WO2013183121A1 - Récipient de culture et dispositif de culture automatique - Google Patents

Récipient de culture et dispositif de culture automatique Download PDF

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Publication number: WO2013183121A1
Authority: WO; WIPO (PCT)
Prior art keywords: container; culture; medium; flow path; lid member
Prior art date: 2012-06-06
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.): Ceased

Application number

PCT/JP2012/064501

Other languages

English (en)

Japanese (ja)

Inventor

貴之野崎

広斌周

志津松岡

亮太中嶌

豊茂小林

直子千田

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

Hitachi Ltd

Original Assignee

Hitachi Ltd

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2012-06-06

Filing date

2012-06-06

Publication date

2013-12-12

2012-06-06 Application filed by Hitachi Ltd filed Critical Hitachi Ltd

2012-06-06 Priority to PCT/JP2012/064501 priority Critical patent/WO2013183121A1/fr

2012-06-06 Priority to JP2014519728A priority patent/JP5866006B2/ja

2013-12-12 Publication of WO2013183121A1 publication Critical patent/WO2013183121A1/fr

2014-12-06 Anticipated expiration legal-status Critical

Status Ceased legal-status Critical Current

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Classifications

- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/02—Membranes; Filters
- C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps

Definitions

the present invention relates to a culture vessel and an automatic culture apparatus for culturing cells or tissues by automatic operation.
regenerative tissue used for regenerative medical treatment is based on GMP (Good Manufacturing Practice), which is the standard for manufacturing management and quality control of pharmaceuticals.
GMP Good Manufacturing Practice
regenerative tissues are manufactured in accordance with SOP (Standard Operational Procedure) by a manufacturing worker with specialized cell culture technology in a CPC (Cell Processing Center) that provides a clean manufacturing environment.
SOP Standard Operational Procedure
CPC Cell Processing Center
the automatic culture apparatus cultures cells as an alternative to manual work, but it is necessary to comply with GMP for manual work contents.
GMP specialized for automatic culture devices
development guidelines on automatic culture devices for clinical use regenerative medicine field (design guidelines for human cell culture processing equipment [revised], 2009)
regenerative medicine field design guidelines for human cell culture processing equipment [revised] 2009
an apparatus for automating the culture process as described in Patent Documents 1 and 2 is required. By performing operations such as medium exchange automatically, the risk of biological contamination is reduced and production efficiency is improved. That.
the cell types to be cultured are epithelial cells such as corneal epithelial cells, oral mucosal epithelial cells, and epidermal cells.
the culture container preferably has a two-layer structure.
Epithelial cells co-culture with feeder cells such as mouse-derived 3T3-J2 cells and grow with growth factors produced by the feeder cells.
feeder cells such as mouse-derived 3T3-J2 cells and grow with growth factors produced by the feeder cells.
Feeder cells are mixed in the regenerated tissue.
the automatic culture device keeps the quality of the regenerated tissue after production in order to keep the quality of the regenerated tissue constant. It is necessary that the old medium is not mixed.
a new medium is mixed in the old medium that is the drainage liquid. Things need to be avoided.
the automatic culture apparatus is required to meet the development guidelines for GMP and automatic culture apparatus that are required for manual work.
One of the requirements is that the quality of the regenerated tissue produced is constant. There is a need to. There are multiple conditions that make the quality constant, but one of them is to replace the old medium and supply a new medium in the medium replacement process in which the medium is replaced in all cases. It is necessary that the old medium is not mixed with the new medium.
the old medium is a medium used for the growth of cells. For example, glucose is consumed and lactic acid is discharged instead. When an old medium is mixed with a new medium, the amount of glucose after the medium exchange is different from the concentration of the new medium, and as a result, the reproducibility of the culture process is lost.
the automatic culture apparatus is required to meet the development guidelines for GMP and automatic culture apparatus that are required for manual work.
GMP development guidelines for GMP
the remaining regenerated tissue needs to be able to continue culturing while maintaining sterility until the time of treatment.
regenerative tissues required for regenerative medical treatment varies depending on the target organ.
esophageal mucosal regeneration clinical research on regenerative medical treatment by using about 8 regenerated tissues, which are oral mucosal sheets with a diameter of 24 mm layered to about 3-5 layers, has already been advanced.
regenerated tissues which are oral mucosal sheets with a diameter of 24 mm layered to about 3-5 layers.
On the day before the treatment it is necessary to inspect whether the manufactured regenerated tissue has good quality, and a regenerated tissue for inspection is further required.
a total of about 10 regeneration tissues are required.
an automatic culture device for regenerative medicine can produce at least a plurality of regenerative tissues for transplantation and inspection, and culture that maintains sterility after taking out the regenerated tissue for inspection the day before. Must.
Patent Document 1 when replacing the medium, a new medium is supplied in a state where the old medium is present in the culture container, and after the state in which the new medium and the old medium are mixed inside the culture container, an excessive amount of liquid is supplied.
the continuous medium exchange method is taken out.
the discharged medium has a large proportion of the old medium, but the old medium is mixed into the new medium although the concentration is low. Since the composition of the old medium varies depending on the cell growth stage (number of cells, degree of differentiation), the concentration of each component of the medium after the medium exchange by this method also varies depending on the cell growth stage. Further, when the medium component analysis is performed using the old medium obtained by this method, there is a problem that the monitoring result is not accurate because the collected old medium contains a new medium.
the device of Patent Document 2 requires a flow channel tube for supplying only air for adjusting the pressure in the culture vessel in addition to a flow channel tube for feeding and discharging a liquid such as a medium. If this method is applied to a closed culture vessel having a two-layer structure, the flow tube required for one closed culture vessel is a flow tube that is responsible for feeding and discharging liquid to and from the upper and lower layers. In addition, a flow path tube for supplying air is required, and a total of five tubes are required. In the method of Patent Document 2, one closed culture vessel is automatically cultured. For example, when 10 closed culture vessels are used for regenerative medical treatment for esophageal mucosal regeneration, the total number of channel tubes is It will be 50, which will be quite a lot. As a result, the flow path circuit becomes complicated.
the regenerated tissue since only one closed culture vessel is cultured in one culture, a part of the regenerated tissue cannot be aseptically removed for the purpose of examination or the like on the day before the regenerative medical treatment is performed.
a regenerative tissue for regenerative medical treatment and a regenerative tissue for examination cannot be extracted separately, and all of them are extracted.
the regenerated tissue is placed in an environment different from the cultured state from the time when the test and the treatment are performed at the same time or after the test until the time of the treatment. In the former case, it is difficult to reflect the test result in the schedule of regenerative medical treatment. In the latter case, there is a risk that the quality of the regenerated tissue will change.
the object of the present invention is to provide a simple flow path circuit so that the old culture medium and the new culture medium do not mix with each other. It is an object of the present invention to provide a culture container and an automatic culture apparatus that make it possible.
Another object of the present invention is to provide a culture container and an automatic culture apparatus capable of aseptically removing one regenerated tissue from a plurality of cell containers in which a plurality are simultaneously cultured.
An example of a representative example of the present invention is as follows.
a culture container for holding and culturing cells, wherein the first container, a plurality of second containers stored and held in the first container, and each of the first container and the second container are sealed.
a container lid member for stopping, each of the first container and the second container, and a connection port for connecting a flow path circuit for supplying the cells and the culture medium to the culture container.
the second container is a container for containing the medium and cells or only the medium, respectively, and a part of the partition between each of the first container and the second container is a liquid and a gas.
a gap that allows gas to flow between each other exists between each of the first container and the second container, and the connection port serves as the first port.
the medium is supplied and the gas is supplied and discharged.
a second supply port and a second discharge port for discharging the medium are connected, and each of the second containers has a first for supplying the medium and supplying and discharging the gas.
a supply port and a first discharge port for discharging the culture medium are connected, and the first supply port and the second supply port are flow paths that always flow the culture medium in one direction with respect to the culture vessel. Function.
the medium always flows in one direction in the cell seeding process and the medium exchange process.
the old medium is not mixed with the new medium, so that the reproducibility of the culture is improved.
Analysis accuracy of medium component analysis using the collected old medium is improved.
the flow path tube attached to the closed culture vessel serves both as a liquid feeding and air feeding function, the flow path circuit is simplified.
one regenerated tissue can be aseptically removed from a cell container in which a plurality of cells are co-cultured. Aseptic culture can be continued for the remaining culture vessels.
FIG. 2 is a plan view of a culture vessel corresponding to the AA ′ cross section of FIG. 1B, in which a plurality of upper-layer culture vessel units according to the first embodiment of the present invention are housed in a lower-layer integrated culture vessel box.
. 1B is a longitudinal sectional view of a culture vessel corresponding to the BB ′ section of FIG. 1A in the first embodiment.
FIG. 3 is a circuit diagram of an entire flow path when 10 regenerated tissues are cultured in the automatic culture apparatus according to the first embodiment.
FIG. 3B is a diagram showing an associated channel circuit extracted from the overall channel circuit diagram of FIG. 3A for one upper layer culture vessel unit in the first example. It is the figure which showed the control mechanism of the automatic culture apparatus which has a culture container which concerns on a 1st Example. In 1st Example, it is the figure which showed a series of protocols which culture
the 1st Example it is the figure which showed an example of the table for controlling cell seeding with an automatic culture apparatus.
1st Example it is the figure which showed the culture medium and the flow of air at the time of cell seeding
1st Example it is the figure which showed the culture medium and the flow of air at the time of cell seeding
Example it is the figure which showed the flow of the culture medium and air at the time of the culture medium exchange of an upper layer with a flow path circuit with respect to one culture container unit for upper layers. It is the figure which showed the culture medium and the flow of air at the time of the culture medium exchange of an upper layer. It is the figure which showed the culture medium and the flow of air at the time of the culture medium exchange of an upper layer. It is the figure which showed the culture medium and the flow of air at the time of the culture medium exchange of an upper layer. According to the first embodiment, the flow of the medium and the air during the exchange of the lower layer medium was shown by the flow path circuit that extracted the relevant part from the entire flow path circuit diagram for one upper culture container unit. FIG.
the culture vessel is a two-layered closed culture vessel having a culture space inside and having a first vessel, a second vessel, and a lid member thereof,
a plurality of second containers can be accommodated in the first container.
the first container includes a second supply port for supplying a medium, supplying and discharging a gas, and a second container for discharging the medium.
Two discharge ports are connected.
Each of the second containers is connected to a first supply port for supplying a medium, supplying and discharging a gas, and a first discharge port for discharging the medium.
a 1st supply port and a 2nd supply port function as a flow path which always flows a culture medium to one direction with respect to a culture container.
a closed culture vessel having a two-layer structure having a culture space inside and having a first vessel and a second vessel, wherein a plurality of second vessels are contained in the first vessel.
a container can be accommodated, and has a first supply port and a second supply port for supplying a cell suspension or a medium and supplying and discharging air to and from the first container and the second container, respectively. And a first discharge port and a second discharge port for discharging the medium and discharging the air from the first container and the second container, respectively.
the culture container of the structure which connected between the container lid member and the 1st container and had the expansion
the medium in an automatic culture apparatus using a closed culture vessel having a two-layer structure including the first container and the second container, the medium is A flow path circuit that can be configured to always flow in one direction and a control protocol for controlling the flow path circuit are employed. That is, an automatic culture apparatus using a closed culture vessel having a culture space inside, a cell bag in which a cell suspension is stored, a medium bag in which a medium is stored, a refrigerator and a medium in which the medium is refrigerated and stored A heater for preliminarily warming the medium to 37 ° C.
a culture vessel for culturing cells
a fluid movement control mechanism for feeding / feeding cell suspension, medium and air, and a source of carbon dioxide and the like
a gas tank for adjusting the gas concentration, a filter for adjusting the atmospheric pressure with the outside, a two-way valve and a three-way valve for opening and closing the flow path.
a flow path circuit including a culture container, a cell bag, a culture medium bag, a fluid movement control mechanism unit, and the like is installed in a thermostat, and the temperature of the entire flow path circuit is controlled.
the culture environment of the culture vessel is controlled by a control device.
a temperature sensor is installed in the device to monitor the internal temperature.
a microscope is installed to monitor the state of cell growth optically as appropriate.
the fluid in the culture container is discharged from the second supply port to the outside while the medium is fed from the medium bag to the first supply port by the fluid movement control mechanism.
the medium that reached the first supply port was refrigerated at 4 ° C. during storage but passed through a heater that warms to 37 ° C., so the temperature is 37 ° C. at this point. Since the medium reaching the first supply port temporarily stands by in a constant temperature bath maintained at 37 ° C., the temperature of the medium is also maintained at 37 ° C.
the air in the culture container is supplied to the inside from the second supply port.
the air in the culture container is discharged to the outside from the second supply port.
This operation is started immediately after the old medium discharged from the first discharge port is completely discharged from the first container. It is not necessary to complete the complete feeding of the old medium discharged from the first discharge port to the drain bag or the like.
a part of the old medium discharged from the first discharge port in the channel tube is collected from the drainage collection bag, and the rest is discharged to the drainage bag.
the old medium can be discharged in the first container without being mixed with the new medium.
the medium in the first container after the medium exchange is only the new medium supplied at the time of the medium exchange.
the discharge port is the first discharge port in the first container, whereas it is the second discharge port in the second container.
the air in the culture vessel is discharged from the first port to the outside. Since the temperature of the culture medium that has reached the second port temporarily stands by as in the case of the first container, it is maintained at 37 ° C. at this time.
the air in the culture container is supplied to the inside from the first supply port.
the air in the culture container is discharged to the outside from the first supply port.
This operation starts immediately after the old medium discharged from the second discharge port is completely discharged from the first container. It is not necessary to complete the complete feeding of the old medium discharged from the second discharge port to the drain bag or the like.
a part of the old medium discharged from the second discharge port in the channel tube is collected from the drainage collection bag, and the rest is discharged to the drainage bag.
the old medium can be discharged in the second container without being mixed with the new medium.
the medium in the second container after the medium exchange is only a new medium supplied at the time of the medium exchange. It should be noted that the order of exchanging the medium for the first container and the second container is arbitrary.
only one second container can be aseptically removed.
the remaining first container and second container can be continuously cultured while maintaining sterility.
the culture medium flows in one direction in the flow path circuit. Old and new media do not mix. As a result, the accuracy of medium component analysis on the collected old medium is also improved.
the first supply port and the second supply port have both the function of feeding the medium and feeding the gas. Therefore, the number of channel tubes to be attached to the closed culture vessel passes as compared with the case where the gas channel tube is provided independently. Therefore, the flow path circuit becomes simpler. Furthermore, it is possible to remove some culture containers aseptically during the culture. Since the remaining culture containers after removal also maintain sterility, the culture can be continued.
one regenerated tissue can be aseptically taken out from the cell container in which a plurality of cells are co-cultured. . Aseptic culture can be continued for the remaining culture vessels.
the medium always flows in one direction. In the medium exchange for exchanging the entire amount, the old medium is not mixed with the new medium, so that the reproducibility of the culture is improved. Analysis accuracy of medium component analysis using the collected old medium is improved. Since the flow path tube attached to the closed culture vessel serves both as a liquid feeding and air feeding function, the flow path circuit is simplified.
FIG. 1A is a plan view of the culture vessel corresponding to the AA ′ cross section of FIG. 1B
FIG. 1B is a vertical cross sectional view of the culture vessel corresponding to the BB ′ cross section of FIG. 1A.
the culture container 100 of this embodiment is composed of a plurality of upper-layer culture container units 101 (10 a to j in this case) for culturing a regenerated tissue, and one lower-layer integrated type for culturing feeder cells. It can be accommodated in the culture container box 102.
Each upper layer culture vessel unit 101 has an inverted truncated cone shape.
Each upper layer culture container unit 101 is housed inside the culture container box 102 through openings 115 provided at a plurality of predetermined positions on the upper surface of the box-shaped culture container box 102, and each upper layer culture container unit 101.
a plurality (10 in this case) of container lid members 103 corresponding to the unit 101 are fixed to the upper surface of the lower layer culture container main body 104 with screws or the like so as to close the openings 115.
a closed space 110 surrounded by the container lid member 103 and the lower layer culture container body (second container) 104 is formed in the culture container 100, and each upper layer culture container unit (first container) 101 is It is held in the enclosed space 110.
the upper layer culture vessel unit 101 is installed on the vessel lid member 103 by a hook structure or the like, and can be easily taken out as necessary.
the plurality of openings 115 are provided in parallel to the upper surface of the culture vessel box 102, but other arrangement methods such as a staggered pattern may be used.
the material of the lower layer-integrated culture vessel box 102 is a plastic having rigidity as well as plasticity such as polycarbonate, polystyrene, and polypropylene.
plasticity such as polycarbonate, polystyrene, and polypropylene.
the case where the shape of the bottom face of the lower-layer integrated culture container box is square is shown.
an example is shown in which ten upper-layer culture container units 101 are accommodated in one culture container box 102 in a state where two vertical and five horizontal culture container units 101 are arranged side by side.
positioning of the culture container unit 101 for upper layers is not limited to this.
the container lid member 103 and the lower layer culture container body 104 constituting the culture container box 102 are formed by injection molding, cutting, or the like.
the upper layer culture container unit 101 is accommodated in the culture container box 102.
a cell culture insert container generally used for cell culture is used.
Cell culture insert containers may be commercially available, such as those manufactured by BD, manufactured by Corning, manufactured by Greiner, etc., and usable products are not limited. Moreover, application to a temperature-responsive cell culture insert container manufactured by Cellseed is also possible.
the bottom surface 120 of the upper-layer culture container unit 101 which is a cell culture insert container or the like, is a porous film, and has many holes with a diameter of about 0.4 ⁇ m, for example. Thereby, a culture medium and a liquid factor can be moved between the upper layer and the lower layer.
the pore diameter is sufficiently smaller than the cells, the cells in the upper layer do not move through the pores to the lower layer. Moreover, since it is the hole of the magnitude
the cells are seeded and cultured on the bottom surface of the upper layer culture container unit 101.
the cells are seeded and cultured on the bottom surface of the lower layer culture container body 104.
the container lid member 103 or the lower layer culture container body 104 is provided with an elastic member 105 such as an O-ring.
the connection of the container lid member 103 to the lower culture container body 104 is fixed by screwing screws provided on the container lid member 103 and the lower culture container body 104 together. In this case, when removing the container lid member 103 from the lower layer culture container body 104, the container lid member 103 is rotated and removed.
the container lid member 103 is fixed by an engagement or fitting engagement portion provided in the container lid member 103 and the lower layer culture vessel main body 104. In this case, the container lid member 103 is lifted and removed in the axial direction without rotating.
the fixing method to the culture container main body 104 for lower layers of the container lid member 103 is not limited to these methods.
the lower layer culture vessel main body 104 includes a lower layer supply flow path 107 having a lower layer supply connection projection structure (second supply port) 106 at one end thereof for supplying a medium and supplying and discharging air / water vapor.
the lower layer discharge passage 109 has a lower layer discharge connection projection structure (second discharge port) 108 at one end thereof for discharging the medium and discharging air / water vapor.
the position of the lower layer supply channel 106 in the lower layer culture vessel main body 104 should be changed depending on the amount of the medium 150 to be introduced, but may be any position above the level of the introduced medium liquid.
the position of the lower layer discharge channel 109 in the lower layer culture vessel body 104 is used for discharging the medium from the lower layer culture vessel body 104.
the discharging efficiency is further improved by discharging the medium while appropriately tilting the culture container by a rotating mechanism for rotating the main culture container shown in FIG. 3A described later.
Each container lid member 103 has an upper layer supply connection projection structure (first supply port) 111 for supplying a culture medium and supplying / discharging air / water vapor to each upper layer culture container unit 101 at one end thereof. And an upper layer discharge flow path 114 having an upper layer discharge connection projection structure (first discharge port) 113 for discharging the culture medium at one end thereof.
first supply port a culture medium
first discharge port an upper layer discharge connection projection structure
the position of the upper layer supply flow path 111 in the container lid member 103 should be changed depending on the amount of the medium to be introduced into the container, but may be any position above the introduced medium liquid level. Since the position of the upper layer culture vessel unit 101 on the bottom surface of the upper layer culture vessel unit 101 is used for discharging the medium from the upper layer culture vessel unit 101, the bottom surface of the upper layer culture vessel unit 101 and the upper layer ejection channel 114 are It is desirable to be close. However, if it is too close, in the production of the regenerated tissue, the upper layer discharge flow path 114 comes into contact with the growth of the cells, and this impedes the growth of the cells.
the cells grow to a height of about several hundred ⁇ m when cultured to regenerated tissue. Therefore, the upper layer discharge channel 114 can be close to the bottom surface of the upper layer culture vessel unit 101 up to a position sufficiently higher than that, for example, up to about 500 ⁇ m.
the distance at which proximity is possible is determined according to the type of cells to be cultured.
the upper layer supply flow path 112 and the upper layer discharge flow path 114 installed in each container lid member 103 are arranged so as not to interfere with cell observation as much as possible.
the lower layer supply flow path 107, the lower layer discharge flow path 109, the upper layer supply flow path 112, and the upper layer discharge flow path 114 are flows made of an elastic body such as silicon having an inner diameter suitable for the protrusion structure size of the flow path.
Road tube is connectable. Thereby, it can connect to the channel circuit which an automatic culture apparatus has.
epithelial cells are seeded in the upper culture container unit 101, and feeder cells are seeded in the lower culture container body 104.
other cells may be seeded or cultured without seeding the cells.
FIG. 2A shows an enlarged state in which one upper layer culture container unit is accommodated in the culture container box 102
FIG. 2B shows one upper layer culture container unit from the culture container box. The separated state is shown.
the upper culture container unit 101 has an outer peripheral edge portion 1010 at the upper end thereof, and the outer peripheral edge portion 1010 is held on the inward protruding portion 1030 of the container lid member 103.
the inward protruding portion 1030 is held on an annular protruding portion 1040 provided around the opening 115 of the lower layer culture vessel body 104.
Screw structures 1050A and 1050B are provided on the outer peripheral portion of each container lid member 103 and the outer peripheral portion of the annular projecting portion 1040 of the lower layer culture vessel main body 104, and the container lid member 103 is provided with the lower layer culture vessel by this screw structure. It is fixed to the main body 104.
the fixing method is not limited to screwing, and any means such as engagement, fitting, or the like may be used.
the culture container 100 for holding and culturing the cells includes the culture container unit 101 for accommodating only the culture medium and the cells, or the culture medium, and the culture medium and cells or the culture medium for accommodating the culture container unit for the upper layer. And a container lid member 103 for sealing each of the upper layer culture container units and the lower layer culture container, and a flow path circuit and an outer surface of the culture container.
the cell suspension / medium always flows in one direction with respect to the culture vessel 100, and the gas flows in both directions.
FIG. 3A is an overall flow path circuit diagram when 10 regenerated tissues are cultured.
FIG. 3B shows an associated channel circuit extracted from the entire channel circuit diagram for one upper-layer culture container unit.
FIG. 3A is used to explain the entire flow path when culturing the lower-layer integrated culture vessel 100 that accommodates the ten upper-layer culture vessel units 101.
the culture target is epithelial cells such as corneal epithelial cells, oral mucosal epithelial cells, and epidermal cells.
the lower layer integrated culture vessel 100 is composed of two layers including a layer for culturing epithelial cells and a layer for culturing feeder cells that produce growth factors for epithelial cells. Structure. Since two types of cells are used, the cell bags 203 and 204 are of two types. Further, in order to prevent two types of cells from being mixed at the time of cell seeding, the flow path circuit for seeding is divided.
epithelial cells are put into the cell bag 202, passed through the first flow path circuit (1) 205 indicated by a broken line, and sequentially seeded in 10 upper culture container units 101 (A to J). .
Feeder cells are put into the cell bag 203, pass through the second flow path circuit 204 indicated by a solid line, and seeded into the lower layer integrated culture container box 102.
each cell suspension is sent from the cell bags 202 and 203 to the culture vessel 100.
the cell bag 202 and the culture medium bag 211 are composed of the first flow path circuit 205, the electromagnetic valves 206B and 206D, the fluid movement control mechanism (tube pump) 208A, the first flow path circuit 221 and the first electromagnetic valve 307. It is connected to the flow path (first supply port) 111 of the upper layer culture vessel unit 101 (A to J) via the (two-way valves A to J).
the first channel circuit 205 is branched to the first channel circuit 221 corresponding to the ten upper-layer culture container units by the multi-branch portion 209A.
the cell bag 203 and the culture medium bag 211 are connected to the culture container box 102 via the second flow path circuit 204, the electromagnetic valves 206C and 206E, the fluid movement control mechanism (tube pump) 208B, and the second flow path circuit 22).
the flow path (second supply port) 106 is connected.
the flow path (first discharge port) 113 of each upper layer culture vessel unit 101 is connected to the drainage bag 213, the drainage collection via the first flow path circuit 223, the fluid movement control mechanism 208C, and the three-way valve 207A. It is connected to the bag 214A.
Ten branch paths (223) constituting the first flow path circuit are combined into one at the multi-branch portion 209B.
the flow path (second discharge port) 108 of the culture container box 102 is connected to the drainage bag 213 and the drainage recovery bag 214B via the second flow path circuit 224, the fluid movement control mechanism 208D, and the three-way valve 207B. It is connected to the.
212 is a heater
215A and 215B are gas supply units
216 is a gas concentration adjusting unit
217 is a filter.
the three-way valve 207 is controlled based on a predetermined sequence by a control protocol given in advance. Thereby, the flow path circuit is controlled so that the culture medium always flows in one direction with respect to the culture container 100 and the new culture medium is supplied after the old culture medium is discharged.
FIG. 3B shows the culture vessel 100 shown in FIG. 3A in which portions related to one upper layer culture vessel unit and the lower layer culture vessel body are extracted.
FIG. 3A the multi-branch portion 209A branches the flow path to ten upper-layer culture container units.
FIG. 3B shows a case where the flow path is connected from the multi-branch portion 209A to one upper culture container unit 101.
FIG. 3B shows the direction of cell suspension / medium and air flow.
the cell suspension / medium flows only in the direction from the outside to the inside of the culture vessel 100 (101, 104).
air flows in both directions.
both the cell suspension / medium and air flow only in the direction from the inside to the outside of the culture vessel 100.
the predetermined two-way valve (206, 307) and three-way valve (207) are opened and closed in advance. Then, the fluid movement control mechanism unit 208 is operated to feed the liquid while controlling the flow rate and the liquid feeding time.
the sent cell suspension branches to the upper layer culture container unit 101 for cell seeding at the multi-branch portion 209A.
the two-way valve and the three-way valve installed in the flow path circuit connected to the culture container unit for the upper layer to be fed are opened so that the feeding can be performed.
the two-way valve and the three-way valve installed in the flow path circuit connected to the culture vessel that is not the liquid feeding target are closed so that the liquid feeding is impossible.
Cell seeding is sequentially performed on the ten upper-layer culture container units 101 and the lower-layer integrated culture container box 102. After all seeding is completed, the rotating mechanism 311 attached to the lower part of the lower layer integrated culture container box 102 is operated. Although the culture container is kept horizontal during cell seeding and cell culture, the culture container is tilted immediately after cell seeding and when changing the medium. By continuously rocking at the time of cell seeding, the distribution of cells after seeding is made uniform. Thereafter, the culture vessel is returned to a horizontal state and cultured in that state.
the medium is changed at a predetermined date and time. In the case of epithelial cells, it is generally performed once every 1-3 days.
the temperature in the thermostatic chamber in which the flow path circuit is installed is maintained at 37 ° C. Thereby, the temperature in a culture container is also maintained at 37 degreeC.
CO 2 or the like is supplied from the gas supply unit 215 as necessary.
the concentration is performed by a gas concentration adjusting unit. For example, a gas containing 5% CO 2 is appropriately sent into the culture vessel.
the gas composition and the air supply schedule are determined according to the cell type to be cultured and the type of medium used.
it is performed through a filter. For example, a filter having a quality that does not pass particles of 0.22 ⁇ m or more is used.
the aseptic desorption part 1103 is attached before and behind the culture container unit 101 for upper layers in a flow path circuit. During culturing, liquid can be fed in the same manner as the channel tube.
the culture container unit for the upper layer is separated from the flow path circuit by using the aseptic detachment part 1103 when the culture is continued while maintaining the sterility.
the upper layer culture container unit is aseptically removed from the lower layer culture container body by the method described above.
the flow path circuit after being removed by the aseptic desorption section maintains the closed state of the flow path tube by the aseptic desorption section 1103 left at the place where the culture container was removed. Thereby, even if it removes a culture container for a test
FIG. 4 is a block diagram showing a control mechanism of an automatic culture apparatus having a closed culture vessel 100.
Each component controlled by the control device 1402 is connected to a culture vessel 1401 arranged inside the thermostatic device 1403. Needless to say, what is placed in the thermostat 1403 is a culture vessel such as the closed culture vessels 101 and 104 described in each embodiment, or the culture vessel installed in an automatic culture apparatus.
the control device 1402 includes a temperature adjustment unit 1404 for controlling the temperature of the thermostatic device 1403, a temperature sensor 1406, and a gas supply unit 1405, and a gas for controlling the gas concentration in the culture vessel.
a microscope 1408 for cell observation and a CO 2 / O 2 sensor (not shown) are connected.
the control device 1402, the display screen 1410, and the database 1412 are processed by a normal computer including a processing unit including a CPU (CentrAl Processing Unit), a storage unit, a display device, an input / output unit including a keyboard, and the like. And the display unit of the display device.
the control device 14022 operates various programs stored in the storage unit on the CPU as a processing unit. Accordingly, the temperature adjustment unit 1404, the gas supply unit 1405, the fluid movement control mechanism unit 1407, the microscope 1408, the CO 2 / O 2 sensor, the gas concentration adjustment unit 1411, the cell / medium / drainage / drainage collection bag 1409, The culture environment in the thermostat 1403 is controlled, and a predetermined culture process in the culture container 1401 can be performed.
the gas concentration adjusting unit 1411 does not need to be directly connected to the culture vessel 1401.
the temperature controller 1404, the gas concentration controller 1411, and the CO 2 / O 2 sensor may be connected to the thermostat 1403.
a part of the lid of the cell culture container 1401 has gas permeability such as polycarbonate, polystyrene, polymethylpentene and the like.
Cell culture can be performed by depositing a transparent thin film to enable gas exchange inside the cell culture vessel 1401.
FIG. 5 is a flowchart for explaining the operation of the automatic culture apparatus.
the operation of the automatic culture apparatus will be described.
it is necessary is just to arrange
cultivation procedure in that case should just perform operation shown below with respect to each culture container in order.
the automatic culture apparatus is activated (step S1), and the schedule is determined (step S2). Further, after opening and closing the appropriate two-way valve and three-way valve, the fluid movement control mechanism is operated, seeding the culture vessel (step S3), culturing the cells in the culture vessel (step S4), and Observation with a microscope is performed (step S5). It is determined whether the cell is in a normal state (step S6). If it is normal, the culture medium in the culture vessel is replaced (step S7). Thereafter, culture and medium exchange just before transplantation are performed (steps S8 to S10). Further, the transplanted tissue is collected (step S11), and the series of processes is terminated (step S12).
step S4 cell culture
step S7 culture immediately before transplantation
step S10 medium exchange
step S9 collection of the test tissue
FIG. 6 is a diagram showing an example of a table 600 for controlling seeding in the culture vessel 100 held in the database 1412.
black circles indicate the opening (ON) of the solenoid valves 206, 207, and 307 and the operating state of the pump 208
X indicates the closing (OFF) of the solenoid valve and the stopped state of the pump (hereinafter the same).
each valve and pump are controlled for 30 seconds for seeding the upper layer culture container 101A, and then seeding to the upper layer culture container 101B. Therefore, a series of processing is performed over 30 seconds, and thereafter, processing for seeding is performed in the same manner up to the upper layer culture vessel 101J.
two-way valves (solenoid valves) 206 and 307, three-way valves (solenoid valves) 207, fluid movement control mechanisms (tube pumps) 208, etc. for seeding in the lower layer, that is, the integrated culture vessel box 102 are controlled in accordance with a predetermined sequence given in the table 600.
a predetermined sequence given in the table 600.
the volume of the culture container main body 104 for the lower layer is 30 ml
a series of processing is performed over 450 seconds
it is 50 ml a series of processing is performed over 750 seconds.
FIG. 7A shows a protocol for seeding cells into the upper layer.
the solution is fed from the cell bag 202 into one upper layer culture vessel 101 via the upper layer supply channel 221.
the two-way valve on the flow path through which the culture medium and air flow is previously opened. Keep everything else closed.
the fluid movement control mechanisms 208A and 208B are operated to carry out liquid feeding and air feeding. That is, the cell bag 202 is cultured in the upper layer via the first flow path circuit (205), the electromagnetic valve 206B, the fluid movement control mechanism 208A, the first flow path circuit (221), and the second electromagnetic valve 307.
the flow path (first supply port) 111 of the container 101 It is connected to the flow path (first supply port) 111 of the container 101, and cell seeding is performed on the upper layer culture container 101.
air in the lower layer culture vessel body 104 is discharged from the lower layer supply channel 107 to the outside of the culture vessel.
the air is finally discharged out of the flow path from the filter 217A.
the flow path (second supply port) 106 has a second flow path circuit (222), a fluid movement control mechanism 208B, a second flow path circuit (204), an electromagnetic valve. It is connected to the filter 217A via 206A.
the gas in the upper layer culture vessel is supplied to the second channel circuit and the filter 217A. It is exhausted through.
the process ends.
Cell seeding is carried out in the same manner for the other upper-layer culture vessels 101.
cell seeding is performed in the order of the upper layer and the lower layer, but the order is arbitrary.
a protocol for seeding cells into the lower layer culture vessel body 104 is shown.
the two-way valve on the flow path through which the culture medium and air flow is opened in advance. Keep everything else closed.
the fluid movement control mechanisms 209A and 209B are operated to carry out liquid feeding and air feeding. That is, as shown in FIG. 7B, from the cell bag 203, the second flow path circuit (204), the electromagnetic valve 206C, the fluid movement control mechanism 208B, and the second flow path circuit (222) are passed through the second flow path circuit (204).
the liquid is fed from the supply port 106 to the culture container main body 104 for the lower layer.
the air in the culture container is supplied from the supply flow path (first supply port) 111 of the upper layer culture container 101 to the first flow path circuit (221), the fluid movement control mechanism 208A, and the electromagnetic valve 206F. Through the culture vessel. The air is finally discharged out of the flow path from the filter 217B. The process ends when the amount of liquid is completely delivered. In this way, cell seeding from the cell bag to the culture container is smoothly performed.
FIG. 8 is a diagram illustrating an example of a table 800 for controlling medium replacement in a closed culture container.
a table 800 for controlling medium replacement in a closed culture container.
two-way valves (solenoid valves) 206 and 307, three-way valves (solenoid valves) 207, fluid movement control mechanisms (tube pumps) 208, and the like are exchanged for predetermined medium given by the table 800. It is controlled according to the sequence.
9A to 9D a protocol for exchanging the medium in the upper layer culture vessel 101 will be described.
9A to 9D show a flow path circuit formed in accordance with the table 800 for one upper layer culture vessel 101 and the flow of the medium and air when the upper layer medium is exchanged.
the medium is fed from the medium bag 202 to the first supply port 111 of the culture container unit for the upper layer through the first flow path circuit (221), and is set in a standby state.
the air in the culture vessel is discharged from the flow channel (second supply port) 106 to the outside of the culture vessel 100 via the second flow channel circuit (222).
the air is finally discharged out of the flow path from the filter 217A.
the two-way valve on the flow path through which the medium and air flow is opened in advance. Keep everything else closed.
the fluid movement control mechanisms 208A and 208B are operated to carry out liquid feeding and air feeding.
the culture medium bag 211 is installed in a refrigerator at 4 ° C.
the culture medium immediately after being fed from the culture medium bag 211 is 4 ° C., but is heated to 37 ° C. by the heater 212, and a culture container and the like are installed. Maintain at 37 ° C. in a thermostat.
the tip of the medium in the first flow path circuit is maintained at the first supply port 111 or in the vicinity thereof.
this stop position suitably according to a use.
the old medium used for the culture in the upper layer culture vessel unit 101 is used for upper layer discharge while maintaining the standby state of the new medium in the first flow path circuit (221).
the liquid is discharged from the flow path 114. That is, the medium in the culture container is discharged to the drainage bag 213 by the fluid movement control mechanism 208C via the first discharge port 113 and the first flow path circuit (223). At that time, a part of the drainage liquid required for the medium component analysis is collected in the drainage collection bag 214A.
the culture container is tilted by the rotation mechanism 311 so that the old medium can be easily discharged from the discharge port side in the culture container.
air is supplied from the second supply port 106 into the culture vessel 100 by the fluid movement control mechanism 208A via the lower layer supply channel, that is, the second channel circuit (222).
the air is finally supplied from the filter 217A into the flow path.
the two-way valve and the three-way valve on the flow path through which the medium and air flow are previously opened. Keep everything else closed.
the fluid movement control mechanisms 208A, 208B and 208C are operated to discharge and supply air.
the old medium is finally discharged into the drainage bag 213 or the drainage collection bag 214A.
liquid is fed from the culture medium bag 211 to the first supply port 111 of the culture container unit 101 for upper layer or the vicinity thereof through the first flow path circuit (221), and the tip is this portion.
the new medium that has been kept at 37 ° C. is supplied into the upper layer culture vessel unit 101.
the air in the culture vessel 100 is discharged out of the culture vessel via the second supply port 106 and the lower layer supply channel (second channel circuit 222).
the air is finally discharged out of the flow path from the filter 217A.
the two-way valve on the flow path through which the medium and air flow is opened in advance. Keep everything else closed. In this state, the fluid movement control mechanisms 208A and 208B are operated to perform liquid feeding and air feeding.
the old medium remaining in the first flow path circuit (223) between the upper layer discharge flow path 114 and the drainage bag 213 or the drainage recovery bag 214A is drained.
the liquid is discharged into the liquid bag 213 or the drainage recovery bag 214A.
air is supplied from the second supply port 106 into the culture vessel 100 via the lower layer supply channel, that is, the second channel circuit (222).
the air is finally supplied from the filter 217A into the flow path.
the two-way valve and the three-way valve on the flow path through which the medium and air flow are previously opened. Keep everything else closed.
the fluid movement control mechanisms 208B and 208C are operated to drain and supply air.
the drainage ends when the entire old medium in the upper layer is discharged to the drainage bag 213 or the drainage collection bag 214A.
FIGS. 10A to 10D show a flow path circuit formed in accordance with the table 800 for the lower culture container box 102 and the flow of the culture medium and air when the lower culture medium is changed.
the medium is fed from the medium bag 211 to the second supply port 106 of the culture vessel 100 or the vicinity thereof through the lower layer supply channel, that is, the second channel circuit (222).
the standby state is set.
the air in the culture vessel 100 is discharged from the first supply port 111 to the outside of the culture vessel via the first flow path circuit (221).
the air is finally discharged out of the flow path from the filter 217B.
the two-way valve on the flow path through which the medium and air flow is opened in advance. Keep everything else closed.
the fluid movement control mechanisms 208A and 208B are operated to carry out liquid feeding and air feeding.
the culture medium bag 211 since the culture medium bag 211 is installed in the refrigerator, the culture medium immediately after being fed from the culture medium bag 211 is 4 ° C., but is heated to 37 ° C. by the heater 212, and a thermostatic device 1403 in which a culture container and the like are installed. Maintain at 37 ° C.
the old medium used for culture in the lower layer integrated culture container box 102 is removed from the lower supply channel while maintaining the standby state of the new medium in the second supply port 106.
the liquid is discharged from 109 through the second flow path circuit (224).
air is supplied from the first supply port 111 into the culture vessel 100 through the first flow path circuit (221).
the air is finally supplied from the filter 217B into the flow path.
the two-way valve and the three-way valve on the flow path through which the medium and air flow are previously opened. Keep everything else closed.
the fluid movement control mechanisms 208A and 208D are operated to drain and supply air.
the drainage ends when the entire amount of the old medium in the lower layer is discharged from the lower layer.
the old medium is finally discharged to the drainage bag 213 or the drainage collection bag 214B, but it is not necessary to complete the total amount at this point.
the solution is fed from the culture medium bag 211 to the second supply port 106 of the culture container box 102 of the lower layer integrated type or the vicinity thereof through the second flow path circuit (224).
a new medium kept at a temperature of 0 ° C. is supplied into the lower culture container box 102.
the air in the culture vessel 100 is discharged from the first supply port 111 to the outside of the culture vessel via the first flow path circuit (221).
the air is finally discharged out of the flow path from the filter 217B.
the two-way valve on the flow path through which the medium and air flow is opened in advance. Keep everything else closed. In this state, the fluid movement control mechanisms 208A and 208B are operated to carry out liquid feeding and air feeding.
the old medium remaining in the second channel circuit (224) between the lower layer discharge channel 109 and the drainage bag 213 or the drainage recovery bag 214B is drained.
the liquid is discharged into the liquid bag 213 or the drainage recovery bag 214B.
air is supplied into the culture vessel 100 from the first supply port 111 via the first flow path circuit (221).
the air is finally supplied from the filter 217B into the flow path.
the two-way valve and the three-way valve on the flow path through which the medium and air flow are previously opened. Keep everything else closed.
the fluid movement control mechanisms 208A and 208D are operated to drain and supply air. The drainage ends when the old medium in the lower layer is entirely discharged into the drainage bag 213 or the drainage collection bag 214B.
a part of the channel tube attached to the closed culture vessel serves both as a liquid feeding function and an air feeding function, so that the entire channel circuit is simplified.
the first supply port, the first discharge port, the second supply port, the second Since it is necessary to connect a flow tube to each of the discharge ports, a total of 40 flow tubes are required.
the present embodiment since only two channel tubes are connected to the lower layer culture vessel body 104 as a whole, a total of 22 channel tubes are sufficient. Also, the number of solenoid valves that control the flow path is reduced. Furthermore, since the seeding to the lower layer culture container main body is performed only once, the treatment time is shortened, and the culture can be promptly transferred.
the culture procedure may be carried out in order for each culture vessel in the following steps S1 to S8 and S10 to S12.
Step S1 Start> First, as shown in FIG. 5, the automatic culture apparatus is activated. The operation is started when the operator presses the start switch of the operation unit in the control device. At this time, the channel circuit and the like are installed in the automatic culture apparatus in advance. On the operation screen of the control unit display, confirm that the value is appropriate for the internal environment of the automatic culture apparatus. For example, it is confirmed that the temperature of the thermostat is 37 ° C. These numerical values are not limited. For example, the temperature can be selected from the range of 0 ° C to 45 ° C. Further, the inside of the apparatus is sterilized with a sterilizing gas or sterilized with ethanol by an appropriate prior operation, and is in a clean state. In addition, sterilization is performed in advance on the flow path portion used for culture.
Step S2 Schedule determination> An automatic culture schedule to be performed by an automatic culture apparatus is determined according to the type and amount of cells to be cultured. Conditions such as date, frequency, fluid volume, etc. for performing operations such as cell seeding, medium exchange, microscopic observation, drainage collection, examination tissue collection, and transplantation tissue collection are input from the operation unit of the control unit.
Step S3 Cell seeding>
the fluid movement control mechanism is operated to suck the cell suspension from the cell bag.
the cell suspension is composed of oral mucosal epithelial cells suspended in KCM medium (keratinocyte culture medium) and 3T3-J2 cells also suspended in KCM medium. is there.
KCM medium keratinocyte culture medium
3T3-J2 cells also suspended in KCM medium.
the fluid movement control mechanism By driving the fluid movement control mechanism, the cell suspension is aspirated while discharging the air in the flow path to the outside of the flow path through the air filter. And it seeds to a culture container.
Cell seeding is sequentially performed on each upper layer and lower layer of each culture vessel. After sowing, the culture vessel is rocked a plurality of times by a rotating mechanism so that the cell distribution on the culture surface becomes uniform.
Step S4 Cell Culture> Culturing is performed for a predetermined time in a state where the culture vessel is left still horizontally.
the stationary period is about 5 days after sowing.
the ambient environment of the culture vessel is maintained at 37 ° C. by a thermostat.
the gas of a predetermined component is sent into the inside of a culture container as needed.
the CO2 concentration is maintained at 5% and the humidity at 100%.
the air inside the automatic culture apparatus is constantly stirred by a fan so that the temperature distribution is always uniform.
⁇ Step S5 Observation with a microscope>
Cell images are acquired using a microscope installed in an automatic culture apparatus.
the light source installed in the automatic culture apparatus is appropriately illuminated, and the cells are focused and imaged by a microscope. If necessary, set a fixed point on the culture surface and photograph.
the acquired cell image is stored in a database so that it can be viewed as necessary on a display installed outside the automatic culture apparatus.
Step S6 Cell State Determination> Judging from the information regarding the growth state of the cells obtained by microscopic observation, the frequency and timing of medium replacement are adjusted. For example, when the cell adhesion is insufficient, the medium exchange in the next step is not performed, and the cell culture in step S4 is continued.
⁇ Step S7 Medium replacement>
the medium exchange is generally performed once every few days.
the frequency is adjusted according to the state of cell growth.
the fluid movement control mechanism is operated, and the fluid movement control mechanism is driven to suck the medium from the medium bag.
the air in the flow path is discharged out of the flow path through the filter.
the medium immediately after being fed from the medium bag is 4 ° C., but the process proceeds to the next step with the temperature of the medium maintained at 37 ° C. due to the gas phase in the heater and thermostatic chamber.
the old medium is discharged from the culture vessel.
the culture vessel is tilted by the rotating mechanism so that the entire amount of the old medium is discharged.
a new medium maintained at 37 ° C. is supplied into the culture vessel. This avoids drying of the cells on the culture surface and a temperature drop on the culture surface.
a part of the old medium discharged from the culture container is sent to the drainage collection bag and the rest is sent to the drainage bag.
the collected old medium is evaluated for cell growth using medium component analysis by a separately prepared medium component analyzer. For example, the amount of glucose used during cell growth and the amount of lactic acid excreted is measured to grasp the cell growth state.
a mycoplasma test or the like is performed to determine whether the medium is contaminated. When there is contamination, the culture is immediately terminated, and the cells are aseptically discarded by appropriate operations so that the place where the automatic culture apparatus is installed is not contaminated.
Step S8 Determination immediately before transplantation> A determination is made as to whether or not the transplant is suitable.
Step S10 Culture and medium exchange just before transplantation> Immediately before the transplantation is performed, the medium is exchanged by the same operation as steps S4 to S7.
Step S11 Collection of transplanted tissue>
the tissue is collected for transplantation and used for regenerative medical treatment. After removing the culture container, it is transported to an operating room where regenerative medical treatment is performed while maintaining sterility and biological quality, and used for treatment.
Step S12 End> Remove the channel used for culture. Subsequently, sterilization with a sterilization gas or disinfection with ethanol is performed by an appropriate operation inside the apparatus to obtain a clean state. The various software of the automatic culture apparatus is terminated, and the operation of the automatic culture apparatus is terminated.
a peristaltic pump is assumed as a fluid movement control mechanism for moving fluid, but it goes without saying that other drive mechanisms such as a syringe pump may be used.
the automatic culture apparatus configured as described above, it is possible to take out a regenerated tissue for examination or the like in advance while maintaining sterility. Since the remaining regenerated tissue after removal maintains the sterility in the same manner, the culture can be continued.
the medium In the cell seeding step and the medium exchange step, the medium always flows in one direction.
the medium exchange for exchanging the entire amount the old medium is not mixed with the new medium, so that the reproducibility of the culture is improved. Analysis accuracy of medium component analysis using the collected old medium is improved.
a new medium warmed to 37 ° C. in advance is supplied immediately.
a part of the flow path tube attached to the closed culture vessel serves both as a liquid feeding function and an air feeding function, so that the entire flow path circuit is simplified.
a peristaltic pump is assumed as a fluid movement control mechanism for moving fluid, but it goes without saying that other drive mechanisms such as a syringe pump may be used.
a peristaltic pump is assumed as a fluid movement control mechanism for moving fluid, but it goes without saying that other drive mechanisms such as a syringe pump may be used.
the medium in the cell seeding step and the medium exchange step, the medium always flows in one direction, so the old medium does not mix with the new medium, and the reproducibility of the culture is improved.
a new medium is quickly supplied after all the old medium is discharged at the time of medium exchange.
a part of the channel tube attached to the closed culture vessel serves both as a liquid feeding and a gas feeding function, so that the entire channel circuit is simplified.
the lower layer is an integrated culture vessel box, it is only necessary to connect two channel tubes in total to the lower layer culture vessel body, the channel circuit is simplified, and an electromagnetic for controlling the channel is provided. There are fewer valves.
the present embodiment provides a culture vessel 100 that meets such needs.
the culture container 100 of Example 2 shown in FIG. 11 has a cell bag, a culture medium bag, a drainage bag, a drainage collection bag, etc. in parallel with the culture container unit 101 for the upper layer and the culture container box 102 integrated with the lower layer. And an upper culture container unit 1101 for test and a test container box 1102 for test.
the upper culture container unit for inspection 1101 and the test culture container box 1102 are co-cultured in the same environment as the culture container 100 of the first embodiment. That is, the test upper layer culture container unit 1101 has an upper layer supply connection projection structure 1111 and an upper layer discharge connection projection structure 1113 corresponding to the upper layer supply connection projection structure 111 and the upper layer discharge connection projection structure 113.
the culture container box for examination 1102 includes a culture container body corresponding to the lower layer culture container body 104 of Example 1, a lower layer supply connection projection structure 1106 corresponding to the lower layer supply connection projection structure 106, and a lower layer discharge connection.
a lower-layer discharge connecting projection structure 1108 corresponding to the projection structure 108 and an opening corresponding to the opening 115 are provided.
a state in which the lower layer integrated culture container box 102 is connected to the test culture container box 1102 by a second flow path circuit including the flow path tube 222 is shown.
the connection relationship between the upper culture container units 1101 for testing is not shown in the figure, but one test is performed in parallel with the plurality of upper culture container units 101 having the same configuration as in Example 1.
the upper layer culture container unit 1101 is placed on the culture container box 1102 for inspection, and the first flow path circuit including the flow path tube is used as a cell bag, a medium bag, a drainage bag, a drainage collection bag, etc. Just connect.
the aseptic desorption part 1103 is installed in the second flow path circuit before and after the culture container box 1102 for testing.
the aseptic desorption part 1103 can supply and supply air in the same manner as the channel tube, and can also cut the channel.
the flow path after cutting maintains sterility.
the three-branch part 1104 is provided in the second channel circuit.
a bypass channel tube 1105 that bypasses the culture vessel box 1102 for inspection is attached in advance.
a bypass channel tube that bypasses the upper-layer culture vessel unit for testing 1101 is also attached in advance to the first channel circuit in the same manner by the three-branch portion.
liquid supply and air feeding are performed to the lower-layer integrated culture container box 102 by the detour channel tube 1105.
liquid supply and air supply are performed to each upper layer culture container unit 101 by the detour channel tube 1105.
step S9 it is possible to perform the processing including the collection of the inspection tissue (step S9) in FIG. 5 as follows (the description of the same processing as in the first embodiment is omitted).
Step S9 Collection of examination tissue> One day out of the culture containers being cultured is collected for examination on the day before the scheduled transplant date. First, open the door of the automatic culture device. In order to avoid a temperature drop in the apparatus, only the flow path may be taken out and the apparatus door may be closed. Subsequently, the flow path tubes of the test culture container box 1102 and the test upper culture container unit 1101 are removed by the aseptic desorption part 1103. Next, the test upper culture container unit 1101 is removed from the test culture container box 1102. The removed upper layer culture container unit is promptly inspected. Similarly, the flow path after removing the upper culture container unit for testing is immediately returned into the apparatus, and the apparatus door is closed.
the flow path is exposed to room temperature and the temperature has dropped, so the air conditioning and heater in the apparatus are controlled and adjusted so that the temperature quickly returns to the culture temperature of 37 ° C.
regenerated tissue in the upper culture container unit 1101 taken out for examination has a quality suitable for regenerative medical treatment.
a regenerated tissue by oral mucosal epithelial cells it has a layered structure of about 3 layers by histological evaluation, or oral mucosal stem cells are present in the basal layer of regenerated tissue by immunohistochemical staining evaluation, or oral mucosa Evaluate whether or not an epithelial cell-specific protein is expressed.
Example 2 According to the culture container of Example 2 configured as described above, it is possible to take out a regenerated tissue for examination or the like in advance while maintaining sterility. Since the remaining regenerated tissue after removal maintains the sterility in the same manner, the culture can be continued. In the cell seeding step and the medium exchange step, the medium always flows in one direction. In the medium exchange for exchanging the entire amount, the old medium is not mixed with the new medium, so that the reproducibility of the culture is improved. Analysis accuracy of medium component analysis using the collected old medium is improved. After collecting the old medium, a new medium warmed to 37 ° C. in advance is supplied immediately. A part of the channel tube attached to the closed culture vessel serves both as a liquid feeding and a gas feeding function, so that the entire channel is simplified.
the present embodiment is another example of the culture container 100 that meets the need to take out one of the cell containers that are co-cultured in the same environment aseptically in advance and test them. It is to provide.
the culture container 100 of the present embodiment is, for example, in the same manner as in the first embodiment, ten upper-layer culture container units 101 (A to J) for culturing a regenerated tissue are integrated with a lower layer for culturing feeder cells. It can be accommodated in the culture container box 102.
a separation membrane 403 for aseptically removing one upper layer culture container unit 101 from the lower layer integrated culture container box 102 is provided. That is, instead of the screw structure shown in FIG. 2A and FIG. 2B, an isolation film 403 is provided between each container lid member 103 and the periphery of the opening 115 of the lower layer culture container main body 104.
the isolation membrane 403 shown in FIGS. 12A to 12D has a bellows shape and is welded to the container lid member 103 and the culture container box 102 integrated with the lower layer. The position is inside the O-ring 105 installed in the container lid member 103 or the lower layer integrated culture container box 102.
a plurality of forms can be considered for the configuration of the isolation film 403.
the separator is fixed by thermal welding that gives high heat during production, but the method is not limited to this method.
Other welding methods for the separator include mechanical pressure, ultrasonic welding, adhesive, light irradiation using a photo-curing resin, and the like.
the inside and outside of the isolation film 403 are isolated by the welded isolation film 403.
the material include an elastic member such as a silicon material used for a film used for a high-pressure steam sterilization, a film used for a clave bag, or a flow tube, but is not limited to these materials. Any material can be used as long as it can be welded, cannot move bacteria, and can be quickly welded and cut when the upper-layer culture container unit is taken out.
FIG. 12A shows a culture state in which the container lid member 103 and the lower layer integrated culture container box 102 are integrated.
a culture medium 150 is contained inside the upper layer culture container unit 101 and the lower layer integrated culture container box 102.
the isolation membrane 403 is accommodated between the container lid member 103 and the lower layer culture container body 104 and in the vicinity of the outer periphery of the container lid member 103.
the intermediate portion of the isolation film 403 is folded or rounded on the lower layer culture vessel main body 104 immediately next to the outer periphery of the vessel lid member 103, and the state is fixed with an adhesive or the like.
FIG. 12B shows a state in which the bellows type isolation membrane 403 is extended to separate the container lid member 103 from the lower layer integrated culture container box 102. That is, the upper culture container unit 101 and the container lid member 103 are lifted. Since the bottom surface portion of the upper layer culture vessel unit 101 is a porous membrane, the medium 150 can be held in the upper layer culture vessel unit for a short time, for example, about several minutes. Since the isolation membrane 403 is welded to the container lid member 103 and the lower layer culture vessel main body 104 in advance, the inside of the isolation membrane 403 maintains sterility.
a plurality of locking pieces 123 are provided at equal intervals along the circumferential direction on the bottom surface of the container lid member 103.
the stop piece 123 is configured to be locked in a lock hole 124 provided outside the annular protrusion 1040 of the lower layer culture vessel main body 104.
the container lid member 103 of the upper layer culture container unit 101 to be taken out and the lower layer integrated culture container box 102 are separated.
the cutting jig 407 is used to squeeze the intermediate portion of the isolation film 403 and press the isolation film 403.
the part of the isolation film 403 sandwiched between the cutting jigs 407 is welded.
heat welding is given as an example.
FIG. 12D shows the state after cutting the welded isolation film 403. That is, as shown in FIG. 12D, one closed space 110 surrounded by the container lid member 103, the lower layer culture container body 104, and the isolation film 403 by welding / cutting is removed from the upper layer culture container unit 101.
step S9 it is possible to perform processing including the collection of the inspection tissue (step S9) in FIG. 5 as follows. (The description of the same processing as in the first embodiment is omitted).
Step S9 Collection of examination tissue> One day out of the culture containers being cultured is collected for examination on the day before the scheduled transplant date.
the channel tube of the upper culture container unit 101 used for the inspection is removed by the aseptic desorption part 1103.
the upper layer culture container unit is detached from the lower layer culture container main body 104.
the isolation membrane 403 installed between the upper layer culture container unit and the lower layer culture container main body removes the upper layer culture container unit from the lower layer culture container main body.
the removed upper layer culture container unit is promptly inspected.
the flow path of the culture container 100 after removing the upper culture container unit for testing is immediately returned to the apparatus, and the apparatus door is closed.
the flow path of the culture vessel 100 is exposed to room temperature and the temperature is lowered. Therefore, the air conditioner and heater in the apparatus are controlled and adjusted so that the temperature quickly returns to the culture temperature of 37 ° C. To do. It is evaluated whether the regenerated tissue in the culture container unit for the upper layer taken out for examination has a quality suitable for regenerative medical treatment. For example, in the case of a regenerated tissue by oral mucosal epithelial cells, it has a layered structure of about 3 layers by histological evaluation, or oral mucosal stem cells are present in the basal layer of regenerated tissue by immunohistochemical staining evaluation, or oral mucosa Evaluate whether or not an epithelial cell-specific protein is expressed.
Step S11 Collection of transplanted tissue>
the tissue is collected for transplantation and used for regenerative medical treatment.
the culture container is removed, and then transported to an operating room where regenerative medical treatment is performed while maintaining sterility and biological quality, and used for treatment.
the timing of taking out the regenerative tissue from the isolation membrane is controlled in accordance with the progress of the regenerative medical treatment. For example, it is possible to wait, transport, etc. in the operating room in a state of being accommodated in the isolation membrane.
Example 4 a method for forming the isolation film 403 of Example 3 will be described. That is, in Example 4, with reference to FIG. 13, the separation membrane 403 is provided between each container lid member 103 holding the upper layer culture container unit 101 and the periphery of the opening 115 of the lower layer culture container main body 104. A manufacturing method to be provided will be described.
the upper layer culture vessel unit 101 is installed on the vessel lid member 103 by a hook structure or the like.
the upper end surface 4033 of the bellows-type isolation film 403 is welded to the lower surface of the inward projecting portion 1030 of the container lid member 103 by means such as thermal welding.
the welding method can also select another method, and is not limited to heat welding.
the other end 4034 of the isolation film 403 is welded around the opening 115 of the lower layer culture vessel body 104 by means such as heat welding.
the welding method is not limited to heat welding.
the container lid member 103 is installed on the lower layer culture container body 104.
⁇ -ray sterilization is performed in a state where the entire closed system flow path connected with a flow path tube or the like is placed in a sterilization bag. At the time of use, the flow path is taken out from the sterilization bag and installed in the automatic culture apparatus to perform automatic culture.
⁇ -ray sterilization is performed only on the applicable material, and the rest is performed by a method other than ⁇ -ray sterilization, such as gas sterilization. Both materials are integrated in a safety cabinet or the like.
the isolation film 403 may have a configuration other than the bellows type described in the third embodiment.
Example 5 with reference to FIGS. 14A to 14C, a flat type separation membrane for aseptically removing one upper layer culture container unit from the layer culture container and a method for manufacturing the same will be described. Similar to the bellows-type isolation membrane described in Example 3, the upper and lower end surfaces of the isolation membrane 403 are welded to the vessel lid member 103 and the lower-layer culture vessel body 104, respectively. Unlike the isolation membrane described in FIG. 12, the example shown in Example 5 accommodates the isolation membrane outside the container lid member 103 and the lower culture vessel body 104.
FIG. 14A holds the upper culture vessel unit 101. The state which removed the container lid
FIG. 14B shows a state in which the container lid member 103 holding the upper layer culture container unit 101 is held in the lower layer culture container main body 104.
the isolation film 403 is positioned in a state where the middle portion thereof is bent and extends to the outside of the upper layer container lid member 101 and the lower layer culture vessel main body 104.
the portion extending to the outside of the isolation film 403 is installed, for example, by being rolled right next to the outer periphery of the container lid member 103. Or you may make it the state which folded the part extended outside.
FIG. 14C shows a method for accommodating the container lid member 103 and the isolation membrane 403 having a portion extending outside the lower layer culture vessel main body 104 in a folded state.
An intermediate portion of the isolation film 403, that is, a portion extending outward, is held between concentric position fixing jigs 801 and 802, and is bent by being pressed by a compression jig 803 from above. In this way, the isolation film 403 is folded compactly on the outer periphery of the container lid member 103 and installed compactly.
each of the upper-layer culture container units 101 can be brought close to the lower-layer culture container main body.
the unit 101 can be arranged.
the isolation membrane is accommodated in the outer lateral portion of the vessel lid member, and accordingly, each of the upper culture container units is separated in the lower culture vessel body.
the number of upper-layer culture container units that can be disposed in the culture container 100 is not accommodated inside the container lid member and the lower layer culture container main body, the lower layer culture container main body does not increase in the vertical direction. Therefore, it does not affect the microscopic observation.
FIG. 15 shows an example of a lid upper surface type separation membrane for aseptically removing one upper layer culture container unit from the lower layer culture container main body.
Example 6 the isolation membrane 403 for taking out one upper culture container unit from the lower culture container main body is installed on the outer periphery of the container lid member 103 and the lower culture container main body 104. Unlike the isolation membranes described in Example 3 and Example 5, the isolation membrane 403 is welded to the outer periphery of the container lid member 103 and the lower layer culture vessel body 104. The upper end surface of the isolation film 403 is welded to the container lid member 103 by the container lid member bonding portion 504 at the outer peripheral upper end surface of the container lid member 103. Further, the lower end surface of the isolation film 403 is welded to the upper surface of the lower layer culture vessel main body 104 by the lower layer lid bonding portion 505.
the separation membrane 403 is accommodated outside the vessel lid member 103 and the lower layer culture vessel main body 104.
the upper end surface of the isolation film 403 may be welded to the container lid member 103 by the container lid member bonding portion 504 on the outer peripheral side surface of the container lid member 103.
the number of upper-layer culture container units that can be placed in the culture vessel 100 is limited because the separation membrane is housed on the outer side of the container lid member as in the fifth embodiment.
the isolation membrane is not housed inside the container lid member and the lower layer culture container main body, the microscopic observation is not affected.
Example 3 Note that the method of cutting the isolation film 403 by the cutting jig 307 described in Example 3 can also be applied to cutting the isolation film of Example 5 and Example 6.
FIG. 16A shows a case in which a container lid member 103 and a lower layer are formed in a culture vessel 100 in which a lid upper surface type isolation membrane 403 is installed between a container lid member 103 holding an upper layer culture container unit and a lower layer culture container main body 104.
the culture vessel main body 104 is fixed by screwing screws 105 (A, B).
the method of cutting the isolation film 403 by the cutting jig 307 described in Example 3 is performed when a fixing method that requires a rotating operation such as a screw for the container lid member and the lower layer culture vessel main body is employed. Is not applicable.
FIG. 16B shows the case of using the method of fixing the container lid member 103 and the lower layer culture vessel body 104 with screws 105 (A, B) when using the isolation membrane of each of the above embodiments.
the state where 101 is removed is shown. Since the container lid member 103 is fixed to the lower layer culture container body 104 by the screws 105 (A, B), when removing from the lower layer culture container body 104, the container lid member 103 is removed while rotating. . Therefore, the isolation film 403 is twisted, and a twisted structure 1004 is generated.
the upper layer culture container unit 101 is aseptically isolated by welding and cutting the twisted structure portion.
the container lid member 103 and the lower layer culture vessel body 104 are fixed by screws, the twisted structure 1004 corresponding to the narrowing is formed in the isolation film 403 when the container lid member 101 is removed while rotating. can get. That is, by separating the upper and lower sides by the twisted structure 1004, one closed space 110 surrounded by the container lid member 103, the lower layer culture container body 104, and the isolation film 403 is removed from the upper layer culture container unit 101.
the present invention is useful as an automatic culture apparatus for culturing cells or tissues by automatic operation using a culture vessel, particularly as an automatic culture apparatus capable of producing a regenerated tissue that can be used for regenerative medicine. .
DESCRIPTION OF SYMBOLS 100 Culture container 101 ... Upper layer culture container unit 102 ... Lower layer integrated culture container box 103 ... Container lid member 104 ... Lower layer culture container main body 105 ... Elastic member (O-ring) 106 ... Connection protrusion structure for lower layer supply (second supply port) 107: Lower layer supply flow path 108: Lower layer discharge connection projection structure (second discharge port) 109 ... lower layer discharge flow path 110 ... closed space 111 ... upper layer supply connection projection structure (first supply port) 112 ... Upper layer supply channel 113 ... Upper layer discharge connection projection structure (first discharge port) 114 ... upper layer discharge channel 115 ... opening 120 ... bottom surface 122 of nutrient container unit ... gap 150 ... culture basin 1010 ...
Aseptic removal part 403 Isolation film 407 ... Cutting jig 600 ... Table 800 ... Table 801 ... Position fixing jig 802 ... Position fixing jig 803 ... Compression jig 1004 ... Twist structure 1102 ... For inspection Culture vessel 1104 ... Three-branch part 1105 ... Detour channel tube 1401 ... Culture container 1402 ... Control device 1403 ... Thermostat 1404 ... Temperature controller 1405 ... Gas supply part 1406 ... Temperature sensor 1407 ... Fluid movement control mechanism part 1408 ... Microscope 1410 Display screen 1411 Gas concentration adjusting unit 1412 Database.

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PCT/JP2012/064501 2012-06-06 2012-06-06 Récipient de culture et dispositif de culture automatique Ceased WO2013183121A1 (fr)

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PCT/JP2012/064501 WO2013183121A1 (fr)	2012-06-06	2012-06-06	Récipient de culture et dispositif de culture automatique
JP2014519728A JP5866006B2 (ja)	2012-06-06	2012-06-06	培養容器及び自動培養装置

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JP2017079633A (ja) *	2015-10-27	2017-05-18	高砂電気工業株式会社	自動灌流培養装置
WO2018186426A1 (fr) *	2017-04-07	2018-10-11	オリンパス株式会社	Dispositif d'échange de milieu de culture et système de culture
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JP2020010683A (ja) *	2018-07-09	2020-01-23	学校法人東京女子医科大学	三次元生体組織の培養方法、並びに三次元生体組織培養デバイス及びシステム
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WO2018186426A1 (fr) *	2017-04-07	2018-10-11	オリンパス株式会社	Dispositif d'échange de milieu de culture et système de culture
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Also Published As

Publication number	Publication date
JPWO2013183121A1 (ja)	2016-01-21
JP5866006B2 (ja)	2016-02-17

Publication	Publication Date	Title
JP5866006B2 (ja)	2016-02-17	培養容器及び自動培養装置
JP5894260B2 (ja)	2016-03-23	培養容器及び自動培養装置
JP5891310B2 (ja)	2016-03-22	細胞培養容器およびそれを用いた細胞培養装置
JP5722329B2 (ja)	2015-05-20	自動培養装置
JP6022674B2 (ja)	2016-11-09	細胞培養装置、及び培養容器
JP5960256B2 (ja)	2016-08-02	培養容器及び自動培養装置
JP6097817B2 (ja)	2017-03-15	細胞培養装置
WO2016157322A1 (fr)	2016-10-06	Récipient de culture en système fermé, procédé de transport, et dispositif de culture automatisé
CN102985526A (zh)	2013-03-20	细胞培养容器及细胞培养装置
JP4845950B2 (ja)	2011-12-28	自動培養装置
JPWO2016016950A1 (ja)	2017-04-27	細胞培養装置、及び閉鎖系培養容器
JP5886455B2 (ja)	2016-03-16	自動培養装置
JP6514952B2 (ja)	2019-05-15	自動培養装置
WO2015107667A1 (fr)	2015-07-23	Dispositif de culture de cellules
TW202242087A (zh)	2022-11-01	細胞培養系統

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