JP2008301746A - Culture apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture apparatus that simply efficiently carries out naturalization culture, in which a part of culture solution is recovered and a culture is performed, by reusing the part of the culture solution in the culture, in a microchip. <P>SOLUTION: The culture apparatus carrying out a cell culture using a minute flow channel has a cell culture chamber; a first flow channel for conveying cells and the culture solution of the cells to the cell culture chamber; a second flow channel connected to the first flow channel; and a liquid storage chamber that is arranged in the second flow channel and stores a part of the culture solution of the cell culture chamber, recovers a part of the culture solution of the cell culture chamber in the liquid storage chamber, returns the culture solution to the cell culture chamber and uses this in the culture. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a culture apparatus that performs conditioned culture of cells in a microchip including a cell culture chamber. More specifically, the first flow path connected to the introduction port, the discharge port, and the cell culture chamber is further provided with a second flow path having a liquid storage chamber, thereby collecting the culture solution from the cell culture chamber; The present invention relates to a culture apparatus that re-feeds a collected culture solution to a cell culture chamber.

  Various animal cells, especially adherent cells, can be cultured and cultured using static culture or cell culture with cells attached to the bottom using petri dishes, culture bottles, test tubes, etc. Continue to supply fresh culture fluid while constantly exchanging and circulating the culture fluid in the cell culture tank by culturing by mechanical stirring using a magnetic stirrer or impeller placed in a Sakaguchi flask or incubator In addition, there is a flow culture that increases the culture efficiency by removing waste products from the system.

  In particular, flow culture is particularly effective in substance production in cells, differentiation control of stem cells, and cell culture in a microchip. A microchip refers to a flow channel structure or array structure having a size of several micrometers to several hundreds of micrometers. Compared with a conventional culture method using a petri dish or a well, It is expected to greatly reduce the amount of reagent. However, a channel-type chip having a channel with a width of several millimeters can be handled as a small chip in comparison with a normal scale. In the present invention, for convenience, the microchip including those having a millimeter-width channel structure is referred to as a microchip.

  As described above, the microchip can reduce the amount of reagents used, etc., and the cells can touch a large amount of medium per unit area during flow culture, so the overall concentration is uniform compared to conventional culture methods. Can be improved. For example, Non-Patent Document 1 and the like discuss in detail about various microchips and various discussions. Examples of cell culture using a microchip include bioassay using cells, production of various pharmacophores such as erythropoietin and G-CSF (granulocyte colony stimulating factor), glycoproteins and antibodies, and regenerative medicine. Application to the functional differentiation control of stem cells and the like is expected.

  On the other hand, there have been reports on devices for recirculating a culture solution collected from a culture tank to a cell culture tank in agitation culture of cells performed while turning a gear or the like. For example, Patent Document 1 reports a bioreactor for performing substance production by cells while performing swirl culture. However, Patent Document 1 is culturing while rotating a stirring blade, and it is necessary to use a very large volume cell culture chamber and to provide a plurality of pumps for controlling the culture solution. In addition, since the cell culture tank is huge, it has been desired to develop an apparatus that can remove cell inactive factors such as waste products.

In addition, Patent Document 2 reports a device related to an apparatus for circulating culture of animal cells that is provided with a heater for temperature control and that continues to be supplied while circulating a culture solution under a microscope. However, in the apparatus, when the culture solution collected from the cell culture chamber is resupplied, it is necessary to store it in a circulating fluid storage bottle outside the system. For this reason, there are still problems such as the complexity of the apparatus, the dead volume, and the contamination of the culture medium due to the incomplete circulation of the culture medium in the apparatus.
Japanese Patent Laid-Open No. 2-117388 JP-A 61-247372 "Technology and application of micro chemical chip", published by Maruzen Co., Ltd. in 2004

  The present invention has been made in view of such a background art, and the cells are secreted from the cells instead of replacing the cultures of the cells with new culture solutions one after another to remove all the wastes and the like from the cells. It is intended to provide a culture apparatus for realizing conditioned culture in a microchip, characterized in that a part of the growth factor and the like collected in a liquid storage chamber is supplied to the cell culture chamber again.

  A culture apparatus that solves the above-described problems is a culture apparatus that performs cell culture using a minute flow path, and includes a cell culture chamber, and a first culture medium that transports cells and a culture solution of the cells to the cell culture chamber. A flow path; a second flow path connected to the first flow path; and a liquid storage chamber provided in the second flow path for storing a part of the culture solution in the cell culture chamber. And after collect | recovering a part of culture solution of the said cell culture chamber to the said liquid storage chamber, the said culture solution is returned to a cell culture chamber, It is characterized by the above-mentioned.

  The present invention can provide a culture apparatus that performs conditioned culture simply and efficiently in a microchip.

Hereinafter, the present invention will be described in detail.
The culture apparatus according to the present invention is a culture apparatus that performs cell culture using a minute flow path, and includes a cell culture chamber, and a first flow path for transporting cells and a culture solution of the cells to the cell culture chamber. And a second flow path connected to the first flow path, and a liquid storage chamber provided in the second flow path for storing a part of the culture solution in the cell culture chamber, A part of the culture solution in the cell culture chamber is collected in the liquid storage chamber, and then the culture solution is returned to the cell culture chamber and used for culture.

  A pump and a valve are connected to at least one of the first flow path and the second flow path, and a part of the culture solution in the cell culture chamber is transferred to the liquid storage chamber by operating the pump and the valve. After recovery, it is preferable to return to the cell culture chamber.

When the cross-sectional area of the first flow path is A and the cross-sectional area of the second flow path is B, the cross-sectional area ratio A / B is preferably 5 <A / B <1000000.
The liquid storage chamber includes a processing means for performing at least one of heating, concentration, stirring, filtration, mixing, chemical reaction, and biochemical reaction on the culture solution collected from the cell culture chamber. It is preferable.

  The present invention relates to a culture device for performing cell culture simply and efficiently in a microchip. The culture apparatus of the present invention includes a cell culture chamber for culturing cells, an inlet and an outlet for the cells and the culture medium of the cells, a cell culture chamber and the inlet and outlet for connecting the cell culture chamber and flowing the culture liquid. A first flow path used in the first flow path, a second flow path disposed in the middle of the first flow path and upstream of the cell culture chamber, and the cell culture chamber provided in the second flow path A liquid storage chamber for storing a part of the culture solution. Further, a valve mechanism including a valve for controlling the collection and resupply of the culture solution is connected to the second channel.

  As described above, by providing a mechanism for collecting and re-supplying the culture medium in the flow path, it becomes possible to re-supply cell growth factors and reduce contamination of the culture medium and dead volume. . In addition, it is known that the culture medium collected from the cell culture chamber contains growth factors produced by cultured cells in addition to impurities. It is also very effective in constructing an extracellular environment. Among them, in the present invention, a culture method called conditioned culture that is used for subsequent culture after recovering the culture medium when cell culture is performed in the microchip and performing appropriate treatment such as removal of impurities, This is an extremely important culture method. In the present invention, it is possible to provide a culture apparatus that performs such conditioned culture in a microchip.

  In the present invention, the cell used in the culture apparatus may be either an animal cell or a plant cell, and the above cell group can be used as it is, or a functionally modified cell subjected to an operation such as genetic modification is also used. be able to. Examples of the function-modified cells herein include transformants in which a reporter gene is linked downstream of a promoter of a candidate gene that is expressed by contact with a test substance, and these transformants are prepared by a conventional method. be able to. Specific examples of the reporter gene include DNA encoding a fluorescent protein such as GFP (green fluorescent protein), luciferases such as firefly luciferase and bacterial luciferase, and enzyme genes such as β-galactosidase. Among them, a DNA encoding a fluorescent protein such as a GFP gene is preferable because of easy detection and confirmation. Such GFP includes derivatives having different fluorescence wavelengths such as EGFP (Enhanced GFP), EYFP (Enhanced Yellow Fluorescent Protein), ECFP (enhanced CYAN fluorescent protein) (blue), and DsRed (red). You can also. The advantage of GFP is that it can be easily analyzed with living cells, so that observation over time becomes easy. In the present invention, animal cells are more preferably used, which are selected from suspension cells or adherent cell groups. Examples of applications of the device include bioassay using cells, production of various lymphokines such as erythropoietin and G-CSF (granulocyte colony stimulating factor), glycoproteins, antibodies and other biopharmaceuticals, stem cells in the field of regenerative medicine, etc. And the like, and an appropriate cell group is selected accordingly.

  An example of the cell type used for substance production is appropriately selected from Escherichia coli, yeast, and animal cell groups. Examples of animal cells include Chinese hamster ovary-derived cells (CHO cells), PER. Substance production using C6 cells, BHK cells, NSO cells, HepG2 cells, hybridomas, insect cell lines, etc. has been widely reported. Regarding selection of the cell group used in substance production, an appropriate cell group is selected according to the purpose. In general, Escherichia coli and yeast are advantageous in terms of cost because they have a higher growth rate and are easier to handle than animal cell groups. The animal cell group has a complex post-translational modification ability close to human type compared to E. coli and yeast, and is used when producing biopharmaceuticals having physiological activity. As specific examples using animal cells, studies have been made on improving productivity of CHO cells, BHK cells, and HepG2 cells by gene recombinants, and on control of glycosylation modification in monoclonal antibody production and protein production. .

  It has also been reported to be effective as a method for culturing functional cells such as stem cells, and even in human stem cell systems, the properties of the liquid are not changed suddenly while gently replacing the culture medium when culturing in vitro. It has also been reported that the culture method is effective (Japanese Patent Laid-Open No. 2000-189157). In the aforementioned report, one of the major features is circulation culture in which the human hematopoietic system-derived progenitor cell composition solution is circulated through the culture medium at a moderate rate such that at least 50% is exchanged per day. It has been reported that human stem cells can be obtained efficiently by using this method.

DESCRIPTION OF EXEMPLARY EMBODIMENTS Hereinafter, preferred embodiments for carrying out the invention will be described with reference to the accompanying drawings.
FIG. 1 is a perspective view showing an embodiment of the culture apparatus of the present invention. FIG. 2 is a plan view of the culture apparatus of the present invention shown in FIG. In FIG. 1, 101 is a substrate, 102 is an inlet, 103 is a discharge port, 104 is a cell culture chamber, 105 is a first channel, 106 is a second channel, 107 is a liquid storage chamber, 108 is a valve, 109 is a hole.

  The culture apparatus of the present invention includes a substrate 101, a cell culture chamber 104 for culturing cells on the substrate 101, a first flow path 105 for conveying cells and a culture solution of the cells to the cell culture chamber 104, A second flow path 106 connected to the flow path 105, a liquid storage chamber 107 provided in the second flow path 106 for storing a part of the culture solution in the cell culture chamber 104, A valve 108 for controlling the flow is provided. And after collect | recovering a part of culture solution of the said cell culture chamber 104 to the said liquid storage chamber 107, the said culture solution is returned to a cell culture chamber again, and is used for culture | cultivation.

  The material of the substrate 101 is not particularly limited as long as it has resistance to the culture solution sample. For example, semiconductor materials such as silicon, glass materials, metal materials, resin materials, and the like can be given. Further, it is desirable that the base material has such a light transmittance that it can be observed with an inverted microscope or the like, and the surface of the substrate can be modified by cleaning, pretreatment or the like. The substrate is cleaned by wet cleaning such as alkali cleaning, acid cleaning, aqueous solvent cleaning, organic solvent cleaning, and RCA cleaning, and dry cleaning such as ultraviolet irradiation, ozone irradiation, and oxygen plasma irradiation.

  The surface modification of the substrate in the present invention is, for example, a method of treating with an acid, plasma, ozone, an organic solvent, an aqueous solvent, a surfactant or the like in advance when using a slide glass, a quartz substrate or the like as a solid substrate. , A method of introducing a substituent into the substrate surface by a treatment such as silane coupling, a method of controlling surface free energy, and the like. In the present invention, the base material of the porous material is selected from those having good moldability, those to which sterilization can be applied, and those having low cytotoxicity. More specifically, for example, polymers of synthetic resins such as cellulose, polyethylene, polypropylene, nylon, polyester, polyacrylamide, and fluororesin, inorganic materials such as glass, alumina, and titania, and metal materials such as gold, titanium, and stainless steel. It is done. As a culture solution sample used in the present invention, an aqueous solvent comprising a cell culture solution, a buffer solution or the like in which cells or drugs are suspended is basically used. Cell culture solutions and buffer solutions having known standard compositions can be used as appropriate.

  In the present invention, the flow path constituting the culture apparatus is configured by adhering or joining the upper and lower substrates, and the flow path, the cell culture chamber, and the liquid storage chamber are formed by the plurality of substrates. . As one example for forming a flow path, a cell culture chamber, and a liquid storage chamber by a plurality of substrates, a flow path is formed by a groove and a through-hole formed in one or both of the opposing surfaces of the substrate, and cell culture The chamber and the liquid storage chamber are formed as a through hole connected to the flow path and penetrating one of the substrates. In that case, it is only necessary to bond or bond the substrates constituting the upper and lower flow paths, and manufacturing is easy.

In the present invention, the bottom surface of the cell culture chamber may be any surface that can promote cell adhesion, such as physical adsorption, chemical adsorption, biological adsorption, electrostatic interaction, hydrophobic interaction, etc. It is particularly preferable that the surface state has an effect of promoting the above. Examples thereof include fibronectin, polylysine, RGD, collagen, laminin, antibodies against cell surface antigens, introduction of amino groups, carboxyl groups, epoxy groups, aldehyde groups, hydroxyl groups and the like onto the surface. The surface can be appropriately selected according to the purpose. In addition, it is preferable that an element for temperature adjustment is attached to the cell culture chamber, and an electrothermal converter such as a minute heater that converts an electrical signal into heat is preferably used as a means for imparting stimulation. . As the electrothermal transducer, for example, an electrothermal transducer including a heating resistor made of a metal such as Ta ₂ N, RuO ₂ , Ta, Ta—Al alloy, an alloy, or a metal compound can be used. The heating resistor can be formed by a known method, such as DC sputtering, RF sputtering, ion beam sputtering, vacuum deposition, or CVD. The electrothermal transducer may be inside the micropore or outside the micropore, and may be on the surface of the substrate or embedded in the substrate. For example, an electrothermal transducer that uses a printed wiring board as a heater and is bonded to the board to set an arbitrary place on the board to a predetermined temperature can be exemplified. On the other hand, as for cooling, an electrothermal transducer that sets an arbitrary place on the substrate to a predetermined temperature can be exemplified by pasting Peltier elements in the same manner.

  In addition to controlling the temperature of the cell culture chamber, an electrode for treating cells can be provided separately. Examples thereof include an apparatus for generating an electric field or a magnetic field for generating a dry force for capturing cells, an electrode for introducing a substance or the like into cells for electroporation, and the like. The substance mentioned here mainly includes fine particles, biomolecules or bioactive molecule groups, labeling agents, and the like. The fine particles are selected from synthetic polymers having a size that can be introduced into cells, degradable polymers, glasses, metals, ceramics, magnetic materials, and the like. The biomolecule or bioactive molecule group includes biomolecules selected from nucleic acids, proteins, saccharides, lipids, and complexes thereof. More specifically, DNA, RNA, aptamer, gene, chromosome, mitochondria, Select from viruses, antigens, antibodies, lectins, haptens, hormones, receptors, enzymes, peptides, antibiotics, preservatives, enzyme inhibitors, antipyretics, anti-inflammatory agents, growth factors, antiproliferative factors, tranquilizers, cytokines, steroids, estrogens, etc. can do. The labeling agent can be selected from fluorescent substances such as fluorescein and GFP, substances containing fluorescent substances, semiconductor fine particles, and the like.

  In the present invention, the culture apparatus includes at least two channels, a first channel for culturing cells, and a second flow for collecting and resupplying the culture solution in the cell culture chamber. Has a road. The width and depth of the first channel in the present invention are set in consideration of the cell diameter (10 μm or more and 30 μm or less). The liquid storage chamber 107 is arranged for temporarily recovering the culture solution used for cell culture in the cell culture chamber 104. The culture solution collected in the liquid storage chamber contains useful active substances such as cytokines produced by the cells, but also contains inactive substances such as waste products, so it is collected from the cell culture chamber. It is preferable to provide a processing means for performing at least one of heating, concentration, stirring, filtration, mixing, chemical reaction, and biochemical reaction for the culture broth. As an example, there may be mentioned a method having a mechanism for removing impurities by providing a filter so as to cross the flow path and filtering the collected culture medium.

  In the present invention, the first channel having a larger channel cross-sectional area than the second channel is selected. Specifically, assuming that the cross-sectional area of the first flow path is A and the cross-sectional area of the second flow path is B, a value in a range where the ratio A / B of the cross-sectional areas is 5 <A / B <1000000. Selected from. More preferably, A / B is selected from the range of 100 <A / B <100,000. As described above, the width and height of the first channel are selected based on the cell size, and the width can be selected from several tens to several hundreds of μm and the height of about 100 μm. The second channel may be selected from those having a smaller cross-sectional area than the first channel, and is defined by the height and width of the channel, but the height is constant, It can be easily produced by controlling only the width of the flow path.

  In the present invention, a pump and a valve are provided connected to at least one of the flow paths, and a part of the culture solution in the cell culture chamber is collected in the liquid storage chamber by the operation of the pump and the valve. 3 can be returned to the cell culture chamber from the first channel.

  The valve used in the present invention is characterized by utilizing a liquid resistance by changing the channel width. Specifically, the cross-sectional area of the second channel is smaller than that of the first channel. And since it is open | released to air | atmosphere, when a culture solution is conveyed, a culture solution is normally conveyed through a 1st flow path. When conveying the culture solution to the second flow path, it is performed by changing the external pressure.

  The valve used in the present invention may use the above-described liquid resistance, but is not limited thereto, and a desired one can be appropriately used from existing methods. Typical examples of the valve include those using a microactuator, those using a stimulus-responsive polymer, those using surface free energy in the flow path, and those using a valve. For example, a valve described in “Technology and Application of Micro Chemical Chip”, published by Maruzen Co., Ltd. in 2004 can be used.

Examples of the present invention will be described below, but the present invention is not limited to these examples.
Example 1
FIG. 3 is a perspective view schematically showing the culture apparatus of Example 1 of the present invention. A slide glass having grooves formed on the bottom substrate of the substrate 310 used in this example was manufactured using polydimethylsiloxane (PDMS) having through holes in the introduction port 302 and the discharge port 303 for the top substrate 301. . The slide glass was masked with photolithography and a metal sacrificial film, and a structure having a depth of 100 μm was formed by wet etching with hydrofluoric acid. The first channel width was 500 μm and the second channel width was 5 μm.

  A PDMS structure having a through hole was prepared by using a resist with a resist on a glass slide using a commercially available negative resist (SU-8; MicroChem Corp.). Next, the PDMS precursor was poured into a mold, and the polymer was solidified by heating in an oven at 90 ° C. for 1 hour with the mold and the prepolymer sandwiched between slide glasses. After allowing to cool, PDMS elastomer was obtained by peeling PDMS from the mold. At this time, in order to facilitate the peeling of the PDMS elastomer from the mold, a water repellent treatment may be performed in advance by introducing a substituent such as a fluoride onto the surface with a silane coupling agent. In addition, when PDMS elastomer is used as a base material, the substrates can be spontaneously bonded to each other. For adhesion between the PDMS elastomer and the slide glass, fusion by oxygen plasma (80 W, 30 seconds) can be used.

  Furthermore, the silicone tube and the inlet, the outlet, and the connector part at the tip of the liquid storage chamber were connected to the inlet, the outlet, and the connector part, and a three-way cock was connected to form a flow path having a valve part. A push-type syringe pump (Harvar Apparatus) was used as the driving force for conveying the culture medium in this example.

  Hereinafter, (1) the cell culturing step, (2) the step of recovering the culture solution from the cell culture chamber, and (3) the step of supplying the culture solution from the liquid storage chamber will be described with reference to FIGS. 4A to 4C. In addition, although a tank for a culture solution or the like is naturally provided between the pump and the inlet, it is omitted in the drawing.

(1) Cell culture process FIG. 4: A is explanatory drawing explaining the conveyance state of the culture solution in the flow path in the culture apparatus of Example 1 of this invention. In FIG. 4A, the state which conveys a cell and a culture solution to a cell culture room is shown.

  The syringe pump 401 is connected to the introduction port 402 and the cells and the culture solution are sent into the first flow path 406. At this time, the connector portions 409 and 410 are open to the atmosphere. The cells and the culture solution are discharged out of the system from the discharge port 403 through the first flow path 406, the cell culture chamber 404, and the first flow path which are main flow paths.

(2) Step of collecting culture solution from cell culture chamber FIG. 4B is an explanatory diagram for explaining the state of conveyance of the culture solution in the flow path in the culture apparatus of Example 1 of the present invention. FIG. 4B shows a state in which a part of the culture solution in the cell culture chamber is transferred to the liquid storage chamber.

  The valve portions 407 and 408 attached to the first flow path 406 are closed, and the valve portion 412 attached to the second flow path 411 is opened. By connecting the pump 401 to the upstream side of the fourth flow path 414 that is a side flow and operating the pump 401, a part of the culture solution remaining in the cell culture chamber 404 is transferred to the cell culture chamber 404, the second flow The liquid is stored in the liquid storage chamber 405 through the path 411.

(3) Step of Resupplying Culture Solution from Liquid Storage Chamber FIG. 4C is an explanatory view for explaining the state of conveyance of the culture solution in the flow path in the culture apparatus of Example 1 of the present invention. FIG. 4C shows a state in which the culture solution in the liquid storage chamber is returned to the cell culture chamber again.

  After the valve portions 407 and 408 attached to the first flow path are opened again and liquid feeding is started, the previous valve portion 412 of the liquid storage chamber 405 is opened. The culture solution stored in the liquid storage chamber 405 is discharged out of the system from the discharge port 403 through the second flow channel 411 and the cell culture chamber 404 together with the culture solution conveyed from the third flow channel 413. Is done. At this time, the culture solution stored in the liquid storage chamber 405 is mixed with the culture solution transported from the first flow path 406 and is transported to the cell culture chamber, whereby acclimation culture is realized. The In addition, by arranging a plurality of liquid storage chambers, it can be used as an apparatus for supplying liquid for a longer time or continuously.

Example 2
Furthermore, it can be used as a sampling device from a reaction tank including a cell culture chamber by arranging a flow path as shown in this embodiment and connecting a detector or a separation device. An outline of the configuration of the culture apparatus in the present example is shown in FIG. 6A and 6B are explanatory views for explaining the state of conveyance of the culture solution in the flow path in the culture apparatus of the second embodiment.

  As shown in the figure, by opening the valve portions 509 and 511 provided at the side end that is normally closed, a part of the culture solution passes through the reaction tank 507, the second flow path 505, and the cell culture chamber 508. 511 can be recovered. Note that the valve unit 504 is a valve disposed in the first flow path, and a desired one of the existing methods can be appropriately used. Specifically, it is appropriately selected from those using a microactuator, those using a stimulus-responsive polymer, those using surface free energy in the flow path, or those using a valve.

  Since the culture apparatus of the present invention can be cultured easily and efficiently in a microchip, it can be used for cell utilization techniques represented by substance production and regenerative medicine.

It is a perspective view which shows one embodiment of the culture apparatus of this invention. It is a top view of the culture apparatus of this invention shown in FIG. It is a perspective view which shows typically the culture apparatus of Example 1 of this invention. It is explanatory drawing explaining the conveyance state of the culture solution in the flow path in the culture apparatus of Example 1 of this invention. It is explanatory drawing explaining the conveyance state of the culture solution in the flow path in the culture apparatus of Example 1 of this invention. It is explanatory drawing explaining the conveyance state of the culture solution in the flow path in the culture apparatus of Example 1 of this invention. It is a perspective view which shows typically the culture apparatus of Example 2 of this invention. It is explanatory drawing explaining the conveyance state of the culture solution in the flow path in the culture apparatus of Example 2 of this invention. It is explanatory drawing explaining the conveyance state of the culture solution in the flow path in the culture apparatus of Example 2 of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 101 Substrate 102 Inlet 103 Outlet 104 Cell culture chamber 105 First channel 106 Second channel 107 Liquid storage chamber 108 Valve 109 Hole 301 Substrate 302 Inlet 303 Outlet 304 Cell culture chamber 305 First channel 306 Second flow path 307 Liquid storage chamber 308 Valve 309 Connector part (atmospheric release part)
310 Substrate 401 Pump 402 Inlet port 403 Discharge port 404 Cell culture chamber 405 Liquid storage chamber 406 First flow path 407 Valve 408 Valve 409 Connector portion (atmospheric release portion)
410 Connector part (atmosphere release part)
411 Second channel 412 Valve 413 Third channel 414 Fourth channel 501 Inlet 502 Ejector 503 First channel 504 Valve 505 Second channel 506 Valve 507 Reaction tank 508 Cell culture chamber 509 Valve 510 Valve 511 Valve 601 Pump

Claims (4)

  A culture apparatus that performs cell culture using a minute channel, a cell culture chamber, a first channel that transports cells and a culture solution of the cell to the cell culture chamber, and a first channel A connected second flow path; and a liquid storage chamber provided in the second flow path for storing a part of the culture liquid in the cell culture chamber. A culture apparatus characterized in that after a part is collected in the liquid storage chamber, the culture solution is returned to the cell culture chamber and used for culture.   A pump and a valve are connected to at least one of the first flow path and the second flow path, and a part of the culture solution in the cell culture chamber is transferred to the liquid storage chamber by operating the pump and the valve. The culture apparatus according to claim 1, wherein the culture apparatus is returned to the cell culture chamber after the collection.   The cross-sectional area ratio A / B is 5 <A / B <1000000, where A is the cross-sectional area of the first flow path and B is the cross-sectional area of the second flow path. The culture apparatus according to 1 or 2.   The liquid storage chamber includes a processing means for performing at least one of heating, concentration, stirring, filtration, mixing, chemical reaction, and biochemical reaction on the culture solution collected from the cell culture chamber. The culture apparatus according to any one of claims 1 to 3, wherein

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