Multilayer Culture Vessel

A multilayer culture vessel, comprising a culture vessel part and a reservoir part attached to the culture vessel part, wherein the culture vessel part comprises at least two culture trays that are stacked; the reservoir part comprises a surrounding wall defining an internal space and a port communicating with the internal space; the surrounding wall comprises a first surrounding wall part provided with the port and a second surrounding wall part facing the first surrounding wall part; the culture vessel part has openings communicating individual culture trays of the at least two culture trays with the internal space of the reservoir part; and the openings of the culture vessel part extend toward a first abutting portion that the first surrounding wall part abuts on the culture vessel part from a second abutting portion that the second surrounding wall part abuts on the culture vessel part.

TECHNICAL FIELD

The invention relates to a culture vessel for culturing cells. In particular, the invention relates to a multilayer culture vessel. The invention also relates to a method for producing a pharmaceutical composition with the multilayer culture vessel.

BACKGROUND

Cells are cultured in various technical fields including pharmaceutical fields relating to cellular medicines and research fields relating to cell science. Cell culturing requires, in general, sterilized culture vessels. Culture vessels are used in sterile environments such as in clean benches. Cell suspension is introduced into the internal space of culture vessel through its port. Ports are covered with caps to keep the internal space sterilized. Culture vessels are placed in culture devices with regulated temperatures for culturing cells. Depending on the condition of cells, culture vessels are transferred to clean benches again to conduct operations such as collecting the cells or changing the culture medium.

Cell culture especially in pharmaceutical fields further requires keeping the inside of cell culture facilities as clean as possible. This requires considerable labor and cost to manage and maintain the sterilized condition. Cell culture vessels capable of culturing cells efficiently are required to effectively utilize the facilities' inside space. For example, culture bags (Patent Literature 1) or multilayer culture flasks in which several flat and hard plates are stacked (Patent Literatures 2 and 3) are used to culture a large number of floating cells or adhesive cells.

CITATION LIST

SUMMARY

Technical Problem

Cell culturing is affected by culture conditions such as the number of cells plated on culture trays and the amount of culture medium. In multilayer culture vessels, it is hard to change culture medium or to subculture cells on each layer separately. A different culture condition on one of the layers may cause the requirement of changing culture medium or subculturing cells on the tray ahead of the other trays. In that case, culture medium change or cell subculturing is required for all the trays, even if it is not necessary for the other trays. This leads to wasted reagents. Reagents for cell culture are relatively expensive. Waste of reagents brings a relatively large economic disadvantage.

Patent literature 2 describes a multilayer culture vessel with a resealable port arranged such that the port axis and the tray surfaces of the culture trays are perpendicular. When cell suspension is injected into the multilayer culture vessel through its resealable port, the cell suspension mostly flows into the bottom culture tray. The cells in the suspension get to accumulate on the tray during the injection process, so that the cell suspension becomes heterogeneous. After completion of the injection, the cell suspension flowed into the bottom culture tray is distributed to the individual culture trays by tilting the multilayer culture vessel. The distribution operation hardly makes the heterogeneous cell suspension homogeneous, and it is difficult to adjust culture conditions for individual culture trays. One object of the present invention is accordingly to provide a multilayer culture vessel comprising at least two culture trays, in which it is easy to adjust culture conditions for individual culture trays in the multilayer culture vessel.

Patent literature 3 describes a multilayer culture vessel comprising a plurality of culture chambers and tracheal spaces, the chambers being covered with filters, and the chambers and the spaces being alternatively stacked with supports. The culture chambers covered with the filters are provided with a manifold enabling to exhaust air from the culture chambers and to supply culture liquid to the culture chambers. The multilayer culture vessel of patent literature 3 is complicated in structure. A multilayer culture vessel with a simple structure was demanded in the field.

A culture vessel with an easy-to-operate shape is advantageous since cellular medical compositions are produced in devices such as clean benches. A multilayer culture vessel with an easy-to-operate shape is demanded in the field.

Solution to Problem

The present invention relates to a multilayer culture vessel and a method for producing a pharmaceutical composition with the multilayer culture vessel, as described below.

[Item 1] A multilayer culture vessel, comprising a culture vessel part and a reservoir part attached to the culture vessel part, wherein the culture vessel part comprises at least two culture trays that are stacked; the reservoir part comprises a surrounding wall defining an internal space and a port communicating with the internal space; the surrounding wall comprises a first surrounding wall part provided with the port and a second surrounding wall part facing the first surrounding wall part; the culture vessel part has openings communicating individual culture trays of the at least two culture trays with the internal space of the reservoir part; and the openings of the culture vessel part extend toward a first abutting portion that the first surrounding wall part abuts on the culture vessel part from a second abutting portion that the second surrounding wall part abuts on the culture vessel part.

[Item 2] A multilayer culture vessel, comprising a culture vessel part and a reservoir part attached to the culture vessel part, wherein the culture vessel part comprises at least two culture trays that are stacked; the reservoir part has an internal space and comprises a port communicating with the internal space; the culture vessel part has openings communicating individual culture trays of the at least two culture trays with the internal space of the reservoir part; and the port is provided in the reservoir part such that an angle formed between an axis of the port and a stacking direction that the at least two trays stack is from not less than 70 degrees to not more than 90 degrees and an angle formed between the axis of the port and a surface that the reservoir part is opposed to the at least two culture trays is from not less than zero degrees to not more than 50 degrees.

[Item 3] A method for producing a pharmaceutical composition, the method comprising: culturing cells with the multilayer culture vessel according to Item 1 or 2; collecting from the multilayer culture vessel cultured cells or a culture fluid containing a component secreted from the cultured cells; and producing the pharmaceutical composition comprising the collected cultured cells, the collected secreted component, or a component isolated and purified from the collected cultured cells.

Effects of Invention

A multilayer culture vessel comprising at least two culture trays according to an embodiment of the present invention allows the culture conditions to be adjusted for the individual culture trays of the multilayer culture vessel, so that waste of reagents may be suppressed. A multilayer culture vessel of another embodiment of the present invention is convenient owing to a simple and/or easy-to-operate structure.

DESCRIPTION OF EMBODIMENTS

A “multilayer culture vessel” as used herein means a culture vessel comprising at least two culture trays. Multilayer culture vessels are made, for example, from transparent, translucent, or non-transparent glass or plastic material. The material of the multilayer culture vessels includes, but is not limited to, plastics conventionally used in pharmaceutical or research fields, such as polystyrene, polycarbonate, polyethylene, polypropylene, and polyethylene terephthalate. The multilayer culture vessels may be produced, for example, by assembling a plurality of parts for the vessels. The multilayer culture vessels may be produced according to known methods. For example, multilayer culture vessels may be produced by individually preparing multiple parts by injection molding and assembling the parts.

The parts for assembling the multilayer culture vessel may all be made from the same material. At least one part may be made from a different material. Alternatively, all the parts may be made from different materials. The multilayer culture vessels are, for example, made from the same transparent or translucent material. Individual parts are prepared, for example, to have a thickness of 1-5 mm. The multilayer culture vessels may be, for example, sterilized. Sterilization treatment may be, for example, radiation sterilization, ethylene oxide gas sterilization, y-ray sterilization, and high-pressure steam sterilization.

A “culture tray” as used herein means a tray providing a storage space capable of being used as a culture vessel. The storage space of the culture tray is defined by a bottom wall and a surrounding wall surrounding the bottom wall. The bottom wall of the culture tray has a tray surface facing the storage space. The tray surface may be treated for the improvement of cell adhesion. The treatment for improving cell adhesion includes, for example, plasma treatment, oxidant treatment, and coating treatment with a hydrophilic material. The surrounding wall of the culture tray is, for example, integrally molded with the bottom wall, is a wall extending from the outer periphery, is a wall attached to the bottom wall, or is a combination thereof.

A “stacking direction” as used herein means a direction that tray surfaces of the bottom walls of culture trays stack. The stacking direction corresponds to, for example, a direction that the liquid levels of the liquid samples injected into culture trays overlay when the multilayer culture vessel is placed in a posture suitable for culturing. For example, the posture of the multilayer culture vessel suitable for culturing is a posture that the liquid sample injected into the culture tray has a maximum area of liquid surface thereof.

A “port axis” as used herein means an axis extending perpendicular to an opening surface of a port from the geometric center of the opening surface. When the opening surface of the port is circular, the port axis is an axis that passes through the center of the opening surface and extends perpendicular to the opening surface.

A “liquid sample” as used herein means a solvent and a solution. The fluid sample is, for example, culture medium, buffer solution, cell suspension, water, and aqueous solution containing a biochemical reagent such as trypsin. Culture media or buffers are commercially available or can be prepared with known reagents. Cell suspensions are liquids containing cells in cell media or buffers. Biochemical reagents such as trypsin are commercially available or can be prepared according to known methods.

A “pharmaceutical composition” as used herein means a composition including an active ingredient and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may be known carriers and can be appropriately used according to types of active ingredients, administration routes, and dosage forms. Pharmaceutical compositions can be prepared according to known methods. Active ingredients may be, for example, components secreted from cells during culturing, components accumulated in cells, or cells themselves.

“Culturing cells” or “cell culture” as used herein means proliferating cells in multilayer culture vessel or producing in cells components of interest. Cell culturing includes, for example, placing cells in multilayer culture vessels under the environment where temperature, humidity and/or concentration of carbon dioxide are controlled. Cell culturing may further include replacing the medium in which cells were cultured and subculturing cultured cells. The cell includes, for example, established cell line, genetically modified cell, cell obtained from living organism.

“Collecting” as used herein means taking out a liquid sample from a multilayer culture vessel. A liquid sample collected from multilayer culture vessels is, for example, cell suspension or culture medium including cultured cells. The collection from multilayer culture vessels can be appropriately carried out according to known methods. For example, a liquid sample can be taken out from a multilayer culture vessel using a dispensing device such as pipette. A cell suspension including cultured cells can be obtained by, for example, injecting a solution containing a reagent such as trypsin and the like with a dispensing device such as pipette to a multilayer culture vessel in which cells are cultured to release cells adhesive to culture trays. The obtained cell suspension can be taken out from the multilayer culture vessel in the same manner as above.

“Separating” used herein means taking out a component of interest from conditions in which the component exists. For example, a component produced in cultured cells can be separated according to known methods including physical methods such as French press and crushing with ultrasonic wave, or chemical methods such as using a disruption solution containing surfactant or the like.

“Purifying” as used herein means an operation for increasing the content rate of a component of interest. Purification is carried out, for example, according to known methods. The known purification methods include, but are not limited to, liquid chromatography, centrifugation, magnetic beads, and flow cytometer.

Hereinafter, embodiments according to aspects of the present invention will be described with reference to the drawings, but these embodiments are examples of the present invention. The embodiments do not limit the inventions described in the appended claims in any way.

FIG. 1shows an exploded perspective view of a multilayer culture vessel of an embodiment according to the present invention (“Embodiment 1”). The multilayer culture vessel1according to Embodiment 1 comprises a culture vessel part2and a reservoir part5attached to the culture vessel part2.

The culture vessel part2according to Embodiment 1 constitutes a culture tray3a, a culture tray3bstacked on the culture tray3a, a culture tray3cstacked on the culture tray3b, a culture tray3dstacked on the culture tray3c, a culture tray3estacked on the culture tray3d, and a cover plate4stacked on the culture tray3e. The reservoir part5is attached to the culture vessel part2, the culture trays3and the cover plate4. The reservoir part5has an internal space and a screw port55communicating with the internal space. The culture trays3, the cover plate4, and the reservoir part5are liquid-tightly bonded by heat bond, melt bond, ultrasonic fusion, or adhesive at joint areas or abutting portions described below.

The culture tray3includes a bottom wall31whose tray surface is substantially rectangular and flat and a surrounding wall32that surrounds the bottom wall and is integrally molded with the bottom wall31. The surrounding wall32of the culture tray comprises peripheral wall parts, which extend substantially vertically upward from each periphery of the bottom wall31and have a predetermined length. The culture tray3has a storage space defined by the bottom wall31and the wall32surrounding the bottom wall31. The five culture trays3a,3b,3c,3d, and3ehave substantially the same shape as each other.

The surrounding wall32of the culture tray3ahas a protrusion33that facilitates properly stacking with the culture tray3b. The outer surface of the bottom wall31of the culture trays3bhas a recess34fitting the protrusion33on the surrounding wall32of the culture tray3a. The culture tray3bcan be easily stacked on the culture tray3aproperly by putting the projection33of the culture tray3ainto the recess34of the culture tray3b. The outer surfaces of the bottom walls31of the culture trays3cto3ealso have recesses34fitting the projections33of the culture trays like the culture tray3b. The culture trays3cto3ecan also be easily stacked properly by putting the projections33into the recesses34. The stacked surrounding walls32of the culture trays3constitute a sidewall22of the culture vessel part2. The bottom wall31of the culture tray3aon which the culture trays are stacked constitutes the bottom of the culture vessel part2. Further, recesses may be provided on each surrounding wall32of the culture trays3ato3e, and protrusions may be provided on the outer surfaces of the bottom walls of the culture trays3bto3eand the surfaces facing the culture trays of a cover wall part41of the cover plate4.

The culture vessel part2is a substantially rectangular parallelepiped and comprises the bottom wall31of the culture tray3a, the cover wall part41that is stacked on the culture tray3eand faces the bottom wall31, and an internal space defined by the sidewall22connecting the bottom wall31and the cover wall part41. The sidewall22has a sidewall part22dto which the reservoir part5is attached, a sidewall part22bfacing the sidewall part22d, and a sidewall part22aand a sidewall part22cwhich face each other and connect the sidewall part22band the sidewall part22d, as described below with reference toFIG. 2. The internal space of the culture vessel part2is partitioned by the culture trays3. Individual culture trays have the partitioned internal space in the culture vessel part2as the internal space of the culture tray. In the case of the culture tray3aon which the culture tray3bstacks, the internal space of the culture tray is a space defined by the bottom wall and the surrounding wall of the culture tray3aand the bottom wall of the culture tray3b. Each of the culture trays3bto3dalso has an internal space defined by its bottom wall31and surrounding wall32, and the bottom wall31of the individual culture trays3cto3ethat are stacked. The culture tray3ehas an internal space defined by its bottom wall31and surrounding wall32, and a first cover wall part41aof the stacked cover plate4. The internal space of the culture tray3can be used as a culture vessel. The internal space of the culture tray3of Embodiment 1 is defined by the bottom wall31of the culture tray3and the surrounding wall32surrounding the bottom wall31.

The cover plate4is stacked on the culture tray3ealong the stacking direction (D1) where the culture trays3ato3eare stacked. The cover plate4comprises a cover wall part41that is substantially flat. The cover wall part41comprises a first cover wall part41astacked on the culture tray3eand a second cover wall part41battached to the reservoir part5. By attaching the reservoir part5to the second cover wall part41bintegrally molded with the first cover wall part41aof the cover plate4, the assembled multilayer culture vessel1can increase its strength. The cover plate stacked on the at least culture trays3ato3econstitutes the upper part of the culture vessel part2.

The first cover wall part41ais substantially flat and comprises a cover surface whose shape is substantially the same as the tray surface of the culture tray3that is substantially rectangular. The first cover wall part41ahas recesses44a, which fit the penetrations33on the surrounding wall32of the culture tray3e, on the cover surface facing the culture tray3e. The cover plate4can be easily stacked on the culture tray3eproperly by putting the projections33of the culture tray3einto the recesses44aof the cover wall part41a.

The second cover wall part41bis substantially flat and comprises a cover surface whose shape is substantially the same as an inner surface of a surrounding wall part52aof a reservoir container50that is substantially rectangular, described below. The second cover wall part41bhas recesses44b, which fit projections53on four surrounding wall parts52b,52c,52d, and52eof the reservoir container50described below, on the cover surface facing the reservoir part5. The cover plate4can be easily attached to the reservoir part5properly by putting the protrusions53of the reservoir container50into the recesses44bof the second cover wall part41b.

The cover plate4has a ridge43formed around an edge. The ridge43facilitates stacking multilayer culture vessels1. When stacking a multilayer culture vessel1on another multilayer culture vessel1whose cover plate4has a ridge43, a bottom wall31of the culture tray3constituting the bottom of the multilayer culture vessel1that stacks may have a positioning protrusion fitting the ridge43. The fitting of the ridge43to the positioning protrusion allows preventing the stacked multilayer culture vessels1from being out of position.

The reservoir part5comprises a reservoir container50that is substantially rectangular parallelepiped and has an internal space. The reservoir part50is attached to the culture trays3ato3eand the cover plate4. The internal space of the reservoir container50is defined by a surrounding wall52and the second cover wall part41bof the cover plate41. The surrounding wall52comprises the surrounding wall part52awhose inner surface is substantially rectangular and parallel to the tray surface (inner surface) of the bottom wall31of the culture tray3aand constitutes a part of the bottom of the culture vessel part2. The surrounding wall52further comprises four surrounding wall parts52b,52c,52d, and52e, which extend substantially vertically upward from each side of the surrounding wall part52aand have predetermined lengths.

The surrounding wall parts52bto52ehave protrusions53that facilitate properly attaching the reservoir part5to the cover plate4. As described above, the reservoir part5can be easily attached properly to the cover plate4by fitting the propagations53of the reservoir container50to the recess of the second cover wall part41b. Further, recesses may be provided on the reservoir part5, and protrusions53may be provided on the second cover wall part41b.

The surrounding wall part52eof the reservoir container50has a socket54that facilitates properly attaching the reservoir part5to the culture trays3. Peripheral wall parts of the surrounding walls32of the culture trays3facing the reservoir container50have locking protrusions35fitting the socket54. The reservoir part5can be easily attached to the culture trays3properly by fitting the socket54of the reservoir container50to the locking projections35of the culture trays3.

The surrounding wall part52eof the reservoir container50abuts on the abutting portions36provided on the peripheral wall parts of the surrounding walls32of the culture trays3. Adhesion of the abutting portions36by, for example, heat bond, melt bond, ultrasonic fusion, or adhesive, can increase the strength of the multilayer culture vessel1after being assembled.

The screw port55is combined with a corresponding screw lid56to constitute an openable port. The screw port55is provided on the surrounding wall part52bof the reservoir container50such that an angle formed between the port axis D2and the stacking direction D1is about 90 degrees. The surrounding wall part52bwith the screw port55is also referred to a first surrounding wall part52b, and the surrounding wall part52dfacing the surrounding wall part52bis also referred to a second surrounding wall part52d. The screw port55is provided on the reservoir part5such that the port axis D2is substantially parallel to a surface where the reservoir container50is opposed to the culture trays3.

Openings are provided on the surrounding walls32of culture trays3, the openings communicating the internal space of the reservoir part5with the internal spaces of the culture trays3. The opening of the culture tray3ais a slit-shaped space formed by the bottom wall31of the culture tray3bstacked on the culture tray3aand a cutout part38provided on the locking protrusion35of the culture tray3adistally from the bottom wall31. The openings of the culture trays3bto3dare slit-shaped spaces formed by the bottom walls31of the stacked culture trays3cto3eand cutout parts38provided on the locking protrusions35of individual culture trays3bto3d. In addition, the opening of the culture tray3eis a slit-shaped space formed by the first cover wall part41aof the cover plate4stacked.

The surrounding wall part52bwith the screw port55abuts on the sidewall22dof the culture vessel part2at the first abutting portion P1, described below with reference toFIG. 2d. The surrounding wall part52dfacing the surrounding wall part52babuts on the sidewall part22dof the culture vessel part2at the second abutting portion P2. The culture vessel part2is liquid-tightly bonded to the reservoir part5at the first abutting portion P1and/or the second abutting portion P2by, for example, heat bond, melt bond, ultrasonic fusion, or adhesive.

An opening provided on the surrounding wall32of the culture tray3is described in detail with reference toFIG. 2.FIG. 2ashows a multilayer culture vessel1according to Embodiment 1, assembled with respective parts shown inFIG. 1. In the multilayer culture vessel1, a sidewall22is constituted by the surrounding walls32of five stacked culture trays3and a reservoir part5is attached to one sidewall part22d. A culture vessel part2has a first sidewall and a second sidewall, both of which are opposed to each other along a first direction where the first surrounding wall part52bis opposed to the second surrounding wall part52d. InFIG. 2a, the first sidewall is the sidewall part22a, and the second sidewall is the sidewall part22c. The port comprising the screw port50and the screw lid56is located between a position of the sidewall part22ain the first direction and a position of the sidewall part22cin the first direction. That is, the port is below the plane including the sidewall part22cand is above the plane including the sidewall part22a, when the sidewall part22ais placed downward inFIG. 2a. The reservoir part5comprises a surrounding wall part52cand a surrounding wall part52e, both of which are opposed to each other along a second direction orthogonal to both the first direction and the stacking direction D1where the at least two trays stack, and the port is located between a position of the surrounding wall part52cin the second direction and a position of the surrounding wall part52ein the second direction. That is, the port is the left side from the plane including the sidewall part22cand is the right side from the plane including the sidewall52e, when the sidewall part22bis placed leftward inFIG. 2a. When the port is provided at the position described above, the port is inside the outer peripheral surfaces of the multilayer culture vessel2and the reservoir part5, so that the vessel can be transported efficiently. In addition, the vessel can reduce the risk of the worker's hands or work equipment touching the port during culture workings, and the operability is good.

FIG. 2bshows a cross-sectional view taken along with the A-A line inFIG. 2a. As shown inFIG. 2b, the internal space of the culture vessel part2of the multilayer culture vessel1is partitioned into five spaces by the five culture trays3ato3e. Each of the five internal spaces is adjacent to the internal space of the reservoir container50of the reservoir part5via the sidewall part22d, which comprises the locking protrusion35of the culture tray3, of the culture vessel part2. In the A-A cross-section, the internal space of the reservoir container50is surrounded by the second cover wall part41b, the surrounding wall parts52ato52c, and the sidewall22d.

FIG. 2cis an enlarged view of the part surrounded by the broken lines inFIG. 2b. It is described that the internal spaces partitioned by the culture trays3are communicated with the internal space of the reservoir container50through the openings39formed on the sidewall part22dof the culture vessel part2with reference toFIG. 2c. As described above, the opening39formed on the sidewall part22dis a space formed between the first cover wall part41aof the cover plate4stacked on the culture tray3or the bottom wall31of the stacked culture tray3and a cutout part38formed on the locking protrusion35, the cutout part being distal to the bottom wall31. As shown inFIG. 2c, each of the internal spaces partitioned by the culture trays3is communicated with the internal space of the reservoir container50through the opening39formed for the individual culture trays3.

FIG. 2dshows a cross-sectional view taken along the D-D line inFIG. 2a, and the cross-section taken along the D-D line corresponds to a surface where the sidewall part22dof the culture vessel part2of the multilayer culture vessel1faces the reservoir container50. As described above, the openings39formed on the sidewall part22dof the culture vessel part are formed between the first abutting portion P1that the surrounding wall part52babuts on the sidewall part22dof the culture vessel part and the second abutting portion P2that the surrounding wall part52dabuts. InFIG. 2d, the openings39formed on the sidewall part22dextend toward the first abutting portion P1from the second abutting portion P2with a length of about one-third of the linear distance between the first abutting portion and the second abutting portion. In the multilayer culture vessel1of Embodiment 1, the openings39formed on the sidewall part22dhave a length corresponding to that of the locking protrusion fitted to the internal wall parts54aand54bof the socket on the surrounding wall part52e(a cross-section thereof is shown). The length of the openings39to be less than two-thirds of the linear distance between P1and P2is preferably set so that the mechanical strength of the multilayer culture vessel1including the culture vessel part2and the reservoir container50can be stronger.

The size of the multilayer culture vessel1of Embodiment 1 is described with reference toFIG. 2. When the short side of the multilayer culture vessel1has a length L1of 100 to 150 mm inFIG. 2a, the long side of the multilayer culture vessel1has, for example, a length L2of 200 to 300 mm, and the long side of the culture vessel part2has, for example, a length L8of 180 to 210 mm. As shown inFIG. 2a, the reservoir container50is attached to the sidewall part22dof the multilayer culture vessel1at an upper position from the horizontal position of the sidewall part22a. The distance L3of the upper position is, for example, 8 to 13 mm when the short side of the multilayer culture vessel1has a length L1of 100 to 150 mm. In the first posture shown inFIG. 3b, lifting the sidewall part22ballows to easily tilt the reservoir part5of the multilayer culture vessel1diagonally downward because of being attached at the upper position, so that users can easily inject or collect a fluid sample through the port. When multilayer culture vessels with relatively large size are used in the limited workspace in clean benches, there is a high possibility that worker's fingers will come into contact with the port, resulting in an increase in the risk of contaminating the inside of the multilayer culture vessel. In addition, when collecting a fluid sample from a relatively large multilayer culture vessel with a relatively short instrument such as a pipette, it is hard to collect the sample from the reservoir part, so that sample waste is likely to occur. The length L1of the multilayer culture vessel is preferably not more than 140 mm to prevent samples waste and decrease work efficiency.

InFIG. 2b, the internal spaces partitioned by the cultures trays3have, for example, a height L4of 5 to 10 mm when the short side of the multilayer culture vessel1has a length L1of 100 to 150 mm. The multilayer culture vessel1has, for example, a height L5of 45 to 55 mm when the short side of the multilayer culture vessel1has a length L1of 100 to 150 mm. In the multilayer culture vessel1of Embodiment 1, the bottom walls31of the culture trays3and the cover wall part41of the cover plate4have, for example, a thickness of 1.5 to 2.5 mm, respectively.

InFIG. 2d, among the contact portions where the reservoir container50abuts on the culture vessel part2, the contact portion at the upper end that the surrounding wall portion52bprovided with the screw port55abuts is referred to a first abutting portion P1ant the contact portion at the lower end is referred to a second contact portion P2. When the distance between the abutting portions is 75 to 90 mm, the openings39formed on the sidewall part22dextend toward the first abutting portion P1from the second abutting portion P2with a length of, for example, 25 to 35 mm (L6). When the openings39formed on the sidewall parts22dhave a longitudinal length L6of 25 to 35 mm, the openings39have, for example, a width L7of 1 to 2 mm. The openings39extend toward the first abutting portion P1through the second abutting portion P2from the sidewall part22aof the culture vessel part2with a distance L10. The distance L10is from the sidewall part22aof the culture vessel part2to the opening edge of the short side, which is located near the first abutting portion P1, among the opening edges of the openings39. In a multilayer culture vessel according to Embodiment 2 described below, the reservoir container is attached to the culture vessel part at the same level as the first sidewall part22a′. Accordingly, the distance L6is the same value as the distance L10. In the multilayer culture vessel according to Embodiment 1, when the openings39have a length L6of 25 to 35 mm in the longitudinal direction, the distance L10is, for example, 33 to 48 mm.

InFIG. 2c, the width L7of the opening39is a distance between a lower edge39aand an upper edge39bof the opening. The upper edge39bof the opening corresponds to the outer surface of the cover wall part41of the stacked cover plate or the outer surface of the bottom wall31of the culture trays. When the height L4of the internal spaces partitioned by the culture trays3is 5 to 10 mm, the distance L9from the bottom wall31of the culture trays to the lower edge39aof the opening is, for example, 3 to 9 mm.

The way to use the multilayer culture vessel1after injecting a fluid sample (for example, culture medium) is described with reference toFIG. 3.FIG. 3ashows a cross-sectional view taken along the E-E line inFIG. 2and shows the multilayer culture vessel1placed in the first posture after the fluid sample is injected. In the first posture of the multilayer culture vessel1, the multilayer culture vessel1is placed so that the sidewall part22aof the culture vessel part2faces downward and the port55provided in the reservoir part5attached to the culture vessel part2faces upward. The multilayer culture vessel1placed in the first posture allows users to easily inject a fluid sample to or collect the fluid sample from the internal space defined by the culture trays of the culture vessel part2through the port55.

As shown inFIG. 3a, the liquid level of the fluid sample in the reservoir container50in the first posture is the same for each of the openings39formed in the sidewall part22d. As described above, each of the internal spaces of the culture vessel part2partitioned by the culture trays3communicates with the internal space of the reservoir container50through the openings39formed in sidewall part22d. When a fluid sample is injected into the internal space of the reservoir container50through the port55, the injected fluid sample flows into each of the internal spaces in the culture vessel part2through each of the openings39formed in the sidewall part22d.

FIG. 3bshows the cross-section of the multilayer culture vessel1in the first posture, taken along with the B-B line inFIG. 2b. As shown inFIG. 3b, the fluid sample flowed into the respective internal spaces of the culture vessel part2through the openings formed in the sidewall part22dof the culture vessel part2has the same liquid level as the fluid sample in the reservoir container50. According to the multilayer culture vessel1, an injected fluid sample evenly flows into the respective internal spaces partitioned by the culture trays3in the culture vessel part2just by performing injection work, as seen fromFIGS. 3aand3b.

FIG. 3cshows the multilayer culture vessel1placed in the second posture so that the sidewall part22bof the culture vessel part2faces downward. The second posture is a posture after the multilayer culture vessel1in the first posture is rotated 90 degrees in the direction along with the arrow R inFIG. 3b. During the transition from the first posture to the second posture, the fluid sample held in the internal space of the reservoir container50flows into the internal spaces partitioned by the culture trays3of the culture vessel part2through the openings39formed in the sidewall part22d. Accordingly, all the fluid sample injected into the reservoir container50are basically distributed to the internal spaces partitioned by the culture trays of the culture vessel part2, so that the waste of fluid samples can be eliminated.

FIG. 3dshows the multilayer culture vessel1placed in the third posture so that the bottom wall31of the culture tray3afaces downward by tilting it 90 degrees toward the back centering on the sidewall part22bfrom the second posture shown inFIG. 3c. As shown inFIGS. 3ato 3d, it is easy to realize the state in which the fluid sample is evenly distributed to the individual culture trays3a,3b,3c,3d, and3e, constituting the culture vessel part2, by changing the postures of the multilayer culture vessel1from the first posture through the second posture to the third posture. The liquid level of the fluid sample distributed to the individual culture trays is lower than the cutout parts forming the openings39of the individual culture trays. As mentioned above, according to the multilayer culture vessel1in the first posture, the fluid sample is evenly distributed to the internal spaces partitioned by the culture trays3of the culture vessel part2just by injecting the fluid sample, so that culture conditions can be easily uniform. This can suppress the waste of reagents caused by the non-uniformity of the state of cultured cells.

Contamination in cell culture using culture vessels generally more likely occurs near the port, which provides communication between the internal space of a culture vessel and the external space. For this reason, cell culture operations are usually performed so that fluid samples contacted with the port do not enter the culture vessel. As seen fromFIG. 3, the multilayer culture vessel1according to Embodiment 1 basically allows the fluid sample injected from the port55to avoid contact with the port until the injected liquid sample is distributed to the individual internal structures partitioned by the culture trays3of the culture vessel part2. The multilayer culture vessel1according to Embodiment 1 is also advantageous from the viewpoint of contamination risk.

A sidewall part constitution is not limited to Embodiment 1 where the sidewall part22of the culture vessel part2is constituted of the surrounding walls32of the stacked at least two culture trays3. A sidewall of the culture vessel part may be, for example, a sidewall of a container constituting the culture vessel part. In the above example, at least two culture trays of the multilayer culture vessel may be stacked such that outer peripheral surfaces of bottom walls or outer surface of surrounding walls of the culture trays are bonded to the internal surface of the sidewall of the container of the culture vessel part. Accordingly, Embodiment 1 of the present invention provides a variation described below.

Another Embodiment

A multilayer culture vessel, comprising a culture vessel part including a storage container and a reservoir part attached to the culture vessel part, wherein the storage container comprises at least two culture trays that are stacked; the reservoir part comprises a surrounding wall defining an internal space and a port communicating with the internal space; the surrounding wall comprises a first surrounding wall part provided with the port and a second surrounding wall part facing the first surrounding wall part; the culture vessel part has openings communicating individual culture trays of the at least two culture trays with the internal space of the reservoir part; and the openings of the culture vessel part extend toward a first abutting portion that the first surrounding wall part abuts on the culture vessel part from a second abutting portion that the second surrounding wall part abuts on the culture vessel part.

The stacking manner is not limited to Embodiment 1 in which at least two culture trays are stacked such that the surrounding wall32of one culture tray3contacts with the bottom wall of another culture tray3. For example, in Another embodiment of Embodiment 1, outer surfaces of the bottom walls or outer surfaces of the surrounding walls of the culture trays are bonded to the internal surface of the sidewall of the storage container that constitutes the culture vessel part. Thereby, the culture trays' bottom walls or tray surfaces are stacked such that the surrounding wall of one culture tray does not contact with the bottom wall of another culture tray.

The stacking order is not limited to that of Embodiment 1 in which the multilayer culture vessel1is assembled by stacking the at least two culture trays3on the culture tray3ain the order of the culture trays3b,3c,3d, and3e. For example, when outer surfaces of the surrounding walls of the culture trays are bonded to the internal surface of the sidewall of the storage container constituting the culture vessel part in Another embodiment of Embodiment 1, the order in which the culture trays stack, that is, the order in which the outer surfaces of the bottom walls or the outer surfaces of the surrounding walls of the culture trays are bonded to the internal surface of the sidewall of the container constituting the culture vessel part is not particularly limited.

The culture tray structure is not limited to that of Example 1 in which the culture tray3comprises a surrounding wall32composed of peripheral wall parts extending from the outer periphery of the bottom wall31and are integrally molded with the bottom wall31. For example, in Another embodiment of Embodiment 1, the culture tray comprises a bottom wall, whose outer peripheral surface is bonded to wall parts of the storage container of the culture vessel part, and the wall parts as a surrounding wall of the culture tray. The surrounding wall of the culture tray is composed of, for example, peripheral wall parts extending from the bottom wall, wall parts attached to the bottom wall, or peripheral wall parts extending from the bottom wall and wall parts attached to the bottom wall.

The culture tray structure is not limited to that of Embodiment 1 in which the surrounding wall32of the culture tray3comprises peripheral wall parts extending substantially vertically upward from the outer peripheral of the bottom wall. The surrounding walls of the culture trays may comprise peripheral wall parts extending diagonally upward from the outer periphery of the bottom wall.

The culture tray structure is not limited to that of Embodiment 1 in which the culture tray3comprises the bottom wall31whose surface is substantially flat and rectangular. The tray surface may be triangular, square, pentagonal, hexagonal, oval, and circular. The tray surface of the bottom wall of a culture tray constitutes, for example, the bottom of the culture vessel when placed in a posture suitable for culturing in a multilayer culture vessel.

The number of trays is not limited to that of Embodiment 1 in which at least culture trays3that are stacked comprise five culture trays3ato3e. The number of the at least culture trays stacked may be appropriately set by those skilled in the art according to the purpose and may be, for example, two, three, four, five, six, or more.

The culture tray structure is not limited to that of Embodiment 1 in which the at least two culture trays3stacked have substantially the same shape as each other. For example, the culture tray constituting the bottom of the multilayer culture vessel, when placed in a posture suitable for culturing in a multilayer culture vessel, may have a different shape than the other culture trays stacked on the tray. For example, the culture tray constituting the bottom surface of the culture vessel part may be provided with protrusions to prevent the bottom of the multilayer culture vessel from directly touching a culture apparatus when the vessel is placed in the culture apparatus.

The cover plate structure is not limited to that of Embodiment 1 in which the cover plate4comprises a substantially flat cover wall part41. The cover plate structure is not limited to that of Embodiment 1 in which the cover plate4comprises the first cover wall part41astacked on the culture tray3eand the second cover wall part41battached to the reservoir part5. The first cover wall part may constitute the cover plate stacked on culture trays.

The cover plate is not limited to that of Embodiment 1 in which the cover plate4is stacked on the surrounding wall32of the at least two culture trays3. For example, when the culture vessel part comprises a storage container like Another embodiment of Embodiment 1, the upper part of the storage container serves as a cover plate.

The reservoir part structure is not limited to that of Embodiment 1, in which the reservoir part5is attached to the sidewall part22dof the culture vessel part and the second cover wall part41bof the cover plate4. For example, the reservoir part may be attached to only the sidewall part22dof the culture vessel part when the reservoir part, instead of cover plate4, has a surrounding wall corresponding to the second cover wall part41b. In another example, when the bottom wall31of the culture tray3, instead of the reservoir part, is integrally formed with an additional bottom wall corresponding to the surrounding wall part52a, the reservoir part may be attached to the sidewall part22dof the culture vessel part, the second cover wall part41bof the cover plate4, and the additional bottom wall of the culture tray3. In this example, the assembled multilayer culture vessel1has a relatively high mechanical strength and is preferable.

The reservoir part structure is not limited to that of Embodiment 1 in which the reservoir part5comprises a reservoir container50that is a roughly rectangular parallelepiped. The shape of the reservoir part5may be, for example, regular hexahedron, truncated cone, polygonal pillar such as a pentagonal pillar.

The assembling manner is not limited to Embodiment 1 in which the culture vessel part2is attached to the reservoir part5by fitting the locking protrusions35formed on the surrounding walls32of the culture trays3to the socket54formed on the reservoir container50and bonding the surrounding wall part52eof the reservoir container50to the abutting portion36of the surrounding wall32of the culture trays. The attachment may be, for example, either fitting the locking protrusion to the socket or bonding at the abutting portion. In another example, the culture vessel part2and the reservoir part5may be integrally molded.

In Embodiment 1, the first surrounding wall part52b, which is provided with port55, of the surrounding wall52constituting the reservoir container50is substantially parallel to the second surrounding wall part52dfacing the first surrounding wall part52b, that is, the angle formed between the first surrounding wall part52band the second surrounding wall part52dfaced to each other is about 0 degrees, but the angle is not limited to about 0 degrees. The angle formed between the first surrounding wall part provided with the port and the second surrounding wall part facing thereto may be, but not limited to, not less than 0 degrees to not more than 50 degrees, for example, not less than 0 degrees to not more than 45 degrees, not less than 0 degrees to not more than 40 degrees, not less than 0 degrees to not more than 35 degrees, not less than 0 degrees to not more than 30 degrees, not less than 0 degrees to not more than 20 degrees, not less than 0 degrees to not more than 10 degrees, or not less than 0 degrees not more than 5 degrees. The angle formed between the first surrounding wall part provided with the port and the second surrounding wall part facing thereto may be, for example, 0 degrees, 10 degrees, 20 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, or 50 degrees.

The port shape is not limited to that of Embodiment 1 in which the shape of the port55is cylindrical convex pipe. The shape of the port is, for example, not particularly limited as long as fluid samples can be injected and collected with a dispensing device such as pipette. The shape of the port may be, for example, convex pipe, concave pipe, or hole. The port has, for example, an opening surface of circular, elliptical, or polygonal (e.g., hexagonal).

The port structure is not limited to that of Embodiment 1 in which the port55is an openable screw port with the screw lid56. The port may be, for example, an opening with a hinge cap. The material of the lid used as a port is, for example, metal or plastic such as stainless steel or polyethylene, commonly used in pharmaceutical fields or research fields. The lid may be, for example, a bent cap having filter with pores or hydrophobic membrane capable of exchanging gas. The pores may have, for example, sizes capable of protecting cells in containers from bacterial or virus contamination. The pores are, for example, less than 0.65 micron, 0.4 micron, or 0.22 micron.

The angle formed between the axis D2of port55and the stacking direction D1is not limited to that of Embodiment 1 in which the angle is about 90 degrees. The angle formed between the port axis D2and the stacking direction D1may be, for example, not less than 70 degrees to not more than 90 degrees, not less than 75 degrees to not more than 90 degrees, not less than 80 degrees to not more than 90 degrees, and not less than 85 degrees to not more than 90 degrees. The angle formed between the port axis D2and the stacking direction D1may be, for example, 90 degrees, 85 degrees, 80 degrees, 75 degrees, or 70 degrees.

In Embodiment 1, the port55is provided on the surrounding wall part52bof the reservoir container50such that the axis D2is substantially parallel to the surface facing the side wall part22dof the culture vessel part2to the surrounding wall part52eof the reservoir container50, that is, the angle formed between the axis D2of port55and the surface facing the sidewall part22dof the culture vessel part2to the surrounding wall part52eof the reservoir container50is about 0 degrees, but the angle is not limited to about 0 degrees. The angle formed between the axis D2of port and the surface facing the sidewall part of the culture vessel part to the surrounding wall part of the reservoir container may be, but not limited to, not less than 0 degrees to not more than 50 degrees, for example, not less than 0 degrees to not more than 45 degrees, not less than 0 degrees to not more than 40 degrees, not less than 0 degrees to not more than 35 degrees, not less than 0 degrees to not more than 30 degrees, not less than 0 degrees to not more than 20 degrees, not less than 0 degrees to not more than 10 degrees, or not less than 0 degrees to not more than 5 degrees. The angle formed between the first surrounding wall part provided with the port and the second surrounding wall part facing it is, for example, 0 degrees, 10 degrees, 20 degrees, 30 degrees, 35 degrees, 40 degrees, 45 degrees, or 50 degrees.

The surface facing the side wall part22dof the culture vessel part2to the side wall52eof the reservoir container50is, for example, a flat surface including two portions at both ends of the second abutting portion P2and the nearest portion between the axis D2of the port55and the first abutting portion P1.

The openings are not limited to that of Embodiment 1 in which the openings39of the culture vessel part2are formed by the cutout parts38provided on the surrounding wall32of the culture tray3. The opening of the culture vessel part may be, for example, a hole or aperture provided in a peripheral wall part extending from the bottom wall. In another example, the openings of the culture vessel part may be spaces formed between culture trays stacked such that a surrounding wall of one of the culture trays does not contact with a bottom wall of the other culture tray like Another embodiment of Embodiment 1.

The openings are not limited to that of Embodiment 1 in which the openings39of the culture vessel part2are formed by stacking the culture trays3such that the cutout parts38formed on the surrounding walls32are aligned with a surface that the sidewall parts22dof the culture vessel parts2are opposed to the surrounding wall part55eof the reservoir container50. Openings whose opening surfaces face toward the stacking direction can be formed by stacking culture trays with locking protrusions whose lengths protruding from the surrounding wall are different and shorter as the stacking direction advances.

The openings are not limited to that of Embodiment 1 in which the culture vessel part2has one opening39per culture tray3. The culture trays may have two or more openings per culture tray. In the case where a fluid sample is injected through the port55into the culture vessel part2of the multilayer culture vessel1placed in the first posture shown inFIG. 3aand the liquid level of the injected fluid is above the surrounding wall part52eof the reservoir container50, the fluid sample does not flow into the culture vessel part2due to the air remaining in the internal space of the culture trays of culture vessel part2. The culture vessel part2may have additional openings on the sidewall part22d, which allow the remaining air to escape so that the fluid sample flows into the culture vessel part2even if the fluid level of the fluid sample is above the surrounding wall part52eof the reservoir container50. The additional openings are formed above the openings39in the multilayer culture vessel1placed in the first posture shown inFIG. 3a.

In the case where no additional openings allowing the remaining air to escape are provided in the culture vessel part2, the surrounding wall part52eof the reservoir container50of the multilayer culture vessel1acts as a regulatory for adjusting the amount of a fluid sample flowing into the culture vessel part2, so that the culture trays hold the fluid sample and the air at a predetermined volume ratio.

The position of the opening is not limited to that of Embodiment 1 in which the openings39of the culture vessel part2are formed on the surrounding walls32of culture trays3such that the lower edges39aof the openings are formed at the position of about four-fifths of the surrounding walls' height. The openings of the culture vessel part2may be formed, for example, on the surrounding walls of the culture trays such that the lower edges39aof the openings are formed at the position of more than half, more than three-fifths, more than two-thirds, more than three-quarters, or more than four-fifths of the surrounding walls' heights.

When the multilayer culture vessel is placed in a posture (third posture) suitable for culturing as shown inFIG. 3d, the distance L9, which is from the tray surface of the bottom wall31of each culture tray3of the culture vessel part2to the lower end39aof the opening formed on the surrounding wall32of said each culture tray3, limits the upper level of the fluid sample that can be held in the culture tray. For example, in the case where a lower end39aof the opening of the culture vessel part is formed at half the height of the surrounding wall32of the culture tray and a fluid sample introduced into the internal space of the culture tray in an amount that the liquid level exceeds the half-height, a part of the fluid sample overflows from the opening. Accordingly, the space formed by the tray surface of the bottom wall31of the culture tray3and the distance L9to the lower end39aof the opening, among the internal spaces partitioned by the culture trays3in the culture vessel parts2, corresponds to the storage space that can be used as a culture vessel. To prevent a part of the fluid sample from overflowing from the internal spaces of the culture trays to the reservoir container50through the openings39when the posture of the multilayer culture vessel1is changed from the first posture through the second posture to the third posture, an indication for the appropriate amount of fluid sample in the multilayer culture vessel1placed in the first posture may be marked on the culture vessel part2and/or the reservoir part5. Such marks may be printed, embossed, or sealed.

A maximum volumetric capacity that the culture vessel part2can hold when the multilayer culture vessel1is placed in the third posture is calculated, for example, by multiplying the area of the tray surface of the bottom wall31of the culture trays (L1×L8, seeFIG. 2a) by the distance L9from the bottom wall31of the culture trays to the lower end39aof the opening to obtain a volumetric capacity (a maximum capacity that one culture tray3can hold) and by multiplying the volumetric capacity by the number of the culture trays3constituting the culture vessel part2(L1×L8×L9×[the number of culture trays]). The above-mentioned mark indicating an appropriate amount of fluid sample in the first posture may be put, for example, at a position corresponding to a volumetric capacity not more than the maximum volumetric capacity that the culture vessel part2can hold. There is a risk that the liquid sample overflows from the culture trays3to the reservoir container50(50) due to vibration or the like, in a particular case where the liquid sample is held up to the maximum volumetric capacity that can be held in the third posture. Accordingly, the amount of liquid sample to be held or the tray area of culture tray3may be appropriately set, preferably such that the liquid volume is less than 50% of the distance L9. The appropriate volume to be held in the culture vessel part may be expressed as L1×L8×L9×0.5×[the number of culture trays].

When air bubbles form on the fluid sample, the amount of the collected fluid sample decreases. Further, when air bubbles burst, fluid sample droplets scatter, which may cause contamination. An internal structure of the multilayer culture vessel may facilitate forming air bubbles on the fluid sample. For example, in the case where the posture of the multilayer culture vessel1is changed from the third posture to the first posture to collect culture medium held in the multilayer culture vessel, air bubbles are likely to be formed if the liquid level of the fluid sample is higher than the position of the opening edge of the short side, which is located near the first abutting portion P1, of the opening formed on the surrounding wall of each culture tray. The opening edge of the short side, which is located near the first abutting portion P1, of the opening formed on the surrounding wall of each culture tray is preferably positioned higher than the liquid level of the appropriate volume to be held in the culture vessel part2in order to suppress the formation of air bubbles, wherein the liquid level corresponds to the appropriate volume when the culture vessel is placed in the first position. In other words, the volumetric capacity obtained by multiplying the area of the bottom surface of the multilayer culture vessel placed in the first posture by the distance to the opening edge located near the first abutting portion P1is preferably larger than the appropriate volume to be held in the culture vessel part2. Such a multilayer culture vessel has, for example, the following relationship:

[volumetric capacity obtained by multiplying the area of the bottom surface of the multilayer culture vessel placed in the first posture by the distance to the opening edge located near the first abutting portionP1]≥[appropriate volume to be held in the culture vessel part]  (1).

The volumetric capacity of the left side in the inequality (1) can be obtained by multiplying the distance L10(seeFIG. 2d), which is from the sidewall part22ato the opening edge located near the first abutting portion P1, by the area that is obtained by multiplying the length L2of the long side of the multilayer culture vessel by the height L5viewed from the sidewall part22aof the multilayer culture vessel (seeFIG. 2b). Accordingly, the volumetric capacity of the left side can be expressed as L10×L2×L5. As described above, the volume of the right side can be expressed as L1×L8×L9×[the number of culture trays]. Substituting these relations into the inequality (1) yields the following formula (2):

As shown inFIG. 2, the height L5of the multilayer culture vessel is almost equal to the value obtained by multiplying the height L4of the internal space of the culture tray by the number of the culture trays constituting the multilayer culture vessel. Accordingly, it can be expressed as L5=L4×[the number of culture trays]. Substituting these relationships into the inequality (2) yields the following formula (3):

L10×L2×L4×[the number of culture trays]≥L1×L8×(L9×0.5)×[the number of culture trays]  (Formula 3).

Dividing both sides of the inequality 3 by L1, L2, L4, and [the number of culture trays] yields formula (4):

[wherein, L10/L1(seeFIG. 2d) is a ratio of the length L10, which is from the sidewall part22ato the opening edge located near the first abutting portion P1, to the length L1of the short side of the multilayer culture vessel; L9/L4(seeFIG. 2c) is a ratio of the distance L9, which is a distance to the opening's lower end39alimiting the upper level of the fluid sample to be held in the culture tray, to the height L4of the internal space used as the culture vessel; and L8/L2(seeFIG. 2a) is a ratio of the length L8of the long side of the culture vessel part2to the length L2of the long side of the multilayer culture vessel1.

The openings are not limited to that of Embodiment 1 in which the openings39of the culture vessel part2are formed on the surrounding walls32of the culture trays3. For example, when the culture vessel part is constituted of a storage container like Another embodiment of Embodiment 1, openings of the culture vessel part may be formed on the sidewall of the storage container. In a way, openings on the area where the reservoir part is attached to the culture vessel part are considered the openings of the culture vessel part, even if the openings are on the surrounding wall of the reservoir part in assembling the multilayer culture vessel.

The shape of the openings is not limited to that of Embodiment 1 in which the openings39of the culture trays3are slit-shaped spaces. The openings of the culture trays may be, for example, triangle or ellipse shape. The openings may be, for example, multiple spaces formed intermittently.

FIG. 4shows a perspective view of a multilayer culture vessel according to another embodiment of the present invention (“Embodiment 2”). Embodiment 2 is described below with reference toFIG. 4. The differences from Embodiment 1 are mainly described, and the descriptions of similar features are omitted. The multilayer culture vessel1of Embodiment 1 comprises five culture trays, while the multilayer culture vessel1′ of Embodiment 2 comprises six culture trays3a′ to3f′.

The port provided in the reservoir part5of Embodiment 1 is a screw port55with an openable screw lid56, while the port provided in a reservoir part5′ of Embodiment 2 is an openable cap type port55′ with a hinge cap56′. The hinge cap56′ is equipped with a gas exchange part having an opening, which communicates the internal space of the reservoir part5′ with the external space and is covered with filter or hydrophobic membrane having pores. For example, the hinge cap56′ of Embodiment 2 shown inFIG. 4has eight gas exchange parts. The eight gas exchange parts are spaced apart from each other around the center of the hinge cap56′ viewed from its thickness direction, and ribs radiate from the center between adjacent gas exchange parts. The gas exchange parts may be provided on the screw lid. A lid or cap may have one or more gas exchange parts. For example, the lid or cap has three gas exchange parts, and the gas exchange parts are spaced around the center of the lid or cap viewed from its thickness direction, preferably at equal angles.

In the multilayer culture vessel1according to Embodiment 1, the surrounding wall part52dof the reservoir container50of the reservoir part5is attached to the surrounding sidewall part22dof the culture vessel part2at an upper position from the horizontal position of the sidewall part22a. In the multilayer culture vessel1′ according to Embodiment 2, the surrounding wall part52e′ of the reservoir container50′ of the reservoir part5′ is attached to the sidewall part22d′ of the culture vessel part2′ at the same level as the sidewall part22a′. This improves stability when the multilayer culture vessel1′ is placed in the first posture. In addition, when a fluid sample is collected in the first posture, openings of the culture tray3′, which extend from the abutting portion where the surrounding wall part52e′ of the reservoir container50′ abuts on the sidewall part22d′ of the culture tray3′, allow reducing the amount of fluid sample left behind.

The multilayer culture vessel1′ according to Embodiment 2 has a drawer part58on the surrounding wall part52c′ and the surrounding wall part52d′ of the reservoir container50′. The drawer part58is convenient for users to hook their fingers and pull the multilayer culture vessel1′ out when delivering the multilayer culture vessel1′ from a culture apparatus after the cell culturing in the culture apparatus. The reservoir container50′ has a surrounding wall part52b′ that is inclined from the surrounding wall part52a′ to form space as the drawer part58that fingers can hook.

The descriptions for members such as the culture tray, the cover plate, the reservoir container, and the openings of the culture trays in the multilayer culture vessel according to Embodiment 1 apply to the corresponding members in the multilayer culture vessel according to Embodiment 2.

Another aspect of the present invention provides a method for producing a pharmaceutical composition, the method comprising: culturing cells with a multilayer culture vessel according to an embodiment of the present invention; collecting from the multilayer culture vessel cultured cells or a culture fluid containing a component secreted from the cultured cells; and producing the pharmaceutical composition comprising the collected cultured cells, the collected secreted component, or a component isolated and purified from the collected cultured cells.

REFERENCE SIGNS