaluronic acid, rhamsan gum, diutan gum, xanthan gum, carrageenan,
fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate,
heparin, heparitin sulfate, keratosulfate, chondroitin sulfate,
dermatan sulfate, rhamnan sulfate, and salts of the foregoing. The
gel medium may also include methyl cellulose. Including methyl
cellulose allows greater control of aggregation between the
cells.
[0280] Alternatively, the gel medium may include at least one
temperature sensitive gel selected from among poly(glycerol
monomethacrylate) (PGMA), poly(2-hydroxypropyl methacrylate)
(PHPMA), poly(N-isopropylacrylamide) (PNIPAM), amine terminated,
carboxylic acid terminated, maleimide terminated,
N-hydroxysuccinimide (NHS) ester terminated, triethoxysilane
terminated, poly(N-isopropylacrylamide-co-acrylamide),
poly(N-isopropylacrylamide-co-acrylic acid),
poly(N-isopropylacrylamide-co-butylacrylate),
poly(N-isopropylacrylamide-co-methacrylic acid),
poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl
acrylate) and N-isopropylacrylamide.
[0281] The gel medium placed in the dialysis tube 75 does not need
to include a ROCK inhibitor. The ROCK inhibitor may be added to the
gel medium placed around the dialysis tube 75 in the vessel 76, to
a final concentration of 1000 .mu.mol/L to 0.1 .mu.mol/L, 100
.mu.mol/L to 1 .mu.mol/L, or 5 .mu.mol/L to 20 .mu.mol/L, for
example. By adding a ROCK inhibitor to the gel medium surrounding
the dialysis tube 75, the ROCK inhibitor will penetrate into the
dialysis tube 75 and colony formation by the cells will be
promoted.
[0282] The gel medium may either include or not include growth
factors such as basic fibroblast growth factor (bFGF) or
TGF-.beta..
[0283] During suspension culturing of the cells in the dialysis
tube 75, the gel medium surrounding the dialysis tube 75 in the
vessel 76 is exchanged. Medium exchange includes partial exchange
of the culture medium, as well as replenishment. In this case, the
gel medium in the dialysis tube 75 does not need to be supplied.
The gel medium may instead be supplied into the dialysis tube 75
during suspension culturing of the cells in the dialysis tube 75.
In this case, the gel medium surrounding the dialysis tube 75 in
the vessel 76 does not need to be supplied.
[0284] As shown in FIG. 21, the cell processing apparatus of this
embodiment uses a supply culture medium solution-feeding pump 77 as
a culture medium supply device to exchange or supply gel medium
surrounding the dialysis tube 75 in the vessel 76. The supply
culture medium solution-feeding pump 77 used may be a pump used for
drip infusion. The supply culture medium solution-feeding pump 77
and the suspension culture vessel 76 are connected by a
solution-feeding tube 78. The supply culture medium
solution-feeding pump 77 feeds gel medium into the suspension
culture vessel 76 through the solution-feeding tube 78. A waste
liquid tube 79 is connected to the suspension culture vessel 76.
The gel medium in the suspension culture vessel 76 is discharged
through the waste liquid tube 79. The gel medium in the suspension
culture vessel 76 may be discharged, for example, by the pressure
of fresh gel medium supplied by the supply culture medium
solution-feeding pump 77, or it may be discharged utilizing
gravity, or it may be discharged by a discharge pump.
[0285] The temperature of the gel medium to be delivered from the
supply culture medium solution-feeding pump 77 to the culturing
vessel is set, for example, so that the temperature of the gel
medium in the culturing vessel does not vary drastically. For
example, when the temperature of the gel medium in the culturing
vessel is 37.degree. C., the temperature of the gel medium
delivered to the culturing vessel is set to 37.degree. C. However,
the culture medium before it is delivered to the culturing vessel
may be set in cold storage at a low temperature of 4.degree. C.,
for example, at the cold storage unit.
[0286] The supply culture medium solution-feeding pump 77 is
controlled so that, for example, the amount of the gel medium fed
into the suspension culture vessel 76 by the supply culture medium
solution-feeding pump 77 and the amount of the gel medium
discharged from the suspension culture vessel 76 are equal. The
supply culture medium solution-feeding pump 77 may feed the gel
medium into the suspension culture vessel 76 constantly, or it may
feed the gel medium at appropriate intervals.
[0287] When the gel medium is delivered constantly, the flow rate
of the gel medium being fed may be either constant or variable. For
example, the culture medium and the cell masses in the culture
medium may be monitored with a photographing device, as explained
below, and the flow rate of the gel medium being fed may be
increased or decreased depending on the state of the culture medium
and the cell mass in the culture medium.
[0288] Also, instead of constant feeding of the gel medium, feeding
of the gel medium may be started and stopped depending on the state
of the culture medium and the cell masses in the culture medium. In
this case as well, the flow rate of the gel medium being fed may be
increased or decreased depending on the state of the culture medium
and the cell masses in the culture medium.
[0289] If the flow rate of the gel medium being fed to the
culturing vessel is too high, the cells in the culturing vessel may
undergo damage by the pressure of the gel medium. Therefore, the
flow rate of the gel medium being delivered to the culturing vessel
is set so that the cells do not suffer damage.
[0290] When culturing of the cells is to be continued without
exchange of the culture medium, accumulation of waste products such
as lactic acid discharged by the cells, or variation in pH, can
adversely affect the cell culture. In addition, proteins including
bFGF or recombinant proteins present in the culture medium may be
degraded, resulting in loss of the components necessary for cell
culturing.
[0291] To counter this, fresh culture medium may be fed to the
culturing vessel by the supply culture medium solution-feeding pump
77, and the old culture medium discharged from the culturing
vessel, to remove waste products from the culturing vessel, to keep
the pH in the culture medium in a suitable range, and to allow
supply of the components necessary for culturing of the cells. This
will allow the state of the culture medium to be kept nearly
constant.
[0292] FIG. 21 shows an example in which the supply culture medium
solution-feeding pump 77 and the suspension culture vessel 76 are
connected by the solution-feeding tube 78. Alternatively, as shown
in FIG. 22, the supply culture medium solution-feeding pump 77 and
the interior of the dialysis tube 75 in the suspension culture
vessel 76 may be connected by the solution-feeding tube 78. By
feeding fresh gel medium into the dialysis tube 75, waste products
present in the culture medium in the dialysis tube 75 are
discharged out of the dialysis tube 75. This also allows the pH of
the culture medium in the dialysis tube 75 to be kept in a suitable
range, and allows the components necessary for culturing of the
cells to be supplied to the culture medium in the dialysis tube
75.
[0293] The cell processing system may further comprise an
initializing culturing photographing device such as a photographing
camera or video camera that photographically records culturing in
the initializing culturing apparatus 50, as shown in FIG. 16. If a
colorless culture medium is used for the culture medium in the
initializing culturing apparatus 50, it will be possible to
minimize diffuse reflection and autologous fluorescence that may be
produced when using a colored culture medium. A pH indicator such
as phenol red may be included however, in order to confirm the pH
of the culture medium. Moreover, since induced cells and
non-induced cells have differences in cellular shape and size, the
cell processing system may further comprise an induced state
monitoring device that calculates the proportion of induced cells
by photographing the cells in the initializing culturing apparatus
50. Alternatively, the induced state monitoring device may
determine the proportion of induced cells by antibody
immunostaining or RNA extraction. In addition, the cell processing
system may comprise a non-induced cell removing device that removes
cells that have not been induced, by magnetic-activated cell
sorting, flow cytometry or the like.
[0294] When the cells are being cultured on a flat dish such as a
plate, the region where the cells are present spreads out in a
planar manner. Thus, if the photographing device and the plate are
oriented so that the optical axis of the lens of the photographing
device is perpendicular to the dish surface, it will be possible to
adjust the focus on essentially all of the cells on the plate.
[0295] When the cells are suspended in the culture medium for
suspension culture, however, the region where the cells are present
will spread out three-dimensionally, and therefore the distance in
the optical axis direction from the photographing device to each of
the cells will vary. It may therefore be difficult to adjust the
focus to all of the cells without using a lens.
[0296] However, by using a bright lens (a lens with a low F value)
or by imaging with as small an aperture as possible for the lens
while illuminating the measuring target with bright lighting, it is
possible to increase the depth of the field.
[0297] Alternatively, a plurality of images may be taken while
gradually varying the focal point of the lens, and the plurality of
images synthesized to obtain a pseudo-deep focused image. Each of
the plurality of images will be a blend of the focused cells and
the blurry non-focused cells. The partial focused images may then
be compiled from the plurality of images to produce a single
synthetic image.
[0298] Alternatively, as shown in FIG. 23, a telecentric lens 172
may be disposed between the initializing culturing photographing
device 171 and the object, such as cells, in the suspension culture
vessel. With the telecentric lens 172, the principal ray running
from the object, such as cells, through the center of the lens
aperture is parallel to the lens optical axis, and therefore the
sizes of the photographed cells do not vary with distance even if
the distances from the initializing culturing photographing device
171 to each of the plurality of cells in the suspension culture
vessel are not uniform.
[0299] FIG. 24 is a schematic view of the suspension culture vessel
shown in FIG. 23, as seen from above. In FIG. 24, the vessel 76
shown in FIG. 23 is omitted. When cells are to be imaged with the
initializing culturing photographing device 171, a scattered light
illumination method may be employed, in which a cell observation
illumination light source 173 is situated in the direction
perpendicular to the optical axis of the initializing culturing
photographing device 171, or a direction nearer the photographing
device than the perpendicular direction, and illumination light is
irradiated on the cells from the cell observation illumination
light source 173. Scattered light from the light illuminated on the
cells will thus reach the initializing culturing photographing
device 171, but the illumination light that has not impacted the
cells passes through the culture medium and does not reach the
initializing culturing photographing device 171. Thus, the culture
medium parts of the image are relatively dark while the cell parts
are relatively light. The illumination method is not limited to
this method, however, so long as the cells can be recognized in the
image. FIG. 25 shows an example of an image of cells taken by a
scattered light illumination method. The culture medium parts are
relatively dark while the cell parts are relatively light.
[0300] As shown in FIG. 26, the cell processing system of this
embodiment may also comprise a central processing unit (CPU) 500
provided with an image processor 501 that carries out image
processing of the image taken by the initializing culturing
photographing device 171. An input device 401 such as a keyboard or
mouse and an output device 402 such as a monitor may be connected
to the CPU 500. The CPU 500 receives the image from the
initializing culturing photographing device 171 via a bus, image
interface or the like.
[0301] The image processor 501 may also comprise an outline
defining unit 511 that defines the outlines of cells or cell masses
in the cell image. FIG. 27 is an example of an enlarged image of
iPS cell masses taken through a macro zoom lens. In the image shown
in FIG. 27, the portions visible as white masses are the iPS cell
masses, and the dark background portions are the culture
medium.
[0302] When the image shown in FIG. 27 is an 8-bit grayscale image,
and the image is subjected to binarization in which the maximum
brightness value of 255, for example, is assigned to the values of
the brightness of pixels having brightness values of at least a
prescribed threshold value, and the minimum brightness value of 0,
for example, is substituted for the values of the brightness of
pixels having brightness values less than the prescribed threshold
value, then not only the culture medium portions but also the
interiors of the cell masses appear as the minimum brightness of
black, as shown in FIG. 28, and contiguous portions appear between
the interiors of the cell masses and the culture medium portions.
Therefore, it may not be possible to extract the cells or cell
masses with binarization.
[0303] However, the outline defining unit 511 of the cell
processing system according to the embodiment shown in FIG. 26
applies a highpass filter which allows passage of high-frequency
components of at least a prescribed frequency in the spatial
frequency while blocking low-frequency components of less than the
prescribed frequency in the image of the cells, with a brightness
value of 0, for example, as the minimum value. Numerous
high-frequency components in the spatial frequency are present in
the cell or cell mass portions of the cell image, while few
high-frequency components in the spatial frequency are present in
the culture medium portions. Consequently, in a cell image
subjected to a highpass filter as shown in FIG. 29, the brightness
values of the culture medium portions are the minimum value of 0,
for example, while the cell or cell mass portions retain their
brightness values. Therefore, the portions that are not at the
minimum value of brightness may be considered to be the cells or
cell masses.
[0304] In the image shown in FIG. 29, the portions where the
brightness was not the minimum value appear as blobs, and even with
detection by blob analysis, two mutually adjacent cell masses, for
example, may appear to be a single cell mass in some cases.
[0305] Therefore, the outline defining unit 511 of the cell
processing system of the embodiment shown in FIG. 26 applies a
watershed algorithm to the image that was subjected to the highpass
filter. A watershed algorithm considers the brightness gradient in
the image as mountainous corrugations, and divides the image so
that zones formed by water flowing from the high locations of the
mountains (the locations of high brightness) to the low locations
(the locations of low brightness) are a single region.
[0306] For example, the outline defining unit 511 of the cell
processing system of this embodiment converts the image by the
distance transform method before applying the watershed algorithm
to the image. The distance transform method is an image
transforming method in which the value of the brightness of each
pixel of an image is substituted based on the distance to the
nearest background pixel. For example, in an image that has been
subjected to a highpass filter, as shown in FIG. 30(a), the
brightness value in the culture medium region is converted to 255
as the maximum brightness value, to produce a white background as
shown in FIG. 30(b). Also, the value of the brightness of each
pixel in the cell region is converted in a range of 0 up to less
than 255, based on the distance to the nearest background pixel.
For example, the brightness value is lowered the further it is from
the nearest background pixel.
[0307] Next, the outline defining unit 511 of the cell processing
system of this embodiment applies a watershed algorithm to the
image that has been transformed by the distance transform method.
In the image shown in FIG. 30(b), with the dark portions of low
brightness being considered to be the mountain ridges, it is
imagined how water that has been poured on the image from the
perpendicular direction will flow, as indicated by the arrows in
FIG. 30(c), and considering the valley to be the location where
water flowing from different directions impacts, as indicated by
the broken line in FIG. 30(c), the cell region is divided at the
bottom of the valley.
[0308] When the pixels in the cell region of the image shown in
FIG. 29 are transformed by the distance transform method, the image
shown in FIG. 31 is obtained. When a watershed algorithm is applied
to the image shown in FIG. 31, the image shown in FIG. 32 is
obtained. When the obtained dividing lines are layered over the
original image shown in FIG. 27, the image shown in FIG. 33 is
obtained. In FIG. 33, the cell masses present in each region
divided by the dividing lines are not masses in which a plurality
of cell masses are adjacent, but instead they may be considered to
be single cell masses. In each region, therefore, the outlines of
the cell masses can be extracted to allow accurate extraction of
single cell masses, as shown in FIG. 34.
[0309] The image processor 501 of the cell processing system of the
embodiment shown in FIG. 26 may further comprise a cell evaluating
unit 512. The cell evaluating unit 512 evaluates the cell mass
size, etc. of each cell mass extracted by the outline defining unit
511. For example, the cell evaluating unit 512 calculates the area
of a single cell mass extracted by the outline defining unit 511.
When the shape of the single cell mass is considered to be
circular, for example, the cell evaluating unit 512 also calculates
the diameter of the single cell mass from the area, using the
following formula (1).
D=2(s/.pi.)1/2 (1)
[0310] Here, D represents the diameter and S represents the
area.
[0311] If the cell mass grows too large, the nutrients and hormones
in the culture medium may fail to reach the interior and the cells
may differentiate. In addition, if cell masses that are too small
are transferred to amplifying culture without using a ROCK
inhibitor, cell death or karyotypic abnormalities may occur.
Consequently, the cell evaluating unit 512 may emit an alert when
the individual cell mass sizes are outside of the suitable range.
In addition, the cell evaluating unit 512 may output a timing for
transfer to amplifying culture when the individual cell mass sizes
are beyond a prescribed threshold value. The supply rate of culture
medium at the initializing culturing apparatus 50 may also be
varied according to the calculated cell mass sizes. For example,
the supply rate of the culture medium may be increased as the cell
mass sizes increase.
[0312] The image processor 501 of the cell processing system of
this embodiment may further comprise a statistical processor 513
that statistically processes data obtained from the image that has
undergone image processing. FIG. 35 is an example of image
processing of the image shown in FIG. 25, with the cell mass
portions extracted and outlined. FIG. 36 is an example of a
histogram of cell mass sizes, drawn based on the image shown in
FIG. 35. By thus continuously and periodically obtaining cell data,
it is possible to quantitatively ascertain the degree of growth,
number and compactness of the cell masses, allowing the results of
culturing to be stabilized. The supply rate of culture medium at
the initializing culturing apparatus 50 may also be varied
according to the calculated number of cell masses. For example, the
supply rate of the culture medium may be increased as the number of
cell masses increases.
[0313] The image processor 501 of the cell processing system
according to the embodiment shown in FIG. 26 may further comprise a
density calculating unit 514 that calculates the turbidity of the
culture medium from the image of the culture medium and calculates
the cell mass density in the culture medium based on the turbidity
of the culture medium.
[0314] For example, a relationship memory unit 403 comprising a
volatile memory or a non-volatile memory may be connected to the
CPU 500. The relationship memory unit 403 stores, for example, the
relationship between the turbidity of the culture medium and the
cell mass density in the culture medium, that have been previously
obtained. The density calculating unit 514 reads out the
relationship between turbidity and density from the relationship
memory unit 403. The density calculating unit 514 also calculates
the density of cell masses in the culture medium, based on the
value of the turbidity of the culture medium that has been
calculated from the image of the culture medium, and the
relationship between turbidity and density. This allows the cell
mass density to be measured in a non-destructive manner without
harvesting the cell masses from the culture medium.
[0315] The density calculating unit 514 may also output a timing
for transfer to the amplifying culturing, when the cell mass
density has reached at least at prescribed threshold value. In
addition, the density calculating unit 514 may calculate the cell
mass density in the culture medium with passing time, and may
calculate the growth rate of the cell masses. An abnormal growth
rate may indicate abnormalities in the cells. For example, the
density calculating unit 514 emits an alert when an abnormal growth
rate has been calculated. Culturing of the cells may be interrupted
when this occurs.
[0316] If the cell mass density in the culture medium is high and
the distance between cell masses is too close, a plurality of cell
masses may adhere together to form a single large cell mass. In a
large cell mass, the nutrients and hormones in the culture medium
may fail to reach the interior and the cells within it may
differentiate. On the other hand, if the cell mass density in the
culture medium is lower than the preferred range, the cell mass
growth rate and cell mass formability may be significantly
reduced.
[0317] However, since the cell mass density can be calculated by
the density calculating unit 514, it is possible to easily
determine whether or not the cell mass density is within the
preferred range. When the cell mass density has become lower than
the preferred range, a judgment may be made to interrupt the
culturing, for example. Furthermore, the supply rate of culture
medium at the initializing culturing apparatus 50 may be varied
according to the calculated cell mass density. For example, the
supply rate of the culture medium may be increased as the cell mass
density increases.
[0318] In addition, in order to observe variation in the culture
medium color that takes place with cell metabolism, a culture
medium observation illumination light source 174 may be situated at
a location facing the initializing culturing photographing device
171 and sandwiching the suspension culture vessel, as shown in FIG.
37. A surface light source, for example, may be used as the medium
observation illumination light source 174, with the medium
observation illumination light source 174 emitting white parallel
rays, for example. The illumination light emitted from the medium
observation illumination light source 174 passes through the
culture medium and impinges on the initializing culturing
photographing device 171, thereby allowing the culture medium color
to be imaged by the initializing culturing photographing device
171.
[0319] Cell culturing is generally carried out with a constant
culture medium pH near 6.8 to 7.2. When the culture medium pH is to
be measured, a pH reagent such as phenol red is added to the
culture medium. Phenol red changes due to the pH of the culture
medium. When the carbon dioxide concentration of the gas contacting
the culture medium is insufficient, carbon dioxide in the air does
not equilibrate with carbon dioxide from bicarbonate in the culture
medium, and therefore the culture medium becomes alkaline and the
culture medium color turns reddish violet. Also, with accumulation
of waste products consisting mainly of lactic acid discharged by
the cells, the culture medium becomes acidic and the culture medium
color turns yellow. Acidity of the culture medium indicates that
the nutrients in the culture medium have been depleted.
[0320] The image processor 501 of the cell processing system
according to the embodiment shown in FIG. 26 may further comprise a
culture medium evaluating unit 515 that evaluates the culture
medium based on the image of the culture medium illuminated by the
medium observation illumination light source. The culture medium
evaluating unit 515 performs image processing of the culture medium
image, for example, and expresses the color of the culture medium
as the three parameters HSV: Hue, chroma (Saturation) and
brightness (Value). Of these, hue is a parameter corresponding to a
concept commonly referred to as "color shade" or "tint". Hue is
commonly represented as angle units.
[0321] FIG. 38 is an example of a graph showing the relationship
between change in culture medium hue and change in culture medium
pH, during long-term culturing of cells without exchange of the
medium. Immediately after the start of culturing, the culture
medium pH was near 7.7, but the culture medium pH decreased to near
7.1 as time progressed. At the same time, the culture medium hue
was near 40 immediately after the start of culturing, but increased
to nearly 60 as time progressed. Thus, culture medium hue is
correlated with culturing time and culture medium pH. Therefore,
the culture medium evaluating unit 515 shown in FIG. 26 judges the
state of the culture medium by monitoring the hue of the culture
medium.
[0322] The relationship memory unit 403 stores, for example, the
relationship between the hue of the culture medium and the pH of
the culture medium, that have been previously obtained. The culture
medium evaluating unit 515 reads out the relationship between hue
and pH from the relationship memory unit 403. The culture medium
evaluating unit 515 also calculates the pH value of the
photographed culture medium based on the value of the hue of the
culture medium that has been calculated from the culture medium
image, and the relationship between hue and pH. For example, the
culture medium evaluating unit 515 may obtain an image of the
culture medium over time and calculate the value of the pH of the
culture medium.
[0323] Incidentally, the culture medium pH may also be measured
with a pH sensor 271, as shown in FIG. 22. The culture medium
temperature may also be measured with a thermometer 272. In this
case, the culture medium evaluating unit 515 may receive the value
of the culture medium pH from the pH sensor 271, and may receive
the value of the culture medium temperature from the thermometer
272.
[0324] When the culture medium hue or the culture medium pH is
outside of the prescribed ranges, the culture medium evaluating
unit 515 judges that exchange of culture medium should be
accelerated, or that contamination has occurred in the culture
medium. Medium exchange includes partial exchange of the culture
medium, as well as replenishment.
[0325] Chemical analysis of culture medium components is costly,
and when the culture medium is taken out of the system for chemical
analysis of the culture medium, there is a risk that the aseptic
state of the culture medium may not be maintained. In contrast,
monitoring the state of a culture medium by monitoring the culture
medium hue has low cost and does not affect the aseptic state of
the culture medium.
[0326] When the culture medium evaluating unit 515 has judged that
the culture medium hue or culture medium pH is outside of the
prescribed range, the culture medium surrounding the dialysis tube
75 of the suspension culture vessel is exchanged by the supply
culture medium solution-feeding pump 77 shown in FIG. 21, for
example. Alternatively, when the culture medium is being constantly
exchanged, the exchange rate of the culture medium surrounding the
dialysis tube 75 of the suspension culture vessel by the supply
culture medium solution-feeding pump 77 increases, and the flow
rate of the exchanged culture medium increases. This allows the
culture medium pH to be maintained within a range suitable for cell
culturing, and allows sufficient nutrients to be supplied to the
culture medium.
[0327] In addition, the culture medium evaluating unit 515 may
calculate the growth rate of the cells from the rate of change of
the culture medium hue. The relationship memory unit 403 stores,
for example, the relationship between the rate of change in the
culture medium hue and the growth rate of the cells, that have been
previously obtained. The culture medium evaluating unit 515 reads
out the relationship between the hue change rate and the growth
rate, from the relationship memory unit 403. In addition, the
culture medium evaluating unit 515 calculates the value for the
growth rate of the cells, based on the calculated value of the hue
change rate and the relationship between the hue change rate and
the growth rate.
[0328] When the culture medium evaluating unit 515 has judged that
the temperature of the culture medium is outside of the prescribed
range, it may control a temperature regulating device so as to
change the temperature surrounding the culturing vessel, or the
temperature of the supplied culture medium. For example, when the
temperature of the culture medium is lower than the prescribed
range, the culture medium evaluating unit 515 regulates the
temperature regulating device so that the temperature of the
culture medium rises. When the temperature of the culture medium is
higher than the prescribed range, the culture medium evaluating
unit 515 regulates the temperature regulating device so that the
temperature of the culture medium falls.
[0329] A first cell mass solution-feeding channel 51 is connected
to the initializing culturing apparatus 50 shown in FIG. 16. The
initializing culturing apparatus 50 employs a pump or the like to
deliver a solution containing trypsin-substituting recombinant
enzyme and the cell masses to the first cell mass solution-feeding
channel 51. When the cell masses are to be physically disrupted,
there is no need for a trypsin-substituting recombinant enzyme.
Also, the first cell mass solution-feeding channel 51 may have an
inner diameter that allows passage of only induced cells of less
than a prescribed size, and it may be connected to a branched fluid
channel that removes non-induced cells of a prescribed size or
larger. As mentioned above, when a gel medium is used, the cell
masses can be collected by suctioning up the gel medium.
[0330] The pump that delivers the cell mass-containing solution to
the first cell mass solution-feeding channel 51 may be driven when,
for example, the value of the cell mass size calculated by the cell
evaluating unit 512 shown in FIG. 26 is at least a prescribed
threshold value. Alternatively, the pump that delivers the cell
mass-containing solution to the first cell mass solution-feeding
channel 51 shown in FIG. 16 may be driven when, for example, the
value of the cell mass density calculated by the density
calculating unit 514 shown in FIG. 26 is at least a prescribed
threshold value.
[0331] The inner wall of the first cell mass solution-feeding
channel 51 shown in FIG. 16 may be coated with poly-HEMA to render
it non-cell-adherent, so that the cells do not adhere.
Alternatively, a material resistant to cell adhesion may be used as
the material for the first cell mass solution-feeding channel 51.
In addition, if a material with good thermal diffusivity and
CO.sub.2 permeability is used as the material for the first cell
mass solution-feeding channel 51, the conditions in the first cell
mass solution-feeding channel 51 will be equivalent to the
controlled temperature and CO.sub.2 concentration in the enclosure
601. A back-flow valve may also be provided in the first cell mass
solution-feeding channel 51 from the viewpoint of preventing
contamination.
[0332] The first cell mass solution-feeding channel 51 is connected
to the first dissociating mechanism 60. The first dissociating
mechanism 60 comprises a mesh, for example. The cell masses in the
solution are dissociated into a plurality of cell masses
corresponding to the sizes of the holes of the mesh, when they pass
through the mesh by water pressure. If the mesh hole sizes are
uniform, for example, the sizes of the plurality of cell masses
after being dissociated will be approximately uniform.
Alternatively, the first dissociating mechanism 60 may comprise a
nozzle. For example, if the interior of an approximately conical
nozzle is micromachined in a step-wise manner, a cell mass in the
solution will be dissociated into a plurality of cell masses when
it passes through the nozzle.
[0333] As another alternative, shown in FIG. 39, the first
dissociating mechanism 60 may comprise a cell mass dissociater
comprising a terminal block 61, a connecting block 62 and a tip
block 63. The terminal block 61, connecting block 62 and tip block
63 are each provided with a through-hole inside them through which
the cell mass-containing culture medium flows. As shown in FIG. 40
and FIG. 41, the terminal block 61, connecting block 62 and tip
block 63 are connected. The cell mass dissociater may comprise a
single connecting block 62, or it may comprise a plurality of
connecting blocks 62.
[0334] As shown in FIG. 39, at the first edge of the connecting
block 62 there is provided a recess 62a, and at the second edge
opposite the first edge of the connecting block 62 there is
provided a protrusion 62b. The protrusion 62b is cylindrical, for
example. As shown in FIG. 40 and FIG. 41, when a plurality of
connecting blocks 62 are used, the protrusions 62b engage with the
recesses 62a of the adjacent connecting blocks 62. The side wall of
the protrusion 62b shown in FIG. 39 may be smooth, or a male screw
form may be provided. The inner wall of the recess 62a may be
smooth, or a female screw form may be provided.
[0335] The through-hole provided in the connecting block 62 has a
first large pore size section 62c that communicates with the recess
62a, a small pore size section 62d that communicates with the first
large pore size section 62c and has a smaller pore size than the
first large pore size section 62c, and a second large pore size
section 62e that communicates with the small pore size section 62d,
has a larger pore size than the small pore size section 62d, and
has an opening at the tip of the protrusion 62b.
[0336] The cross-sectional shapes of the first large pore size
section 62c, small pore size section 62d and second large pore size
section 62e are circular, for example. The pore size of the first
large pore size section 62c and the pore size of the second large
pore size section 62e are the same, for example. Thus, when a
plurality of connecting blocks 62 are used and the plurality of
connecting blocks 62 are connected, as shown in FIG. 40 and FIG.
41, the second large pore size section 62e will smoothly
communicate with the first large pore size section 62c of the
adjacent connecting block 62.
[0337] The pore sizes of the first and second large pore size
sections 62c, 62e shown in FIG. 39 are, for example, between 2.0 mm
and 4.0 mm, inclusive, without any particular restriction to this
range. The pore size of the small pore size section 62d is, for
example, between 0.4 mm and 1.2 mm, inclusive, without any
particular restriction to this range. A step is formed at the
section connecting from the first large pore size section 62c to
the small pore size section 62d. A step is also formed at the
section connecting from the small pore size section 62d to the
second large pore size section 62e. The side walls of the steps may
be perpendicular to the central axis of the through-hole, or they
may be inclined at less than 90.degree..
[0338] The central axes of the first and second large pore size
sections 62c, 62e and the central axis of the small pore size
section 62d in the connecting block 62 may match. Alternatively,
the central axes of the first and second large pore size sections
62c, 62e and the central axis of the small pore size section 62d in
the connecting block 62 may be offset, as shown in FIG. 42.
[0339] A recess 63a is provided at the first edge of the tip block
63 shown in FIG. 39, and a nozzle section 63b is provided at the
second edge opposite the first edge of the tip block 63. When the
tip block 63 and the connecting block 62 are connected, the recess
63a of the tip block 63 engages with the protrusion 62b of the
connecting block 62. The inner wall of the recess 63a may be
smooth, or a female screw form may be provided.
[0340] The through-hole provided in the tip block 63 has a large
pore size section 63c that communicates with the recess 63a, and a
small pore size section 63d that communicates with the large pore
size section 63c, has a smaller pore size than the large pore size
section 63c, and has an opening at the tip of the nozzle section
63b.
[0341] The cross-sectional shapes of the large pore size section
63c and the small pore size section 63d are circular, for example.
The pore size of the large pore size section 63c of the tip block
63 and the pore size of the second large pore size section 62e of
the connecting block 62 are the same, for example. This will allow
the second large pore size section 62e of the connecting block 62
and the large pore size section 63c of the adjacent tip block 63 to
smoothly communicate when the connecting block 62 and the tip block
63 have been connected, as shown in FIG. 40 and FIG. 41.
[0342] The pore size of the large pore size section 63c shown in
FIG. 39 is, for example, between 2.0 mm and 4.0 mm, inclusive,
without any particular restriction to this range. The pore size of
the small pore size section 63d is, for example, between 0.4 mm and
1.2 mm, inclusive, without any particular restriction to this
range. A step is formed at the section connecting from the large
pore size section 63c to the small pore size section 63d. The side
walls of the steps may be perpendicular to the central axis of the
through-hole, or they may be inclined at less than 90.degree..
[0343] A recess 61a is provided at the first edge of the terminal
block 61, and a protrusion 61b is provided at the second edge
opposite the first edge of the terminal block 61. When the terminal
block 61 and the connecting block 62 are connected, the protrusion
61b of the terminal block engages with the recess 62a of the
connecting block 62. The side wall of the protrusion 61b of the
terminal block may be smooth, or a male screw may be provided.
[0344] The through-hole provided in the terminal block 61 has at
least a large pore size section 61c that communicates with the
recess 61a and has an opening at the tip of the protrusion 61b.
[0345] The cross-sectional shapes of the recess 61a and the large
pore size section 61c are circular, for example. The pore size of
the large pore size section 61c of the terminal block 61 and the
pore size of the second large pore size section 62e of the
connecting block 62 are the same, for example. This will allow the
large pore size section 61c of the terminal block 61 and the large
pore size section 62c of the adjacent connecting block 62 to
smoothly communicate when the terminal block 61 and the connecting
block 62 have been connected, as shown in FIG. 40 and FIG. 41.
[0346] The pore size of the large pore size section 61c shown in
FIG. 39 is, for example, between 2.0 mm and 4.0 mm, inclusive,
without any particular restriction to this range.
[0347] The materials of the terminal block 61, the connecting block
62 and the tip block 63 may be, but are not restricted to, resins
such as polypropylene.
[0348] As shown in FIG. 40, FIG. 41 and FIG. 42, an insertion
nozzle 64, for example, is inserted in the recess 61a of the
terminal block 61. A suction drainer that suction drains the cell
mass-containing culture medium, either directly or through a tube
or the like, is connected to the insertion nozzle 64. When the
terminal block 61, connecting block 62 and tip block 63 are
connected, the nozzle section 63b of the tip block 63 is thrust
into the cell mass-containing culture medium, and the suction
drainer carries out suction drainage of the culture medium once or
repeats suction drainage of the culture medium, the cell
mass-containing culture medium is reciprocated in the through-holes
in the connecting block 62 and the tip block 63. Because steps are
provided in the through-holes of the connecting block 62 and tip
block 63, the cell masses in the culture medium are dissociated
into small cell masses in an efficient manner.
[0349] Conventionally, dissociation of cell masses has been carried
out by a technician using a Pipetman or the like. However, as shown
in FIG. 47(a), cell mass sizes dissociated by the conventional
method have been non-uniform. Moreover, the obtained cell mass
sizes have been variable depending on the technician. If the
dissociated cell masses are too large, the nutrients and hormones
in the culture medium may fail to reach the interior and the cells
may differentiate. If the cell masses are too small and a ROCK
inhibitor is not used, cell death or karyotypic abnormalities may
occur. By using the cell mass dissociater illustrated in FIG. 40,
FIG. 41 and FIG. 42, however, it is possible to dissociate cell
masses into cell masses of uniform sizes, as shown in FIG. 47(b).
When the cell mass dissociater is used to dissociate cell masses,
the culture medium may include enzymes such as trypsin, or TrypLE
Express.RTM. (Thermo Fisher Scientific), TrypLE Select.RTM. (Thermo
Fisher Scientific) or TrypLE Select.RTM. (Thermo Fisher
Scientific). Also, by increasing the number of connecting blocks 62
or raising the pressure during suction drainage of the culture
medium, it is possible to degrade the cell masses into single
cells.
[0350] If a suitable number and suitable lengths of repeating large
pore size sections and small pore size sections have been
determined, the cell mass dissociater does not need to be composed
of a plurality of blocks. For example, as shown in FIG. 43, the
cell mass dissociater may have an integral cylindrical shape with a
through-hole in the interior, the through-hole through which the
cell mass-containing culture medium flows having, in an alternating
manner, large pore size sections 65a, 65c, 65e, 65g, and small pore
size sections 65b, 65d, 65f that communicate with the large pore
size sections 65a, 65c, 65e, 65g and have smaller pore sizes than
the large pore size sections 65a, 65c, 65e, 65g. In this case as
well, as shown in FIG. 44, the central axes of the large pore size
sections 65a, 65c, 65e, 65g and the central axes of at least some
of the small pore size sections 65b, 65d, 65f may be offset.
[0351] The culture medium may pass through the cell mass
dissociater only once to dissociate the cell masses in the culture
medium into small cell masses. In this case, as shown in FIG. 45,
insertion sections 66a, 66b may be provided to allow insertion of a
tube or the like at both ends of the cell mass dissociater. The
culture medium passes from the insertion section 66a through the
through-hole and is discharged from the insertion section 66b,
during which time the cell masses in the culture medium are
dissociated. In this case as well, as shown in FIG. 46, the central
axes of the large pore size sections 65a, 65c, 65e, 65g and the
central axes of at least some of the small pore size sections 65b,
65d, 65f may be offset.
[0352] The amplifying culturing apparatus 70 is connected to the
first dissociating mechanism 60 shown in FIG. 16. The solution
including cell masses that have been dissociated at the first
dissociating mechanism 60 is fed to the amplifying culturing
apparatus 70.
[0353] The amplifying culturing apparatus 70 can house a well plate
in its interior. The amplifying culturing apparatus 70 also
comprises a pipetting machine. The amplifying culturing apparatus
70 receives the solution including the plurality of cell masses
from the first dissociating mechanism 60, and the solution is
allocated into the wells with a pipetting machine. After allocating
the cell masses into the wells, the amplifying culturing apparatus
70 cultures the cell masses for about 8 days, for example, at
37.degree. C., 5% CO.sub.2. The amplifying culturing apparatus 70
also carries out appropriate exchange of the culture medium.
[0354] The amplifying culturing apparatus 70 then adds a
trypsin-substituting recombinant enzyme such as TrypLE Select.RTM.
(Life Technologies Corp.) to the cell masses. In addition, the
amplifying culturing apparatus 70 places a vessel containing the
collected cell masses in an incubator, and reacts the cell masses
with the trypsin-substituting recombinant enzyme for 1 minute at
37.degree. C., 5% CO.sub.2. When the cell masses are to be
physically disrupted, there is no need for a trypsin-substituting
recombinant enzyme. For example, the amplifying culturing apparatus
70 may disrupt the cell masses by pipetting with a pipetting
machine. Alternatively, the amplifying culturing apparatus 70 may
disrupt the cell masses by passing the cell masses through a pipe
provided with a filter, or a pipe that intermittently varies the
inner diameter, similar to the introduced cell solution-feeding
channel 31 shown in FIG. 17 or FIG. 18. The amplifying culturing
apparatus 70 then adds culture medium such as maintenance culture
medium to the solution containing the cell masses. Furthermore,
when the amplifying culturing apparatus 70 carries out adhesion
culture, the cell masses are scraped from the vessel with an
automatic cell scraper or the like, and the cell mass-containing
solution is fed to the first dissociating mechanism 60 through an
amplifying culturing solution-feeding channel 71.
[0355] Culturing in the amplifying culturing apparatus 70 may be
carried out in a CO.sub.2-permeable bag instead of a well plate.
The culturing may also be by adhesion culture, or by suspension
culture, or by hanging drop culture. Agitation culture may be
carried out in the case of a suspension culture. The culture medium
may also be in the form of agar. Agar culture media include gellan
gum polymers and deacylated gellan gum polymers. When agar culture
medium is used, there is no settling or adhesion of cells, and
therefore agitation is not necessary even though it is suspension
culture.
[0356] The amplifying culturing apparatus 70 may also comprise a
second culture medium supply device that supplies culture solution
to the well plate or CO.sub.2-permeable bag. The second culture
medium supply device collects the culture solution in the well
plate or CO.sub.2-permeable bag, and it may use a filter or
dialysis membrane to filter the culture solution, to allow reuse of
the purified culture solution. During this time, growth factors or
the like may be added to the culture solution that is to be reused.
The amplifying culturing apparatus 70 may also comprise a
temperature regulating device that regulates the temperature of the
culture medium, and a humidity regulating device that regulates the
humidity in the vicinity of the culture medium.
[0357] In the amplifying culturing apparatus 70, the cells may be
placed in a culture solution-permeable bag 301 such as a dialysis
membrane as shown in FIG. 19, for example, and the culture
solution-permeable bag 301 may be placed in a culture
solution-impermeable CO.sub.2-permeable bag 302, so that the
culture solution is placed in bags 301, 302. The initializing
culturing apparatus 50 may have multiple bags 302 prepared
containing fresh culture solution, and the bag 302 in which the
cell-containing bag 301 is placed may be replaced by a bag 302
containing fresh culture solution, at prescribed intervals of
time.
[0358] The culturing method in the amplifying culturing apparatus
70 is not limited to the method described above, and may employ a
suspension culture vessel such as shown in FIG. 20, similar to the
culturing method in the initializing culturing apparatus 50. In the
amplifying culturing apparatus 70, the plurality of cell masses are
to be placed in the dialysis tube 75 of the suspension culture
vessel shown in FIG. 20. The details regarding the suspension
culture vessel are as explained above. In the amplifying culturing
apparatus 70 as well, a supply culture medium solution-feeding pump
77 may be used as shown in FIG. 21, for exchange and supply of the
gel medium surrounding the dialysis tube 75 in the vessel 76.
Alternatively, as shown in FIG. 22, the supply culture medium
solution-feeding pump 77 and the interior of the dialysis tube 75
in the suspension culture vessel 76 may be connected by the
solution-feeding tube 78, to supply the components necessary for
culturing of cells in the culture medium in the dialysis tube
75.
[0359] The cell processing system may further comprise an
amplifying culturing photographing device that photographically
records culturing in the amplifying culturing apparatus 70, as
shown in FIG. 16. If a colorless culture medium is used for the
culture medium in the amplifying culturing apparatus 70, it will be
possible to minimize diffuse reflection and autologous fluorescence
that may be produced when using a colored culture medium. In order
to confirm the pH of the culture medium, however, a pH indicator
such as phenol red may be included. Moreover, since induced cells
and non-induced cells have differences in cellular shape and size,
the cell processing system may further comprise an induced state
monitoring device that calculates the proportion of induced cells
by photographing the cells in the amplifying culturing apparatus
70. Alternatively, the induced state monitoring device may
determine the proportion of induced cells by antibody
immunostaining or RNA extraction. In addition, the cell processing
system may comprise a non-induced cell removing device that removes
cells that have not been induced, by magnetic-activated cell
sorting, flow cytometry or the like.
[0360] The amplifying culturing photographing device is similar to
the initializing culturing photographing device 171 shown in FIG.
23, and it may photograph culturing in the amplifying culturing
apparatus 70 through a telecentric lens 172. The illumination
method during photography by the amplifying culturing photographing
device may also be the same as the illumination method during
photography by the initializing culturing photographing device 171,
which is as described above.
[0361] The amplifying culturing photographing device is also
connected to a CPU 500 comprising an image processor 501, as shown
in FIG. 26. The image processor 501 comprising the outline defining
unit 511, cell evaluating unit 512, statistical processor 513,
density calculating unit 514 and culture medium evaluating unit
515, performs image processing on the image taken by the amplifying
culturing photographing device, in the same manner as for the image
taken by the initializing culturing photographing device 171. The
details regarding the image processor 501 are as described
above.
[0362] For example, if the cell mass grows too large during
amplifying culturing, the nutrients and hormones in the culture
medium may fail to reach the interior and the cells may
differentiate. In addition, if cell masses that are too small are
subcultured, without using a ROCK inhibitor, cell death or
karyotypic abnormalities may occur. The cell evaluating unit 512
may therefore emit an alert when the individual cell mass sizes are
outside of the suitable range. In addition, the cell evaluating
unit 512 may output a timing for subculturing when the individual
cell mass sizes are beyond a prescribed threshold value. In this
case, the cell masses may be fragmented to reduce the sizes of the
individual cell masses, and subcultured by resuming culturing in
the culturing vessel. In addition, if the individual cell mass
sizes after fragmentation of the cell masses are calculated during
the subculturing, it is possible to judge whether or not the
fragmentation has been adequate. The supply rate of culture medium
at the amplifying culturing apparatus 70 may also be varied
according to the calculated cell mass sizes. For example, the
supply rate of the culture medium may be increased as the cell mass
sizes increase.
[0363] The supply rate of culture medium at the amplifying
culturing apparatus 70 may also be varied according to the number
of cell masses calculated by the statistical processor 513. For
example, the supply rate of the culture medium may be increased as
the number of cell masses increases.
[0364] The density calculating unit 514 may also output a timing
for subculturing, when the cell mass density has reached at least a
prescribed threshold value. When the cell mass density has become
higher than the suitable range, the cell mass density may be
adjusted to within the suitable range by subculturing, for example.
In addition, if the cell mass density after fragmentation of the
cell masses is calculated during the subculturing, it is possible
to judge whether or not the fragmentation has been adequate. The
supply rate of culture medium at the amplifying culturing apparatus
70 may also be varied according to the calculated cell mass
density. For example, the supply rate of the culture medium may be
increased as the cell mass density increases.
[0365] When the culture medium evaluating unit 515 has judged that
the culture medium hue or the culture medium pH is outside of the
prescribed range, the culture medium surrounding the dialysis tube
75 of the suspension culture vessel is exchanged by the supply
culture medium solution-feeding pump 77 shown in FIG. 21, for
example, at the amplifying culturing apparatus 70 as well.
Alternatively, when the culture medium is being constantly
exchanged, the exchange rate of the culture medium surrounding the
dialysis tube 75 of the suspension culture vessel by the supply
culture medium solution-feeding pump 77 increases, and the flow
rate of the exchanged culture medium increases. This allows the
culture medium pH to be maintained within a range suitable for cell
culturing, and allows sufficient nutrients to be supplied to the
culture medium.
[0366] The cell masses that have been dissociated by the first
dissociating mechanism 60 shown in FIG. 16 are again cultured in
the amplifying culturing apparatus 70. Dissociation of the cell
masses at the first dissociating mechanism 60 and culturing of the
cell masses in the amplifying culturing apparatus 70 are repeated
until the necessary cell volume is obtained.
[0367] The pump that delivers the cell mass-containing solution in
the amplifying culturing apparatus 70 to the first dissociating
mechanism 60 through the amplifying culturing solution-feeding
channel 71 may be driven when, for example, the value of the cell
mass size calculated by the cell evaluating unit 512 shown in FIG.
26 is at least a prescribed threshold value. Alternatively, the
pump that delivers the cell mass-containing solution to the first
cell mass solution-feeding channel 51 shown in FIG. 16 may be
driven when, for example, the value of the cell mass density
calculated by the density calculating unit 514 shown in FIG. 26 is
at least a prescribed threshold value.
[0368] A second cell mass solution-feeding channel 72 is connected
to the amplifying culturing apparatus 70. The amplifying culturing
apparatus 70 delivers the cell mass-containing solution, that has
been amplifying cultured and detached from the vessel, to the
second cell mass solution-feeding channel 72 using a pump or the
like. Detachment is not necessary, however, in the case of
suspension culture. The second cell mass solution-feeding channel
72 may have an inner diameter that allows passage of only induced
cells of less than a prescribed size, and it may be connected to a
branched fluid channel that removes non-induced cells of a
prescribed size or larger.
[0369] The inner wall of the second cell mass solution-feeding
channel 72 may be coated with poly-HEMA to render it
non-cell-adherent, so that the cells do not adhere. Alternatively,
a material resistant to cell adhesion may be used as the material
for the second cell mass solution-feeding channel 72. Also, by
using a material with good thermal diffusivity and CO.sub.2
permeability as the material for the second cell mass
solution-feeding channel 72, the conditions in the second cell mass
solution-feeding channel 72 will be equivalent to the controlled
temperature and CO.sub.2 concentration in the enclosure 601. A
back-flow valve may also be provided in the second cell mass
solution-feeding channel 72 from the viewpoint of preventing
contamination.
[0370] The second cell mass solution-feeding channel 72 is
connected to the second dissociating mechanism 80. The second
dissociating mechanism 80 comprises a mesh, for example. The cell
masses in the solution are dissociated into a plurality of cell
masses corresponding to the sizes of the holes of the mesh, when
they pass through the mesh by water pressure. If the mesh hole
sizes are uniform, for example, the sizes of the plurality of cell
masses after being dissociated will be approximately uniform.
Alternatively, the second dissociating mechanism 80 may comprise a
nozzle. For example, if the interior of an approximately conical
nozzle is micromachined in a step-wise manner, a cell mass in the
solution will be dissociated into a plurality of cell masses when
it passes through the nozzle.
[0371] Alternatively, the second dissociating mechanism 80, similar
to the first dissociating mechanism 60, may comprise a cell mass
dissociater comprising a terminal block 61, connecting block 62 and
tip block 63 as shown in FIG. 39 to FIG. 42, or an integral cell
dissociater as shown in FIG. 43 to FIG. 46. The details regarding
the cell mass dissociater are as explained above.
[0372] The cell mass transport mechanism 90 that sends the
plurality of cell masses in order to the packaging device 100 is
connected to the second dissociating mechanism 80 shown in FIG. 16.
A pre-packaging cell channel 91 is connected between the cell mass
transport mechanism 90 and the packaging device 100. The cell mass
transport mechanism 90 employs a pump or the like to send each of
the cell masses that have been dissociated by the second
dissociating mechanism 80, to the packaging device 100 through the
pre-packaging cell channel 91.
[0373] The pre-packaging cell channel 91 is coated with poly-HEMA
so that the cells do not adhere. Alternatively, a material
resistant to cell adhesion may be used as the material for the
pre-packaging cell channel 91. Also, by using a material with good
thermal diffusivity and CO.sub.2 permeability as the material of
the pre-packaging cell channel 91, the conditions in the
pre-packaging cell channel 91 will be equivalent to the controlled
temperature and CO.sub.2 concentration in the enclosure 601. A
back-flow valve may also be provided in the pre-packaging cell
channel 91 from the viewpoint of preventing contamination.
[0374] A cryopreservation liquid-feeding mechanism 110 is connected
to the pre-packaging cell channel 91. The cryopreservation
liquid-feeding mechanism 110 feeds a cell cryopreservation liquid
into the pre-packaging cell channel 91. As a result, the cell
masses are suspended in the cell cryopreservation liquid inside the
pre-packaging cell channel 91.
[0375] The packaging device 100 freezes each of the plurality of
cell masses in order, that have been fed through the pre-packaging
cell channel 91. For example, each time it receives cell masses,
the packaging device 100 places the cell masses in a
cryopreservation vessel such as a cryotube, and immediately freezes
the cell mass solution at -80.degree. C. or below, for example.
When using a cryopreservation vessel with a small surface area per
volume, more time will tend to be necessary for freezing, and
therefore it is preferred to use a cryopreservation vessel with a
large surface area per volume. By using a cryopreservation vessel
with a large surface area per volume it is possible to increase the
survival rate of the cells after thawing. The shape of the
cryopreservation vessel may be capillary-like or spherical, without
any particular restrictions. Immediate freezing is not necessarily
essential, depending on the survival rate required for the cells
after thawing.
[0376] Vitrification, for example, may be employed for the
freezing. In this case, the cell cryopreservation liquid used may
be DAP213 (Cosmo Bio Co., Ltd.) or Freezing Medium (ReproCELL,
Inc.). The freezing may also be carried out by a common method
other than vitrification. In this case, the cell cryopreservation
liquid used may be CryoDefend-Stem Cell (R&D Systems) or
STEM-CELLBANKER.RTM. (Zenoaq). The freezing may be carried out with
liquid nitrogen, or it may be carried out with a Peltier element.
When a Peltier element is used, temperature changes can be
controlled and temperature variation can be minimized. When the
frozen cells are to be used in the clinic, the cryopreservation
vessel is preferably a completely closed system. However, the
packaging device 100 may package the stem cells in a storage vessel
without freezing.
[0377] Alternatively, in the packaging device 100, the cell mass
solution may be exchanged from the culture medium to the
cryopreservation liquid using a solution exchanger 101 as
illustrated in FIG. 48. Inside the solution exchanger 101 there is
provided a filter 102 having at the bottom a fine hole which does
not permit passage of cell masses. In the solution exchanger 101
there is also provided a cell mass introduction hole where a first
solution-feeding channel 103 that feeds cell mass-containing
culture medium onto the internal filter 102 is connected, an
exchange solution introduction hole where a second solution-feeding
channel 104 that feeds cell mass-free frozen solution onto the
internal filter 102 is connected, and a cell mass outflow hole
where a first discharge channel 105 that discharges cell
mass-containing frozen solution onto the internal filter 102 is
connected. There is also provided in the solution exchanger 101 a
waste liquid outflow hole wherein there is connected a second
discharge channel 106 that discharges solution that has passed
through the filter 102. Tubes or the like may be used for each of
the first solution-feeding channel 103, second solution-feeding
channel 104, first discharge channel 105 and second discharge
channel 106.
[0378] First, as shown in FIG. 48(a) and FIG. 48(b), cell
mass-containing culture medium is placed inside the solution
exchanger 101 from the first solution-feeding channel 103, while
flow of the solution in the second discharge channel 106 is
stopped. Next, as shown in FIG. 48(c), a state is formed allowing
flow of the solution in the second discharge channel 106, and the
culture medium is discharged from the solution exchanger 101. The
cell masses remain on the filter 102 during this time, as shown in
FIG. 48(d). As shown in FIG. 48(e) and FIG. 48(f), the
cryopreservation liquid is first placed inside the solution
exchanger 101 from the second solution-feeding channel 104, while
flow of the solution in the second discharge channel 106 is
stopped, and the cell masses are dispersed in the cryopreservation
liquid. Next, as shown in FIG. 48(g), the cell mass-containing
cryopreservation liquid is discharged from the first discharge
channel 105. The cell mass-containing cryopreservation liquid is
sent to a cryopreservation vessel or the like through the first
discharge channel 105.
[0379] The solution exchanger 101 shown in FIG. 48 may be used not
only for exchange from culture medium to cryopreservation liquid,
but also for exchange from old culture medium to fresh culture
medium. In this case, the second solution-feeding channel 104 feeds
fresh culture medium. Alternatively, when dissociating the cell
masses, the solution exchanger 101 may be used for exchange of the
culture medium with solution containing a cell mass dissociating
enzyme. Examples of cell mass dissociating enzymes include trypsin,
and trypsin-substituting recombinant enzymes such as TrypLE
Select.RTM. (Life Technologies Corp.). In this case, the second
solution-feeding channel 104 feeds solution containing a cell mass
dissociating enzyme.
[0380] The cell processing system may further comprise a packaging
step photographing device in which the packaging step is
photographed at the packaging device 100, as shown in FIG. 16.
[0381] The cell processing system may also record the behavior of
the separating device 10, preintroduction cell solution-feeding
channel 20, inducing factor solution-feeding mechanism 21, factor
introducing device 30, cell mass preparation device 40 and
packaging device 100, and may transmit the image taken by the
photographing device to an external server, in either a wired or
wireless manner. At the external server, factors such as the
conditions including the inducing factor introduction conditions,
the culturing conditions and the freezing conditions, and results
such as incomplete initialization of the stem cells, failed
differentiation and growth of the stem cells and chromosomal
aberrations, for example, are analyzed by a neural network, and the
conditions leading to results may be extracted and results
predicted. In addition, the external server may control the
separating device 10, inducing factor solution-feeding mechanism
21, factor introducing device 30, cell mass preparation device 40
and packaging device 100 of the cell processing system based on a
standard operating procedure (SOP), monitor whether or not each
device is running based on the SOP, and automatically produce a
running record for each device.
[0382] With the cell processing system described above, it is
possible to carry out induction, establishment, amplifying
culturing and cryopreservation of stem cells such as iPS cells,
fully automatically in a single process.
[0383] The cell processing apparatus of the cell processing system
according to this embodiment is not limited to the construction
illustrated in FIG. 16. For example, in the cell processing
apparatus shown in FIG. 49, blood is delivered from the blood
storing unit 201 to the mononuclear cell separating unit 203,
through a blood solution-feeding channel 202. Tubes, for example,
may be used as the blood storing unit 201 and mononuclear cell
separating unit 203. The blood solution-feeding channel 202 may be
a resin tube or silicon tube, for example. This also applies for
the other solution-feeding channels described below. An identifier
such as a barcode is attached to the blood storing unit 201 for
control of the blood information. A pump 204 is used for feeding of
the solution. The pump 204 that is used may be a
positive-displacement pump. Examples of positive-displacement pumps
include reciprocating pumps including piston pumps, plunger pumps
and diaphragm pumps, and rotating pumps including gear pumps, vane
pumps and screw pumps. Examples of diaphragm pumps include tubing
pumps and piezoelectric pumps. Examples of tubing pumps include
Perista Pump.RTM. (Atto Corp.) and RP-Q1 and RP-TX (Takasago
Electric, Inc.). Examples of piezoelectric pumps include SDMP304,
SDP306, SDM320 and APP-20KG (Takasago Electric, Inc.). A microflow
chip module (Takasago Electric, Inc.) comprising a combination of
various different pumps may also be used. When a sealed pump such
as a Perista Pump.RTM., tubing pump or diaphragm pump is used,
feeding can be accomplished without direct contact of the pump with
the blood inside the blood solution-feeding channel 202. The same
also applies to the other pumps described below. Alternatively,
syringe pumps may be used for the pump 204, and for the pump 207,
pump 216, pump 222, pump 225, pump 234, pump 242 and pump 252
described below. Even pumps other than sealed pumps may be
reutilized after heat sterilization treatment.
[0384] An erythrocyte coagulant is fed to the mononuclear cell
separating unit 203 from the separating agent storing unit 205,
through a solution-feeding channel 206 and the pump 207. Tubes, for
example, may be used as the separating agent storing unit 205. An
identifier such as a barcode is attached to the separating agent
storing unit 205 for control of the separating agent information.
The erythrocyte coagulant used may be, for example, HetaSep.RTM.
(STEMCELL Technologies) or an Erythrocyte Coagulant (Nipro Corp.).
In the mononuclear cell separating unit 203, the erythrocytes
precipitate by the erythrocyte coagulant and the mononuclear cells
are separated. The mononuclear cell-containing supernatant in the
mononuclear cell separating unit 203 is sent to a mononuclear cell
purifying filter 210 through a mononuclear cell solution-feeding
channel 208 and pump 209. At the mononuclear cell purifying filter
210, components other than the mononuclear cells are removed to
obtain a mononuclear cell-containing solution. The mononuclear cell
purifying filter 210 used may be Purecell.RTM. (PALL), Cellsorba E
(Asahi Kasei Corp.), SEPACELL PL (Asahi Kasei Corp.),
ADACOLUMN.RTM. (Jimro), or a separation bag (Nipro Corp.).
[0385] In FIG. 49, the mononuclear cell separating unit 203,
separating agent storing unit 205, mononuclear cell purifying
filter 210 and pumps 204, 207, 209 constitute a separating
device.
[0386] The mononuclear cell-containing solution is sent to a factor
introducing device 213 through a preintroduction cell
solution-feeding channel 211 and pump 212. Tubes, for example, may
be used as the factor introducing device 213. Pluripotency inducing
factor is fed to the factor introducing device 213 from a factor
storing unit 214 including pluripotency inducing factor, through a
factor solution-feeding channel 215 and the pump 216. Tubes, for
example, may also be used as the factor storing unit 214. An
identifier such as a barcode is attached to the factor storing unit
214 for control of the pluripotency inducing factor information.
The factor storing unit 214 and the pump 216 constitute the
inducing factor solution-feeding mechanism. In the factor
introducing device 213 as the factor introducing device, the
pluripotency inducing factor is introduced into cells by RNA
lipofection, for example, and inducing factor-introduced cells are
prepared. The method of transfection of the inducing factor,
however, is not limited to RNA lipofection. For example, Sendai
virus vector including a pluripotency inducing factor may be used.
Alternatively, the pluripotency inducing factor may be a
protein.
[0387] The inducing factor-introduced cells are sent through an
introduced cell solution-feeding channel 217 and pump 218 to an
initializing culturing vessel 219 as a part of the cell mass
preparation device. The introduced cell solution-feeding channel
217 may be temperature-permeable and CO.sub.2-permeable, for
example. The suspension culture vessel shown in FIG. 20 may be used
as the initializing culturing vessel 219. In this case, the
inducing factor-introduced cells are placed in a dialysis tube. For
the first few days after introduction of the pluripotency inducing
factor to the cells, blood cell culture medium is supplied to the
initializing culturing vessel 219 shown in FIG. 49 from a blood
cell culture medium storing unit 220 including blood cell culture
medium, through a culture medium solution-feeding channel 221 and
pump 222. The culture medium solution-feeding channel 221 may be
temperature-permeable and CO.sub.2-permeable, for example. An
identifier such as a barcode is attached to the blood cell culture
medium storing unit 220 for control of the blood cell culture
medium information. The blood cell culture medium storing unit 220,
culture medium solution-feeding channel 221 and pump 222 constitute
the culture medium supply device. The pump 222 may continuously
supply blood cell culture medium, or it may supply blood cell
culture medium at a prescribed timing, according to directions by
the CPU 500 shown in FIG. 26.
[0388] Next, stem cell culture medium is supplied to the
initializing culturing vessel 219 shown in FIG. 49, from a stem
cell culture medium storing unit 223 including stem cell culture
medium, through a culture medium solution-feeding channel 224 and
pump 225. An identifier such as a barcode is attached to the stem
cell culture medium storing unit 223 for control of the stem cell
culture medium information. The culture medium solution-feeding
channel 224 may be temperature-permeable and CO.sub.2-permeable,
for example. The stem cell culture medium storing unit 223, culture
medium solution-feeding channel 224 and pump 225 constitute the
culture medium supply device. The pump 225 may continuously supply
stem cell culture medium, or it may supply stem cell culture medium
at a prescribed timing, according to directions by the CPU 500
shown in FIG. 26.
[0389] The blood cell culture medium storing unit 220 and stem cell
culture medium storing unit 223 may be placed in cold storage in
the cold storage unit 260 at a low temperature of 4.degree. C., for
example. The culture medium fed from the blood cell culture medium
storing unit 220 and the stem cell culture medium storing unit 223
may be fed to the culturing vessel, for example, after having the
temperature raised to 37.degree. C. with a heater outside the cold
storage unit 260. Alternatively, the temperature surrounding the
solution-feeding channel may be set so that the culture medium
stored at low temperature increases in temperature to 37.degree. C.
while it progresses through the solution-feeding channel. The used
culture medium in the initializing culturing vessel 219 is sent to
a waste liquid storage section 228 through a waste liquid
solution-feeding channel 226 and pump 227. An identifier such as a
barcode is attached to the waste liquid storage section 228 for
control of the waste liquid information.
[0390] The cell masses that have been cultured at the initializing
culturing vessel 219 are sent to a first amplifying culturing
vessel 232 as a part of the cell mass preparation device, through
an introduced cell solution-feeding channel 229, pump 230 and cell
mass dissociater 231. The cell mass dissociater 231 may also
comprise the construction shown in FIG. 45 or FIG. 46, for example.
By passing through the cell mass dissociater 231, the cell masses
are dissociated into smaller cell masses. The suspension culture
vessel shown in FIG. 20 may be used as the first amplifying
culturing vessel 232 shown in FIG. 49. In this case, the cell
masses are placed in a dialysis tube. Stem cell culture medium is
supplied to the first amplifying culturing vessel 232 shown in FIG.
49, from the stem cell culture medium storing unit 223 including
stem cell culture medium, through a culture medium solution-feeding
channel 233 and pump 234. The introduced cell solution-feeding
channel 229 and culture medium solution-feeding channel 233 may be
temperature-permeable and CO.sub.2-permeable, for example. The stem
cell culture medium storing unit 223, culture medium
solution-feeding channel 233 and pump 234 constitute the culture
medium supply device. The pump 234 may continuously supply stem
cell culture medium, or it may supply stem cell culture medium at a
prescribed timing, according to directions by the CPU 500 shown in
FIG. 26.
[0391] The used culture medium in the first amplifying culturing
vessel 232 shown in FIG. 49 is sent to the waste liquid storage
section 228 through a waste liquid solution-feeding channel 235 and
pump 236.
[0392] The cell masses that have been cultured at the first
amplifying culturing vessel 232 are sent to a second amplifying
culturing vessel 240 as a part of the cell mass preparation device,
through an introduced cell solution-feeding channel 237, pump 238
and cell mass dissociater 239. The cell mass dissociater 239 may
also comprise the construction shown in FIG. 45 or FIG. 46, for
example. By passing through the cell mass dissociater 239, the cell
masses are dissociated into smaller cell masses. The suspension
culture vessel shown in FIG. 20 may be used as the second
amplifying culturing vessel 240 shown in FIG. 49. In this case, the
cell masses are placed in a dialysis tube. Stem cell culture medium
is supplied to the second amplifying culturing vessel 240 shown in
FIG. 49, from the stem cell culture medium storing unit 223
including stem cell culture medium, through a culture medium
solution-feeding channel 241 and pump 242. The introduced cell
solution-feeding channel 237 and culture medium solution-feeding
channel 241 may be temperature-permeable and CO.sub.2-permeable,
for example. The stem cell culture medium storing unit 223, culture
medium solution-feeding channel 241 and pump 242 constitute the
culture medium supply device. The pump 242 may continuously supply
stem cell culture medium, or it may supply stem cell culture medium
at a prescribed timing, according to directions by the CPU 500
shown in FIG. 26.
[0393] The used culture medium in the second amplifying culturing
vessel 240 shown in FIG. 49 is sent to the waste liquid storage
section 228 through a waste liquid solution-feeding channel 243 and
pump 244.
[0394] The cell masses that have been cultured in the second
amplifying culturing vessel 240 are sent to a solution exchanger
247 through an introduced cell solution-feeding channel 245 and
pump 246. The solution exchanger 247 comprises the construction
shown in FIG. 48, for example. In the solution exchanger 247 shown
in FIG. 49, the cell masses are held at a filter while the culture
medium is sent to the waste liquid storage section 228 through a
waste liquid solution-feeding channel 248 and pump 249.
[0395] After stopping flow of the solution in the waste liquid
solution-feeding channel 248 by stopping driving of the pump 249,
or after closing the waste liquid solution-feeding channel 248 with
a valve or the like, cryopreservation liquid is placed in the
solution exchanger 247 from a cryopreservation liquid storing unit
250, that contains cryopreservation liquid, through a
solution-feeding channel 251 and pump 252. This disperses the cell
masses in the cryopreservation liquid.
[0396] The cryopreservation liquid that has dispersed the cell
masses is fed into a cryopreservation vessel 255 through a
solution-feeding channel 253 and pump 254, as parts of the
packaging device. The cryopreservation vessel 255 is situated in a
low-temperature repository 256. Liquid nitrogen at -80.degree. C.,
for example, is fed to the low-temperature repository 256 from a
liquid nitrogen repository 257, through a solution-feeding channel
258. The cell masses in the cryopreservation vessel 255 are thus
frozen. Freezing of the cell masses does not need to be by liquid
nitrogen, however. For example, the low-temperature repository 256
may be a freezer such as a compression freezer, an absorption
freezer or a Peltier freezer.
[0397] Back-flow valves may also be provided in the
solution-feeding channels as appropriate. The solution-feeding
channels, mononuclear cell separating unit 203, mononuclear cell
purifying filter 210, factor introducing device 213, initializing
culturing vessel 219, first amplifying culturing vessel 232, second
amplifying culturing vessel 240 and solution exchanger 247 are
situated in an element 259, for example. The material of the
element 259 may be, but is not limited to, a resin, for example.
The element 259 is made of a sterilizable heat-resistant material,
for example. The element 259 may be in the form of a plate or a
cassette. The element 259 may also be a flexible bag. The
solution-feeding channel through which the culture medium flows is
made of a CO.sub.2-permeable material, for example. The
solution-feeding channels, mononuclear cell separating unit 203,
mononuclear cell purifying filter 210, factor introducing device
213, initializing culturing vessel 219, first amplifying culturing
vessel 232, second amplifying culturing vessel 240 and solution
exchanger 247 may also be housed in a plurality of separate
cases.
[0398] The element 259, and the enclosure 601 shown in FIG. 1,
comprise, for example, engaging parts that mutually engage.
Therefore, the element 259 shown in FIG. 49 is disposed at a
prescribed location inside the enclosure 601 shown in FIG. 1.
Furthermore, the pump, blood storing unit 201, separating agent
storing unit 205, factor storing unit 214, blood cell culture
medium storing unit 220, stem cell culture medium storing unit 223,
waste liquid storage section 228, cryopreservation vessel 255,
low-temperature repository 256 and liquid nitrogen repository 257
shown in FIG. 49 are also disposed at prescribed locations inside
the enclosure 601. When the element 259 is disposed at the
prescribed location inside the enclosure 601 shown in FIG. 1, the
solution-feeding channels in the element 259 shown in FIG. 49 are
in contact with the pump, blood storing unit 201, separating agent
storing unit 205, factor storing unit 214, blood cell culture
medium storing unit 220, stem cell culture medium storing unit 223,
waste liquid storage section 228, cryopreservation vessel 255,
low-temperature repository 256 and liquid nitrogen repository
257.
[0399] The element 259 and the channel provided in the element 259
may be disposable, for example, and upon completion of freezing of
the cell masses, they may be discarded and exchanged with new ones.
Alternatively, when the element 259 and the channel provided in the
element 259 are to be reused, an identifier such as a barcode may
be attached to the element 259 to manage the number of times used,
etc.
[0400] With the cell processing system of the embodiment described
above, it is possible to automatically process cryopreserved stem
cells such as iPS cells from blood.
[0401] Moreover, as shown in FIG. 50, FIG. 51 and FIG. 52, the cell
processing apparatus may also comprise a cell culture device 900,
an embedding member 2000 that embeds the cell culture device 900,
and a communicating solution-feeding channel that allows
communication between the outside of the embedding member 2000 and
the cell culture device 900 that is inside the embedding member
2000. The embedding member 2000 is omitted in FIG. 51 and FIG.
52.
[0402] The material used for the embedding member 2000 may be a
resin, glass, a metal, or the like. If the embedding member 2000 is
transparent it will be possible to observe the cell culture device
900 disposed inside the embedding member 2000, but it may also be
opaque.
[0403] In the cell processing apparatus, blood is fed from a blood
storing unit 1201, disposed outside the embedding member 2000, that
stores blood, to a mononuclear cell separating unit 1203 disposed
inside the embedding member 2000, through a blood solution-feeding
channel 1202. Tubes, for example, may be used as the blood storing
unit 1201 and mononuclear cell separating unit 1203. The blood
solution-feeding channel 1202 may be a resin tube or silicon tube,
for example. This also applies for the other solution-feeding
channels described below. An identifier such as a barcode is
attached to the blood storing unit 1201 for control of the blood
information.
[0404] A driving unit 1204 such as a pump that is attachable to the
outer wall of the embedding member 2000 is used for the feeding.
The driving unit 1204 that is used may be a positive-displacement
pump. Examples of positive-displacement pumps include reciprocating
pumps including piston pumps, plunger pumps and diaphragm pumps,
and rotating pumps including gear pumps, vane pumps and screw
pumps. Examples of diaphragm pumps include tubing pumps and
piezoelectric pumps. Examples of tubing pumps include Perista
Pump.RTM. (Atto Corp.) and RP-Q1 and RP-TX (Takasago Electric,
Inc.). Examples of piezoelectric pumps include SDMP304, SDP306,
SDM320 and APP-20KG (Takasago Electric, Inc.). A microflow chip
module (Takasago Electric, Inc.) comprising a combination of
various different pumps may also be used. When a sealed pump such
as a Perista Pump.RTM., tubing pump or diaphragm pump is used,
feeding can be accomplished without direct contact of the pump with
the blood inside the blood solution-feeding channel 1202. The same
also applies to the other pumps described below. Alternatively, a
syringe pump may be used for the driving unit 1204, as well as for
the driving units mentioned below. Even pumps other than sealed
pumps may be reutilized after heat sterilization treatment.
[0405] For example, a slave unit 2204 to which driving force from
the driving unit 1204 is transmitted, may be provided inside the
embedding member 2000 as shown in FIG. 53 and FIG. 54, with the
slave unit 2204 being connected to the blood solution-feeding
channel 1202. The driving unit 1204 and the slave unit 2204 may
also be connected by magnetic force. The driving unit 1204 may
comprise an electromagnet that generates magnetic force. This also
applies to the driving units other than the driving unit 1204.
[0406] A separating agent for separation of mononuclear cells of
the erythrocyte coagulant is fed from the separating agent storing
unit 1205 disposed on the outside of the embedding member 2000, to
the mononuclear cell separating unit 1203 disposed inside the
embedding member 2000, shown in FIG. 50, FIG. 51 and FIG. 52,
through a separating agent solution-feeding channel 1206 and a
driving unit 1207 that is attachable to the outer wall of the
embedding member 2000. Tubes, for example, may be used as the
separating agent storing unit 1205. An identifier such as a barcode
is attached to the separating agent storing unit 1205 for control
of the separating agent information. The erythrocyte coagulant used
may be, for example, HetaSep.RTM. (STEMCELL Technologies) or an
Erythrocyte Coagulant (Nipro Corp.).
[0407] In the mononuclear cell separating unit 1203, the
erythrocytes precipitate by the erythrocyte coagulant and the
mononuclear cells are separated. The supernatant containing the
mononuclear cells in the mononuclear cell separating unit 1203 is
fed to a mononuclear cell purifying filter 1210, through a
mononuclear cell solution-feeding channel 1208 disposed inside the
embedding member 2000 and a driving unit 1209 that is attachable to
the outer wall of the embedding member 2000. The precipitated
portion produced in the mononuclear cell separating unit 1203 is
fed to a waste liquid storage section 4228 disposed on the outside
of the embedding member 2000, through a waste liquid
solution-feeding channel that allows communication from the inside
to the outside of the embedding member 2000.
[0408] At the mononuclear cell purifying filter 1210, components
other than the mononuclear cells are removed to obtain a
mononuclear cell-containing solution. The mononuclear cell
purifying filter 1210 used may be Purecell.RTM. (PALL), Cellsorba E
(Asahi Kasei Corp.), SEPACELL PL (Asahi Kasei Corp.),
ADACOLUMN.RTM. (Jimro), or a separation bag (Nipro Corp.).
[0409] In FIG. 52, the mononuclear cell separating unit 1203,
separating agent storing unit 1205, mononuclear cell purifying
filter 1210 and driving units 1204, 1207, 1209 constitute a
separating device. The solution containing the mononuclear cells,
separated at the mononuclear cell purifying filter 1210, may be
fed, through a mononuclear cell channel 2001 which is disposed
inside the embedding member 2000 and connected to the mononuclear
cell purifying filter 1210, to a mononuclear cell storage unit 2002
which is disposed inside the embedding member 2000 and connected to
the mononuclear cell channel 2001. The solution containing the
separate components other than mononuclear cells separated at the
mononuclear cell purifying filter 1210, may be fed, through a
non-mononuclear cell component channel 2003 which is disposed
inside the embedding member 2000 and connected to the mononuclear
cell purifying filter 1210, to a non-mononuclear cell component
storage unit 2004 which is disposed inside the embedding member
2000 and connected to the separate component channel 2003.
[0410] The solution containing mononuclear cells is fed to a factor
introducing device 1213 disposed inside the embedding member 2000,
through a preintroduction cell solution-feeding channel 1211
disposed inside the embedding member 2000 and a driving unit 1212
that is attachable to the outer wall of the embedding member 2000.
Mononuclear cells separated at the mononuclear cell separating unit
1203 may also be fed to the factor introducing device 1213 without
passing through the mononuclear cell purifying filter 1210. Tubes,
for example, may be used as the factor introducing device 1213.
Pluripotency inducing factor is fed to the factor introducing
device 1213 from a factor storing unit 1214 that contains
pluripotency inducing factor, disposed outside the embedding member
2000, through a factor solution-feeding channel 1215 provided at
least partially inside the embedding member 2000 and a driving unit
1216 disposed outside the embedding member 2000. Tubes, for
example, may also be used as the factor storing unit 1214. An
identifier such as a barcode is attached to the factor storing unit
1214 for control of the pluripotency inducing factor information.
The factor storing unit 1214 and the driving unit 1216 constitute
the inducing factor solution-feeding mechanism.
[0411] In the factor introducing device 1213, as at least part of
the factor introducing device, the pluripotency inducing factor is
introduced into cells by RNA lipofection, for example, and inducing
factor-introduced cells are prepared. The method of transfection of
the inducing factor, however, is not limited to RNA lipofection.
For example, Sendai virus vector including a pluripotency inducing
factor may be used. Alternatively, the pluripotency inducing factor
may be a protein. Another method of transfection is transfection or
electroporation using episomal plasmids. The waste liquid produced
at the factor introducing device 1213 is fed to a waste liquid
storage section 5228 disposed on the outside of the embedding
member 2000, through a waste liquid solution-feeding channel that
allows communication from the inside to the outside of the
embedding member 2000.
[0412] The inducing factor-introduced cells are fed to an
initializing culturing vessel 1219, as part of the cell mass
preparation device, through an introduced cell solution-feeding
channel 1217 provided inside the embedding member 2000 and a
driving unit 1218 disposed on the outside of the embedding member
2000. The initializing culturing vessel 1219 is embedded in the
embedding member 2000. The suspension culture vessel shown in FIG.
20 may be used as the initializing culturing vessel 1219. In this
case, the inducing factor-introduced cells are placed in a dialysis
tube. For the first few days after introduction of the pluripotency
inducing factor to the cells, blood cell culture medium is supplied
to the initializing culturing vessel 1219 shown in FIG. 52, from a
blood cell culture medium storing unit 1220 disposed outside of the
embedding member 2000 and including blood cell culture medium,
through a culture medium solution-feeding channel 1221 provided at
least partially inside the embedding member 2000 and a driving unit
1254 disposed on the outside of the embedding member 2000. An
identifier such as a barcode is attached to the blood cell culture
medium storing unit 1220 for control of the blood cell culture
medium information. The blood cell culture medium storing unit
1220, culture medium solution-feeding channel 1221 and driving unit
1254 constitute the culture medium supply device. The driving unit
1254 may continuously supply blood cell culture medium, or it may
supply blood cell culture medium at a prescribed timing according
to directions by the CPU 500 shown in FIG. 26.
[0413] Next, stem cell culture medium is supplied to the
initializing culturing vessel 1219 shown in FIG. 52, from a stem
cell culture medium storing unit 1223 disposed outside of the
embedding member 2000 and including stem cell culture medium,
through a culture medium solution-feeding channel provided inside
the embedding member 2000 and a driving unit disposed on the
outside of the embedding member 2000. An identifier such as a
barcode is attached to the stem cell culture medium storing unit
1223 for control of the stem cell culture medium information. The
stem cell culture medium storing unit 1223, culture medium
solution-feeding channel and driving unit constitute the culture
medium supply device. The driving unit may continuously supply stem
cell culture medium, or it may supply stem cell culture medium at a
prescribed timing, according to directions by the CPU 500 shown in
FIG. 26.
[0414] The blood cell culture medium storing unit 1220 and stem
cell culture medium storing unit 1223 may be placed in cold storage
in the cold storage unit at a low temperature of 4.degree. C., for
example. The culture medium fed from the blood cell culture medium
storing unit 1220 and the stem cell culture medium storing unit
1223 may be fed to the culturing vessel, for example, after having
the temperature raised to 37.degree. C. with a heater outside the
cold storage unit. Alternatively, the temperature surrounding the
solution-feeding channel may be set so that the culture medium
stored at low temperature increases in temperature to 37.degree. C.
while it progresses through the solution-feeding channel. The used
culture medium in the initializing culturing vessel 1219 is fed to
a waste liquid storage section 1228 disposed on the outside of the
embedding member 2000, through a waste liquid solution-feeding
channel 1226 that allows communication from the inside to the
outside of the embedding member 2000. An identifier such as a
barcode is attached to the waste liquid storage section 1228 for
control of the waste liquid information.
[0415] The cell masses that have been cultured at the initializing
culturing vessel 1219 are fed to an amplifying culturing vessel
1232, as part of the cell mass preparation device, through an
introduced cell solution-feeding channel 1229 provided inside the
embedding member 2000, a driving unit 1230 disposed outside the
embedding member 2000, a cell mass dissociater 1231 provided inside
the embedding member 2000, a driving unit 1218 disposed outside the
embedding member 2000, and an introduced cell solution-feeding
channel 2229 provided inside the embedding member 2000. The cell
mass dissociater 1231 provided inside the embedding member 2000 may
have the construction shown in FIG. 45 or FIG. 46, for example. By
passing through the cell mass dissociater 1231, the cell masses are
dissociated into smaller cell masses.
[0416] The suspension culture vessel shown in FIG. 20 may be used
as the first amplifying culturing vessel 1232 shown in FIG. 52. In
this case, the cell masses are placed in a dialysis tube. Stem cell
culture medium is supplied to the amplifying culturing vessel 1232
shown in FIG. 52, from the stem cell culture medium storing unit
1223 disposed outside the embedding member 2000 and including stem
cell culture medium, through a culture medium solution-feeding
channel 1224 that allows communication from the outside to the
inside of the embedding member 2000, and a driving unit 1222
disposed outside the embedding member 2000. The stem cell culture
medium storing unit 1223, culture medium solution-feeding channel
1224 and driving unit 1222 constitute the culture medium supply
device. The driving unit may continuously supply stem cell culture
medium, or it may supply stem cell culture medium at a prescribed
timing according to directions by the CPU 500 shown in FIG. 26.
[0417] The used culture medium in the amplifying culturing vessel
1232 shown in FIG. 52 is fed to a waste liquid storage section 2228
disposed outside the embedding member 2000, through a waste liquid
solution-feeding channel 1235 that allows communication from the
inside to the outside of the embedding member 2000.
[0418] The cell masses cultured in the amplifying culturing vessel
1232 are fed to a solution exchanger 1247 provided inside the
embedding member 2000, through an introduced cell solution-feeding
channel 1245 provided inside the embedding member 2000, a driving
unit 1246 that is attachable to the outer wall of the embedding
member 2000 and a cell mass dissociater 2231 provided inside the
embedding member 2000. The solution exchanger 1247 comprises the
construction shown in FIG. 48, for example. In the solution
exchanger 1247 shown in FIG. 52, the cell masses are held at a
filter while culture medium is fed to a waste liquid storage
section 3228 disposed outside the embedding member 2000, through a
waste liquid solution-feeding channel 1248 that allows
communication from the inside to the outside of the embedding
member 2000.
[0419] After the waste liquid solution-feeding channel 1248 has
been closed with a valve or the like, a cryopreservation liquid is
placed in the solution exchanger 1247 from a cryopreservation
liquid storing unit 1250 disposed outside the embedding member 2000
and containing the cryopreservation liquid, through a
cryopreservation liquid-feeding channel 1251 that allows
communication from the outside to the inside of the embedding
member 2000 and a driving unit 1252 that is attachable to the outer
wall of the embedding member 2000. This disperses the cell masses
in the cryopreservation liquid.
[0420] The cryopreservation liquid in which the cell masses have
been dispersed is fed into a cryopreservation vessel 1255 through a
freezing cell solution-feeding channel 1253 which allows
communication between the inside and outside of the embedding
member 2000, and a driving unit 1209 that is attachable to the
outer wall of the embedding member 2000. The solution-feeding
channel 1253, driving unit 1254 and cryopreservation vessel 1255
form part of a packaging apparatus. The cryopreservation vessel
1255 is then transferred into the low-temperature repository.
[0421] For the cell processing apparatus illustrated in FIG. 50 to
FIG. 52, after the factor introducing device 1213, initializing
culturing vessel 1219 and amplifying culturing vessel 1232 of the
cell culture device 900 have been connected, the embedding member
2000 is molded so as to embed them and they are processed.
Alternatively, the factor introducing device 1213, initializing
culturing vessel 1219 and amplifying culturing vessel 1232 of the
cell culture device 900 of the cell processing apparatus may be
formed inside the embedding member 2000 by an apparatus such as a
3D printer. Embedding the factor introducing device 1213,
initializing culturing vessel 1219 and amplifying culturing vessel
1232 of the cell culture device 900 with the embedding member 2000
can minimize outward diffusion of the processed cells or the
inducing factor introduced into the cells. Likewise, it can
minimize contact of outside contaminants with the treated
cells.
[0422] An embodiment of the invention has been described above, but
the description and pertinent drawings that are intended merely to
constitute part of the disclosure are not to be understood as
limiting the invention. Various alternative embodiments,
embodiments and operating technologies will be readily apparent to
a person skilled in the art from this disclosure. For example, the
factor introducing device 30 may induce the cells not by
electroporation or RNA lipofection, but rather by a viral vector
such as retrovirus, lentivirus or Sendai virus, or by transfection
using plasmids, or by protein transfection. Cells may also be
induced by introduction of compounds as factors. The
preintroduction cell solution-feeding channel 20, introduced cell
solution-feeding channel 31, cell mass solution-feeding channel 51,
amplifying culturing solution-feeding channel 71, cell mass
solution-feeding channel 72 and pre-packaging cell channel 91 may
be provided on a substrate by a microfluidics technique. It will
therefore be understood that the invention encompasses various
embodiments not described herein.
Example 1
(Preparation)
[0423] Human blood cells were acquired from a healthy adult male.
There were also prepared modified mRNA (TriLink), a non-adherent
dish, a 15 mL tube, a 50 mL tube, Ficoll, a Cytoflowmeter (BD),
anti-CD34 antibody (Miltenyi Biotec), anti-CD3 antibody (Miltenyi
Biotec), MACS.RTM. buffer (Miltenyi Biotec), T cell culture medium,
low serum culture medium (Opti-MEM.RTM., Gibco), siRNA introducing
reagent (Lipofectamine.RTM., RNAiMAX, Thermo Fisher Scientific) and
anti-TRA-1-60 antibody (BD).
[0424] The T cell (CD3-positive cell) culture medium was a liquid
mixture of the following culture medium A and culture medium B.
Culture medium A was a liquid mixture of 15 mL of X vivo-10 (Lonza,
04-743Q) and IL-2 (10 .mu.g/mL). Culture medium B was prepared by
mixing X vivo-10 and 50 .mu.L of Dynabeads CD3/CD28 (Life
Technologies, 111-31D) in a 1.5 mL tube, vortexing the mixture for
5 seconds, allowing spin-down, stationing the mixture in a
DynaMag-2 (Thermo fisher Scientific), and removing the supernatant
after one minute of stationing.
[0425] There was additionally prepared a blood cell culture medium
(blood stem/precursor cell medium) by adding 10 .mu.L of IL-6 (100
.mu.g/mL), 10 .mu.L of SCF (300 .mu.g/mL), 10 .mu.L of TPO (300
.mu.g/mL), 10 .mu.L of Flt3 ligand (300 .mu.g/mL) and 10 .mu.L of
IL-3 (10 .mu.g/mL) to 10 mL of serum-free medium (StemSpan H3000,
STEMCELL Technologies).
[0426] There were further prepared an OCT3/4 mRNA-containing
solution, SOX2 mRNA-containing solution, KLF4 mRNA-containing
solution, c-MYC mRNA-containing solution, LIN28A mRNA-containing
solution and green fluorescent protein (GFP) mRNA-containing
solution, each to a concentration of 100 ng/.mu.L. Next, 385 .mu.L
of the OCT3/4 mRNA-containing solution, 119 .mu.L of the SOX2
mRNA-containing solution, 156 .mu.L of the KLF4 mRNA-containing
solution, 148 .mu.L of the c-MYC mRNA-containing solution, 83 .mu.L
of the LIN28A mRNA-containing solution and 110 .mu.L of the GFP
mRNA-containing solution were mixed to obtain an initializing
factor mixture. The obtained initializing factor mixture was
dispensed into 1.5 mL-volume RNase-Free tubes (Eppendorf Tube.RTM.,
Eppendorf AG) at 50 .mu.L each, and preserved in a freezer at
-80.degree. C.
(Preparation of Mononuclear Cells)
[0427] A centrifuge was set to 18.degree. C. Blood was sampled in
amounts from 5 mL to 50 mL, EDTA was added to the blood, and each
mixture was gently mixed. Also, medium for human lymphocyte
separation Ficoll-Paque PREMIUM, GE Healthcare, Japan) was
dispensed into two 15 mL tubes at 5 mL each. After adding 5 mL of
PBS to the blood for dilution, 5 mL of each was overlaid onto the
human lymphocyte separation medium in the tubes. During this time,
the diluted blood was slowly added onto the medium while causing it
to slide on the tube wall, so as not to disturb the interface.
[0428] The solutions in the tubes were centrifuged at 400.times.g,
18.degree. C. for 30 minutes. Acceleration and deceleration were
carried out slowly during the procedure. After centrifugation, a
white cloudy intermediate layer appeared in the tube. The white
cloudy intermediate layer included mononuclear cells. The white
cloudy intermediate layer in each tube was slowly collected with a
Pipetman and transferred to a new 15 mL tube. The lower layer was
not handled during this time. Approximately 1 mL of the white
cloudy intermediate layer could be collected from each tube. The
intermediate layers of two tubes were combined and transferred to a
single tube.
[0429] After adding 12 mL of PBS to the collected mononuclear
cells, the solution was further centrifuged at 200.times.g,
18.degree. C. for 10 minutes. Next, an aspirator was used to remove
the supernatant of the solution by aspiration, and 3 mL of
serum-free hematopoietic cell culture medium of known composition
(X-VIVO.RTM. 10, Lonza) was added, forming a suspension, thereby
obtaining a mononuclear cell suspension. A 10 .mu.L portion of the
mononuclear cell suspension was stained with Trypan blue and the
count was determined with a hemocytometer.
(Separation of CD34 or CD3-Positive Cells)
[0430] Reaction was performed between 1.times.10.sup.7 mononuclear
cells and CD34 antibody or CD3 antibody for 15 minutes in 100 .mu.L
of solution at 4.degree. C. Following the reaction, 5 mL of
MACS.RTM. buffer (Miltenyi Biotec) was added to the solution, and
centrifugation was performed at 270 g. After centrifugation, the
supernatant was removed and 1 mL of MACS buffer was added. Next,
utilizing the separation program of an automatic magnetic cell
separator (autoMACS, Miltenyi Biotec), CD34-positive cells and
CD3-positive cells were separated from among the mononuclear
cells.
(Culturing of Separated Cells)
[0431] After suspending 5.times.10.sup.6 of the separated
mononuclear cells in 1 mL of T cell culture medium or blood
stem/precursor cell culture medium, they were seeded in a 12-well
plate and cultured. The culturing conditions were 5% CO.sub.2
concentration, 19% oxygen concentration, 37.degree. C.
temperature.
(Lipofection of Initializing Factor)
[0432] A first mixture was prepared by mixing 100 .mu.L of low
serum culture medium (Opti-MEM.RTM., Gibco) and 25 .mu.L of
initializing factor mixture. A second mixture was also prepared by
mixing 112.5 .mu.L of low serum culture medium (Opti-MEM.RTM.,
Gibco) and 12.5 .mu.L of siRNA introducing reagent
(Lipofectamine.RTM., RNAiMAX, Thermo Fisher Scientific). Next, the
first mixture and second mixture were combined and allowed to stand
at room temperature for 15 minutes, to prepare a lipofection
reaction mixture.
[0433] After gently adding 60 .mu.L of the obtained lipofection
reaction mixture to the 12-well plate in which the mononuclear
cells were being cultured, the mononuclear cells were then cultured
in a feeder-free manner at 37.degree. C. for 18 hours. The
culturing conditions were 5% CO.sub.2 concentration, 19% oxygen
concentration, 37.degree. C. temperature. The mononuclear cell
density upon addition of the lipofection reaction mixture was
3.times.10.sup.6. After 18 hours, the mononuclear cells were
collected in a 15 mL tube and centrifuged at 300 g, and the
supernatant was removed. Next, 1.25 mL of CD34 blood cell culture
medium was added to a 15 mL tube, the mononuclear cell suspension
was returned to the same 12-well plate, and feeder-free culturing
of the mononuclear cells was carried out overnight at 37 degrees.
The culturing conditions were 5% CO.sub.2 concentration and 19%
oxygen concentration. The steps described above were repeated once
every 2 days for 7 days.
(Confirmation of GFP Expression)
[0434] On the 7th day after the start of lipofection, the density
of cells after a total of 4 lipofections was 3.times.10.sup.6. When
a portion of the cells was removed from the 12-well plate and GFP
expression was examined with a fluorescent microscope, expression
of GFP was confirmed, as shown in FIG. 55. This confirmed that mRNA
had been transfected in the mononuclear cells, and that protein had
been synthesized from the transfected mRNA.
(Confirmation of TRA-1-60 Expression)
[0435] On the 7th day after the start of lipofection, a portion of
the cells were removed from the 12-well plate, and the removed
cells were stained with antibody for TRA-1-60 as a surface antigen
specifically expressed on the iPS cells that had begun to be
initialized, the antibody being labeled with Allophycocyanin (APC)
fluorescent dye. Next, the ratio of TRA-1-60-positive cells was
determined with a fluorescence activated cell sorter (FACS.RTM.,
BD), to confirm that reprogramming of the cells had been initiated,
iPS cell genes had been expressed and iPS cells had emerged.
[0436] A dot plot was drawn with autologous fluorescence intensity
on the x-axis and fluorescent labeled anti-TRA-1-60 antibody
fluorescence intensity on the y-axis, as shown in FIG. 56. No
TRA-1-60-positive cells were detected in a negative control without
gene introduction. In contrast, TRA-1-60-positive cells were
detected in Experiments 1, 2 and 3. Experiment 1 represents the
results of induction from all of the mononuclear cells without
separation by markers, Experiment 2 represents the results of
induction from cells separated as CD3-positive, and Experiment 3
represents the results of induction from cells separated as
CD34-positive. It was thus demonstrated that iPS cells can be
induced by using lipofection of initializing factor RNA to
introduce the initializing factor into blood-derived cells.
Example 2
[0437] A bFGF-containing human iPS culture medium was prepared by
mixing 500 mL of Primate ES Cell Medium (ReproCELL) and 0.2 mL of
bFGF (Gibco PHG0266) at a 10 .mu.g/mL concentration.
[0438] Also, deacylated gellan gum (Nissan Chemical Industries,
Ltd.) was added to the bFGF-containing human iPS culture medium to
a concentration of 0.02 wt %, to prepare a bFGF-containing human
iPS gel medium. In addition, 5 mL of trypsin at 2.5 wt %
concentration, 5 mL of collagenase IV at 1 mg/mL concentration, 0.5
mL of CaCl.sub.2) at 0.1 mol/L concentration, 10 mL of KnockOut
Serum Replacement.RTM. (Invitrogen 10828-028) and 30 mL of purified
water were mixed to prepare a dissociation solution, commonly known
as CTK solution.
[0439] After adding 300 .mu.L of the CTK solution to a 6-well dish
(Thermoscientific 12-556-004) in which iPS cells were being
cultured on feeder cells, the mixture was incubated for 3 minutes
in a CO.sub.2 incubator. After 3 minutes, the dish was removed from
the incubator, detachment of the feeder cells alone was confirmed,
and an aspirator was used to remove the CTK solution. After
removing the CTK solution, 500 .mu.L of PBS (Santa Cruz Biotech
sc-362183) was added to the 6-well dish to rinse the iPS cells, and
then the PBS was removed from the 6-well dish and 0.3 mL of
dissociation solution (Accutase.RTM.) was added to the 6-well dish,
which was placed in a CO.sub.2 incubator and incubated for 5
minutes. Next, 0.7 mL of bFGF-containing iPS culture medium was
added to the 6-well dish and the iPS cells were suspended until
single cells were obtained.
[0440] After suspension of the iPS cells, 4 mL of bFGF-containing
human iPS culture medium was added to a 15 mL centrifugation tube,
and the iPS cell suspension was centrifuged at 270 g using a
centrifuge. After centrifugation, the supernatant was removed, 1 mL
of bFGF-containing human iPS culture medium was added to a 15 mL
centrifugation tube, and a blood cell counting chamber was used to
calculate the cell count. After cell counting, 5.times.10.sup.5 of
iPS cells each were seeded in a 15 mL Falcon Tube.RTM. (Corning
352096) or a non-adherent dish, and suspension culture was carried
out without agitation.
[0441] A 2 mL portion of bFGF-containing human iPS gel medium was
used in a 15 mL tube. A 2 mL portion of non-gelled bFGF-containing
human iPS culture medium was used in the non-adherent dish. ROCK
inhibitor (Selleck S1049) was added at 10 .mu.mol/L to each medium.
Thereafter, 500 .mu.L of bFGF-containing human iPS gel medium was
added each day to the 15 mL tube and non-adherent dish and 500
.mu.L of bFGF-containing human iPS culture medium was added each
day to the non-adherent dish. Also, ROCK inhibitor was added to the
15 mL tube and non-adherent dish each day to a final concentration
of 10 .mu.mol/L, and suspension culture was continued for 7
days.
[0442] The results are shown in FIG. 57. As shown in FIG. 57(b),
when iPS cells were cultured in the non-adherent dish using
non-gelled bFGF-containing human iPS culture medium, notable
aggregation of the iPS cell colonies was observed. In contrast, as
shown in FIG. 57(a), when iPS cells were cultured using
bFGF-containing human iPS gel medium in the 15 mL tube, no such
conspicuous aggregation was observed. FIG. 58(a) is a photograph on
the 1st day after culturing of iPS cells using bFGF-containing
human iPS gel medium in the 15 mL tube, and FIG. 58(b) is a
photograph on the 9th day after culturing of iPS cells using
bFGF-containing human iPS gel medium in the 15 mL tube. The
photographs of FIG. 58(a) and FIG. 58(b) confirmed colony formation
without aggregation between iPS cells of different lines.
[0443] FIG. 59(a) is a photograph immediately before reseeding of
the iPS cell colonies that had been suspension cultured for 7 days
in gel medium, onto feeder cells. FIG. 59(b) is a photograph taken
when confirming the forms of the colonies after 3 days. As shown in
FIG. 60, the results confirmed that at least 95% of the colonies
were undifferentiated. It was thus demonstrated that iPS cells can
be cultured in gel medium while maintaining their undifferentiated
state.
Example 3
[0444] The same bFGF-containing human iPS culture medium and
bFGF-containing human iPS gel medium were prepared as in Example 2.
After adding 300 .mu.L of the CTK solution to a 6-well dish in
which iPS cells were being cultured on feeder cells, the mixture
was incubated for 3 minutes in a CO.sub.2 incubator. After 3
minutes, the dish was removed from the incubator, detachment of the
feeder cells alone was confirmed, and an aspirator was used to
remove the CTK solution. After removing the CTK solution, 500 .mu.L
of PBS was added to the dish to rinse the iPS cells, and then the
PBS was removed from the dish and 0.3 mL of Accumax was added to
the dish, after which the dish was placed in a CO.sub.2 incubator
and incubated for 5 minutes. Next, 0.7 mL of bFGF-containing iPS
culture medium was added to the dish and the iPS cells were
suspended until single cells were obtained.
[0445] After suspension of the iPS cells, 4 mL of bFGF-containing
human iPS culture medium was added to a 15 mL centrifugation tube,
and the iPS cell suspension was centrifuged at 270 g using a
centrifuge. After centrifugation, the supernatant was removed, 1 mL
of bFGF-containing human iPS culture medium was added to a 15 mL
centrifugation tube, and a hemocytometer was used to calculate the
cell count. The cells were counted, and then 5.times.10.sup.5 iPS
cells were seeded in each 15 mL tube and suspension culture was
carried out without agitation.
[0446] A 2 mL portion of bFGF-containing human iPS gel medium was
used in a 15 mL tube. ROCK inhibitor was added at 10 .mu.mol/L to
each medium. A 500 .mu.L portion of bFGF-containing human iPS gel
medium was added to the 15 mL tube each day thereafter. A 500 .mu.L
portion of gel medium includes 0.5 .mu.L of ROCK inhibitor. As a
control, iPS cells were also suspension cultured for 7 days under
the same conditions, but without addition of a ROCK inhibitor.
[0447] As shown in FIG. 61(a), no iPS cell colonies formed when a
ROCK inhibitor was not added to the bFGF-containing human iPS
culture medium. In contrast, as shown in FIG. 61(b), iPS cell
colonies formed when a ROCK inhibitor was added to the
bFGF-containing human iPS culture medium. These results
demonstrated that a ROCK inhibitor is effective for suspension
culturing of iPS cells from single cells.
Example 4
[0448] Using a CO.sub.2-non-permeable vessel, Falcon 50 mL Conical
Tube.RTM., and a CO.sub.2-permeable vessel, G-Rex.RTM. (Wilson
Wolf), as dialysis tube-housing vessels, cells were suspension
cultured under the same conditions, other than the vessels. As a
result, culturing using the CO.sub.2-permeable vessel had higher
cell viability, as shown in FIG. 62.
Example 5
[0449] Gel medium containing iPS cells was added to each of two
dialysis modules (Spectrum G235035) comprising a dialysis tube with
a 100 kDa molecular cutoff. The dialysis modules were each placed
in a 50 mL centrifugation tube, and gel medium was placed around
the dialysis tubes in the centrifugation tubes. The gel medium
containing the iPS cells was also directly placed in a separate 50
mL centrifugation tube.
[0450] Next, a pump was connected to one of the centrifugation
tubes of the two centrifugation tubes in which dialysis tubes had
been placed, as shown in FIG. 21, and the gel medium in the
centrifugation tube was continuously exchanged for several days.
The gel medium was stored at 4.degree. C., and set so as to be at
37.degree. C. when reaching the centrifugation tube. No pump was
connected to the other centrifugation tube of the two
centrifugation tubes in which a dialysis tube had been placed, and
the gel medium in the centrifugation tube was not exchanged. The
gel medium was also not exchanged in the centrifugation tube in
which a dialysis tube had not been placed.
[0451] When the cells cultured in each vessel were observed after
culturing for the same period, numerous cell masses formed when the
cell masses were cultured in a dialysis tube and the gel medium
surrounding the dialysis tube was continuously exchanged with a
pump, as shown in FIG. 63 and FIG. 64. The number of differentiated
cells was also very low. However, when the cell masses were
cultured in a dialysis tube and the gel medium surrounding the
dialysis tube was not continuously exchanged with a pump, the
number of cell masses was low and the number of differentiated
cells increased. Moreover, when the cell masses were cultured
without using a dialysis tube and the gel medium was not
continuously exchanged with a pump, virtually no cell masses were
formed.
EXPLANATION OF SYMBOLS
[0452] 2: Tube, 10: separating device, 20: preintroduction cell
solution-feeding channel, 21: inducing factor solution-feeding
mechanism, 30: factor introducing device, 31: introduced cell
solution-feeding channel, 40: cell mass preparation device, 50:
initializing culturing apparatus, 51: cell mass solution-feeding
channel, 60: dissociating mechanism, 61: terminal block, 61a:
recess, 61b: protrusion, 61c: large pore size section, 62:
connecting block, 62a: recess, 62b: protrusion, 62c: large pore
size section, 62d: small pore size section, 62e: large pore size
section, 63: tip block, 63a: recess, 63b: nozzle section, 63c:
large pore size section, 63d: small pore size section, 64:
insertion nozzle, 65a: large pore size section, 65b: small pore
size section, 66a: insertion section, 66b: insertion section, 70:
amplifying culturing apparatus, 71: amplifying culturing
solution-feeding channel, 72: cell mass solution-feeding channel,
75: dialysis tube, 76: vessel, 77: supply culture medium
solution-feeding pump, 78: solution-feeding tube, 79: waste liquid
tube, 80: dissociating mechanism, 90: cell mass transport
mechanism, 91: pre-packaging cell channel, 100: packaging
apparatus, 101: solution exchanger, 102: filter, 103:
solution-feeding channel, 104: solution-feeding channel, 105:
discharge channel, 106: discharge channel, 110: cryopreservation
liquid-feeding mechanism, 171: initializing culturing photographing
device, 172: telecentric lens, 173: cell observation illumination
light source, 174: culture medium observation illumination light
source, 201: blood storing unit, 202: blood solution-feeding
channel, 203: mononuclear cell separating unit, 204: pump, 205:
separating agent storing unit, 206: solution-feeding channel, 207:
pump, 208: mononuclear cell solution-feeding channel, 209: pump,
210: mononuclear cell purifying filter, 211: preintroduction cell
solution-feeding channel, 212: pump, 213: factor introducing
device, 214: factor storing unit, 215: factor solution-feeding
channel, 216: pump, 217: introduced cell solution-feeding channel,
218: pump, 219: initializing culturing vessel, 220: blood cell
culture medium storing unit, 221: culture medium solution-feeding
channel, 222: pump, 223: stem cell culture medium storing unit,
224: culture medium solution-feeding channel, 225: pump, 226: waste
liquid solution-feeding channel, 227: pump, 228: waste liquid
storage section, 229: introduced cell solution-feeding channel,
230: pump, 231: cell mass dissociater, 232: amplifying culturing
vessel, 233: culture medium solution-feeding channel, 234: pump,
235: waste liquid solution-feeding channel, 236: pump, 237:
introduced cell solution-feeding channel, 238: pump, 239: cell mass
dissociater, 240: amplifying culturing vessel, 241: culture medium
solution-feeding channel, 242: pump, 243: waste liquid
solution-feeding channel, 244: pump, 245: introduced cell
solution-feeding channel, 246: pump, 247: solution exchanger, 248:
waste liquid solution-feeding channel, 249: pump, 250:
cryopreservation liquid storing unit, 251: solution-feeding
channel, 252: pump, 253: solution-feeding channel, 254: pump, 255:
cryopreservation vessel, 256: low-temperature repository, 257:
liquid nitrogen repository, 258: solution-feeding channel, 259:
member, 260: cold storage unit, 271: sensor, 272: thermometer, 301:
bag, 302: bag, 401: input device, 402: output device, 403:
relationship memory unit, 501: image processor, 511: outline
defining unit, 512: cell evaluating unit, 513: statistical
processor, 514: density calculating unit, 515: culture medium
evaluating unit, 601: enclosure, 602: intake air purification
filter, 603: exhaust purification filter, 605: exhaust system, 606:
exhaust purification filter, 608: injector, 613: gas discharger,
651: returning member, 652: base, 653: opening, 654: cover, 655:
opening, 656: cover, 701: outer enclosure, 702: pressure adjustment
hole, 703: occluding member, 800: shielding member, 801: enclosure
side shielding member, 802: exhaust system side shielding member,
811: guide, 812: guide, 900: cell culture device, 1201: blood
storing unit, 1202: blood solution-feeding channel, 1203:
mononuclear cell separating unit, 1204: driving unit, 1205:
separating agent storing unit, 1206: separating agent
solution-feeding channel, 1207: driving unit, 1208: mononuclear
cell solution-feeding channel, 1210: mononuclear cell purifying
filter, 1211: preintroduction cell solution-feeding channel, 1212:
driving unit, 1213: factor introducing device, 1214: factor storing
unit, 1215: factor solution-feeding channel, 1216: driving unit,
1217: introduced cell solution-feeding channel, 1218: driving unit,
1219: initializing culturing vessel, 1220: blood cell culture
medium storing unit, 1221: culture medium solution-feeding channel,
1222: driving unit, 1223: stem cell culture medium storing unit,
1224: culture medium solution-feeding channel, 1226: waste liquid
solution-feeding channel, 1228: waste liquid storage section, 1229:
introduced cell solution-feeding channel, 1230: driving unit, 1231:
cell mass dissociater, 1232: amplifying culturing vessel, 1235:
waste liquid solution-feeding channel, 1245: introduced cell
solution-feeding channel, 1246: driving unit, 1247: solution
exchanger, 1248: waste liquid solution-feeding channel, 1250:
cryopreservation liquid storing unit, 1251: cryopreservation
liquid-feeding channel, 1252: driving unit, 1253: freezing cell
solution-feeding channel, 1253: solution-feeding channel, 1254:
driving unit, 1255: cryopreservation vessel, 2000: embedding
member, 2001: mononuclear cell channel, 2002: mononuclear cell
storage unit, 2003: non-mononuclear cell component channel, 2004:
non-mononuclear cell component storage unit, 2204: slave unit,
2228: waste liquid storage section, 2229: introduced cell
solution-feeding channel, 2231: cell mass dissociater, 3228: waste
liquid storage section, 4228: waste liquid storage section, 5228:
waste liquid storage section
* * * * *