CN105154329A - Automatic culture apparatus - Google Patents

Automatic culture apparatus Download PDF

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Publication number
CN105154329A
CN105154329A CN201510428228.5A CN201510428228A CN105154329A CN 105154329 A CN105154329 A CN 105154329A CN 201510428228 A CN201510428228 A CN 201510428228A CN 105154329 A CN105154329 A CN 105154329A
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CN
China
Prior art keywords
mentioned
cell
cell suspension
stream
culture vessel
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CN201510428228.5A
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Chinese (zh)
Inventor
野崎贵之
小林丰茂
松冈志津
中岛亮太
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Hitachi Ltd
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Hitachi Ltd
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Priority claimed from CN201080068572.3A external-priority patent/CN103068962B/en
Publication of CN105154329A publication Critical patent/CN105154329A/en
Pending legal-status Critical Current

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    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • 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
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/10—Rotating vessel
    • 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
    • C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/14—Incubators; Climatic chambers
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure

Abstract

The invention provides an automatic culture apparatus, which is capable of uniformly inoculating cells in a culture surface of automatic culture. The automatic culture apparatus makes use of a culture container which has a culture space therein, wherein the automatic culture apparatus comprises an accommodating part for accommodating a cell suspension; a flow path which is connected to the accommodating part; and a fluid control mechanism part which is used for controlling in a mode of moving the cell suspension towards the culture container by virtue of the flow path; the cell suspension is moved towards the side of the flow path by virtue of the fluid moving control mechanism; and when the cell suspension is conveyed, a stirring flow is generated in the cell suspension inside the accommodating part by an action of repeatedly injecting the cell suspension which is conveyed from the interior of the accommodating part into the accommodating part.

Description

Automatic culture apparatus
The divisional application that the application is application number is 201080068572.3, international filing date is on August 12nd, 2010, denomination of invention is the application for a patent for invention of " automatic culture apparatus ".
Technical field
The present invention relates to and use the automatic culture apparatus that the closed culture vessel of cell type is cultivated cell or tissue by automatic operation, the automatic culture apparatus that such as can manufacture the regenerating tissues that can be used for regenerative medicine.
Background technology
Manufacture as regenerative medicine treatment for the regenerating tissues to transplant etc. is based on the GMP (GoodManufacturingPractice as the manufacturing management of medicine etc. and the benchmark of qualitative control; Good manufacturing standards).Usually, regenerating tissues is by having the manufacturer of special cell culture technology according to SOP (StandardOperationalProcedure; Standard Operations Manual) CPC (CellProcessingCenter of clean manufacturing environment is being provided; Cell process is arranged) in manufacture.In Japan, with regard to GMP, the such as Health and human services department that specified by MHLW makes No. 179, medicine sends out the regulation such as No. 480 implementing.Outside Japan, also centered by the such as American-European mechanism such as food and drug administration, the Europe council, issue relevant regulations.
Manufacture under the environment that regenerating tissues is managed in the security to above-mentioned regulation defined, degree of cleaning by the manufacturer with special cell culture technology.Therefore, a large amount of personal expenditures, labour, utilization cost is produced.In addition, manually complete all manufacturing processes owing to utilizing, so the manufacture of regenerating tissues is limited.As a result, the manufacturing cost for the manufacture of regenerating tissues uprises, and what regenerative medicine can be hindered to treat is universal.Therefore, require to import to break such present situation will cultivate a part for operation and even all carry out the automatic culture apparatus of automatization.Utilize automatic culture apparatus instead of manually implement to cultivate operation, thus Labor-saving can be realized and reduce costs, and realizing batch production.In addition, because the operation of automatic culture apparatus is constant, so the stabilization contributing to realizing manufacturing the regenerating tissues quality obtained also can be expected.Herein, automatic culture apparatus replaces manually carrying out culturing cell, but thinks that it is identical with artificial manufacturing process, must meet GMP.That is, must show according to scientific basis, automatic culture apparatus reproducibility can manufacture the regenerating tissues of high-quality well under the state keeping degree of cleaning.
But, the culture vessel that automatic culture apparatus uses mainly is divided into: easily switch and the open culture vessel larger with the contact area in the external world, such as there is the culture vessel of lid, and be not easy switch and the closed culture vessel less with the contact area in the external world, such as cell type culture vessel.The culture technique employing open culture vessel is established, and is generally used for manufacture sales stage of comprising from development to regenerating tissues all by the cultivation manually carried out.But open culture vessel is the structure that substratum easily spills, larger with the contact area in the external world becoming biological contamination reason.Degree of cleaning are kept to carry out unchangeably cultivating the special culture technique of needs.That to have disclosed with open culture vessel be object, that there is the such drive unit of robots arm or conveying machine people etc. automatic culture apparatus.
On the other hand, for closed culture vessel, be located as and suppose the culture vessel of new generation that uses in automatic culture apparatus and develop in various research institution.The culture technique employing closed culture vessel and automatic culture apparatus is not yet fully established, but has proposed several parts of reports (patent documentation 1 and 2).Usually, closed culture vessel is the structure that substratum not easily spills, less with the contact area in the external world, therefore has the advantage keeping degree of cleaning easier than open culture vessel.As the example of automatic culture apparatus employing closed culture vessel, disclose and use the closed culture vessel of cell type and the automatic culture apparatus (patent documentation 3 and 4) linking closed for this cell type culture vessel and automatic culture apparatus to carry out cell inoculation, substratum exchange etc. via the connector portions based on crimping mode.The closed culture vessel of this cell type has valve constitution, only carries out inflow, the outflow of substratum relative to outside via this valve constitution.In addition, the culture tank of the closed culture vessel of cell type carries out O via gas-exchange membrane and outside 2, CO 2these cultivate needed for the exchange of gas.In addition, as the automatic culture apparatus employing closed culture vessel, patent documentation 5,6 discloses other devices.
Prior art document
Patent documentation
Patent documentation 1: Japanese Unexamined Patent Publication 2008-113600 publication
Patent documentation 2: Japanese Unexamined Patent Publication 2004-089138 publication
Patent documentation 3: Japanese Unexamined Patent Publication 2006-320304 publication
Patent documentation 4: Japanese Unexamined Patent Publication 2006-149237 publication
Patent documentation 5: Japanese Unexamined Patent Publication 2006-141328 publication
Patent documentation 6: Japanese Unexamined Patent Publication 2004-208665 publication
Summary of the invention
The problem that invention will solve
As mentioned above, require that automatic culture apparatus meets GMP.Particularly need the degree of cleaning in culture environment.When cultivating, need to prevent bacterium from invading the flow path portion that directly may contact via substratum and cell.In addition, in order to stably carry out regenerative medicine treatment, uniform regenerating tissues must be manufactured.As the condition manufacturing uniform regenerating tissues, need at culture surface inoculating cell equably when starting to cultivate.For this reason, the ununiformity of the cell distribution that the reason such as precipitation due to cell produces must be eliminated when attracting cell suspension, thus the cultivation under realizing uniform state.Further, if inoculate with the state being mixed into bubble in cell suspension, then the skewness of cell is made due to capillary reason.That is, need from cell suspension removing bubble.Finally, such as in the manufacture of the regenerating tissues for cornea regeneration, the cell quantity started when cultivating is a small amount of compared to large regenerating tissues such as skins, but need to utilize automatic culture apparatus to cultivate a small amount of cell efficiently, cell bags must be utilized to attract whole cell efficiently, and not celliferous loss in each parts of the stream passed through before inoculation.
About the automatic culture apparatus employing closed culture vessel, in above-mentioned patent documentation 5, be used in the closed culture vessel that inside has gas phase, therefore, as long as tilt repeatedly just can make being evenly distributed of cell by closed culture vessel when inoculating.For the method, such as, when utilizing the closed culture vessel be only made up of liquid phase as the closed culture vessel of cell type to cultivate, even if tilt, liquid is also almost motionless, and therefore cell distribution is uneven.In addition, when patent documentation 5, can discharge from the gas phase portion of closed culture vessel the air be mixed in stream because of certain reason.On the other hand, not there is gas phase when the closed culture vessel of cell type, therefore, particularly when being mixed into micro-bubble, being difficult to discharge above-mentioned micro-bubble.As relating in the patent documentation 6 of other documents of the automatic culture apparatus employing closed culture vessel, being provided with between the Storage Department of culture vessel and substratum etc. can the inoculation unit of diluting cells suspension liquid.But, due to for the purpose of the use that ether is aerial, so become the MIN function needed for can only realizing cultivating.Accordingly, in the method for the literature, the homogenizing being difficult to realize cell distribution, the removing of bubble be mixed into.
As mentioned above, in the automatic culture apparatus employing the culturing cell of closed culture vessel, tissue, in order to carry out good regenerative medicine treatment, need to manufacture uniform regenerating tissues.For this reason, must when starting to cultivate to culture surface inoculating cell equably.In addition, cultivate under the state needing to there is not bubble in the inside of closed culture vessel.But, when inoculating cell, when exchanging substratum and when carrying various solution, being mixed into of bubble may be produced.Need the function removing bubble when being mixed into bubble.Further, particularly cultivate when only having a small amount of cell, need the high efficiency conveying, the inoculation that do not produce loss cell, the whole cells contained by the cell suspension being injected into cell bags are inoculated in culture surface.
The object of the present invention is to provide and can to inoculate equably in the culture surface of the closed culture vessel of cell and the state that there is not bubble with the inside of closed culture vessel carries out the automatic culture apparatus cultivated.
Solve the method for problem
In order to realize above-mentioned object, the following automatic culture apparatus formed is provided in the present invention, namely, be used in the closed culture vessel of cell type that inside has culture space, and possess: first flow path, its can with a side end of the closed culture vessel of cell type near link, and to culture space delivering fluids; Second stream, its can with the end of the opposite side of the closed culture vessel of cell type near link, and from culture space displacement fluids; And fluid moving control mechanism portion, it is connected respectively with first flow path, the second stream, and control in the mode making fluid move to the closed culture vessel of cell type, the control mode in fluid moving control mechanism portion is: under the state being full of the inside of the closed culture vessel of cell type in the cell suspension as fluid, repeatedly carry out a small amount of cell suspension to be supplied to first flow path and carry out the action that attracts, thus the cell suspension in the closed culture vessel of cell type being produced stir stream.
In addition, in order to realize above-mentioned object, provide the following automatic culture apparatus formed in the present invention, that is, be used in the closed culture vessel of cell type that inside has culture space, and have: cell bags, it receives cell suspension; Culture medium bag, it receives substratum; Casing, it is arranged between the closed culture vessel of cell type and cell bags, and temporarily can store cell suspension; First flow path, it is arranged at the top of casing, and links cell bags and culture medium bag and casing; Second stream, it discharges the gas in casing; 3rd stream, it is arranged at the below of casing, and links the closed culture vessel of cell type and casing; Valve portion, it can distinguish switch first flow path, the second stream and the 3rd stream; And fluid moving control mechanism portion, it is to control to casing supply cell suspension and substratum the mode of diluting cells suspension liquid, and controlling organization portion is with the bubble of discharging from the second stream in the cell suspension in casing and control from the mode of the cell suspension removing bubble in casing.
Further, in order to realize above-mentioned object, provide the following automatic culture apparatus formed in the present invention, that is, be used in the closed culture vessel of cell type that inside has culture space, and possess: cell bags, it receives cell suspension, stream, it is connected with cell bags, and fluid moving control mechanism portion, it controls in the mode making cell suspension and move to the closed culture vessel of cell type via stream, fluid moving control mechanism portion is to make to become decompression state in stream and to produce pressure change relative to the cell suspension in cell bags and make cell suspension control to the mode of stream side movement, cell suspension is being delivered to the closed culture vessel of cell type by fluid moving control mechanism portion, during casing, repeatedly carry out the liquid that cell suspension in delivery ratio cell bags is a small amount of, the action of the cell suspension transported is injected to cell bags, thus make the cell suspension in cell bags produce stirring stream.
Namely, state in realization the of the present invention preferred mode of object, employ and have in the automatic culture apparatus of the closed culture vessel of cell type of culture space in inside, possess to culture space supply gas and (or) stream of liquid, i.e. fluid and the stream from culture space displacement fluids.In addition, each stream is connected with syringe respectively.These two syringes pass through Beng Deng mechanism with synchronous state action.Further, be connected with the flow limitation of air vacuum breaker in one direction between each syringe and valve.By making two syringe actions, decompression state is become in the stream that culture vessel closed with cell type is connected, make to become pressurized state in another stream, produce pressure change relative to the liquid being full of the closed culture vessel of cell type from both sides, thus liquid is moved.By applying pressure in the mode switching pressurized state and decompression state, liquid can also be made to move in the opposite direction.
Further, in preferred mode of the present invention, the controlling organization of following action when there is inoculating cell suspension liquid in the culture space to the closed culture vessel of cell type, is implemented.Namely, with during at inoculating cell suspension liquid, the state that the mode being positioned at downside with the stream that the stream of the discharge carrying out the fluid of the closed culture vessel of cell type is positioned at upside, carry out the supply of the fluid of the closed culture vessel of cell type makes the closed culture vessel of cell type erect, makes cell suspension be full of the inside of the closed culture vessel of cell type.Then, after cell suspension is full of the inside of the closed culture vessel of cell type, the closed culture vessel of cell type is made to become level.In this condition, the cell suspension that further supply is a small amount of, again attract the liquid of equivalent, by the action repeatedly carrying out repeatedly this supply and attraction, cell suspension in the closed culture vessel of cell type is produced and stir stream, carry out eliminating cell when to inject cell suspension under the state erected by closed for cell type culture vessel and produce the action of the uneven state of the cell caused by precipitation because of the reason of deadweight.Culture space in the closed culture vessel of cell type arranges diffuser, and this diffuser can impel cellular invasion and to culture space inoculating cell equably when cell suspension flows into.
In addition, the automatic culture apparatus of preferred mode of the present invention has the casing of diluting cells suspension liquid between the closed culture vessel of cell type and the cell bags of storage cell suspension.This casing has the well heater as heating part, the temperature that cell suspension is heated to be applicable to cultivating by this well heater, such as 37 DEG C, and can maintain this temperature.By supplying substratum from culture medium bag to casing, thus there is the function of the concentration of adjustment cell suspension.There is the second stream linking and be accommodated with the cell bags of cell suspension and the first flow path of casing and discharge the air in casing above casing, there is in the below of casing the stream of the link closed culture vessel of cell type and casing.There is the magnetic valve forming and can close the valve portion of first flow path, the second stream and each stream of the 3rd stream respectively.Pass through said structure, there is the control texture being implemented as follows action: when carrying cell suspension from the cell bags being accommodated with cell suspension to casing, cell suspension containing the bubble be mixed into because of certain reason is accommodated in casing, is come from cell suspension removing bubble by the bubble contained by discharging from the second stream.
In addition, in the automatic culture apparatus of preferred mode of the present invention, there is the controlling organization being implemented as follows action: the micro-bubble that the liquid level in cell suspension is produced, under the state utilizing closed electromagnetic valve first flow path and the 3rd stream, air is attracted to make to become decompression state in casing by utilizing the second stream, keep this state, the micro-bubble of the liquid level of removing cell suspension, then makes the pressure in casing revert to normal pressure gradually.
In addition, in the automatic culture apparatus of preferred mode of the present invention, there is the controlling organization being implemented as follows action: when to cell type closed culture vessel conveying cell suspension, delivery ratio is stored in a small amount of liquid of the cell suspension of casing, the cell suspension transported again is injected to casing, by repeatedly carrying out repeatedly the action of this conveying and injection, cell suspension in casing is produced and stirs stream, eliminate cell when to store cell suspension in casing and produce the uneven state of the cell caused by precipitation because of the reason of deadweight.The driving method of liquid used herein is identical with the method used above-mentioned box.
In the automatic culture apparatus of preferred mode of the present invention, there is the controlling organization being implemented as follows action: have in cell bags at automatic culture apparatus, during to casing conveying cell suspension, the liquid that cell suspension in delivery ratio cell bags is a small amount of, re-inject the cell suspension transported, by the action repeatedly carrying out repeatedly this conveying and injection, cell suspension in cell bags is produced and stir stream, eliminate the uneven state of the cell caused by precipitation that cell produces because of the reason of deadweight when to store cell suspension in cell bags.The driving method of liquid used herein is identical with the method used above-mentioned box.
In the automatic culture apparatus of preferred mode of the present invention, it is used in the culture vessel that inside has culture space, possesses: the incorporating section of storage cell suspension; The stream (stream herein is not defined as a stream) be connected with above-mentioned incorporating section; And to make above-mentioned cell suspension carry out the flow control mechanism portion controlled to the mode of above-mentioned culture vessel movement via an above-mentioned stream,
Above-mentioned fluid moving control mechanism portion makes above-mentioned cell suspension move to above-mentioned first-class trackside, when carrying above-mentioned cell suspension, by repeating the action of the above-mentioned cell suspension transported from the above-mentioned cell suspension in above-mentioned incorporating section being injected to above-mentioned incorporating section, come to produce in the above-mentioned cell suspension in above-mentioned incorporating section to stir stream
The effect of invention
According to automatic culture apparatus involved in the present invention, can carry out carrying and inoculating with the uniform state of cell distribution.In addition, the removing of the bubble of the reason of the ununiformity as cell distribution can be carried out.Thereby, it is possible to manufacture uniform regenerating tissues.
Accompanying drawing explanation
Fig. 1 is the one-piece construction figure representing the first embodiment, be provided with the automatic culture apparatus of flow path portion.
Fig. 2 is the integrally-built figure representing the first embodiment, unloaded the automatic culture apparatus of flow path portion.
Fig. 3 A is the figure of the integrality of the flow path portion representing the first embodiment, be arranged at automatic culture apparatus.
Fig. 3 B is the figure of the base unit representing the first embodiment, keep the part relevant to cultivation of the flow path portion of automatic culture apparatus.
Fig. 3 C is the figure of the base unit representing the first embodiment, keep the part relevant to cultivation of the flow path portion of automatic culture apparatus.
Fig. 4 is the figure of the structure representing the closed culture vessel of the first embodiment, cell type.
Fig. 5 is the figure of that represent the variation of the first embodiment, be provided with the diffuser for making cellular invasion in the culture space of the closed culture vessel of cell type state.
Fig. 6 is the first embodiment, the overall diagram of stream when cultivating the closed culture vessel of two cell type.
Fig. 7 A is the figure of the flowing (1) of when representing the carrying to the closed culture vessel of cell type from casing of the first embodiment, the solution corresponding with the action of syringe and air.
Fig. 7 B is the figure of the flowing (2) of when representing the carrying to the closed culture vessel of cell type from casing of the first embodiment, the solution corresponding with the action of syringe and air.
Fig. 8 is the figure of a series of flow processs of the cultivation representing the first embodiment, use automatic culture apparatus enforcement cell.
Fig. 9 A is the figure of the summary (1) of the flow process representing the first embodiment, implement at the casing of flow path portion.
Fig. 9 B is the figure of the summary (2) of the flow process representing the first embodiment, implement at the casing of flow path portion.
Fig. 9 C is the figure of the summary (3) of the flow process representing the first embodiment, implement at the casing of flow path portion.
Fig. 9 D is the figure of the summary (4) of the flow process representing the first embodiment, implement at the casing of flow path portion.
Fig. 9 E is the figure of an example with heater structure of the casing representing the first embodiment, flow path portion.
Figure 10 A is the figure of the summary (1) of flow process representing the first embodiment, implement in the closed culture vessel of the cell type of flow path portion.
Figure 10 B is the figure of the summary (2) of flow process representing the first embodiment, implement in the closed culture vessel of the cell type of flow path portion.
Figure 10 C is the figure of the summary (3) of flow process representing the first embodiment, implement in the closed culture vessel of the cell type of flow path portion.
Figure 10 D is the figure of the summary (4) of flow process representing the first embodiment, implement in the closed culture vessel of the cell type of flow path portion.
Embodiment
Below, be described in detail with reference to the embodiment of accompanying drawing to automatic culture apparatus of the present invention.In addition, in this manual, sometimes the gas flowed in the stream of automatic culture apparatus, liquid, gas and liquid are referred to as fluid.In addition, sometimes the mechanisms such as the syringe of the fluid movement made in this stream and syringe pump (being referred to as " syringe "), the also rotation control unit comprised as the rotating mechanism making the closed culture vessel of cell type etc. rotate are referred to as fluid moving control mechanism portion.
Embodiment 1
Fig. 1 illustrates the one-piece construction of the automatic culture apparatus being provided with flow path portion.The one-piece construction of the automatic culture apparatus before Fig. 2 illustrates and arranges flow path portion.Fig. 3 A, Fig. 3 B, Fig. 3 C show respectively in the integrality of the flow path portion being arranged at automatic culture apparatus, flow path portion to cultivate relevant part, keep the base unit of the part of being correlated with cultivation of flow path portion.The flow path portion herein represented as an example, is structure when the closed culture vessel of cultivation two cell type.
Fig. 4 illustrates the structure of the closed culture vessel of cell type of the first embodiment.Fig. 5 illustrates the state being provided with diffuser in the culture space of the closed culture vessel of the cell type of the distortion of the first embodiment, and this diffuser can impel cellular invasion and to culture space inoculating cell equably when cell suspension flows into.The overall diagram of stream when Fig. 6 illustrates cultivation two cell type closed culture vessel.Fig. 7 A, Fig. 7 B illustrate the action of an example of the flowing as the solution and air employing the flow circuit diagram represented in Fig. 6, syringe when carrying from casing to the closed culture vessel of cell type.Fig. 8 illustrates a series of flow processs using the automatic culture apparatus of the present embodiment to implement cell cultures.Fig. 9 A-9E represents the summary of the flow process implemented at the casing of flow path portion.Figure 10 A-10D illustrates the summary of the flow process implemented in the closed culture vessel of the cell type of flow path portion.
Fig. 1, Fig. 2 basic structure key element to the automatic culture apparatus of the first embodiment is used to be described.Automatic culture apparatus by cell culture chamber 101,201, the refrigerator of keeping substratum etc. 102,202, control part 103,203 and clean air circulation portions 104,204 that inner air is circulated form.This automatic culture apparatus has cell culture chamber door 105,205 and refrigerator door 106,206, by opening the inside that these can access automatic culture apparatus.In cell culture chamber 101,201, when cultivating, closed for cell type culture vessel 107 and culture vessel pedestal 108 are arranged at culture vessel driving part 109,209.When exchanging substratum in the training period, by by rotating mechanism 110,210, the rotation control unit that forms such as motor 111, the 211 and closed culture vessel 107 of cell type, culture vessel pedestal 108, culture vessel driving part 109,209 are rotated integrally, thus the closed culture vessel of cell type 107,108 can erect along roughly vertical, vertical direction.There is in flow path portion the magnetic valve 112, motor 113, casing 114, stream pedestal 115, the stream driving part 116,216 that form valve portion, various solution can be carried.
When cultivating, the inside of cell culture chamber 101,201 such as keeps temperature 37 DEG C, CO 2the state of concentration 5%, humidity 100%.Therefore, have and produce mechanism 121 and humidity sensor 122 as the well heater 117 of heating part, temperature sensor 118, carbonic acid gas feed mechanism 119, carbon dioxide sensor 120, humidity.Fan 123 is utilized to make inner air cycle in order to make inner environment homogenizing.Refrigerator 102,202 keeps the temperature of 4 DEG C in order to keeping substratum etc.Substratum etc. is put into medium container etc., and they are positioned over substratum pedestal 124 in the lump.Culture vessel pedestal 108, stream pedestal 115, substratum pedestal 124 are set to pedestal 125 jointly.In addition, culture vessel driving part 109,209 and stream driving part 116,216 are set to driving part 126,226 jointly.
Sealing member 127 is set between cell culture chamber 101,201 and refrigerator 102,202, to make the movement that heat does not occur between two spaces.In clean air circulation portions 104,204, the cleaning and filterings such as HEPA strainer (HighEfficiencyParticulateAirFilter, high efficiency particle air filter) are used to keep the degree of cleaning of cell culture chamber 101,201.In addition, there is microscope 128, take the inside cultured cells at the closed culture vessel 107 of cell type, and evaluate the development condition of cell.Further, by medium component analytical equipment and outer ties, be recovered in the old substratum that obtains as waste liquid when exchanging substratum, and carry out the mensuration of amount of development condition medium component of glucose, these reflection cells of lactic acid.
Control part 103,203 has: display part, the information such as temperature, gas concentration lwevel, humidity that its display is inner; Input part, it is for implementing the operation relevant to the control of automatic culture apparatus; And communication unit, it is for carrying out the access with external mechanical such as recording units.There is the function in order to keep the degree of cleaning of automatic culture apparatus inside to be carried out to sterilization or sterilization at the end of each cultivation.That is, when implementing sterilization, the sterilization gas such as ethylene oxide gas, hydrogen peroxide gas, ozone gas are utilized to carry out inner sterilization.In this case, the material of automatic culture apparatus has tolerance for the sterilization gas used, and can also guarantee security and sterilization gas can not be spilt to the outside of automatic culture apparatus.When not used by sterilization by sterilization, utilize the sterilizing agents such as alcohol to carry out spraying and (or) cleaning disinfection.Inner shape is preferably easy wiping, concavo-convex less shape.
Then, the flow path portion of the automatic culture apparatus of the present embodiment is described.Fig. 3 A represents the integrality of the flow path portion being arranged at automatic culture apparatus.This flow path portion is arranged in the automatic culture apparatus shown in Fig. 2.Fig. 3 B represents part relevant to cultivation in flow path portion.Fig. 3 C represents the pedestal of the part relevant to cultivation keeping the flow path portion shown in Fig. 3 B.If by the part integration shown in Fig. 3 B and Fig. 3 C, then become the state shown in Fig. 3 A.
Form primarily of the closed culture vessel 301 of cell type, stream 302, the cell bags 303 putting into cell suspension, the culture medium bag 304 putting into substratum, the cleaning bag 305 putting into scavenging solution, the waste fluid bag 306 putting into waste liquid, casing 307 to the structure of cultivating relevant part.As mentioned above, pedestal is made up of culture vessel pedestal 308, stream pedestal 309, substratum pedestal 310.In addition, also having by carrying the syringe 311 of various solution by changing the pressure of stream inside, forming the fluid moving control mechanism portion that the magnetic valve 312 etc. in valve portion forms.
The detailed construction of later use Fig. 6 flow path is described.This flow path portion is arranged on together with possessing the pedestal of the mechanism of carrying flow path portion (such as in CPC) under the higher environment of degree of cleaning.Implement cellular segregation process etc. in advance, in safety cabinet, inject to the cell bags 303 of flow path portion that be formed as becoming can cultivation conditions cell suspension.The state that degree of cleaning are very high is maintained in safety cabinet.Accordingly, carry out stream inside between this operational period can not be contaminated.In addition, flow path portion is closed, and for the structure that outside bacterium etc. invades can not be there is.Even if there is a small amount of bacterium etc. at the cell culture chamber of automatic culture apparatus, the inside of flow path portion also can be made to keep high degree of cleaning from putting into during cultivation terminates of cell.
An example of the closed culture vessel of the cell type of the present embodiment is represented at Fig. 4.In this example, cell type culture vessel 400 entirety in rectangular-shaped, but also can be the arbitrary shapes such as drum.In the closed culture vessel 400 of cell type, as the cylindrical shape of culture tank 401 of culture space, the upper surface of drum and bottom surface by gas can from outside through air penetrating film 402 and 403 formed.Air penetrating film 402 and 403 is fixed on the closed culture vessel parts 404 of cell type by methods such as ultrasonic wave coatings.The material of air penetrating film 402 and 403 is such as polycarbonate, silicone-polycarbonate.The exchangeability of the gas of air penetrating film 402 and 403 is lower than general open culture vessel, but when cultivating, carries out oxygen, the inflow of carbonic acid gas, outflow via air penetrating film 402 and 403 between outside and inside.In addition, even if carry out cultivating undesirable material at the outside particle-bound bacteria etc. of cell type culture vessel, also inside can not be entered by air penetrating film 402 and 403, the closed culture vessel parts 404 of cell type.
At the closed culture vessel of cell type, by methods such as adhesivess, junctor portion 405,406 is installed.And, be connected with stream 407,408 in junctor portion 405,406.Culture vessel after using sterilization in a coupled condition starts to cultivate, until at the end of cultivating, be always connected with stream.Thus, the possibility that bacterium etc. invades from outside is reduced significantly.In this example, illustrate the closed culture vessel 400 of cell type that culture tank 401 is one deck, but also can have the intermediate coat in the hole of the circulation that can realize substratum etc. in the setting of the inside of culture tank 401 and culture tank 401 is separated into two-layer.In this case, cultivate different separately cell in the upper and lower, can separation on implementation space.Such as when cornea regeneration, carry out the cultivation of the corneal epithelial stem cells deriving from people on upper strata, carry out in lower floor the fibroblastic cultivation deriving from mouse, thus the cultivation that is spatially separated by heterologous cells can be carried out.
As the variation of the embodiment of the container of Fig. 4, Fig. 5 illustrates the container being provided with diffuser in the culture space of the closed culture vessel of cell type, and this diffuser can impel cellular invasion and to culture space inoculating cell equably when cell suspension flows into.Illustrate the sectional view of cell type culture vessel 500 and the figure from top view cell type culture vessel 500.The parts for making cellular invasion are provided with in the inside of cell type culture vessel 500.Cell type culture vessel has culture space 501 and junctor portion 502,503.Stream 504,505 is there is between culture space 501 and junctor portion 502,503.The boundary member of stream 504,505 and culture space 501 is provided with diffuser 506,507.Diffuser 506,507 is when cell suspension flows into culture space 501 from stream 504,505, the flowing of cell suspension is divided into both direction, and diffuser 506,507 is arranged at the position that the cell contributed to contained by cell suspension is distributed in culture surface equably.In addition, although manufacture regenerating tissues in culture surface, the size of diffuser 506,507 be can maintain size required when regenerating tissues is for transplanting degree and do not hinder cultivation.The shape of diffuser meets such condition, and the shape shown in Fig. 5 is one of them example.
Use above structure, Fig. 6 represent in the present embodiment can the example of stream of actual culturing cell.Be configured to the structure that two closed culture vessels 608,609 of cell type are cultivated in figure 6.In addition, take cornea regeneration as model, the culture tank of the closed culture vessel 608,609 of cell type is made up of the upper and lower, cultivates different cells at each layer.
As shown in Figure 6, there is cell bags 601 and 602, culture medium bag 603, scavenging solution bag 604, waste fluid bag 605, casing 606,607,610 and 611, the closed culture vessel 608 and 609 of cell type, syringe 612 and 613, magnetic valve 614-639, gas vacuum breaker 640-643, liquid vacuum breaker 644 and 645, air filter 646 and 647, substratum returnable 648 and 649.The magnetic valve 614-639 forming valve portion controls the switch of stream.Substantially there are two by the stream of magnetic valve 614-639, always open for one when magnetic valve is not energized, and another always closed.After powered up, the switch of two streams is contrary.
In figure 6, by the stream of the magnetic valve 614-639 as valve portion, the side that is labeled as NC (NormallyClosed) is at the non-closed in electrified state of magnetic valve.On the other hand, the side being labeled as NO (NormallyOpen) opens for during energising at magnetic valve.Vacuum breaker 640-645 makes gas or liquid only towards a direction flowing, does not flow in the opposite direction.Air filter 646 and 647 sterilely suitably attracts relative to the outside of stream as required, exhausted air.Thus, the pressure in flow path controls.Substratum returnable 648 and 649 carries out the recovery of the old substratum obtained when exchanging substratum.Use the medium component analytical equipment configured in addition relative to automatic culture apparatus to measure the amount of substance of glucose, these reflection cell development situations of lactic acid, and the index of measurement result as the development condition judging cell is used.Utilize the stream shown in Fig. 6 suitably to carry out the switch of magnetic valve, utilize the action of syringe to make to change in the mode of suitably pressurizeing, reducing pressure in stream, thus carry out the conveying of solution.
As an example, Fig. 7 A and Fig. 7 B represent in Fig. 6 from casing 607 to the switch of magnetic valve during a layer delivered solution of the closed culture vessel 608 of cell type and the flowing of solution and air.As mentioned above, when not being energized the NO side of magnetic valve open, NC side closure.When carrying, first magnetic valve 621,622,624 and 629 is energized.So, magnetic valve, the stream that is labeled as NC in Fig. 6 opens, on the contrary, the stream being labeled as NO is closed.In this condition, make syringe 612 and 613 towards the direction action of arrow 650.Syringe 612 and 613 can synchronously action, when syringe 612 is supplied gas, and syringe 613 air-breathing.On the contrary, when syringe 613 is supplied gas, syringe 612 air-breathing.In addition, the gas pushing quantity in respective action is suitable with inspiratory capacity.Two syringes 612,613 are by repeatedly carrying out supplying gas and air-breathing carries out Liquid transfer continuously.For the liquid in stream, pull liquid from one-sided, promote liquid from opposition side, by two power combinations, thus efficient conveying can be implemented.
Fig. 7 A illustrate the direction injector-actuated 612,613 towards arrow 700 and make syringe 612 air-breathing, the solution of syringe 613 when supplying gas and the flowing of air.The working direction of solution and air is represented by black arrow 701 and white arrow 702 respectively.The vacuum breaker 640-643 being configured at the surrounding of syringe makes air not flow relative to reverse direction relative to the flowing of some directions.Be used for limiting the flowing of air, result, as illustrated delivered solution by this vacuum breaker.On the other hand, Fig. 7 B illustrates the effect by vacuum breaker 640-643, towards arrow 703 direction injector-actuated 612,613 thus make that syringe 612 is supplied gas, syringe 613 air-breathing time solution and the flowing of air.Repeatedly carry out the action of Fig. 7 A and Fig. 7 B, before the solution of specified amount completes and to move from casing 607 to a layer of the closed culture vessel 608 of cell type, repeatedly carry out supplying gas and the action of air-breathing of syringe.By suitably carrying out the switch of magnetic valve and utilizing injector delivery air, the conveying of other solution can be realized.
The automatic culture apparatus with the present embodiment of above structure is used to be described a series of cultivation orders during culturing cell.Fig. 8 is the schema of the action for illustration of automatic culture apparatus.Below, the action of action diagram to automatic culture apparatus of the overall diagram of the automatic culture apparatus of Fig. 1, the flow circuit diagram of Fig. 6, the action diagram of the casing of Fig. 9 A-Fig. 9 E, the closed culture vessel of cell type of Figure 10 A-Figure 10 E is used to be described.In addition, when used cell type close be culture vessel quantity increase, arrange side by side in stream cell type close be culture vessel.In addition, for the cultivation order in this situation, closing each cell type is that culture vessel implements operation shown below in order.
< step S1: start >
First, as shown in Figure 8, automatic culture apparatus is started.Operator by pressing be positioned at the operating portion of the control part 103 of Fig. 1 eliminate illustrated starting switch to start.In addition, in this moment, CO 2bomb, flow path portion, substratum, cell solution are arranged at automatic culture apparatus.Confirm in the operation screen of illustrated indicating meter that the internal medium of automatic culture apparatus is suitable value eliminating of control part 103.Such as, as mentioned above, temperature 37 DEG C, CO 2concentration 5%, humidity 100%.Not be defined in these numerical value, such as temperature can be selected in the scope of 0 DEG C to 45 DEG C.In addition, implement the sterilization utilizing sterilization gas to carry out or the sterilization utilizing alcohol to carry out by proper handling in advance, and make the state that the inside of device becomes clean.In addition, sterilization is implemented to cultivating used flow path portion in advance.
< step S2: determine timetable >
The automatic incubation time table implemented by automatic culture apparatus is decided corresponding to the kind of cultured cells and amount.The date and time of the operations such as cell inoculation, substratum exchange, microscopic examination, the recovery of inspection tissue, the recovery of transplanting tissue is carried out, the condition such as frequency, liquid measure by the operating portion input of control part 103.
< step S3: attract cell suspension > from cell bags
The step S3-S7 of Fig. 8 is the operation of cell inoculation.After the switch suitably carrying out magnetic valve, make syringe 612 and 613 action, attract cell suspension from cell bags 601 and 602.In the example of cornea regeneration, cell suspension is the corneal epithelial cell suspended in KCM substratum (keratinocyteculturemedium) and the 3T3 cell suspended in KCM substratum equally.By injector-actuated 612 and 613 via air filter 647 to the air in discharge duct outside stream, and utilize produce decompression state attract cell suspension.From injecting cell suspension to the cell bags 601 and 602 in safety cabinet, needing the time to making automatic culture apparatus action, therefore, cell precipitates to the bottom in cell bags 601 and 602 due to the reason of deadweight.Accordingly, following operation is carried out in order to carry out attracting after making uneven cell suspension evenly again.
That is, if before attracting whole cell suspension from cell bags 601 and 602, repeatedly carry out following two actions, that is: switching solenoid valve 614-639, a small amount of cell suspension is attracted by producing decompression state; Switching solenoid valve 614-639, injects the cell suspension of equivalent by producing pressurized state.Thus, stirring stream is produced in the inside of cell bags 601 and 602.Repeatedly carry out attraction and the injection of enough number of times, eliminating cell precipitation and the whole cell suspension making the stage after being evenly distributed attract in cell bags 601 and 602, and carry respectively to casing 606 and 607.By implementing this operation, can attract from cell bags 601 and 602 with uniform cell distribution state.Consequently, in cell bags 601 and 602, the cell of precipitation is suspended in solution again, therefore, it is possible to attract complete soln and do not have loss cell.Particularly when manufacturing little regenerating tissues as cornea regeneration, this operation is that a small amount of situation needing as far as possible to avoid losing is effective for the cell collected.
< step S4: from casing conveying cell suspension >
The cell suspension transported by cell bags 601 and 602 is temporarily accommodated in casing 606 and 607.Fig. 9 A-Fig. 9 D illustrates the summary of the flow process implemented at casing 606 and 607.First, cell bags 601 and 602 is accommodated in the refrigerator 102 that temperature is such as 4 DEG C.In addition, the substratum used when exchanging substratum, scavenging solution are also stored in refrigerator with 4 DEG C.Particularly exchanging in the process of cultivating, between the substratum commutation period implemented by S10-S14, need in advance substratum to be heated to 37 DEG C, the casing 606 and 607 of use is general in whole operation process.When inoculating cell, need the heating temperatures to 37 of cell suspension DEG C.
Therefore, as illustrated, utilize the well heater being installed on the periphery of casing 606 and 607 by the heating temperatures to 37 of cell suspension DEG C later.Due to be accommodated in casing after re-use the mechanism of heater heats, so with such as utilize heat block to be heated to compared with the situation of 37 DEG C, can temperature be made efficiently to increase with the state entering stream.In addition, by temporarily receiving cell suspension at casing 606 and 607, cell bags 601 and 502, strainer 646 and 647 etc. can be utilized to discharge to be mixed into because of certain reason the air in cell suspension.
Carry out the injection of the cell suspension 900 shown in Fig. 9 A, and, under the state moving specified amount as shown in Figure 9 B, the cell suspension of the bubble 901 containing the air be mixed into arrives casing 606 and 607, then lodge in the bottom of casing 606 and 607, air bubble 901 is removed by stream.Further, when liquid level creates small bubble 902, by driving the syringe 612 and 613 shown in Fig. 6 to come decompression in casing 606 and 607, thus the micro-bubble 902 being present in the liquid level of cell suspension 900 is eliminated.To time of decompression state be kept to be set to the very short time, disappear but the dissolved gases amount that is discharged into liquid external is less as condition using the bubble 902 of liquid level.Thereby, it is possible to carry various solution with bubble-free state to the closed culture vessel of cell type 608,609.
When supposing to there is bubble in the closed culture vessel 608,609 of cell type, due to the surface tension of bubble liquid level, cell is concentrated, become the reason of the uneven homogenize of cell distribution.In addition, the cell drying with bubble contact is made because bubble exists.Like this, the removing of bubble is very important.
In casing 606 and 607, carry out the dilution of cell suspension as required.After cell suspension being put into casing 606 and 607, attract the substratum of specified amount from culture medium bag 603 and carry to casing 606 and 607, thus diluting.In the example of cornea regeneration, substratum is KCM substratum (keratinocyteculturemedium).Can inoculate in the closed culture vessel of multiple cell type and cultivate the cell suspension after diluting.Further, can concentration be changed according to the closed culture vessel of each cell type and inoculate.In this case, because the concentration of the closed culture vessel of each cell type is different, so cell proliferation and to become time of transplantable regenerating tissues different.Thus, when carrying out the transplanting of regenerating tissues, can from the different regenerating tissues of developmental condition, the regenerating tissues being most suitable for the state of transplanting be selected to use.
After diluting as required, as implementing in cell bags 601 and 602, generation in casing 606 and 607 was made to stir stream and make the distribution uniformity of cell.That is, before attracting whole cell suspension from casing 606 and 607, the attraction repeatedly carrying out a small amount of cell suspension and these two actions of injection of the cell suspension of equivalent producing malleation by switching solenoid valve and carry out.Produce in the inside of casing 606 and 607 thus and stir stream.Such as, syringe 612 produces decompression state by attracting, and pulls solution.Meanwhile, syringe 613 discharges the air of equivalent, produces pressurized state and is extruded by solution.Pull solution from certain direction, extrude solution from contrary direction, easily move under the state that these two actions act on simultaneously.Compared with only using the situation of a syringe, solution can be made to move efficiently by use two syringes as shown in this embodiment.
Eliminate the precipitation of cell in the attraction and injection of repeatedly carrying out enough number of times and make distribution become the uniform stage, attract the whole cell suspension in casing 606 and 607, and carry respectively to the closed culture vessel 608 and 609 of cell type.By implementing this operation, can attract from casing 606 and 607 under the state of uniform cell distribution.Consequently, the cell of precipitation again at solution inner suspension, therefore, it is possible to attract complete soln and do not have loss cell.In addition, due to can to the cell suspension of cell type closed culture vessel 608 and 609 conveying uniform, so contribute to the closed culture vessel of cell type inoculating cell equably.
< stirs the cell suspension > in box and casing simultaneously
As mentioned above, in casing 606 and 607 and the closed culture vessel 608 and 609 of cell type, the attraction repeatedly carrying out a small amount of cell suspension before inoculating cell and these two actions of injection of the cell suspension of equivalent producing malleation by switching solenoid valve and carry out, stir stream in inside generation and make the distribution uniformity of cell, but also can carry out the operation of casing 606 and 607 and the closed culture vessel 608 and 609 of cell type simultaneously.Such as, when inoculating in the closed culture vessel 608 of cell type, the cell suspension that the closed culture vessel 609 of pre-directed cell type is inoculated has entered in casing 606 or 607.In this condition, by suitably carrying out the switch of valve, the attraction of a small amount of cell suspension being carried out repeatedly to the cell suspension of the both sides in the closed culture vessel of cell type 608 and casing 606 or 607 and carrys out malleation by switching solenoid valve and produce and these two actions of injection of the cell suspension of equivalent of carrying out.Compared to situation about implementing respectively, by implementing to cut down flow chart simultaneously.
As mentioned above, in casing 606 and 607, carry out the heating of substratum when exchanging substratum.Fig. 9 E is the figure representing the structure of mounting heater 903 and temperature sensor 904 around casing as the function for heating substratum.The cultivation of cell is carried out usually at 37 DEG C.On the other hand, as previously described, the substratum keeping before using is in refrigerator.Therefore, need in advance substratum to be heated to 37 DEG C before enforcement substratum exchanges, in the present embodiment, implement in casing 606,607.Utilize well heater 903 heating compartment, and control well heater 903, the homo(io)thermism measured to make temperature sensor 904 is 37 DEG C.After the time enough that heating compartment 904 makes substratum reach needed for 37 DEG C, use overheated substratum to implement substratum and exchange.
< step S5: to cell type closed culture vessel inoculation >
Then, in the treatment scheme of Fig. 8, the cell suspension transported is seeded to the closed culture vessel 608 and 609 of cell type from casing 606 and 607.
The summary of the flow process implemented in the closed culture vessel 1000 of cell type is illustrated in Figure 10 A-Figure 10 D.At first, as shown in Figure 10 A, the state erected with the container 1000 corresponding with the closed culture vessel of cell type 608 and 609 is carried.The closed culture vessel 1000 of cell type is rotated by using the rotating mechanism 110,210 shown in Fig. 1 and motor 111,211 and vertically erects.The cell suspension come from casing conveying is injected from the stream 1001 of the below being installed on the closed culture vessel 1000 of the cell type erected.On the other hand, extrude from the stream 1002 of the top being installed on the closed culture vessel of the cell type erected 1000 air being present in the closed culture vessel 1000 of cell type.
Thus, shown in Figure 10 B, cell suspension can be made to be full of container 1000 corresponding to culture vessel 608 and 609 closed with cell type.As above described in S4, in casing 606 and 607, implement decompression operation, therefore there is not small bubble.In this moment, under the state that the closed culture vessel 1000 of cell type erects, inject cell suspension, therefore cell precipitates because of the deadweight of cell, shows the distribution representing that cell is uneven in culture surface.
Then, as illustrated in figure 10 c, cell type closed culture vessel 1000 level is made.And, as shown in Figure 10 D, as the operation implemented in cell bags and casing, make 1000 generations in the closed culture vessel of cell type stir stream and make the distribution uniformity of cell.That is, the attraction repeatedly carrying out a small amount of cell suspension and these two actions of injection of the cell suspension of equivalent producing malleation by switching solenoid valve and carry out.Produce in the inside of the closed culture vessel 1000 of cell type thus and stir stream.Repeatedly carry out the attraction of enough number of times and injection and make being evenly distributed of cell.Then, the container 1000 corresponding with the closed culture vessel of cell type 608 and 609 is left standstill.
< step S6: utilize scavenging solution to clean stream >
Be back to the flow processing of Fig. 8, use the scavenging solution cleaning stream of specified amount from scavenging solution bag 604.In the example of cornea regeneration, scavenging solution uses pure water, PBS (phosphatebufferedsaline) solution.In the former case, even if remain drop after cleaning in stream, also any material can not be remained after moisture evaporation, therefore the preferred pure water of scavenging solution.On the other hand, PBS solution is also habitual uses as scavenging solution.In this case, when during residual drop, having after moisture evaporation and salt out in stream after cleaning.When implementing substratum exchange etc. afterwards, residual salt is dissolved in substratum and the composition of molten substratum is changed.According to this reason, preferably do not use PBS solution.
When cleaning, first attract scavenging solution from scavenging solution bag 604, and carry to casing 606 and 607.Owing to cleaning whole casing 606 and 607, so liquid measure is preferably equal with the capacity of casing.Then, scavenging solution is delivered to the immediately front of the closed culture vessel of cell type.Walk around the closed culture vessel of cell type from the magnetic valve 624,625,626 and 627 immediately front of the closed culture vessel of cell type and scavenging solution be delivered to the magnetic valve 628,629,630 and 631 immediately rear being positioned at the closed culture vessel of cell type.Then, whole scavenging solution is made to move to casing 610 and 611.Then, scavenging solution is made to move from casing 610 and 611 to waste fluid bag 605.Identical operation is implemented to all casings, all streams.By cleaning stream so above, the effect reducing the probability that developmental biology pollutes can be expected.
< step S7: the air-dry > of stream
Then, air-dry stream after stream cleaning.The well heater 117 shown in Fig. 1 is used to heat whole piece stream, to make to remain unnecessary liquid.In addition, air is flowed in stream.Thus, when carrying out substratum exchange etc. in ensuing operation, the liquid remained can be avoided to be the change in concentration that reason makes substratum.
< step S8: the cultivation > of cell
The specified time is cultivated under the state making cell type closed culture vessel 608 and 609 horizontal rest.When for corneal epithelial cell, leave standstill period for latter about five days of inoculation.In culturing process, utilize well heater 117 that internal temperature is maintained 37 DEG C.By CO 2concentration maintains 5%, humidity maintains 100%.Above-mentioned each value is by temperature sensor 118, CO 2sensor 119, humidity sensor 120 monitor.In addition, always utilize fan 123 to stir the air of automatic culture apparatus inside, be always uniformly distributed to make temperature, CO2, humidity.
< step S9: utilize microscope to carry out observation >
The microscope 128 be arranged in automatic culture apparatus is used to obtain the image of cell.Make the light source that is arranged in automatic culture apparatus suitably luminous, utilize microscope focus is aimed at cell and takes.Determine arbitrarily fix a point and take in culture surface as required.The cell image of acquisition is stored in database, and makes to read on the indicating meter of outside being arranged at automatic culture apparatus as required above-mentioned cell image.The information relevant to the developmental condition of cell according to being obtained by microscopic examination is judged, and carries out the frequency of substratum exchange, the adjustment in period.Such as when the adhesion of cell is insufficient, the substratum not implementing S10 ~ S14 exchanges, but proceeds the cell cultures of S8.
< step S10: attract substratum > from culture medium bag
Step S10-S14 is the sequence of operations of the substratum exchanged in the closed culture vessel 608 and 609 of cell type.Usually implement substratum with several days frequency once to exchange.Development condition according to cell carries out frequency adjustment.
After the switch suitably carrying out magnetic valve, make syringe 612 and 613 action, attract substratum from culture medium bag 603.Come via air filter 647 to the air in discharge duct outside stream by injector-actuated, and utilize the decompression state produced to attract substratum.Then, substratum is carried to casing 606 and 607.Because substratum is cooled to 24 DEG C in refrigerator 102, so use well heater to be heated to 37 DEG C in casing 606 and 607.
< step S11: the exchange > of the substratum in the closed culture vessel of cell type
From casing 606 and 607, substratum is delivered to the closed culture vessel of cell type.Under the state that closed for cell type culture vessel is erected, inject from the stream of the below being installed on the closed culture vessel 608 and 609 of the cell type erected and carry and next substratum from casing 606 and 607.On the other hand, from the stream of the top being installed on the closed culture vessel of the cell type erected 608 and 609, the old substratum being present in the closed culture vessel of cell type 608 and 609 is extruded.Thereby, it is possible to make new substratum be full of the closed culture vessel of cell type.Be filled with the stage of new substratum at the closed culture vessel of cell type, close the magnetic valve 628,629,630 and 631 be positioned near the closed culture vessel of cell type.And, whole old substratum is moved to casing.
< step S12: the recovery > of old substratum
Old substratum to casing 610 and 611 movement is moved to waste fluid bag 605.Time mobile, make magnetic valve 638 and 639 action, a part for old substratum is moved to returnable 648 and 649.Vacuum breaker 644 and 645 is set in the discharge portion of old substratum, makes solution only to a direction flowing.Thus, bacterium can not be produced enter from outside and pollute the situation of stream inside.For the old substratum reclaimed, use the medium component analytical equipment prepared in addition to analyze the composition of substratum.Such as, the amount of the glucose used when measuring cell development and the lactic acid of discharge, grasps the developmental condition of cell.In addition, implement mycoplasma test etc. and judge that whether substratum is uncontaminated.Terminate immediately when existing and polluting to cultivate, sterilely abandon cell by suitable operation, to make the setting place of automatic culture apparatus not contaminated.
< step S13: the cleaning > of stream
Utilize the method identical with step S6, use the scavenging solution cleaning stream of specified amount from scavenging solution bag 604.First, attract scavenging solution and carry to casing 606 and 607.Owing to cleaning whole casing, so liquid measure is preferably equal with the capacity of casing.Then, scavenging solution is delivered to the immediately front of the closed culture vessel of cell type 608 and 609.Scavenging solution is walked around the closed culture vessel 608 and 609 of cell type from the magnetic valve 624,625,626 and 627 immediately front of the closed culture vessel of cell type and is delivered to the magnetic valve 628,629,630 and 631 immediately rear being positioned at the closed culture vessel of cell type.Then, whole scavenging solution is made to move to casing 610 and 611.Then, scavenging solution is made to move from casing 610 and 611 to waste fluid bag 605.Identical operation is implemented to all casings, all streams.
< step S14: the air-dry > of stream
Utilize the method identical with step S7, air-dry stream after cleaning stream.Heater heats whole piece stream is used to make to remain unnecessary liquid.
< step S15: the recovery > of inspection tissue
Transplanting the day before yesterday on pre-settled date, in closed for cell type culture vessel 608 and 609 is being used for check and reclaim.Cut off the stream flowed out from the closed culture vessel of cell type, and unload the closed culture vessel of cell type.Two close places on stream use clip to close closed channel.Then, cut off by clip clamp two between stream.In the closed culture vessel of cell type after recovery, whether the state of carrying out cell wherein has the inspection being applicable to the character of transplanting.Such as when cornea regeneration, whether the cell obtained has the evaluation of the multiple stratification structure of about three layers by histological evaluation, carry out by immunohistochemical staining evaluation the evaluation whether corneal epithelial stem cells be present in stratum basale etc.
< step S16: the cultivation before transplanting and substratum exchange >
Cultivated by the operation identical with step S8.Then, before implementation step S17, carry out substratum exchange by the operation identical with step S10 ~ S14.
< step S17: the recovery > of transplanting tissue
When the result that step S15 evaluates is the judgement obtaining turning out the regenerating tissues being suitable for transplanting, setup action is transplanted with and reclaims, and treating for regenerative medicine.Identical with S15, cut off the stream flowed out from the closed culture vessel of cell type, and unload the closed culture vessel of cell type.Two close places on stream use clip to close closed channel.Then, cut off by clip clamp two between stream.Afterwards, reclaim cell type closed culture vessel, it is delivered to the state maintaining sterility biological property the Operation theatre that carries out regenerative medicine treatment and is used for the treatment of.
< step S18: terminate >
Unload carry out cultivation use flow path portion.Then, the sterilization utilizing sterilization gas to carry out to device internal implementation by suitable operation or the sterilization utilizing alcohol to carry out, become clean state.Terminate the various softwares of automatic culture apparatus, terminate the action of automatic culture apparatus.
Above, with reference to accompanying drawing, embodiments of the present invention example is illustrated, but known the present invention is not limited to these embodiments.Such as, in an embodiment, illustrate syringe pump to be illustrated as fluid moving control mechanism portion, but also can replace syringe pump and use other driving mechanisms such as peristaltic pump.
According to the preferred implementation of the automatic culture apparatus formed like that above, in order to manufacture uniform regenerating tissues, when starting to cultivate, cell can be inoculated in equably the culture surface of the closed culture vessel of cell type.In addition, the cell suspension that can distribute equably from cell bags and casing conveying cell, consequently, can inoculate to cell culturing surfaces in the mode of not celliferous loss.In addition, the removing of the bubble of the reason becoming cell ununiformity can be carried out.
The present invention has good effect as the automatic culture apparatus using cell type closed culture vessel by automatic operation culturing cell or tissue, the automatic culture apparatus that particularly can manufacture the regenerating tissues that can be used for regenerative medicine.
The explanation of symbol
101,201 ... cell culture chamber; 102,202 ... refrigerator; 103,203 ... control part; 104,204 ... clean air circulation portions; 105,205 ... cell culture chamber door; 106,206 ... refrigerator door; 107 ... the closed culture vessel of cell type; 108,308 ... culture vessel pedestal; 109,209 ... culture vessel driving part; 110,210 ... rotating mechanism; 111,113,211 ... motor; 112,312,614-639 ... magnetic valve; 114,307,606,606,610,611 ... casing; 115 ... stream pedestal; 116,216 ... stream driving part; 117,901 ... well heater; 118,902 ... temperature sensor; 119 ... carbonic acid gas feed mechanism; 120 ... carbon dioxide sensor; 121 ... humidity produces mechanism; 122 ... humidity sensor; 123 ... fan; 124,310 ... substratum pedestal; 125 ... pedestal; 126,226 ... driving part; 127 ... sealing member; 128 ... microscope; 301,400,500,608,609 ... the closed culture vessel of cell type; 302,407,408 ... stream; 303,601,602 ... cell bags; 304,603 ... culture medium bag; 305,604 ... scavenging solution bag; 306,605 ... waste fluid bag; 309 ... stream pedestal; 311,612,613 ... syringe; 401 ... culture tank; 402,403 ... air penetrating film; 404 ... the closed culture vessel parts of cell type; 405,406 ... junctor portion; 501 ... culture space portion; 502,503 ... junctor portion; 504,505 ... stream; 506,507 ... diffuser; 640-643 ... gas vacuum breaker; 644,645 ... liquid vacuum breaker; 646,647 ... air filter; 648,649 ... substratum returnable; 900 ... air bubble; 902 ... temperature sensor.

Claims (11)

1. an automatic culture apparatus, it is used in the culture vessel that inside has culture space,
The feature of above-mentioned automatic culture apparatus is,
Possess:
The incorporating section of storage cell suspension;
The stream be connected with above-mentioned incorporating section; And
Carry out the flow control mechanism portion controlled to the mode of above-mentioned culture vessel movement to make above-mentioned cell suspension via an above-mentioned stream,
Above-mentioned fluid moving control mechanism portion makes above-mentioned cell suspension move to above-mentioned first-class trackside, when carrying above-mentioned cell suspension, by repeating the action of the above-mentioned cell suspension transported from the above-mentioned cell suspension in above-mentioned incorporating section being injected to above-mentioned incorporating section, come to produce in the above-mentioned cell suspension in above-mentioned incorporating section to stir stream.
2. automatic culture apparatus according to claim 1, is characterized in that,
Above-mentioned fluid moving control mechanism portion makes above-mentioned cell suspension be full of the inside of above-mentioned culture vessel, and repeats above-mentioned cell suspension to the above-mentioned supply on first-class road and the action of attraction.
3. automatic culture apparatus according to claim 2, is characterized in that,
Above-mentioned fluid moving control mechanism portion makes pressure change in above-mentioned culture vessel, and the above-mentioned cell suspension in above-mentioned culture vessel is moved to another stream side.
4. automatic culture apparatus according to claim 3, is characterized in that,
Above-mentioned culture vessel possesses diffuser therein, this diffuser, when above-mentioned cell suspension flows into above-mentioned culture space via an above-mentioned stream or another stream above-mentioned, makes the cellular invasion in above-mentioned cell suspension and is seeded in above-mentioned culture space equably.
5. automatic culture apparatus according to claim 1, is characterized in that,
Above-mentioned incorporating section is made up of the casing being connected to an above-mentioned stream,
Also possess and carry out heating the heating part that also can maintain heated condition to above-mentioned casing.
6. automatic culture apparatus according to claim 5, is characterized in that,
Above-mentioned fluid moving control mechanism portion, by repeating the action of the above-mentioned cell suspension of carrying to above-mentioned culture vessel being injected to above-mentioned casing, comes to produce in the above-mentioned cell suspension in above-mentioned casing to stir stream.
7. automatic culture apparatus according to claim 1, is characterized in that,
Above-mentioned incorporating section is made up of the cell bags being connected to an above-mentioned stream.
8. automatic culture apparatus according to claim 1, is characterized in that,
Above-mentioned fluid moving control mechanism portion comprises the syringe connected via an above-mentioned stream.
9. automatic culture apparatus according to claim 1, is characterized in that,
Above-mentioned fluid moving control mechanism portion comprises:
The first syringe connected via an above-mentioned stream; And
With the state action synchronous with above-mentioned first syringe and via being connected to another stream of above-mentioned culture vessel and the second syringe connected.
10. automatic culture apparatus according to claim 1, is characterized in that,
Above-mentioned fluid moving control mechanism portion comprises rotation control unit, this rotation control unit controls in the mode obtaining any one state in following two states, above-mentioned two states is, make the state that above-mentioned culture vessel generally perpendicularly erects, and substantially horizontally keep the state of above-mentioned culture vessel.
11. automatic culture apparatuses according to claim 9, is characterized in that,
Above-mentioned fluid moving control mechanism portion makes above-mentioned first syringe and above-mentioned second syringe action,
Make to become decompression state in an above-mentioned stream by above-mentioned first syringe, make to become pressurized state in another stream above-mentioned by above-mentioned second syringe, pressure change is produced from both sides relative to the above-mentioned cell suspension being full of above-mentioned culture vessel, above-mentioned cell suspension is moved to above-mentioned first-class trackside
Make to become pressurized state in an above-mentioned stream by above-mentioned first syringe, make to become decompression state in another stream above-mentioned by above-mentioned second syringe, produce pressure change relative to the above-mentioned cell suspension being full of above-mentioned culture vessel from both sides, above-mentioned cell suspension is moved to another stream side above-mentioned.
CN201510428228.5A 2010-08-12 2010-08-12 Automatic culture apparatus Pending CN105154329A (en)

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