Cell culture device and cell culture method

Abstract

To provide a technique that can coat a surface of a support with a proper amount of a scaffold material without excess and shortage, in a cell culture device.SOLUTION: A cell culture device includes a control unit, a flow path mechanism, a culture vessel including a support, and a measurement vessel equipped with a measurement device that measures an adsorption amount of a scaffold material, and performs a coating step of coating the support with the scaffold material and a culture step of seeding cells on the support and culturing them. The flow path mechanism supplies a scaffold material coating liquid to both the measurement vessel and the culture vessel in the coating step, and the control unit controls a supply state of the scaffold material coating liquid based on a measurement result of the measurement device in the coating step.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a cell culture device and the like that is capable of coating a scaffolding agent onto a support used for cell culture. [Background technology]

[0002] In general, when culturing adherent cells such as human iPS cells, the cells are attached to a support such as a petri dish or culture plate, and the surface of the support is coated with a scaffolding material to facilitate the adhesion of the adherent cells to the support.

[0003] Patent Document 1 proposes an apparatus configuration for coating a support with a scaffold material in a culture device. That is, the apparatus is described with a scaffold material solution delivery pipe for delivering the scaffold material from a scaffold material container storing the scaffold material solution to a culture container, and a waste liquid delivery pipe for discharging the scaffold material solution from the culture container to a drainage container. In the apparatus of Patent Document 1, the liquid delivery pump is stopped when a predetermined amount of scaffold material solution is supplied from the scaffold material container to the culture container, and the culture container is left to stand and the scaffold material is coated. After the coating on the culture container is completed by leaving it to stand for a predetermined time, the scaffold material solution is discharged from the culture container and a cell suspension is injected into the culture container.

[0004] Patent Document 2 describes a device for growing cells in a hollow fiber bioreactor, in which a coating solution containing a cell adhesive protein (scaffold) is circulated in a flow path to attach the scaffold to the bioreactor before the cells are grown. A component analysis unit is provided downstream of or within the culture vessel to detect changes in the concentration of scaffold components contained in the coating solution. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2019-59 A [Patent Document 2] Patent Publication No. 2021-48773 Summary of the Invention [Problem to be solved by the invention]

[0006] In order to perform proper cell culture, it is necessary to coat the surface of the support with an appropriate amount of scaffold material, without excess or deficiency. However, with conventional methods, the amount of scaffold material coated was not sufficiently accurate.

[0007] In the device of Patent Document 1, the liquid supply pump is stopped when a predetermined amount of scaffold solution is supplied from the scaffold container to the culture container, and the culture container is left to stand for a predetermined time to coat the culture container, but the reproducibility of the coating amount was not always sufficient. For example, when the concentration or temperature of the scaffold solution supplied fluctuates, the amount of coating of the scaffold may fluctuate in a management method that keeps the liquid supply amount and the standing time constant.

[0008] In the device of Patent Document 2, when a scaffold adheres to a hollow fiber bioreactor (support), the concentration of the scaffold component in the coating liquid decreases as a result. Therefore, a component analysis unit that detects the change in the concentration of the scaffold contained in the coating liquid is arranged downstream of the culture vessel or in the culture vessel, and the amount of the scaffold adhered to the bioreactor is estimated based on the component analysis result. However, even if an attempt is made to measure the change in the concentration of the scaffold component remaining in the coating liquid, high measurement accuracy is generally not obtained, so it is difficult to accurately estimate the amount of the scaffold adhered to the bioreactor based on the measurement result. In addition, if the component analysis unit is arranged near the hollow fiber bioreactor in the culture vessel, it is not practical because the component analysis unit may adversely affect the cell culture after the scaffold is coated.

[0009] Therefore, there was a need for a technology that could coat the surface of a support with an appropriate amount of scaffolding material, without excess or deficiency. [Means for solving the problem]

[0010] A first aspect of the present invention is a culture apparatus comprising a control unit, a flow path mechanism, a culture vessel equipped with a support, and a measurement vessel equipped with a measurement device for measuring the adsorption amount of a scaffold material, and which performs a coating step of coating the support with a scaffold material, and a culture step of seeding cells onto the support and culturing them, wherein in the coating step, the flow path mechanism supplies a scaffold material coating liquid to both the measurement vessel and the culture vessel, and the control unit controls the supply state of the scaffold material coating liquid based on the measurement results of the measurement device in the coating step.

[0011] In addition, a second aspect of the present invention is a cell culture method using a culture apparatus comprising a control unit, a flow path mechanism, a culture vessel equipped with a support, and a measurement vessel equipped with a measurement device for measuring the adsorption amount of a scaffold material, the cell culture method comprising a coating step of coating the support with a scaffold material, and a culture step of seeding cells onto the support and culturing them, wherein in the coating step, the flow path mechanism supplies a scaffold material coating liquid to both the measurement vessel and the culture vessel, and the control unit controls the supply state of the scaffold material coating liquid based on the measurement results of the measurement device. Effect of the Invention

[0012] According to the present invention, it is possible to coat the surface of a support with an appropriate amount of scaffold material, without excess or deficiency. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic block diagram for explaining the overall configuration of a culture system according to an embodiment. [Diagram 2] FIG. 1 is a schematic block diagram for explaining the configuration of a culture device according to embodiment 1. [Diagram 3] 1 is a flowchart for explaining a processing procedure of a cell culture method according to an embodiment. [Figure 4] 1 is a flowchart for explaining the processing procedure of a scaffold material coating process according to an embodiment. [Diagram 5] Graph showing a schematic representation of the progression of the adsorption state of the scaffold material. [Figure 6] FIG. 11 is a schematic block diagram for explaining the configuration of a culture device according to a second embodiment. [Figure 7] FIG. 11 is a schematic block diagram for explaining the configuration of a culture device according to embodiment 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The cell culture device, the scaffold coating method, and the like, which are embodiments of the present invention, will be described with reference to the drawings. The embodiments shown below are merely examples, and those skilled in the art can appropriately modify and implement the detailed configurations, for example, without departing from the spirit and scope of the present invention.

[0015] In the drawings referred to in the following description of the embodiments, elements denoted by the same reference numerals have the same functions unless otherwise noted. In the case where a plurality of identical elements are arranged in a drawing, the reference numerals and their explanations may be omitted. In addition, since the drawings may be represented diagrammatically for convenience of illustration and explanation, the shapes, sizes, arrangements, etc. of elements depicted in the drawings may not strictly correspond to the actual objects.

[0016] [Embodiment 1] (Overall configuration of the culture system) 1 is a schematic block diagram for explaining the overall configuration of a culture system according to an embodiment. The culture system 100 includes a culture device 101, a temperature adjustment mechanism 401, a humidity adjustment mechanism 501, an atmosphere adjustment mechanism 601, containers 301 to 304, and a switching valve 185.

[0017] The culture device 101 is a device for culturing adherent cells, such as human iPS cells. The culture device 101 can be supplied with various liquids, such as a scaffold coating liquid, a diluting liquid, a cell suspension, a culture medium, a cell detachment liquid, and a cleaning liquid, from containers 301 and 302. After a scaffold is coated on a support in the culture device 101, cells are adhered and cultured. The culture device 101 is provided with inlets 201 and 202 for introducing various liquids, and an outlet 203 for removing waste liquid and cultured cells. The culture device 101 will be described in detail later.

[0018] The container 301 is a container that contains, for example, a diluting solution, a cell suspension, a culture medium, a cell detachment solution, and a cleaning solution. The container 302 is a container that contains, for example, a scaffold coating solution. The container 303 is a container that contains waste liquid discharged from the culture device 101. The container 304 is a container that contains cells detached from the support after culture. The switching valve 185 is a valve that switches whether the liquid discharged from the culture device 101 is stored in the container 303 or the container 304.

[0019] The temperature adjustment mechanism 401 is a device that controls the temperature inside the culture device 101. The humidity adjustment mechanism 501 is a device that controls the humidity inside the culture device 101. The atmosphere adjustment mechanism 601 is a device that adjusts the partial pressures of oxygen and carbon dioxide inside the culture device 101.

[0020] A container 301 is connected to the inlet 201 of the culture device 101, and the container 301 storing different liquids is connected to the inlet 201 depending on the cell culture process. When a diluent for diluting the scaffold agent coating liquid is supplied to the culture device 101, for example, a container 301 storing phosphate buffered saline is connected to the inlet 201. When a cell suspension is supplied to the culture device 101, a container 301 storing a cell suspension in which cells to be cultured (e.g., iPS cells, ES cells) are suspended in phosphate buffered saline is connected to the inlet 201. When a culture medium is supplied to the culture device 101, a container 301 storing a culture medium suitable for the cells to be cultured is connected to the inlet 201. For example, when the cells to be cultured are iPS cells, the Stem Fit (registered trademark) series manufactured by Ajinomoto Co., Inc. or Stem Span (registered trademark) manufactured by STEMCELL Technology Co., Ltd. can be used as the culture medium. When a cell detachment liquid is supplied to the culture device 101, for example, a container 301 storing trypsin or ethylenediaminetetraacetic acid is connected to the inlet 201. When a cleaning liquid is supplied to the culture device 101, for example, a container 301 storing phosphate buffered saline is connected to the inlet 201.

[0021] A container 302 is connected to the inlet 202 of the culture device 101, and in the scaffold coating step, the container 302 storing a scaffold coating liquid is connected to the inlet 202. The scaffold is not particularly limited, but may contain at least one selected from the group consisting of full-length or fragmented laminin, collagen, gelatin, fibronectin, vitronectin, elastin, albumin, polylysine, polyornithine, and fragments thereof. The liquid for dissolving the scaffold is not particularly limited, but may be, for example, phosphate buffered saline.

[0022] The containers 301 and 302 are desirably made of a material to which cells or scaffolds are unlikely to adhere, and polypropylene is a suitable material for the containers. The shape of the containers 301 and 302 is not particularly limited, and examples of the containers include tanks, bottles, vials, and flasks.

[0023] Container 303 and container 304 are connected to the outlet 203 of the culture device 101 via a switching valve 185. Container 303, which contains the waste liquid discharged from the culture device 101, is preferably made of a material to which scaffolding material or cells are unlikely to adsorb, for example, polypropylene. Container 303 is preferably a sealed container to prevent the discharged solution from leaking into the atmosphere, for example, a plastic tank with a lid is preferably used. Container 304, which contains the cells detached from the support after culture, is preferably an openable and closable container.

[0024] (Configuration of the culture device) 2 is a schematic block diagram for explaining the configuration of the culture device 101. The culture device 101 includes a culture section 130, a measurement section 120, a control section 190, and a flow path mechanism.

[0025] The control unit 190 is a computer that controls the operation of each part of the culture device 101. The control unit 190 may be a lower-level computer that operates under the management of a higher-level computer included in the culture system 100, or may be a computer that performs integrated control of the entire culture system 100. The control unit 190 is provided with a computer-readable recording medium that stores a program for the culture device 101 to execute a predetermined process in each step of cell culture. For example, a hard disk, a flexible disk, an optical disk, a magneto-optical disk, a magnetic tape, a USB memory, an SSD, etc. can be used as the recording medium. The control unit 190 acquires the measurement values ​​of various sensors included in the culture unit 130 and the measurement unit 120, and controls control elements such as pumps and valves to execute each step of cell culture including the scaffold coating step.

[0026] The flow path mechanism provided inside the culture device 101 can be connected to the outside via an inlet 201, an inlet 202, and an outlet 203. For convenience, the side of the flow path mechanism closer to the inlet 201 or the inlet 202 is called the upstream side, and the side closer to the outlet 203 is called the downstream side. As described below, in the flow path mechanism, various parts including valves and pumps are connected by flow paths. For the flow paths, tubes or pipes made of a material such as polyolefin to which scaffolding material or cells to be cultured are unlikely to adsorb are used.

[0027] The inlet 201 and the inlet 202 are connected to the three-way valve 181 via a flow path. From the three-way valve 181 toward the downstream side, the on-off valve 182, the temperature control unit 170, and the supply pump 150 are connected in series in this order via a flow path. At the downstream side of the supply pump 150, the flow path branches into the flow path 11 and the flow path 13, the flow path 11 is connected to the inlet of the measurement unit 120, and the flow path 13 is connected to the inlet of the culture unit 130. The outlet of the measurement unit 120 is connected to the downstream side via the flow path 12, and the outlet of the culture unit 130 is connected to the downstream side via the flow path 14. The flow path 12 and the flow path 14 merge and are connected to the inlet of the discharge pump 160. The outlet of the discharge pump 160 is connected to the discharge port 203 via a flow path. In this way, in the flow path mechanism inside the culture device 101, the measurement unit 120 and the culture unit 130 are connected in parallel.

[0028] The flow rate ratio of the liquids introduced from inlet 201 and inlet 202 is adjusted by three-way valve 181. For example, in a scaffold coating step, a scaffold coating liquid containing scaffold components is supplied from inlet 202, and a diluting liquid is supplied from inlet 201, and the three-way valve 181 can adjust these flow rate ratios in response to a control command from control unit 190. It is desirable for the three-way valve 181 to be capable of varying the flow rate ratio stepwise between 100:0 and 0:100, but in some cases it may be a switching valve that is only capable of switching the flow path connection.

[0029] The on-off valve 182 controls the opening and closing of the flow path according to a control command from the control unit 190. When liquid is to be sent only to the culture unit 130 or to both the culture unit 130 and the measurement unit 120, the on-off valve 182 is opened. When liquid is not to be sent to either the culture unit 130 or the measurement unit 120, the on-off valve 182 is closed.

[0030] The temperature control unit 170 adjusts the temperature of the liquid sent to the culture unit 130 (both the culture unit 130 and the measurement unit 120 in the scaffold coating process) according to a control command from the control unit 190 so that the temperature of the liquid is suitable for the process. The temperature control unit 170 preferably includes both a mechanism for heating the liquid, such as a heater, and a mechanism for cooling the liquid, such as a Peltier element. The control unit 190 can control the operation of the temperature control unit 170 based on the liquid temperature measured by the sensor unit 141 or sensor unit 142 described below.

[0031] The supply pump 150 is a pump that sends liquid to the culture section 130 (both the culture section 130 and the measurement section 120 in the scaffold material coating process) in response to a control command from the control section 190. The control section 190 can control the operation of the supply pump 150 based on the flow rate measured by the sensor section 141 or the sensor section 142 described below so as to send liquid at an optimal flow rate for each process.

[0032] The discharge pump 160 is a pump that discharges liquid from the culture section 130 (both the culture section 130 and the measurement section 120 in the scaffold coating step) in response to a control command from the control section 190.

[0033] The culture section 130 is a section where cells are cultured, and includes a culture vessel 132, a support 131, a flow path opening / closing valve 184, and a sensor section 142. The support 131 that supports cells is provided inside the culture vessel 132. For example, a member separate from the culture vessel 132, such as a culture plate, may be provided inside the culture vessel 132 as the support 131, or the inner surface of the culture vessel 132 itself may serve as the support 131.

[0034] Various liquids are introduced into the culture vessel 132 through the flow channel 13 and discharged through the flow channel 14 in each step of cell culture, and a flow channel opening / closing valve 184 controls the opening and closing of the flow channel and the flow rate according to a control command from the control unit 190. The sensor unit 142, which is disposed downstream of the culture vessel 132, is equipped with a temperature sensor that measures the temperature of the liquid discharged from the culture vessel 132 and a flow rate sensor that measures the flow rate, and the measurement results are transmitted to the control unit 190.

[0035] The culture vessel 132 is detachably attached to the culture device 101. As the culture vessel 132, a petri dish-type, tray-type, or well-type vessel may be suitably used to enable observation and movement of cells, but is not limited thereto. The culture vessel 132 is preferably openable and closable to facilitate control of the carbon dioxide partial pressure and oxygen partial pressure in the medium. When the inner surface of the culture vessel 132 itself is made to function as the support 131, the material of the culture vessel 132 is preferably a material suitable for attaching a scaffolding material, such as a thermoplastic resin such as polystyrene or polycarbonate. There is no particular limitation on the cross-sectional shape, number, or length of the culture vessel 132.

[0036] The measurement section 120 is a device for measuring the adsorption state of the scaffold in order to allow the scaffold to be adsorbed just enough onto the surface of the support 131 of the culture vessel 132. The measurement section 120 includes a measurement vessel 122, a measurement device 121, a flow path opening / closing valve 183, a sensor section 141, a measurement processing section 123, and a communication section 124.

[0037] In order to measure the amount of adsorption of the scaffold material, it is conceivable to provide a measurement mechanism such as a sensor near the support 131 in the culture vessel 132, but this is not practical because the measurement mechanism may adversely affect the subsequent cell culture. Therefore, in this embodiment, when supplying the scaffold material coating liquid to the culture vessel 132, a flow path configuration is adopted in which the measurement vessel 122 is arranged so as to be connected in parallel with the culture vessel 132, and the scaffold material coating liquid is supplied to both the culture vessel 132 and the measurement vessel 122.

[0038] A measuring device 121 is disposed in the measurement container 122, and the measuring device 121 includes an adsorption surface to which the scaffold is adsorbed, and a measuring section for measuring the amount of the scaffold adsorbed to the adsorption surface. In the scaffold coating step, the scaffold coating liquid is introduced from the flow path 11 into the measurement container 122 and discharged from the flow path 12, and a flow path opening / closing valve 184 controls the opening and closing of the flow path and the flow rate of the liquid according to a control command from the control section 190. For example, in a step in which it is not necessary to introduce liquid into the measurement container 122, such as a step of introducing a cell suspension into the culture container 132, the flow path opening / closing valve 183 is closed. A sensor section 141 disposed downstream of the measurement container 122 includes a temperature sensor for measuring the temperature of the liquid discharged from the culture container 132 and a flow rate sensor for measuring the flow rate.

[0039] In a first aspect of this embodiment, each part is configured so that adsorption of the scaffold material progresses in substantially the same manner on the adsorption surface of the support 131 in the culture vessel 132 and the measuring device 121 in the measurement vessel 122. The amount of adsorption of the scaffold material measured by the measuring device 121 is treated as the amount of adsorption on the support 131 in the culture vessel 132, and the coating process of the scaffold material is controlled based on the measurement result.

[0040] In order to ensure that the adsorption of the scaffold material proceeds in substantially the same manner on the adsorption surfaces of the support 131 and the measuring device 121, it is desirable to make the shapes (outer shape and surface roughness) and materials of the adsorption surfaces of the support 131 and the measuring device 121 substantially identical. Making them substantially identical means making them identical except for differences due to unavoidable reasons such as manufacturing errors and mounting constraints.

[0041] In addition, it is preferable that the culture vessel 132 and the measurement vessel 122 have substantially the same vessel size (internal volume), shape, and material. In addition, it is preferable that the control unit 190 controls the flow rate of the scaffold coating liquid supplied to the culture vessel 132 and the measurement vessel 122 so that the flow rates are substantially the same. Specifically, it is preferable to control the opening degree of the flow path opening / closing valve 184 and the flow path opening / closing valve 183 based on the flow rate measurement value by the sensor unit 142 and the sensor unit 141. In addition, it is preferable that the control unit 190 monitors the liquid temperature measurement value by the sensor unit 142 and the sensor unit 141, and confirms that the adsorption is proceeding at substantially the same temperature.

[0042] For example, when using a polystyrene culture vessel 132 and support 131, the measurement vessel 122 is a polystyrene vessel of the same shape as the culture vessel 132, and the measurement device 121 is a member of the same shape as the support 131 and having an adsorption surface coated with polystyrene.

[0043] In a second aspect of this embodiment, the relationship between the amount of adsorption on the adsorption surface of the measuring device 121 and the amount of adsorption on the support 131 is obtained in advance by experiment or simulation, and stored in the control unit 190. The control unit 190 can store the relationship between the two amounts of adsorption as, for example, a lookup table or a formula. Then, the amount of adsorption of the scaffold material measured by the measuring device 121 is converted into the amount of adsorption on the support 131 based on the stored relationship, and the coating process of the scaffold material is controlled based on the conversion result.

[0044] In this embodiment, since the adsorption amounts in the support 131 and the measuring device 121 do not necessarily need to be physically the same, for example, the measurement container 122 and the culture container 132 can be made different in size and shape. By reducing the volume of the measurement container 122, it is possible to miniaturize the culture device 101. Even if the measured values ​​of the flow rate and temperature of the scaffold material coating liquid in the sensor unit 142 and the sensor unit 141 are not the same, if the relationship between the adsorption amounts under those conditions is stored in the control unit 190, it is possible to find the adsorption amount in the support 131 based on the measured values.

[0045] The measuring section 120 of this embodiment is configured to be capable of measuring the total adsorption amount of the scaffold material and the change over time in the adsorption amount (or the adsorption rate). The measuring section 120 includes a measuring device 121 that measures the adsorption amount of the scaffold material, a measurement processing section 123 that processes measurement data from the measuring device 121, and a communication section 124 that transmits the processed measurement results to the control section 190. The measurement processing section 123 calculates the total adsorption amount and the change over time in the adsorption amount (or the adsorption rate) on the support 131 of the culture section 130 based on the adsorption amount measured by the measuring device 121.

[0046] A suitable example of the measuring device 121 is the QCM method (Quartz crystal microbalance method) that uses a quartz oscillator to measure the mass change due to the adsorption of a scaffolding material. The QCM method is a method that can quantify the amount of adsorption of a substance (e.g., a scaffolding material) on the sensor surface at the nano [g] level in real time. By analyzing the progress of adsorption, it is possible to measure the film thickness, surface adsorption amount, and surface adsorbed material viscoelasticity of a substance with a certain mass, from low molecules to proteins, nucleic acids, and polymer thin films. In the QCM method, a sensor made of a slice of a quartz crystal on which a metal thin film is formed, and a circuit that applies an AC electric field to the sensor can be used. When a voltage is applied to the sensor, the sensor vibrates at a certain frequency. When a substance (e.g., a scaffolding material) is adsorbed on the sensor, the frequency decreases according to its mass, so even a small amount of adsorption can be measured.

[0047] The measuring device 121 may be a device equipped with a laser light source, an optical waveguide, an SPR (Surface Plasmon Resonance), an optical interferometer, an ellipsometer, or the like, which optically measures the thickness of the adsorption layer, the bonding state between the adsorption layer and the sensor surface, and the like. In a method using SPR, a sensor made of a metal thin film, and a laser and a photodetector that measure the reflectance of the light from the sensor may be used. When a molecule is adsorbed to the sensor, the angle of incidence at which the reflectance is minimized when light is incident on the sensor changes, and the amount of adsorption can be measured from the change in the angle of incidence at which the reflectance is minimized. As the measuring device 121, a method using a QCM or a method using SPR is preferable in that a minute amount of adsorption can be measured with high time resolution.

[0048] Glasses made of soda lime, silicate, synthetic quartz, or various metal oxides, metals such as silicon and aluminum, and resins such as acrylic, polycarbonate, polystyrene, and polycycloolefin may be used for the adsorption surface of the measuring device 121. Although these materials may be used in combination, it is preferable to use resins that are easy to process and thermally deformable, as with the support 131 in the culture vessel 132.

[0049] The measurement processing unit 123 calculates the total adsorption amount and the change over time of the adsorption amount (or the adsorption rate) of the scaffold material on the support 131 in the culture vessel based on the measurement result of the measuring device 121. The measurement processing unit 123 can store the measurement result of the measuring device 121 and the calculation result based on it. For example, the measurement processing unit 123 calculates the total adsorption amount by integrating the measurement result of the measuring device 121, and calculates the change over time of the adsorption amount (or the adsorption rate) by differentiating it. Note that, when a measuring device capable of measuring the total adsorption amount and the change over time of the adsorption amount (or the adsorption rate) without performing calculations such as integration or differentiation is used, the measurement result may be used as it is. The measurement processing unit 123 transmits the calculation result to the communication unit 124. The communication unit 124 transmits the calculation result transmitted from the measurement processing unit 123 to the control unit 190.

[0050] (Cell culture method) A description will be given of a cell culture method using the cell culture system according to embodiment 1. Fig. 3 is a flow chart for explaining the processing procedure of the cell culture method according to this embodiment.

[0051] When cell culture is started, a cleaning step (step S11) is first performed. In the culture device 101, a container 301 that stores a cleaning liquid is connected to the inlet 201, and a container 303 that stores waste liquid is connected to the outlet 203. To clean the culture unit, the measurement unit, and the flow path mechanism in the culture device with the cleaning liquid, the control unit 190 controls each part of the culture device 101 as follows.

[0052] The three-way valve 181 connects only the inlet 201 of the inlet 201 and the inlet 202 to the flow path in the culture device 101, and the on-off valve 182, the flow path on-off valve 183, and the flow path on-off valve 184 are all opened. The supply pump 150 and the discharge pump 160 are driven to flow the cleaning liquid supplied from the container 301 into the culture section, the measurement section, and the flow path mechanism, and wash the inside while pushing out air bubbles. The used cleaning liquid is collected in the container 303 as waste liquid. At that time, the supply pump 150, the discharge pump 160, the flow path on-off valve 183, and the flow path on-off valve 184 are controlled so that the flow rate measured by the sensor section 141 and the sensor section 142 becomes a predetermined speed. The temperature control section 170 is driven so that the temperature of the cleaning liquid measured by the sensor section 141 and the sensor section 142 becomes a predetermined temperature. When the cleaning is sufficiently performed, the supply pump 150 and the discharge pump 160 are stopped and the valves are closed, completing the cleaning step (step S11).

[0053] Next, a scaffold coating step (step S12) is performed. In the culture device 101, a container 301 storing a diluent is connected to the inlet 201, a container 302 storing a scaffold coating liquid containing scaffold components is connected to the inlet 202, and a container 303 storing waste liquid is connected to the outlet 203. Details of the scaffold coating step (step S12) will be described later with reference to FIG. 4. After the scaffold coating step (step S12) is completed, a step (not shown) of cleaning the culture vessel and the flow path mechanism may be performed before starting the cell seeding step (step S13). In this case, the diluent is introduced from the inlet 201 and caused to flow, and the culture vessel and the flow path mechanism (and the measurement vessel in some cases) are cleaned.

[0054] Next, a cell seeding step (step S13) is performed. A container 301 in which a cell suspension is stored is connected to the inlet 201, and a container 303 for storing waste liquid is connected to the outlet 203. Generally, a liquid in which cells are dispersed in a medium or diluent is used as the cell suspension. The cell suspension contains at least adherent cells. The adherent cells may be pluripotent stem cells such as human iPS cells (human induced pluripotent stem cells). The three-way valve 181 connects only the inlet 201 of the inlet 201 and the inlet 202 to a flow path in the culture device 101. The opening / closing valve 182 and the flow path opening / closing valve 184 are opened, and the flow path opening / closing valve 183 is closed. The supply pump 150 and the exhaust pump 160 are driven to introduce the cell suspension supplied from the container 301 into the culture container 132 of the culture unit 130, and the cells are seeded on the support 131 coated with the scaffold material.

[0055] Next, a medium replacement step (step S14) is performed. A container 301 storing a medium is connected to the inlet 201, and a container 303 storing a waste liquid is connected to the outlet 203. The three-way valve 181 connects only the inlet 201 of the inlet 201 and the inlet 202 to a flow path in the culture device 101. The opening / closing valve 182 and the flow path opening / closing valve 184 are opened, and the flow path opening / closing valve 183 is closed. The supply pump 150 and the discharge pump 160 are driven to introduce the medium supplied from the container 301 into the culture container 132 of the culture unit 130, replacing the medium. The culture of cells is continued in the culture container 132 in which the medium has been replaced. The replacement of the medium may be performed multiple times until the culture is completed. For example, when culturing human iPS cells, media such as Stemspan (manufactured by STEMCELL Technology), StemFit series (manufactured by Ajinomoto Co., Inc.), and NutriStemXF (manufactured by Cosmo Bio Co., Ltd.) are preferably used.

[0056] When the cell culture is completed, a cell detachment step (step S15) is performed. A container 301 in which a cell detachment solution is stored is connected to the inlet 201, and a container 304 (FIG. 1) for collecting the detached cells is connected to the outlet 203. The three-way valve 181 connects only the inlet 201 of the inlet 201 and the inlet 202 to a flow path in the culture device 101. The opening / closing valve 182 and the flow path opening / closing valve 184 are opened, and the flow path opening / closing valve 183 is closed. The supply pump 150 and the discharge pump 160 are driven to introduce the cell detachment solution supplied from the container 301 into the culture container 132 of the culture unit 130, and the cultured cells are detached from the support 131 and collected in the container 304. For example, trypsin or EDTA (ethylenediaminetetraacetic acid) can be used as the cell detachment solution. In this manner, cells can be cultured and collected in the culture device 101. Next, the scaffold coating step (step S12) will be described in detail.

[0057] (Scaffolding coating process) 4 is a flow chart for explaining the procedure of the scaffold coating step according to this embodiment. When the scaffold coating step is started, a container 301 storing a dilution liquid is connected to the inlet 201 of the culture device 101, and a container 302 storing a scaffold coating liquid is connected to the inlet 202. In addition, a container 303 for storing waste liquid is connected to the outlet 203.

[0058] In this embodiment, the adsorption state of the scaffold is monitored using the measuring unit 120 in the scaffold coating step, but in order to ensure measurement accuracy, the following process (not shown) may be performed at the beginning of the scaffold coating step. That is, first, the diluent stored in the container 301 is introduced into the culture device and allowed to flow, while measuring the amount of adsorption by the measuring unit 120. While allowing only the diluent to flow, the measuring unit 120 waits for the measurement result to stabilize, and when the drift of the measurement value has subsided, step S21 is started.

[0059] In step S21, the scaffold coating liquid is sent to the culture section 130 and the measurement section 120, which are connected in parallel. The control section 190 uses the three-way valve 181 to control the flow rate ratio so that the flow rate of the scaffold coating liquid supplied from the container 302 and the flow rate of the dilution liquid supplied from the container 301 become a predetermined ratio. Then, when coating starts, the scaffold coating liquid containing the scaffold component at a preset initial concentration value is sent to the culture section 130 and the measurement section 120. Note that step S21 can be repeatedly executed by a control loop, but when it is executed via step S25 described later, the concentration value of the scaffold component contained in the scaffold coating liquid can be changed from the initial concentration value.

[0060] In order to send the liquid to the culture section 130 and the measurement section 120 connected in parallel to the flow path, the opening / closing valve 182, the flow path opening / closing valve 183, and the flow path opening / closing valve 184 are opened at appropriate openings. The supply pump 150 and the discharge pump 160 are driven to supply the scaffold coating liquid, the concentration of which is adjusted and supplied from the three-way valve 181, to the culture container 132 and the measurement container 122 connected in parallel to the flow path. The openings of the supply pump 150, the discharge pump 160, the flow path opening / closing valve 183, and the flow path opening / closing valve 184 are adjusted so that the flow rate measured by the sensor section 141 and the sensor section 142 becomes a predetermined speed. The temperature control section 170 is driven so that the liquid temperature of the scaffold coating liquid measured by the sensor section 141 and the sensor section 142 becomes a predetermined temperature. When the scaffold coating liquid is introduced into the culture container 132 and the measurement container 122, the scaffold is adsorbed to the support 131 and the measurement device 121.

[0061] Next, in step S22, the measurement unit 120 measures the adsorption amount of the scaffolding material using the measurement device 121, and the measurement processing unit calculates the adsorption state of the scaffolding material on the support 131 and transmits the measurement result to the control unit 190 via the communication unit 124. The calculated adsorption state of the scaffolding material includes, for example, the total adsorption amount and the change over time in the adsorption amount (or the adsorption speed).

[0062] Next, in step S23, the control unit 190 determines whether the total adsorption amount of the scaffold material on the support 131 has reached a predetermined value (target adsorption amount) based on the measurement result received from the measurement unit 120. Step S23 may be repeatedly executed by a control loop, but if it is determined that the total adsorption amount has reached the predetermined value (target adsorption amount) (step S23: Yes), the process proceeds to step S26.

[0063] In step S26, the control unit 190 sends a control signal to each unit to stop the supply of the scaffold coating liquid, and ends the scaffold coating process. When ending the scaffold coating process, for example, the opening / closing valve 182, the flow path opening / closing valve 183, and the flow path opening / closing valve 184 are closed, and the supply pump 150 and the discharge pump 160 are stopped.

[0064] When it is determined that the total adsorption amount has not reached the predetermined value (target adsorption amount) (step S23: No), the process proceeds to step S24. Based on the measurement result received in step S22, the control unit 190 determines whether the adsorption speed of the scaffolding material on the support 131 is within a predetermined range.

[0065] 5 is a graph showing a schematic diagram of the transition of the adsorption state of the scaffold material. The horizontal axis shows time, and the vertical axis shows the amount of adsorption. When the supply of the scaffold material coating solution starts at time T0, the amount of adsorption of the scaffold material increases with time. The goal is to have the target amount of scaffold material adsorbed onto the support 131 just right within a specified time TE.

[0066] Graph A, indicated by a solid line, shows the progression of adsorption under certain adsorption conditions (concentration, temperature, and flow rate of scaffold material components). Graph A shows that at time T1, which is earlier than a predetermined time TE, the amount of adsorption reaches a target amount of adsorption and reaches a saturated state, and the state in which the target amount of scaffold material is adsorbed is maintained after time T1. When executing step S21 for the first time after starting the scaffold material coating process, the control unit 190 controls each unit so that adsorption progresses according to the progression shown in graph A. That is, the control unit 190 stores in advance the conditions for the result to be as shown in graph A, and sets the concentration of the scaffold material components with the three-way valve 181, the temperature of the scaffold material coating liquid with the temperature control unit 170, and the flow rate with the supply pump 150, etc.

[0067] However, due to setting errors and fluctuations in various conditions, the adsorption does not necessarily progress as shown in graph A in reality. For example, when the concentration of the scaffold component is low, the increase rate of the adsorption amount is slow as shown in graph B indicated by the dashed line, and the adsorption may reach saturation at a level lower than the target adsorption amount. The same thing can also occur due to setting errors or fluctuations in the temperature and flow rate of the scaffold coating solution. In addition, although not shown in Figure 5, depending on the conditions such as the concentration and temperature of the scaffold component, the adsorption may reach saturation at a level higher than the target adsorption amount, and an excessive amount of scaffold may be coated.

[0068] In this embodiment, step S24 is repeatedly executed by a control loop until coating is completed, so that it is possible to determine in a timely manner whether the adsorption speed of the scaffold material on the support 131 is within a predetermined range.

[0069] In step S24, if it is determined that the adsorption rate is within the predetermined range (step S24: Yes), the process returns to step S21, and the scaffold coating liquid is continuously sent to the culture section 130 and the measurement section 120 under the same conditions.

[0070] If it is determined in step S24 that the adsorption rate is not within the predetermined range (step S24: No), the process proceeds to step S25, where the control unit 190 controls each unit so that the adsorption rate falls within the predetermined range. Specifically, if the measured adsorption rate is low and deviates from the predetermined range, the three-way valve 181 is controlled to reduce the mixing ratio of the diluent and increase the concentration of the scaffold material component. Conversely, if the measured adsorption rate is high and deviates from the predetermined range, the three-way valve 181 is controlled to increase the mixing ratio of the diluent and decrease the concentration of the scaffold material component.

[0071] Note that step S25 may be any process that controls the supply state of the scaffold-material coating liquid so that the adsorption rate falls within a predetermined range, and is not limited to adjusting the concentration of the scaffold-material components using the three-way valve 181. For example, control may be performed to adjust the temperature or flow rate of the scaffold-material coating liquid so that the adsorption rate falls within a predetermined range, or adjustment may be performed by appropriately combining the concentration, temperature, and flow rate.

[0072] The predetermined range (desirable range) of the adsorption speed can be set to a different numerical range as appropriate depending on the time elapsed after the time T0 when the scaffold coating liquid started to be fed. For example, the period A1 in Fig. 5 is a stage in which the adsorption of the scaffold is progressing rapidly, whereas the period A2 is a stage after the adsorption has progressed to a certain extent. For this reason, it is appropriate to set different numerical ranges as the predetermined range of the adsorption speed between the periods A1 and A2.

[0073] In the initial stage after the start of the adsorption process, such as in period A1, the adsorption speed (the slope of the adsorption amount graph) changes rapidly in a short time, so it is appropriate to set the predetermined range of the adsorption speed with a relatively large width. In the stage after the adsorption process has progressed to a certain extent, such as in period A2, it is necessary to accurately determine whether the target adsorption amount is reached within the predetermined time TE, so it is appropriate to set the predetermined range of the adsorption speed with a relatively small width.

[0074] For example, at the end of period A2, in step S24, the control unit 190 can calculate the future adsorption amount based on the adsorption amount and adsorption speed during period A2, and extend graph B to extrapolate graph D. In this way, the control unit 190 can determine that the target adsorption amount will not be reached within the specified time TE (i.e., step S24: No). Therefore, in step S25, the control unit 190 adjusts the supply state of the scaffold coating liquid (e.g., the concentration of the scaffold component) to obtain the dotted line graph C.

[0075] As described above, in the present embodiment, in the scaffold coating step, the scaffold is coated by flowing the scaffold coating liquid through a flow path configuration in which the culture section 130 and the measurement section 120 are connected in parallel. This allows the measurement section 120 to measure the coating status of the scaffold under the same conditions as those of the culture section 130, using the same scaffold coating liquid as that supplied to the culture section 130, without adversely affecting the culture section 130. By controlling the supply state of the scaffold coating liquid according to the measured adsorption amount and adsorption speed of the scaffold, it is possible to coat the support 131 with an appropriate amount of scaffold, neither too much nor too little, within a predetermined time.

[0076] [Embodiment 2] A description will be given of embodiment 2. Regarding matters common to embodiment 1, the description will be simplified or omitted.

[0077] (Overall configuration of the culture system) The overall configuration of the culture system according to this embodiment is similar to that of the first embodiment described with reference to Fig. 1. However, the culture system according to this embodiment differs from the first embodiment in that the culture system includes a culture device 102 described below instead of the culture device 101.

[0078] (Configuration of the culture device) FIG. 6 is a schematic block diagram for explaining the configuration of the culture device 102. The culture device 102 is common to the culture device 102 according to the first embodiment in that it includes a culture section 130, a measurement section 120, a control section 190, and a flow path mechanism. In the first embodiment, the flow path mechanism is configured so that the culture section 130 and the measurement section 120 are connected in parallel, but in the present embodiment, the flow path mechanism is configured so that the measurement section 120 is connected in series upstream of the culture section 130. In addition, a three-way valve 284 capable of switching the flow path is provided at the inlet section of the culture section 130 instead of the flow path opening / closing valve 184. The three-way valve 284 can switch whether the liquid injected from the measurement section 120 flows into the culture vessel 132 or into the bypass flow path 15. The three-way valve 284 connects the outlet of the measurement section 120 to the culture vessel 132 when, for example, a scaffold coating liquid, a cell suspension, a medium, or the like is supplied to the culture vessel 132. On the other hand, when discharging the used liquid from the measurement section 120 at the end of each process, the measurement section 120 is connected to the bypass flow path 15 so as not to contaminate the culture container 132 with the used liquid.

[0079] (Cell culture method) In this embodiment, cell culture is performed in the same process as in embodiment 1 described with reference to the flowchart in Fig. 3. In this embodiment, when various liquids are supplied to the culture container 132 in each of steps S11 to S15, the liquids are supplied to the culture container 132 via the measurement container 122.

[0080] (Scaffolding coating process) In this embodiment, the scaffold-coating step is performed in the same procedure as in embodiment 1 described with reference to the flowchart in Fig. 4. In this embodiment, the measurement unit 120 is connected in series to the upstream side of the culture unit 130, and the scaffold-coating liquid that has passed through the measurement unit 120 is supplied to the culture unit 130.

[0081] As already mentioned, in order to measure the amount of adsorption of the scaffold material, it is conceivable to provide a measuring mechanism such as a sensor near the support 131 in the culture vessel 132, but this is not practical since the measuring mechanism may adversely affect the subsequent cell culture. In this embodiment, when supplying the scaffold material coating liquid to the culture vessel 132, a flow path configuration is adopted in which a measuring vessel 122 is connected in series to the upstream side of the culture vessel 132, and the scaffold material coating liquid is supplied to the culture vessel 132 via the measuring vessel 122. A measuring device 121 is disposed in the measuring vessel 122, and the measuring device includes an adsorption surface to which the scaffold material is adsorbed and a measuring unit that measures the amount of the scaffold material adsorbed to the adsorption surface.

[0082] In this embodiment, the culture section 130 is supplied with the scaffold coating liquid after a certain amount of scaffold components have been adsorbed to the measurement section 120. It is guaranteed that no more than the amount of adsorption measured by the measurement section 120 has occurred in the culture section 130. When the scaffold coating process is completed, the scaffold coating liquid in the measurement container 122 is discharged via the bypass flow path 15, thereby preventing excess adsorption from occurring in the culture container 132.

[0083] In this embodiment, the coating state of the scaffold material in the culture area can also be measured using the measurement unit without affecting the culture area. By controlling the supply state of the scaffold material coating liquid according to the measured adsorption amount and adsorption speed of the scaffold material, it is possible to coat the culture area with an appropriate amount of scaffold material, neither too much nor too little, within a specified time.

[0084] [Embodiment 3] A description will be given of embodiment 3. Regarding matters common to embodiment 1 or embodiment 2, description will be simplified or omitted.

[0085] (Overall configuration of the culture system) The overall configuration of the culture system according to this embodiment is similar to that of the first embodiment described with reference to Fig. 1. However, the culture system according to this embodiment differs from the first embodiment in that the culture system includes a culture device 103 described below instead of the culture device 101.

[0086] (Configuration of the culture device) FIG. 7 is a schematic block diagram for explaining the configuration of the culture device 103. The culture device 103 is common to the culture devices described as the first or second embodiment in that it includes a culture section 130, a measurement section 120, a control section 190, and a flow path mechanism. This embodiment also adopts a flow path configuration in which a measurement container 122 is connected in series to the upstream side of the culture container 132 when supplying the scaffold coating liquid to the culture container 132, as in the second embodiment. In this embodiment, a three-way valve 283 capable of switching the flow path is provided at the inlet of the measurement section 120, and a three-way valve 285 capable of switching the flow path is provided at the inlet of the culture section 130. The three-way valve 283 can switch whether the liquid sent from the supply pump 150 flows into the measurement container 122 or into the bypass flow path 16. The three-way valve 285 can switch whether the measurement section 120 or the bypass flow path 16 is connected to the inlet of the culture container 132.

[0087] (Cell culture method) In this embodiment, cell culture is performed in the same procedure as in the embodiment 1 described with reference to the flowchart in Fig. 3. In this embodiment, for example, when the scaffold coating liquid is supplied to the culture vessel 132 in step S12, the three-way valve 283 introduces the liquid sent from the supply pump 150 into the measurement vessel 122, and the three-way valve 284 connects the measurement unit 120 and the culture vessel 132. On the other hand, when the cell suspension, the culture medium, the cell detachment liquid, etc. are supplied to the culture vessel 132 in steps S13 and S14, the three-way valve 283 flows the liquid sent from the supply pump 150 into the bypass flow path 16, and the three-way valve 284 connects the bypass flow path 16 and the culture vessel 132. That is, the scaffold coating liquid is introduced into the culture vessel 132 via the measurement vessel 122, but the cell suspension, the culture medium, the cell detachment liquid, etc. are introduced into the culture vessel 132 without passing through the measurement vessel 122.

[0088] (Scaffolding coating process) In this embodiment, the scaffold coating step is also carried out in the same procedure as in embodiment 1 or embodiment 2 described with reference to the flow chart of FIG.

[0089] As already mentioned, in order to measure the amount of scaffold adsorption, it is conceivable to provide a measuring mechanism such as a sensor near the support 131 in the culture vessel 132, but this is not practical because the measuring mechanism may adversely affect the subsequent cell culture. In this embodiment, when the scaffold coating liquid is supplied to the culture vessel 132, a flow path configuration is adopted in which the measurement vessel 122 is arranged upstream so as to be connected in series with the culture vessel 132, and the scaffold coating liquid is supplied to the culture vessel 132 via the measurement vessel 122. A measuring device 121 is arranged in the measurement vessel 122, and the measuring device is provided with an adsorption surface to which the scaffold is adsorbed and a measuring unit that measures the amount of the scaffold adsorbed to the adsorption surface. In this embodiment, the scaffold coating liquid after a certain amount of scaffold components are adsorbed to the measuring unit 120 is supplied to the culture unit 130. Therefore, it is guaranteed that the adsorption in the culture unit 130 is not greater than the amount of adsorption measured by the measuring unit 120.

[0090] Also, unlike the second embodiment, when supplying a cell suspension, culture medium, cell detachment solution, and the like to the culture container 132, they are supplied via the bypass flow path 16 without passing through the measurement container 122. Therefore, the cell suspension and culture medium are not consumed in the measurement container 122, and unexpected cultured cells produced in the measurement container 122 are not detached and mixed in.

[0091] In this embodiment, the coating state of the scaffold material in the culture area can be measured using the measurement unit without affecting the culture area. By controlling the supply state of the scaffold material coating liquid according to the measured adsorption amount and adsorption speed of the scaffold material, it is possible to coat the culture area with an appropriate amount of scaffold material, neither too much nor too little, within a specified time.

[0092] [Other embodiments] The present invention is not limited to the above-described embodiment, and many modifications are possible within the technical spirit of the present invention. For example, the above-described different embodiments may be combined in whole or in part.

[0093] For example, in the culture system shown in Fig. 1, liquid is supplied from container 301 and container 302 to the culture device, and liquid is collected from the culture device to container 303 and container 304, so to speak, the liquid flows from the starting point to the end point. However, for example, a circulation flow path may be formed by a container that stores culture medium and the culture device, and the culture medium may be circulated and supplied to the culture device. Furthermore, the elements that constitute the flow path mechanism are not limited to those illustrated. It is possible to constitute the flow path mechanism using various types of pumps and valves.

[0094] This specification discloses at least the following: [Matter 1] The present invention comprises a control unit, a flow path mechanism, a culture vessel having a support, and a measurement vessel having a measurement device for measuring the amount of adsorption of a scaffold material, A culture device that performs a coating step of coating a scaffold material on the support and a culture step of seeding cells on the support and culturing the cells, In the coating step, the flow path mechanism supplies a scaffold coating liquid to both the measurement container and the culture container; The control unit controls a supply state of the scaffold-material coating liquid based on a measurement result of the measuring device in the coating step. A cell culture device characterized by: [Matter 2] The flow path mechanism connects the measurement container and the culture container in parallel in the coating step. 2. The cell culture device according to item 1, [Matter 3] The flow path mechanism connects the measurement container to the upstream side of the culture container in series in the coating step. 2. The cell culture device according to item 1, [Matter 4] The flow path mechanism delivers a cell suspension and / or a medium to the culture container in the culture step, but does not deliver the cell suspension and / or a medium to the measurement container. 4. The cell culture device according to any one of claims 1 to 3, [Matter 5] The control unit controls a concentration of a scaffold component contained in the scaffold coating solution based on a measurement result of the measuring device in the coating step. 5. The cell culture device according to any one of claims 1 to 4, [Matter 6] The control unit controls a flow rate of the scaffold coating liquid supplied to the culture vessel based on a measurement result of the measuring device in the coating step. 6. The cell culture device according to any one of claims 1 to 5, [Matter 7] The control unit controls a temperature of the scaffold coating solution supplied to the culture vessel based on a measurement result of the measuring device in the coating step. 7. The cell culture device according to any one of claims 1 to 6, [Matter 8] the control unit controls the flow path mechanism so that the flow rates of the scaffold-material coating solution supplied to the measurement container and the culture container are substantially the same in the coating step. 8. The cell culture device according to any one of claims 1 to 7, [Matter 9] The measurement container and the culture container have substantially the same internal volume. 9. The cell culture device according to any one of claims 1 to 8, [Matter 10] The support and the adsorption surface of the measuring device have substantially the same shape. 10. The cell culture device according to any one of claims 1 to 9, [Matter 11] The support and the adsorption surface of the measuring device are made of substantially the same material. 11. The cell culture device according to any one of claims 1 to 10, [Matter 12] The control unit controls a supply state of the scaffold-material coating liquid to the culture vessel based on a relationship between a previously obtained measurement result of the measuring device and an adsorption amount of the support. 12. The cell culture device according to any one of claims 1 to 11, [Matter 13] The control unit controls the supply state of the scaffold-material coating liquid based on the time-dependent change in the amount of adsorption or the adsorption rate measured by the measuring device so that the total amount of adsorption reaches a predetermined value within a predetermined time. 13. The cell culture device according to any one of claims 1 to 12, [Matter 14] The control unit stops supplying the scaffold-material coating liquid when the total adsorption amount measured by the measuring device reaches a predetermined value. 14. The cell culture device according to any one of claims 1 to 13, [Matter 15] The scaffold comprises at least one selected from the group consisting of laminin, collagen, gelatin, fibronectin, vitronectin, elastin, albumin, polylysine, polyornithine, and fragments thereof; 15. The cell culture device according to any one of claims 1 to 14, [Matter 16] The flow path mechanism supplies a cell suspension containing pluripotent stem cells to the culture vessel in the culture step. 16. The cell culture device according to any one of claims 1 to 15, [Matter 17] A cell culture method using a culture device including a control unit, a flow path mechanism, a culture vessel including a support, and a measurement vessel including a measurement device for measuring an adsorption amount of a scaffold material, comprising: A coating step of coating the support with a scaffold material; A culture step of seeding cells on the support and culturing the cells, In the coating step, the flow path mechanism supplies a scaffold-material coating liquid to both the measurement container and the culture container, and the control unit controls a supply state of the scaffold-material coating liquid based on a measurement result of the measurement device. A cell culture method comprising: [Matter 18] The flow path mechanism connects the measurement container and the culture container in parallel in the coating step. 18. The cell culture method according to item 17, [Matter 19] The flow path mechanism connects the measurement container to the upstream side of the culture container in series in the coating step. 18. The cell culture method according to item 17, [Matter 20] The flow path mechanism delivers a cell suspension and / or a medium to the culture container in the culture step, but does not deliver the cell suspension and / or a medium to the measurement container. 20. The method for culturing cells according to any one of items 17 to 19, [Matter 21] The control unit controls a concentration of a scaffold component contained in the scaffold coating solution based on a measurement result of the measuring device in the coating step. 21. The cell culture method according to any one of items 17 to 20, [Matter 22] The control unit controls a flow rate of the scaffold coating liquid supplied to the culture vessel based on a measurement result of the measuring device in the coating step. 22. The method for culturing cells according to any one of items 17 to 21, [Matter 23] The control unit controls a temperature of the scaffold coating solution supplied to the culture vessel based on a measurement result of the measuring device in the coating step. 23. The method for culturing cells according to any one of items 17 to 22, characterized in that [Matter 24] the control unit controls the flow path mechanism so that the flow rates of the scaffold-material coating solution supplied to the measurement container and the culture container are substantially the same in the coating step. 24. The method for culturing cells according to any one of items 17 to 23, [Matter 25] The control unit controls a supply state of the scaffold-material coating liquid to the culture vessel based on a relationship between a previously obtained measurement result of the measuring device and an adsorption amount of the support. 25. The method for culturing cells according to any one of items 17 to 24, [Matter 26] The control unit controls the supply state of the scaffold-material coating liquid based on the time-dependent change in the amount of adsorption or the adsorption rate measured by the measuring device so that the total amount of adsorption reaches a predetermined value within a predetermined time. 26. The cell culture method according to any one of items 17 to 25, characterized in that [Matter 27] The control unit stops supplying the scaffold-material coating liquid when the total adsorption amount measured by the measuring device reaches a predetermined value. 27. The method for culturing cells according to any one of items 17 to 26, [Matter 28] The scaffold comprises at least one selected from the group consisting of laminin, collagen, gelatin, fibronectin, vitronectin, elastin, albumin, polylysine, polyornithine, and fragments thereof; 28. The method for culturing cells according to any one of items 17 to 27, [Matter 29] The flow path mechanism supplies a cell suspension containing pluripotent stem cells to the culture vessel in the culture step. 29. The method for culturing cells according to any one of items 17 to 28, [Explanation of symbols]

[0095] 11-14···flow path / 100···culture system / 101-103···culture apparatus / 120···measurement section / 121···measurement device / 122···measurement container / 123···measurement processing section / 124···communication section / 130···culture section / 131···support / 132···culture container / 141-142···sensor section / 150···supply pump / 160···discharge pump / 170···temperature control section / 181···three-way valve / 182···on-off valve / 183~184···flow path on-off valve / 185···switching valve / 190···control section / 201~202···inlet / 203···outlet / 283···three-way valve / 284···three-way valve / 285···three-way valve / 301~304···container / 401···temperature control mechanism / 501···humidity control mechanism / 601···atmosphere control mechanism

Claims

1. The present invention comprises a control unit, a flow path mechanism, a culture vessel having a support, and a measurement vessel having a measurement device for measuring the amount of adsorption of a scaffold material, A culture device that performs a coating step of coating a scaffold material onto the support and a culture step of seeding cells onto the support and culturing the cells, In the coating step, the flow path mechanism supplies a scaffold coating liquid to both the measurement container and the culture container; The control unit controls a supply state of the scaffold-material coating liquid based on a measurement result of the measuring device in the coating step. A cell culture device characterized by:

2. The flow path mechanism connects the measurement container and the culture container in parallel in the coating step. The cell culture device according to claim 1 .

3. The flow path mechanism connects the measurement container to the upstream side of the culture container in series in the coating step. The cell culture device according to claim 1 .

4. The flow path mechanism delivers a cell suspension and / or a medium to the culture vessel in the culture step, but does not deliver the cell suspension and / or a medium to the measurement vessel. The cell culture device according to any one of claims 1 to 3.

5. The control unit controls a concentration of a scaffold component contained in the scaffold coating solution based on a measurement result of the measuring device in the coating step. The cell culture device according to any one of claims 1 to 3.

6. The control unit controls a flow rate of the scaffold coating liquid supplied to the culture vessel based on a measurement result of the measuring device in the coating step. The cell culture device according to any one of claims 1 to 3.

7. The control unit controls a temperature of the scaffold coating solution supplied to the culture vessel based on a measurement result of the measuring device in the coating step. The cell culture device according to any one of claims 1 to 3.

8. the control unit controls the flow path mechanism so that the flow rates of the scaffold-material coating solution supplied to the measurement container and the culture container are substantially the same in the coating step. The cell culture device according to any one of claims 1 to 3.

9. The measurement container and the culture container have substantially the same internal volume. The cell culture device according to any one of claims 1 to 3.

10. The support and the adsorption surface of the measuring device have substantially the same shape. The cell culture device according to any one of claims 1 to 3.

11. The support and the adsorption surface of the measuring device are made of substantially the same material. The cell culture device according to any one of claims 1 to 3.

12. The control unit controls a supply state of the scaffold-material coating liquid to the culture vessel based on a relationship between a previously obtained measurement result of the measuring device and an adsorption amount of the support. The cell culture device according to any one of claims 1 to 3.

13. The control unit controls the supply state of the scaffold-material coating liquid based on the time-dependent change in the amount of adsorption or the adsorption rate measured by the measuring device so that the total amount of adsorption reaches a predetermined value within a predetermined time. The cell culture device according to any one of claims 1 to 3.

14. The control unit stops supplying the scaffold-material coating liquid when the total adsorption amount measured by the measuring device reaches a predetermined value. The cell culture device according to any one of claims 1 to 3.

15. The scaffold comprises at least one selected from the group consisting of laminin, collagen, gelatin, fibronectin, vitronectin, elastin, albumin, polylysine, polyornithine, and fragments thereof; The cell culture device according to any one of claims 1 to 3.

16. The flow path mechanism supplies a cell suspension containing pluripotent stem cells to the culture vessel in the culture step. The cell culture device according to any one of claims 1 to 3.

17. A cell culture method using a culture device including a control unit, a flow path mechanism, a culture vessel including a support, and a measurement vessel including a measurement device for measuring an adsorption amount of a scaffold material, comprising: A coating step of coating the support with a scaffold material; A culture step of seeding cells on the support and culturing the cells, In the coating step, the flow path mechanism supplies a scaffold-material coating liquid to both the measurement container and the culture container, and the control unit controls a supply state of the scaffold-material coating liquid based on a measurement result of the measurement device. A cell culture method comprising:

18. The flow path mechanism connects the measurement container and the culture container in parallel in the coating step. The cell culture method according to claim 17 .

19. The flow path mechanism connects the measurement container to the upstream side of the culture container in series in the coating step. The cell culture method according to claim 17 .

20. The flow path mechanism delivers a cell suspension and / or a medium to the culture vessel in the culture step, but does not deliver the cell suspension and / or a medium to the measurement vessel. The cell culture method according to any one of claims 17 to 19.

21. The control unit controls a concentration of a scaffold component contained in the scaffold coating solution based on a measurement result of the measuring device in the coating step. The cell culture method according to any one of claims 17 to 19.

22. The control unit controls a flow rate of the scaffold coating liquid supplied to the culture vessel based on a measurement result of the measuring device in the coating step. The cell culture method according to any one of claims 17 to 19.

23. The control unit controls a temperature of the scaffold coating solution supplied to the culture vessel based on a measurement result of the measuring device in the coating step. The cell culture method according to any one of claims 17 to 19.

24. the control unit controls the flow path mechanism so that the flow rates of the scaffold-material coating solution supplied to the measurement container and the culture container are substantially the same in the coating step. The cell culture method according to any one of claims 17 to 19.

25. The control unit controls a supply state of the scaffold-material coating liquid to the culture vessel based on a relationship between a previously obtained measurement result of the measuring device and an adsorption amount of the support. The cell culture method according to any one of claims 17 to 19.

26. The control unit controls the supply state of the scaffold-material coating liquid based on the time-dependent change in the amount of adsorption or the adsorption rate measured by the measuring device so that the total amount of adsorption reaches a predetermined value within a predetermined time. The cell culture method according to any one of claims 17 to 19.

27. The control unit stops supplying the scaffold-material coating liquid when the total adsorption amount measured by the measuring device reaches a predetermined value. The cell culture method according to any one of claims 17 to 19.

28. The scaffold comprises at least one selected from the group consisting of laminin, collagen, gelatin, fibronectin, vitronectin, elastin, albumin, polylysine, polyornithine, and fragments thereof; The cell culture method according to any one of claims 17 to 19.

29. The flow path mechanism supplies a cell suspension containing pluripotent stem cells to the culture vessel in the culture step. The cell culture method according to any one of claims 17 to 19.

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