Culturing apparatus and culturing method

Abstract

A culture device 1 comprises: a culture vessel 2 for housing cell aggregates S and a liquid culture medium W; a suction tube 4 for sucking the liquid culture medium W in the culture vessel 2; a suction unit 8 for generating a suction force in the suction tube 4; and a control unit 14 for controlling the suction unit 8 in accordance with the opposing suction flow velocity of the aggregates S, said opposing suction flow velocity being the velocity at which the sedimentation of the aggregates S opposes the surfacing of the aggregates S due to the suction of the liquid culture medium W.

Description

Technical Field

[0001] The present invention relates to a culture device and a culture method.

Background Art

[0002] Conventionally, as a method for culturing cells in large quantities, suspension stirring culture of cell aggregates using a culture tank is known. Regarding such a culture method, Patent Document 1 discloses a cell culture device provided with an aspirator for sucking a culture solution. By sucking the culture solution with the aspirator and performing operations such as replacement of the culture solution, it is possible to improve the efficiency of cell culture.

[0003] The aspirator of Patent Document 1 has a double-tube structure composed of an outer tube and an inner tube. Further, the outer tube is provided with a filter through which the culture solution passes, and the inner tube sucks the culture solution that has passed through the filter. Furthermore, the outer tube is provided with air holes that communicate the inner cavity of the outer tube with the outside, and excessive negative pressure inside the outer tube can be released to the outside. In Patent Document 1, by providing such a structure, it was possible to suppress clogging of the filter by cell aggregates larger than the mesh size.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Even if excessive negative pressure is prevented from occurring inside the outer tube, as long as the outer tube is provided with a filter, it is inevitable that the aggregates will come into contact with the filter. As long as there is a chance for the aggregates to contact the filter, there is a risk that the aggregates will adhere to the filter. If the aggregates adhere to the filter, the suction of the culture solution will be inhibited, and the efficiency of cell culture may decrease. In addition, the growth and proliferation of cells may also be inhibited, and in this regard as well, the efficiency of cell culture may decrease.

[0006] The present invention has been made in view of such circumstances, and one of its objects is to provide a technique for improving the efficiency of cell culture.

Means for Solving the Problems

[0007] A culture apparatus according to an aspect of the present invention includes a culture vessel that houses cell aggregates and a culture solution, a suction tube that sucks the culture solution in the culture vessel, a suction unit that generates a suction force in the suction tube, and a control unit that controls the suction unit according to the counteracting suction flow rate of the aggregates, which is a rate that counteracts the sedimentation of the aggregates and the floating of the aggregates accompanying the suction of the culture solution.

[0008] Another aspect of the culture method of the present invention includes sucking the culture solution from a culture vessel that houses cell aggregates and a culture solution at a flow rate corresponding to the counteracting suction flow rate of the aggregates, which is a rate that counteracts the sedimentation of the aggregates and the floating of the aggregates accompanying the suction of the culture solution.

[0009] In addition, any combination of the above components, or those obtained by mutually substituting the components and expressions of the present invention among methods, apparatuses, systems, etc. are also effective as aspects of the present invention.

Effects of the Invention

[0010] According to the present invention, it is possible to improve the efficiency of cell culture.

Brief Description of the Drawings

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Embodiments for Carrying Out the Invention

[0012] Hereinafter, the present invention will be described with reference to the drawings based on preferred embodiments. The embodiments are illustrative and not restrictive of the invention, and all features and combinations thereof described in the embodiments are not necessarily essential to the invention. The same or equivalent components, members, and processes shown in each drawing are denoted by the same reference numerals, and repeated explanations are appropriately omitted. Also, the scales and shapes of the respective parts shown in each figure are set for convenience in order to facilitate the explanation, and are not to be construed restrictively unless otherwise specified. Further, when terms such as "first" and "second" are used in this specification or claims, these terms do not represent any order or importance, but are for distinguishing one configuration from another. Also, in each drawing, some members that are not important for explaining the embodiments are omitted from the display.

[0013] (Embodiment 1) FIG. 1 is a schematic diagram of a culture device 1 according to Embodiment 1. The culture device 1 of the present embodiment includes a culture container 2, a suction tube 4, an auxiliary filter 6, a suction unit 8, a management unit 10, a discharge tube 12, and a control unit 14.

[0014] The culture container 2 is a bioreactor that houses cell aggregates S and a culture solution W. The culture container 2 is not particularly limited as long as it can perform suspension culture of the aggregates S. As an example, the culture container 2 is a so-called spinner flask having a stirring blade 16 for stirring the culture solution W. Note that the culture container 2 may be a culture tank, a cell culture bottle, a cell culture bag, or the like. The volume of the culture container 2 is, for example, 100 mL to 100 L. The culture container 2 of the present embodiment may be a closed system in which the atmosphere does not substantially enter from the outside, or an open system in which the atmosphere can enter from the outside. When the culture container 2 is a closed system, it is necessary to control the concentrations of oxygen and carbon dioxide in the culture container 2. This concentration control is performed, for example, by the management unit 10 or a gas supply / discharge unit (not shown). The oxygen concentration is controlled to, for example, 1-6 ppm. The carbon dioxide concentration is controlled so that the pH of the culture solution W is 6.8-7.6, for example. When the culture container 2 is an open system, the above-described concentration control is not essential, but it is preferably performed.

[0015] The cells constituting the aggregates S are not particularly limited. For example, as the cells, pluripotent stem cells such as human iPS cells, human ES cells, and human Muse cells; differentiation-induced cells derived from pluripotent stem cells such as human iPS cell-derived nephron progenitor cells; somatic stem cells such as mesenchymal stem cells (MSCs); progenitor cells derived from somatic stem cells such as nephron progenitor cells; tissue cells such as human proximal tubular epithelial cells, human distal tubular epithelial cells, and human collecting duct epithelial cells; antibody-producing cell lines such as human fetal kidney cells (HEK293 cells); antibody-producing cell lines derived from animals other than humans such as Chinese hamster ovary cells (CHO cells) and insect cells (SF9 cells), etc. can be mentioned.

[0016] The culture medium W is not particularly limited and can be appropriately selected from known culture media according to the type of cells to be cultured and the like. For example, when culturing proximal tubular cells, REGM (Lonza), EpiCM (ScienCell), KeratinocyteSFM (Life Technologies), etc. can be used.

[0017] The suction tube 4 sucks the culture medium W in the culture vessel 2. One end of the suction tube 4 is inserted into the culture vessel 2 and contacts the culture medium W in the culture vessel 2. The other end of the suction tube 4 is connected to the inlet port of the management unit 10. The tube diameter of the suction tube 4 is not particularly limited as long as the culture medium W can be sucked by the suction unit 8.

[0018] An auxiliary filter 6 is provided in the middle of the suction tube 4. The auxiliary filter 6 is arranged at a predetermined interval from one end of the suction tube 4. The length of the suction tube 4 from the auxiliary filter 6 to the tip is preferably such that the suction flow rate of the culture medium W in at least the region in front of the auxiliary filter 6 is not affected by the flow of the culture medium W caused by stirring in the culture vessel 2, or is minimized to the extent that the invention can be implemented. At least in the region of this length from the tip of the suction tube 4, the suction flow rate of the culture medium W is adjusted to a flow rate corresponding to the antagonistic suction flow rate described later. Hereinafter, this region is referred to as the flow rate adjustment region. The flow rate adjustment region can be appropriately set based on the designer's empirical knowledge or experiments by the designer. Also, when the auxiliary filter 6 is omitted, the flow rate adjustment region is defined as the region from the tip of the suction tube 4 where the suction flow rate of the culture medium W is not affected by the flow of the culture medium W caused by stirring in the culture vessel 2, or is minimized as much as possible. For example, in the case of the culture vessel 2 using the rotary stirring method, the length from the auxiliary filter 6 to the tip is preferably 1 cm or more, for example. As an example, the auxiliary filter 6 is arranged in the region of the suction tube 4 that extends above the liquid level of the culture medium W, or in the region that extends outside the culture vessel 2. Note that the auxiliary filter 6 may be omitted. Also, the pore diameter of the auxiliary filter 6 is preferably less than the particle size of at least the aggregate S to be cultured, that is, the aggregate S that will ultimately be the recovery target, for example, 1 μm to 1000 μm.

[0019] Between the auxiliary filter 6 and the management unit 10 in the suction tube 4, a suction unit 8 is provided. The suction unit 8 generates a suction force in the suction tube 4. As the suction unit 8, a known suction device such as a peristaltic pump, a diaphragm pump, or an aspirator can be used. By driving the suction unit 8, the culture solution W is sucked from one end of the suction tube 4. The suction unit 8 can suck the culture solution W at a flow rate of, for example, 0.1 mL / min to 1 L / min. Note that the arrangement of the suction unit 8 is not particularly limited as long as a suction force can be generated in the suction tube 4.

[0020] The culture solution W sucked from the culture vessel 2 flows through the inside of the suction tube 4 toward the other end side, and flows into the management unit 10 via the auxiliary filter 6 and the suction unit 8. The management unit 10 performs at least one of analysis and adjustment of the components of the sucked culture solution W. For example, the management unit 10 has known analyzers such as a pH meter, a dissolved oxygen meter (DO meter), and a spectroscopic device, and can analyze the components of the culture solution W. Further, for example, the management unit 10 has a module for supplementing the culture solution W with medium components such as glucose and protein and oxygen. Further, for example, the management unit 10 has a column filled with an adsorbent for waste products such as lactic acid and ammonia, and can remove waste products in the culture solution W.

[0021] One end of the discharge tube 12 is connected to the outlet port of the management unit 10. The other end of the discharge tube 12 is inserted into the culture vessel 2. The culture solution W subjected to analysis processing and regeneration processing in the management unit 10 is returned to the culture vessel 2 via the discharge tube 12.

[0022] The control unit 14 controls the driving of the suction unit 8. The control unit 14 is realized by elements and circuits including a computer CPU and memory as a hardware configuration, and is realized by a computer program or the like as a software configuration. However, in FIG. 1, it is depicted as a functional block realized by the cooperation of these as appropriate. It is understood by those skilled in the art that this functional block can be realized in various forms by a combination of hardware and software.

[0023] For example, the user of the culture device 1 can select the operation mode of the culture device 1 via an operation panel (not shown) or the like. As an example, the operation mode includes a culture solution regeneration mode. The control unit 14 receives a signal indicating the selected operation mode from the operation panel and controls the suction unit 8 according to the operation mode. The control unit 14 controls the suction unit 8 according to the counteracting suction flow rate of the aggregate S. The counteracting suction flow rate is the rate at which the sedimentation of the aggregate S due to its own weight or the like and the floating of the aggregate S accompanying the suction of the culture solution W counteract each other. That is, the control unit 14 selects the magnitude of the suction flow rate of the culture solution W with respect to the counteracting suction flow rate of the aggregate S according to the purpose of the operation mode.

[0024] Generally, as the particle size of the aggregate S increases, the sedimentation rate of the aggregate S increases. Also, as the sedimentation rate increases, the suction rate that balances the sedimentation rate also increases. Therefore, there is a positive correlation between the particle size of the aggregate S and the counteracting suction flow rate. In addition, the particle size of the aggregate S has a positive correlation with the number of culture days (culture time). The relationship between the particle size of the aggregate S and the number of culture days can be appropriately set based on the designer's empirical knowledge or experiments by the designer according to the cell type, culture conditions, etc.

[0025] Therefore, the control unit 14 can specify the particle size of the aggregate S accommodated in the culture vessel 2, and thus the counteracting suction flow rate that serves as the reference for suction force adjustment, by counting the number of culture days. For example, the control unit 14 incorporates a timer and can count the number of culture days when a signal indicating the start timing of culture is input from the operation panel or the like. Also, the control unit 14 pre-holds a conversion table associating the number of culture days with the counteracting suction flow rate, and can determine the counteracting suction flow rate from the number of culture days using this conversion table.

[0026] Note that the particle size of the aggregate S in the culture solution W may be measured by known optical methods or the like. Further, the particle size of the aggregate S can also be determined based on parameters other than the culture days having a correlation with the particle size. Examples of such parameters include the consumption rate of glucose, the production rate of lactic acid, the consumption rate of oxygen, and the like. The control unit 14 can determine the counteracting suction flow rate using a conversion table associating the measured value of the particle size and the parameters with the counteracting suction flow rate. As an example, the particle size of the aggregate S is grasped as the average of the maximum value and the minimum value of the distance between two points located on the contour of the aggregate S in, for example, a microscopic image of the aggregate S.

[0027] When the culture solution regeneration mode is selected, the control unit 14 controls the suction unit 8 to suction the culture solution W at a flow rate equal to or lower than the counteracting suction flow rate of the aggregate S contained in the culture vessel 2. As a result, while the culture solution W is sent to the management unit 10, the suction of the aggregate S contained in the culture vessel 2 into the suction pipe 4 is suppressed. Note that the control unit 14 may always execute the analysis process and the regeneration process of the culture solution W regardless of the mode selection. Further, the control unit 14 may execute feedback control for switching the interruption and restart of the suction of the culture solution W based on the analysis result, receiving a signal indicating the analysis result of the culture solution W from the management unit 10 in either the case of executing the culture solution regeneration mode or always executing.

[0028] As described above, the antagonistic suction flow rate of the aggregate S is specified according to the number of culture days. On the other hand, there may be a distribution in the particle size of the aggregate S. That is, in the culture vessel 2 at a certain number of culture days, not only the main group of the aggregate S having the particle size to be taken at that number of culture days, but also the aggregate S with a smaller particle size, the aggregate S with a larger particle size, single cells, debris, etc. may be mixed. When the control unit 14 sucks the culture solution W at a flow rate equal to the antagonistic suction flow rate corresponding to the particle size of the main group, or at a flow rate with a small difference from the antagonistic suction flow rate, the aggregate S with a smaller particle size, etc. may be sucked into the suction pipe 4. The aggregate S, etc. sucked into the suction pipe 4 are suppressed from advancing to the management unit 10 side by the auxiliary filter 6. The pore size of the auxiliary filter 6 is preferably smaller than the target particle size of the aggregate S in cell culture, for example, 1 μm to 1000 μm. The target particle size of the aggregate S is, for example, 100 μm to 1500 μm.

[0029] Subsequently, the antagonistic suction flow rate as a reference for the suction force adjustment will be described in detail. The present inventors specified the antagonistic suction flow rate by the following procedure.

[0030] (Sedimentation test of aggregate S) First, a sedimentation test of the aggregate S was carried out to measure the sedimentation rate of the aggregate S. Specifically, mouse fetal-derived nephron progenitor cells (mNPC) were seeded at 50 μl (5000 cells) in a 96-well plate (PrimeSurface (registered trademark) plate 96U, Sumitomo Bakelite Co., Ltd.) and cultured using an mNPC medium. Thereby, aggregates S with various particle sizes were obtained. The particle size of the obtained aggregate S was measured using a microscope (BZ-X710, Keyence Corporation). Then, the aggregate S was gently sedimented in the mNPC medium, and the time t for passing through a predetermined drop L was measured. And the sedimentation rate w s (w s = L / t) was calculated. FIG. 2 is a diagram showing the relationship between the particle size and the sedimentation rate of the aggregate S. As shown in FIG. 2, it was confirmed that the sedimentation rate increases with the increase in the particle size.

[0031] (Suction test of aggregate S) Next, an aspiration test of the aggregate S was conducted, and the antagonistic aspiration flow rate of the aggregate S was measured. Specifically, The above sedimentation test in the experiment With the aggregates S of various particle sizes obtained placed in the suction tube 4, the culture solution W was aspirated at various aspiration flow rates. As the suction tube 4, those with tube diameters of φ6, φ8, φ10 (6 mm, 8 mm, 10 mm) were used. The medium temperature was set at 25°C. A peristaltic pump (WPX1, manufactured by WELCO) was used for aspiration. Then, the state of the aggregate S while aspirating the culture solution W was visually observed and evaluated. In the evaluation of the state, when the aggregate S settled, it was evaluated as "A", when the aggregate S remained, that is, when sedimentation and floating were antagonistic, it was evaluated as "B", and when the aggregate S floated, it was evaluated as "C". The aspiration flow rate obtained in evaluation B corresponds to the antagonistic aspiration flow rate for each particle size of the aggregate S.

[0032] FIG. 3 is a diagram showing the state of the aggregate S when the culture solution W is aspirated at various aspiration flow rates. As shown in FIG. 3, it was confirmed that for aggregates S of the same particle size, the same antagonistic aspiration flow rate was obtained regardless of the tube diameter of the suction tube 4. Therefore, it was confirmed that regardless of the tube diameter of the suction tube 4, by adjusting the aspiration flow rate of the culture solution W, the aggregate S can be made to settle or be in an antagonistic state, or be floated. That is, it was shown that by adjusting the aspiration flow rate of the culture solution W, it is possible to take out the culture solution W outside the culture container 2 without aspirating the aggregate S to be cultured. Note that the aspiration flow rate of the culture solution W is the value obtained by multiplying the aspiration flow rate by the tube diameter (inner diameter) of the suction tube 4. Therefore, by increasing the tube diameter of the suction tube 4, the aspiration flow rate can be increased while suppressing the aspiration of the aggregate S to be cultured.

[0033] (Comparison between sedimentation rate and antagonistic aspiration flow rate) From the results of the sedimentation test and aspiration test described above, the relationship between the sedimentation rate of the aggregate S and the antagonistic aspiration flow rate was derived. FIG. 4 is a diagram showing the relationship between the sedimentation rate of the aggregate S and the antagonistic aspiration flow rate. As shown in FIG. 4, by plotting the results of the aggregates S of each particle size on the coordinates with the sedimentation rate set on the first axis (horizontal axis) and the antagonistic aspiration flow rate set on the second axis (vertical axis) and performing linear approximation, a linear approximation formula showing the relationship between the sedimentation rate and the antagonistic aspiration flow rate was obtained.

[0034] Theoretically, if the suction flow rate of the culture solution W and the sedimentation rate of the aggregate S are the same value, the aggregate S should be in an antagonistic state without sedimenting or floating. However, in reality, although the sedimentation rate of the aggregate S and the antagonistic suction flow rate have a positive correlation, it has been confirmed that the antagonistic suction flow rate is lower than the sedimentation rate. This is presumably due to the influence of the pulsation of the pump constituting the suction unit 8 and the like.

[0035] (Creation of sedimentation rate prediction curve) If the sedimentation rate of the aggregate S for each particle size can be predicted and the antagonistic suction flow rate can be predicted based on this, for aggregates S of any particle size, the suction flow rate of the culture solution W that meets the purpose can be accurately determined. Therefore, the aggregate S of mNPC was regarded as spherical particles, and a sedimentation rate prediction curve of the aggregate S was created. For this creation, the method proposed in the paper: Jimenez, J. A. and Madsen, O. S., A simple formula to estimate settling velocity of natural sediments. Journal of Waterway, Port,Coastal, and Ocean Engineering Vol.129, No. 2, pp.70-78,2003 was used.

[0036] First, the following formula (1) [Number] [In formula (1), W * is the dimensionless sedimentation rate, w s is the sedimentation rate of the aggregate S [m / s], s is the specific gravity given by ρ s / ρ w , ρ s is the density of the aggregate S [kg / m 3 , ρ w is the density of the culture solution W [kg / m 3 , g is the acceleration due to gravity [m / s 2 , and d is the particle size of the aggregate S [m]] Using this, the sedimentation velocity of each particle size obtained in the sedimentation test described above was converted into the dimensionless sedimentation velocity W * Note that Equation (1) is a transformation of the first equation (balance equation) in the paper and is the second equation in the paper

[0037] Also, the following Equation (2) [Number] [In Equation (2), S * is a dimensionless particle parameter, ν is the kinematic viscosity of the culture solution W given by μ / ρ w [m 2 / s], μ is the viscosity of the culture solution W [Pa·s], ρ w is the density of the culture solution W [kg / m 3 , and d, s, and g are the same as in Equation (1)] Using this, the dimensionless particle parameter S * was calculated for the aggregate S of each particle size. Note that Equation (2) is the fourth equation in the paper

[0038] And the following Equation (3) [Number] [In Equation (3), W * is the same as in Equation (1), S * is the same as in Equation (2), and A and B are constants determined from a straight line obtained by plotting the values for each particle size of the aggregate S in a coordinate system with 1 / W * set as the first axis and 1 / S * set as the second axis] Using this, W * and S * for the aggregate S of each particle size were substituted into Equation (3) to obtain the constants A and B. Note that Equation (3) is the sixth equation in the paper. Specifically, as shown in Figure 5, with 1 / S * set as the first axis and 1 / W *By plotting the values of the aggregates S of each particle size at the coordinates set on the second axis and performing linear approximation, a linear approximation formula is obtained. Then, the constant A can be obtained from the intercept of the linear approximation formula, and the constant B can be obtained from the slope of the linear approximation formula. Figure 5 shows 1 / S * and 1 / W * and the relationship between them.

[0039] In the above series of calculations, the gravitational acceleration g was taken as 9.80655 m / s2, the density ρs of the aggregate S was taken as 1030 kg / m3, the density ρw of the culture solution W at 25°C was taken as 1005.294 kg / m3, and the viscosity μ of the culture solution W at 25°C was taken as 1.027 mPa·s. culture solution W The viscosity μ can be measured using a tuning fork vibration viscometer (SV-A, AND). As a result, in the example shown in Figure 5, A in Equation (3) was 0.89 and B was 3.82.

[0040] Note that in calculating the density ρ w of the culture solution W, the specific gravity s w of the culture solution W at t°C was calculated by the following method. That is, the weight W 1 [g] of a dry specific gravity bottle (1-4566-01, AS ONE), the weight W 2 [g] of the specific gravity bottle filled with pure water at t°C, and the weight W 3 [g] of the specific gravity bottle filled with the culture solution W at t°C were measured using an electronic balance (HR251-AZ, AND). Then, taking the density ρ air [g / cm 3 of air at t°C and the density ρ 0 [g / cm 3 of water at 4°C, the specific gravity susing the following formula (F1) published in "Measurement, Vol. 9, No. 10" of the "Liquid Specific Gravity Measurement Method (JIS) (Draft)"

Equation

[0041] Also, in calculating the density ρ s of the aggregate S, the specific gravity s swas calculated by the following method. That is, the weight W 4 [g] of a dry microtube (130 - 806C, WATSON) was measured with an electronic balance (HR251 - AZ, AND). Then, a plurality of aggregates S were collected in the microtube. After subjecting the microtube to centrifugation, the supernatant medium was removed, and the weight W 5 [g] was measured. The region occupied by the cells was replaced with pure water at t°C, and the weight W 6 [g] was measured. The density of water at t°C was ρ 1 [g / cm 3 , and the specific gravity s

Equation

[0042] Formula (F2) is based on the basic method of obtaining specific gravity, that is, the relational formula (specific gravity of an object) = (mass of the object) / (mass of water at 4°C with the same volume as the volume of the object). It is difficult to measure the weight of water under the condition of being controlled at 4°C. Therefore, by dividing the weight of water measured in a stabilized state at room temperature by the density of water at the same temperature, the volume of water at room temperature is calculated. Then, by dividing the weight of the aggregate S by the volume of water at room temperature and then dividing by the density of water at 4°C, the specific gravity s s of the aggregate S can be calculated.

[0043] Subsequently, substituting Formula (1) and Formula (2) into Formula (3), the following formula (4)

Equation

[0044] (Creation of the antagonistic suction flow rate prediction curve) Then, using the linear approximation formula showing the relationship between the sedimentation rate and the antagonistic suction flow rate described above, the sedimentation rate prediction curve obtained from formula (4) is converted into an antagonistic suction flow rate prediction curve. The control unit 14 adjusts the suction flow rate force of the culture solution W generated by the suction unit 8 based on the antagonistic suction flow rate determined based on this sedimentation rate prediction curve. FIG. 7 is a diagram showing the antagonistic suction flow rate prediction curve. Further, FIG. 8 is a diagram showing the predicted value of the sedimentation rate, the predicted value of the antagonistic suction flow rate, and the antagonistic flow rate at each pipe diameter for each particle size of the aggregate S. When the measured value of the antagonistic suction flow rate obtained in the suction test of the aggregate S described above was compared with the antagonistic suction flow rate prediction curve, it was confirmed that the two approximated with high precision.

[0045] As described above, the culture apparatus 1 according to the present embodiment includes a culture container 2 that houses the cell aggregate S and the culture solution W, a suction pipe 4 that sucks the culture solution W in the culture container 2, a suction unit 8 that generates a suction force in the suction pipe 4, and a control unit 14 that controls the suction unit 8 according to the antagonistic suction flow rate of the aggregate S, which is the rate at which the sedimentation of the aggregate S and the floating of the aggregate S accompanying the suction of the culture solution W are antagonistic. Thereby, operations such as suction of only the culture solution W (that is, separation of the culture solution W and the aggregate S) and classification of the aggregate S can be performed without using a filter. Therefore, it is possible to suppress the adhesion of the aggregate S to the filter, and thus the efficiency of cell culture can be improved.

[0046] Further, the control unit 14 of the present embodiment controls the suction unit 8 to suck the culture solution W at a flow rate below the antagonistic suction flow rate. Thereby, separation of the aggregate S and the culture solution W becomes possible without using a filter. Therefore, the efficiency of cell culture can be improved.

[0047] Further, in the present embodiment, the antagonistic suction flow rate is determined based on the sedimentation velocity prediction curve of the aggregate S obtained from the above formulas (1) to (4). Thereby, for aggregates S of any particle size, it is possible to suck the culture solution W while suppressing the suction of the aggregates S. Further, according to the purpose of sucking the culture solution W, a more suitable suction flow rate of the culture solution W can be set.

[0048] Further, the culture apparatus 1 of the present embodiment includes a control unit 10 that is connected to the suction pipe 4 and performs at least one of analysis and adjustment of the components of the sucked culture solution W. Thereby, the state of the cells and the culture solution W can be grasped more easily, and a better culture environment can be created. Therefore, the efficiency of cell culture can be further improved.

[0049] (Embodiment 2) Embodiment 2 has the same configuration as Embodiment 1 except for a part of the configuration of the culture apparatus 1 and the control content of the control unit 14. Hereinafter, the configuration different from Embodiment 1 in the present embodiment will be mainly described, and the common configuration will be briefly described or the description will be omitted. FIG. 9 is a schematic diagram of the culture apparatus 1 according to Embodiment 2. The culture apparatus 1 of the present embodiment includes a switching valve 18, a recovery pipe 20, and a recovery container 22 in addition to the culture container 2, the suction pipe 4, the suction unit 8, the control unit 10, the discharge pipe 12, and the control unit 14. The auxiliary filter 6 is omitted.

[0050] One end of the discharge pipe 12 and one end of the recovery pipe 20 are connected to the outlet port of the management unit 10 via a switching valve 18. The other end of the discharge pipe 12 is inserted into the culture vessel 2. The other end of the recovery pipe 20 is inserted into the recovery vessel 22. The switching valve 18 is composed of, for example, a known solenoid valve, and can switch the destination of the culture solution W flowing out from the outlet port of the management unit 10 between the discharge pipe 12 and the recovery pipe 20. When the switching valve 18 sets the destination of the culture solution W to the discharge pipe 12, the culture solution W that has passed through the management unit 10 is returned to the culture vessel 2 via the discharge pipe 12. When the switching valve 18 sets the destination of the culture solution W to the recovery pipe 20, the culture solution W that has passed through the management unit 10 is sent to the recovery vessel 22 via the recovery pipe 20. The culture vessel 2 and the recovery vessel 22 of the present embodiment may be a closed system in which the atmosphere does not substantially enter from the outside, or may be an open system in which the atmosphere can enter from the outside. When the culture vessel 2 and the recovery vessel 22 are closed systems, the above-described concentration control is performed on oxygen and carbon dioxide in the culture vessel 2 and the recovery vessel 22. When the culture vessel 2 and the recovery vessel 22 are open systems, the above-described concentration control is not essential, but it is preferable to perform it.

[0051] In addition, the culture apparatus 1 has a pipe 24 connecting the culture vessel 2 and the recovery vessel 22. The supernatant in the culture solution W contained in the recovery vessel 22 and not containing the aggregate S is returned to the culture vessel 2 via the pipe 24. Thereby, the amount of the culture solution W in the culture vessel 2 can be ensured. The movement from the recovery vessel 22 to the culture vessel 2 via the pipe 24 may be realized by utilizing the height difference of the liquid levels of the culture solution W in the recovery vessel 22 and the culture vessel 2 as shown in FIG. 9, or may be realized by providing a suction part such as a pump in the pipe 24. When a suction part is provided in the pipe 24, the arrangement of the pipe 24 is not limited to that shown in the figure.

[0052] The control unit 14 controls the drive of the suction unit 8 and the switching of the switching valve 18. The culture apparatus 1 of the present embodiment can select, as an example, a medium regeneration mode, an aggregate disposal mode, and an aggregate recovery mode. The control unit 14 controls the suction unit 8 and the switching valve 18 according to each operation mode.

[0053] When the culture solution regeneration mode is selected, as described in the first embodiment, the control unit 14 controls the suction unit 8 to suck the culture solution W at a flow rate equal to or lower than the antagonistic suction flow rate of the aggregate S contained in the culture vessel 2. Further, the control unit 14 controls the switching valve 18 so that the culture solution W flows into the discharge pipe 12. Thereby, the culture solution W subjected to the regeneration process by the management unit 10 is returned to the culture vessel 2.

[0054] When the aggregate disposal mode is selected, the control unit 14 controls the suction unit 8 to suck the culture solution W at a first flow rate that exceeds the antagonistic suction flow rate for the aggregate S of a predetermined first particle size and is equal to or lower than the antagonistic suction flow rate for the aggregate S of a second particle size larger than the first particle size. The first particle size is the particle size of the largest aggregate S among the aggregates S to be discarded. The second particle size is the particle size of the aggregate S to be cultured. Further, the control unit 14 controls the switching valve 18 so that the culture solution W flows into the recovery pipe 20. Thereby, the aggregate S to be cultured and the aggregate S to be discarded can be sorted (classified), and the aggregate S to be discarded can be moved from the culture vessel 2 to the recovery container 22. The aggregate S to be discarded accommodated in the recovery container 22 is discarded outside the apparatus.

[0055] In the culture solution regeneration mode, it is desirable not to suck even the aggregate S to be discarded as much as possible. On the other hand, in the aggregate disposal mode, it is desired to actively suck the aggregate S to be discarded. Therefore, as an example, the control unit 14 holds in advance a predetermined first margin M1 and a second margin M2 smaller than the first margin M1 with respect to the flow rate. Then, the margin selected according to the mode to be executed is subtracted from the reference antagonistic suction flow rate determined from the number of culture days or the like, and the flow rate to be generated by the suction unit 8 is determined.

[0056] Specifically, when the culture solution regeneration mode is selected, the suction unit 8 is controlled to suck the culture solution W at a flow rate obtained by subtracting a larger first margin M1 from the counteracting suction flow rate. Thereby, the amount of the aggregate S to be discarded that is sucked can be reduced. On the other hand, in the aggregate discard mode, the suction unit 8 is controlled to suck the culture solution W at a flow rate obtained by subtracting a smaller second margin M2 from the counteracting suction flow rate. That is, the second margin M2 is subtracted from the counteracting suction flow rate to determine the first flow rate. Thereby, the aggregate S to be discarded can be sucked more reliably. The first margin M1 and the second margin M2 can be appropriately set based on the designer's empirical knowledge or experiments by the designer, etc.

[0057] Also, when the aggregate recovery mode is selected, the control unit 14 controls the suction unit 8 to suck the culture solution W at a second flow rate that exceeds the counteracting suction flow rate for the aggregate S of the second particle size after sucking the culture solution W at the first flow rate. That is, the aggregate recovery mode includes, in part, the process executed in the aggregate discard mode. Further, the control unit 14 controls the switching valve 18 so that the culture solution W flows into the recovery pipe 20. Thereby, the aggregate S (aggregate S of the second particle size) that has grown until it becomes a recovery target can be moved from the culture container 2 to the recovery container 22. Note that the aggregate S of the second particle size can be isolated by moving the culture solution W sucked at the first flow rate and the culture solution W sucked at the second flow rate to separate recovery containers 22. The aggregate S accommodated in the recovery container 22 is used for the intended use. The second flow rate is determined as a value obtained by adding a predetermined third margin M3 to the counteracting suction flow rate corresponding to the aggregate S to be recovered so that the aggregate S to be recovered is sucked more reliably. The third margin M3 can be appropriately set based on the designer's empirical knowledge or experiments by the designer, etc.

[0058] In addition, in the aggregate recovery mode, it is preferable to suck the culture solution W by the suction unit 8 composed of an aspirator instead of a pump. Thereby, it is possible to suppress the aggregates S to be recovered from collapsing during the movement to the recovery container 22. Further, in the culture device 1 for discarding or recovering aggregates, the diameter of the suction pipe 4 is preferably larger. Thereby, the suction resolution of the aggregates S can be enhanced. For example, the sedimentation rate of the aggregates S having a particle size of several tens of μm to 2 mm is approximately 0.01 cm / second to 2 cm / second. When classifying with other aggregates S at a flow rate of 0.1 mL / min or more, the pipe diameter is preferably φ4 or more. When classifying with other aggregates S at a flow rate of 1 L / min or less, the pipe diameter is preferably φ50 or less.

[0059] The flow path structure for sending the sucked culture solution W to the recovery container 22 is not limited to that shown in FIG. 9. Further, the auxiliary filter 6 may be provided in the suction pipe 4 without being omitted. In this case, as an example, the culture device 1 separately includes a suction pipe 4 used in the culture solution regeneration mode, a suction pipe 4 used in the aggregate discard mode, and a suction pipe 4 used in the aggregate recovery mode. And each suction pipe 4 is provided with an auxiliary filter 6 having a pore diameter corresponding to the operation mode. The suction pipe 4 used in the culture solution regeneration mode is provided with the same auxiliary filter 6 as in the first embodiment. The suction pipe 4 used in the aggregate discard mode is provided with an auxiliary filter 6 through which the aggregates S to be discarded can pass. The pore diameter of this auxiliary filter 6 is set, for example, in the range of 100 μm to 1500 μm. The suction pipe 4 used in the aggregate recovery mode is provided with an auxiliary filter 6 through which the aggregates S to be recovered can pass. The pore diameter of this auxiliary filter 6 is set, for example, in the range of 100 μm to 1500 μm.

[0060] As described above, the control unit 14 according to the present embodiment controls the suction unit 8 to suck the culture solution W at a first flow rate that exceeds the antagonistic suction flow rate for the aggregate S having a predetermined first particle size and is equal to or less than the antagonistic suction flow rate for the aggregate S having a second particle size larger than the first particle size. Thereby, the aggregate S having the first particle size and the aggregate S having the second particle size can be classified. Therefore, if the aggregate S having the first particle size is determined as the object to be discarded and the aggregate S having the second particle size is determined as the object to be cultured, the aggregate S to be discarded can be discarded more easily. As a result, the aggregate S to be cultured can be cultured in a better environment, so that the efficiency of cell culture can be improved.

[0061] Further, after sucking the culture solution W at the first flow rate, the control unit 14 controls the suction unit 8 to suck the culture solution W at a second flow rate that exceeds the antagonistic suction flow rate for the aggregate S having the second particle size. Thereby, the target aggregate S can be recovered more easily. Therefore, the efficiency of cell culture can be improved.

[0062] (Embodiment 3) Embodiment 3 has the same configuration as Embodiment 1 except that the culture apparatus 1 includes a plurality of culture vessels 2 and a plurality of suction tubes 4. Hereinafter, the configuration different from that of Embodiment 1 in the present embodiment will be mainly described, and the common configuration will be briefly described or the description will be omitted. FIG. 10 is a schematic diagram of the culture apparatus 1 according to Embodiment 3. The culture apparatus 1 of the present embodiment includes a first culture vessel 2a, a second culture vessel 2b, a third culture vessel 2c, a first suction tube 4a, a second suction tube 4b, a third suction tube 4c, a suction unit 8, a management unit 10, a discharge tube 12, and a control unit 14. The auxiliary filter 6 is omitted.

[0063] One end of the first suction tube 4a is inserted into the first culture vessel 2a. The other end of the first suction tube 4a is inserted into the second culture vessel 2b. Also, one end of the second suction tube 4b is inserted into the second culture vessel 2b. The other end of the second suction tube 4b is inserted into the third culture vessel 2c. Also, one end of the third suction tube 4c is inserted into the third culture vessel 2c. The other end of the third suction tube 4c is connected to the inlet port of the control unit 10. A suction unit 8 is provided in the middle of the third suction tube 4c. Each culture vessel is a closed system where the atmosphere does not substantially enter from the outside. Therefore, by driving the suction unit 8, a suction force can be generated in the first suction tube 4a to the third suction tube 4c. One end of a discharge tube 12 is connected to the outlet port of the control unit 10. The other end of the discharge tube 12 is inserted into the first culture vessel 2a. Since each culture vessel is a closed system, the above-described concentration control is performed for oxygen and carbon dioxide in each culture vessel.

[0064] By driving the suction unit 8, the first suction tube 4a sucks the culture solution W from the first culture vessel 2a. The culture solution W sucked from the first culture vessel 2a moves through the first suction tube 4a to the second culture vessel 2b. Also, by driving the suction unit 8, the second suction tube 4b sucks the culture solution W from the second culture vessel 2b. The culture solution W sucked from the second culture vessel 2b moves through the second suction tube 4b to the third culture vessel 2c. Also, by driving the suction unit 8, the third suction tube 4c sucks the culture solution W from the third culture vessel 2c. The culture solution W sucked from the third culture vessel 2c flows into the control unit 10 through the third suction tube 4c. The culture solution W that has passed through the control unit 10 returns to the first culture vessel 2a through the discharge tube 12. Note that the arrangements of the suction unit 8 and the control unit 10 can be changed as appropriate. For example, the suction unit 8 and the control unit 10 may be provided in the first suction tube 4a or the second suction tube 4b.

[0065] The first suction tube 4a, the second suction tube 4b, and the third suction tube 4c have different diameters from each other, at least in the flow rate adjustment region. Therefore, by driving the suction unit 8, different suction flow rates (or linear velocities) are generated in each suction tube 4. For example, the suction tube 4 located on the upstream side of the flow of the culture solution W has a smaller diameter. That is, the diameter of the first suction tube 4a is the smallest, the diameter of the second suction tube 4b is intermediate, and the diameter of the third suction tube 4c is the largest. Therefore, the fastest suction flow rate is generated in the first suction tube 4a, an intermediate suction flow rate is generated in the second suction tube 4b, and the slowest suction flow rate is generated in the third suction tube 4c. Thereby, the first suction tube 4a can suck aggregates S with a larger particle size than the second suction tube 4b and the third suction tube 4c. Also, the second suction tube 4b can suck aggregates S with a larger particle size than the third suction tube 4c.

[0066] Therefore, aggregates S with a relatively large particle size gather in the first culture vessel 2a, aggregates S with a relatively intermediate particle size gather in the second culture vessel 2b, and aggregates S with a relatively small particle size gather in the third culture vessel 2c. The particle size of the aggregates S to be aggregated in each culture vessel 2 can be appropriately changed by adjusting the diameter of each suction tube 4, the output of the suction device constituting the suction unit 8, and the like.

[0067] That is, the culture device 1 of the present embodiment has a plurality of combinations of the culture vessels 2 and the suction tubes 4. These plurality of combinations are connected in series with each other. Also, the diameters of the respective suction tubes 4 are different from each other. Then, by driving the suction unit 8, the culture solution W is sucked by each suction tube 4 and sequentially moves from the upstream culture vessel 2 to the downstream culture vessel 2. Thereby, aggregates S having a particle size corresponding to the diameter of the corresponding suction tube 4 can be aggregated in each of the plurality of culture vessels 2. Therefore, the efficiency of cell culture can be further improved. Note that the combination of the culture vessel 2 and the suction tube 4 may be two sets or four or more sets.

[0068] (Embodiment 4) Embodiment 4 has the same configuration as Embodiment 1 except for including a recovery unit constituted by the suction tube 4. Hereinafter, the configuration different from that of Embodiment 1 in this embodiment will be mainly described, and the common configuration will be briefly described or the description will be omitted. FIG. 11 is a schematic diagram of the culture apparatus 1 according to Embodiment 4. The culture apparatus 1 of this embodiment includes a culture vessel 2, a suction unit 8, a management unit 10, a discharge tube 12, a control unit 14, a connection pipe 26, and a recovery unit 28 (trap chamber).

[0069] The connection pipe 26 connects the culture vessel 2 and the recovery unit 28. One end of the connection pipe 26 is inserted into the culture vessel 2. The culture vessel 2 of this embodiment may be a closed system in which the atmosphere does not substantially enter from the outside, or may be an open system in which the atmosphere can enter from the outside. When the culture vessel 2 is a closed system, the above-mentioned concentration control is performed on oxygen and carbon dioxide in the culture vessel 2. When the culture vessel 2 is an open system, the above-mentioned concentration control is not essential, but it is preferable to implement it. The other end of the connection pipe 26 is connected to the inlet port of the recovery unit 28. One end of the discharge tube 12 is connected to the outlet port of the recovery unit 28. The other end of the discharge tube 12 is inserted into the culture vessel 2. The management unit 10 is provided in the middle of the connection pipe 26. The suction unit 8 is provided in the middle of the discharge tube 12. Further, an auxiliary filter 6 is provided on the downstream side of the suction unit 8 in the discharge tube 12.

[0070] By driving the suction unit 8, the culture solution W in the culture vessel 2 moves through the connection pipe 26 to the recovery unit 28. In this process, the management unit 10 performs regeneration processing and the like on the culture solution W. The culture solution W that has reached the recovery unit 28 is subjected to a recovery process of aggregates S described later in the recovery unit 28. Then, the culture solution W is returned to the culture vessel 2 through the discharge tube 12. Note that the arrangements of the suction unit 8 and the management unit 10 can be changed as appropriate. For example, the suction unit 8 may be provided in the connection pipe 26, or the management unit 10 may be provided in the discharge tube 12.

[0071] However, it is preferable that the suction unit 8 is provided on the downstream side of the recovery unit 28, that is, in the discharge pipe 12. Thereby, it is possible to avoid the aggregate S from passing through the suction unit 8. In the flow path structure where the aggregate S passes through the suction unit 8, there is a possibility that some of the aggregates S may be destroyed when passing through the suction unit 8. On the other hand, by arranging the suction unit 8 on the downstream side of the recovery unit 28, it is possible to avoid the destruction of the aggregate S by the suction unit 8. Alternatively, in the flow path structure where the aggregate S passes through the suction unit 8, measures may be required to prevent the destruction of the aggregate S by the suction unit 8, but by arranging the suction unit 8 on the downstream side of the recovery unit 28, such measures can be omitted.

[0072] The recovery unit 28 has a suction pipe 4. The recovery unit 28 of the present embodiment has a first suction pipe 4a, a second suction pipe 4b, and a third suction pipe 4c. Each suction pipe 4 has its upper end and lower end closed by caps in a state where its posture is determined to extend in the vertical direction. Further, an inlet port 30 is provided near the lower end on the side surface of each suction pipe 4, and an outlet port 32 is provided near the upper end. Therefore, the outlet port 32 is arranged above the inlet port 30. The other end of the connection pipe 26 is connected to the inlet port 30 of the first suction pipe 4a. Therefore, the inlet port 30 of the first suction pipe 4a corresponds to the inlet port of the recovery unit 28. The outlet port 32 of the first suction pipe 4a is connected to the inlet port 30 of the second suction pipe 4b via a pipe. The outlet port 32 of the second suction pipe 4b is connected to the inlet port 30 of the third suction pipe 4c via a pipe. One end of the discharge pipe 12 is connected to the outlet port 32 of the third suction pipe 4c. Therefore, the outlet port 32 of the third suction pipe 4c corresponds to the outlet port of the recovery unit 28.

[0073] Similar to Embodiment 3, the first suction pipe 4a, the second suction pipe 4b, and the third suction pipe 4c have different diameters at least in the flow rate adjustment region. Therefore, different suction flow rates are generated in each suction pipe 4 due to the driving of the suction unit 8. For example, the suction pipe 4 located on the upstream side of the flow of the culture solution W has a smaller diameter. That is, the diameter of the first suction pipe 4a is the smallest, the diameter of the second suction pipe 4b is intermediate, and the diameter of the third suction pipe 4c is the largest. Therefore, the fastest suction flow rate is generated in the first suction pipe 4a, the intermediate suction flow rate is generated in the second suction pipe 4b, and the slowest suction flow rate is generated in the third suction pipe 4c. As a result, in the first suction pipe 4a, the aggregates S with relatively intermediate particle sizes and the aggregates S with relatively small particle sizes are sucked up to the outlet port 32 together with the culture solution W and sent to the second suction pipe 4b. Also, in the second suction pipe 4b, the aggregates S with relatively small particle sizes are sucked up to the outlet port 32 together with the culture solution W and sent to the third suction pipe 4c. In the third suction pipe 4c, even the aggregates S with relatively small particle sizes are not sucked up to the outlet port 32, and only the culture solution W flows into the discharge pipe 12.

[0074] For this reason, the aggregates S with relatively large particle sizes are trapped at the bottom of the first suction pipe 4a, the aggregates S with relatively intermediate particle sizes are trapped at the bottom of the second suction pipe 4b, and the aggregates S with relatively small particle sizes are trapped at the bottom of the third suction pipe 4c. The particle size of the aggregates S to be trapped in each suction pipe 4 can be appropriately changed by adjusting the diameter of each suction pipe 4, the output of the suction device constituting the suction unit 8, and the like.

[0075] That is, the culture device 1 of the present embodiment includes a recovery unit 28 including the suction pipe 4. The suction pipe 4 has an inlet port 30 and an outlet port 32 disposed above the inlet port 30, and the inlet port 30 is connected to the culture vessel 2. Then, by driving the suction unit 8, the culture solution W is sucked by the suction pipe 4, flows into the suction pipe 4 from the inlet port 30, rises in the suction pipe 4, and is discharged from the outlet port 32. As a result, the aggregates S with particle sizes corresponding to the pipe diameter are recovered in the suction pipe 4.

[0076] Furthermore, the recovery unit 28 of the present embodiment includes a plurality of suction tubes 4 having different pipe diameters. One outlet port 32 and the other inlet port 30 in two adjacent suction tubes 4 are connected, and the plurality of suction tubes 4 are connected in series with each other. Also, the inlet port 30 of the most upstream suction tube 4 is connected to the culture vessel 2. Then, by driving the suction unit 8, the culture solution W is sucked by each suction tube 4 from the upstream suction tube 4 to the downstream suction tube 4 and moves in order. The culture solution W rises in each suction tube 4 from the inlet port to the outlet port and moves to the downstream suction tube 4. As a result, aggregates S having a particle size corresponding to the pipe diameter are collected in each suction tube 4. Thereby, aggregates S having a desired particle size can be recovered more easily, and the efficiency of cell culture can be further improved. Note that the number of suction tubes 4 provided in the recovery unit 28 is not particularly limited and may be one or more.

[0077] As described above, the embodiments of the present invention have been described in detail. The above-described embodiments are merely specific examples for carrying out the present invention. The content of the embodiments does not limit the technical scope of the present invention, and many design changes such as changes, additions, and deletions of components are possible without departing from the idea of the invention defined in the claims. The new embodiments to which design changes are applied have the effects of the combined embodiments and the modifications respectively. In the above-described embodiments, with regard to the content in which such design changes are possible, notations such as "in the present embodiment" and "in the present embodiment" are added and emphasized, but design changes are also allowed for the content without such notations. Any combination of the above components is also effective as an aspect of the present invention.

[0078] The embodiment may be specified by the items described below. (Item 1) A culture vessel (2) for accommodating cell aggregates (S) and a culture solution (W); A suction tube (4) for sucking the culture solution (W) in the culture vessel (2); A suction unit (8) for generating a suction force in the suction tube (4); A control unit (14) that controls the suction unit (8) according to the antagonistic suction flow rate of the aggregate (S), which is the rate that antagonizes the sedimentation of the aggregate (S) and the floating of the aggregate (S) due to the suction of the culture solution (W). Culturing apparatus (1). (Item 2) The antagonistic suction flow rate is determined based on the sedimentation rate prediction curve of the aggregate (S) obtained from the above formula (1), the above formula (2), the above formula (3), and the above formula (4). The culturing apparatus (1) according to Item 1. (Item 3) The control unit (14) controls the suction unit (8) to suck the culture solution (W) at a flow rate below the antagonistic suction flow rate. The culturing apparatus (1) according to Item 1 or Item 2. (Item 4) The control unit (14) controls the suction unit (8) to suck the culture solution (W) at a first flow rate that exceeds the antagonistic suction flow rate for the aggregate (S) of a predetermined first particle size and is below the antagonistic suction flow rate for the aggregate (S) of a second particle size larger than the first particle size. The culturing apparatus (1) according to Item 1 or Item 2. (Item 5) After sucking the culture solution (W) at the first flow rate, the control unit (14) controls the suction unit (8) to suck the culture solution (W) at a second flow rate that exceeds the antagonistic suction flow rate for the aggregate (S) of the second particle size. The culturing apparatus (1) according to Item 4. (Item 6) Comprising a management unit (10) connected to the suction pipe (4) and performing at least one of analysis and adjustment of the components of the sucked culture solution (W). The culturing apparatus (1) according to any one of Items 1 to 5. (Item 7) Having a plurality of combinations of culture vessels (2a, 2b, 2c) and suction pipes (4a, 4b, 4c). The plurality of combinations are connected to each other. The pipe diameters of the respective suction pipes (4a, 4b, 4c) are different from each other. By driving the suction unit (8), the culture solution (W) is sucked by each suction pipe (4a, 4b, 4c) from the upstream culture vessel (2a) to the downstream culture vessel (2c) and moves in order. The culture apparatus (1) according to any one of Items 1 to 6. (Item 8) A recovery unit (28) for aggregates (S) including a suction pipe (4) is provided. The suction pipe (4) has an inlet port (30) and an outlet port (32) disposed above the inlet port (30). The inlet port (30) is connected to the culture vessel (2). By driving the suction unit (8), the culture solution (W) is sucked by the suction pipe (4), flows into the suction pipe (4) from the inlet port (30), rises in the suction pipe (4), and is discharged from the outlet port (32). The suction pipe (4) recovers aggregates (S) having a particle size corresponding to the pipe diameter. The culture apparatus (1) according to any one of Items 1 to 6. (Item 9) The recovery unit (28) includes a plurality of suction pipes (4a, 4b, 4c) having different pipe diameters. One outlet port (32) and the other inlet port (30) in two adjacent suction pipes (4a, 4b, 4c) are connected, and the plurality of suction pipes (4a, 4b, 4c) are connected to each other. The inlet port (30) of the most upstream suction pipe (4a) is connected to the culture vessel (2). By driving the suction unit (8), the culture solution (W) is sucked by each suction pipe (4a, 4b, 4c) from the upstream suction pipe (4a) to the downstream suction pipe (4c) and moves in order. Each suction pipe (4a, 4b, 4c) recovers aggregates (S) having a particle size corresponding to its respective pipe diameter. The culture apparatus (1) according to Item 8. (Item 10) From the culture vessel (2) containing cell aggregates (S) and the culture solution (W), sucking the culture solution (W) at a flow rate corresponding to the antagonistic suction flow rate of the aggregates (S), which is a rate at which the sedimentation of the aggregates (S) and the floating of the aggregates (S) accompanying the suction of the culture solution (W) are antagonistic. Culture method. (Item 11) The antagonistic suction flow rate is the sedimentation of the aggregate (S) obtained from the above formula (1), the above formula (2), the above formula (3), and the above formula (4). speed Determined based on the predicted curve The culture method according to item 10.

Example

[0079] Hereinafter, examples of the present invention will be described. However, the examples are merely illustrative for preferably explaining the present invention and do not limit the present invention in any way.

[0080] [Cell culture test] (Comparative Example 1) Using a known culture vessel with a suction tube, the stirring suspension culture of mNPC was carried out at an initial cell density of 1×10 5 cells / mL. The inner diameter of the suction tube was φ10. On the second day of culture when the particle size of many aggregates became 200 μm or more, a filter with a pore diameter of 100 μm was installed at the tip of the suction tube. Then, the suction of the culture solution at a flow rate of 1 mL / min was started, and the culture was continued while returning the suctioned culture solution to the culture vessel. As shown in FIG. 8, when the inner diameter of the suction tube is φ10, when the flow rate is 2.2 mL / min, the flow rate of the culture solution becomes the antagonistic suction flow rate of the aggregate with a particle size of 200 μm. Therefore, in Comparative Example 1 where the flow rate is 1 mL / min, the flow rate of the culture solution is lower than the antagonistic suction flow rate.

[0081] On the fourth day of culture, container since the growth and proliferation of the aggregates in the culture could no longer be confirmed, the culture was stopped and the cells were observed. FIG. 12(A) is an optical microscope image of the culture solution according to Comparative Example 1. FIG. 12(B) is an optical microscope image of the filter according to Comparative Example 1. As shown in FIG. 12(A), in Comparative Example 1, the number of aggregates floating in the culture solution was small. The cell density in the culture vessel was 9.4×104 cells / mL. Also, as shown in FIG. 12(B), many aggregates adhered to the filter.

[0082] In Comparative Example 1, although the flow rate of the culture solution was lower than the counteracting suction flow rate, the filter was clogged by aggregates. This was because a filter was installed at the tip of the suction tube. That is, it is considered that the aggregates riding on the flow of the culture solution due to stirring came into contact with the filter and were captured. Thus, when the filter is exposed in the culture vessel and in contact with the culture solution, even if the flow rate of the culture solution is lower than the counteracting suction flow rate, the aggregates can be captured by the filter and the survival rate of the cells can be significantly reduced.

[0083] (Example 1) Using the culture apparatus shown in FIG. 1, stirring suspension culture of mNPC was carried out at an initial cell density of 1×10 5 cells / mL. The inner diameter of the suction tube was φ4. From the 5th day of culture when the particle size of many aggregates became 400 μm or more, suction of the culture solution at a flow rate of 1 mL / min was started, and the culture was continued while returning the aspirated culture solution to the culture vessel. As shown in FIG. 8, when the inner diameter of the suction tube is φ4, when the flow rate is 1.1 mL / min, the flow rate of the culture solution becomes the counteracting suction flow rate of aggregates with a particle size of 400 μm. Therefore, in Example 1 where the flow rate is 1 mL / min, the flow rate of the culture solution is lower than the counteracting suction flow rate. In addition, in order to prevent suction of aggregates and debris with a size less than 400 μm, a mesh filter with a pore size of 100 μm was installed at the upper part inside the suction tube.

[0084] FIG. 12(C) is an optical microscope image of the culture solution according to Example 1. As shown in FIG. 12(C), in Example 1, no decrease in aggregates was observed compared to Comparative Example 1. Also, the culture was continued with the target number of days set to 8 days. As a result, it was confirmed that the aggregates could be cultured in a good state for at least 8 days. The cell density in the culture vessel on the 8th day of culture was 4.1×10 6 cells / mL. Also, the survival rate of the cells was 91%. From the above, it can be understood that according to the culture apparatus and culture method according to the embodiment, the culture solution can be aspirated without aspirating the aggregates of the culture target, and thus the efficiency of cell culture is improved.

[0085] [Aggregate removal test] In this test, it was verified that the culture apparatus and culture method according to the embodiment can remove aggregates of a specific particle size from aggregates of various particle sizes in the culture solution. That is, using the culture apparatus shown in FIG. 1, the stirring suspension culture of mNPC was carried out at an initial cell density of 1×10 5 cells / mL. The culture period was 8 days. For the replacement of the culture solution, after stopping the stirring of the culture solution and confirming that the aggregates had settled, the supernatant was aspirated and fresh culture solution was added. The replacement of the culture solution was carried out every day.

[0086] After 8 days of stirring suspension culture, a suction tube with a diameter of φ10 was installed in the culture apparatus. In order to remove aggregates and debris with a particle size smaller than the target particle size, a mesh filter with a pore size of 100 μm was installed at the upper part inside the suction tube. The suction of the culture solution was started at a flow rate of 8.0 mL / min, and the perfusion treatment of returning the aspirated culture solution to the culture vessel was continued for 6 hours. As shown in FIG. 8, when the diameter of the suction tube is φ10 and the flow rate is 8.0 mL / min, the flow velocity of the culture solution becomes the counteracting suction flow velocity of aggregates with a particle size of 450 μm. Therefore, it is expected that aggregates with a particle size of less than 450 μm will be aspirated by the suction tube and removed by the mesh filter.

[0087] Also, after the perfusion treatment at a flow rate of 8.0 mL / min for 6 hours was completed, the flow rate was increased to 14.1 mL / min, and the perfusion treatment was continued for 6 hours. As shown in FIG. 8, when the diameter of the suction tube is φ10 and the flow rate is 14.1 mL / min, the flow velocity of the culture solution becomes the counteracting suction flow velocity of aggregates with a particle size of 650 μm. Therefore, it is expected that aggregates with a particle size of less than 650 μm will be aspirated by the suction tube and removed by the mesh filter.

[0088] After the stirring suspension culture, after the perfusion treatment at a flow rate of 8.0 mL / min, and after the perfusion treatment at a flow rate of 14.1 mL / min, the culture solution was imaged with an optical microscope (BZ-X500, Keyence Corporation) to obtain an optical microscope image. Then, using measurement software (BZ-X Analyzer, Keyence Corporation), the particle size of each aggregate shown in the optical microscope image was measured to obtain the particle size distribution of the aggregates. The results are shown in FIG. 13.

[0089] Figure 13 is a diagram showing the particle size distribution of aggregates. "A" in Figure 13 is the result after agitation suspension culture. "B" in Figure 13 is the result after perfusion treatment at a flow rate of 8.0 mL / min. "C" in Figure 13 is the result after perfusion treatment at a flow rate of 14.1 mL / min. As shown in Figure 13, after agitation suspension culture, the proportion of aggregates with a particle size of less than 450 μm was 15.3%, and the proportion of aggregates with a particle size of 450 μm or more was 84.7%. On the other hand, after perfusion treatment at a flow rate of 8.0 mL / min, the proportion of aggregates with a particle size of less than 450 μm was 2.1%, and the proportion of aggregates with a particle size of 450 μm or more was 97.9%.

[0090] Also, after perfusion treatment at a flow rate of 8.0 mL / min, the proportion of aggregates with a particle size of less than 650 μm was 52.6%, and the proportion of aggregates with a particle size of 650 μm or more was 47.4%. On the other hand, after perfusion treatment at a flow rate of 14.1 mL / min, the proportion of aggregates with a particle size of less than 650 μm was 10.8%, and the proportion of aggregates with a particle size of 650 μm or more was 89.2%. From the above, according to the culture apparatus and culture method according to the embodiment, it was confirmed that aggregates having a particle size less than the target particle size can be sucked and removed from an aggregate population having various particle sizes. Thus, it can be understood that the yield of aggregates having a desired particle size can be increased.

[0091] [Aggregate Recovery Test] In this test, it was verified that specific-sized aggregates can be recovered from aggregates of various particle sizes in the culture solution by the culture apparatus and culture method according to the embodiment. That is, using the culture apparatus shown in Figure 1, agitation suspension culture of mNPC was carried out at an initial cell density of 1×10 5 cells / mL. The culture period was 8 days. For the replacement of the culture solution, after confirming that the aggregates had settled by stopping the agitation of the culture solution, the supernatant was sucked and fresh culture solution was added. The replacement of the culture solution was carried out daily.

[0092] After the 8-day agitation suspension culture was completed, a suction tube with a diameter of φ10 was installed in the culture device. In order to remove aggregates and debris with a particle size smaller than the target particle size, a mesh filter with a pore size of 100 μm was installed at the upper part inside the suction tube. Suction of the culture solution at a flow rate of 6.7 mL / min was started, and the perfusion process of returning the aspirated culture solution to the culture vessel was continued for 10 hours. As shown in Figure 8, when the diameter of the suction tube is φ10 and the flow rate is 6.7 mL / min, the flow velocity of the culture solution becomes the antagonistic suction flow velocity of aggregates with a particle size of 400 μm. Therefore, it is expected that aggregates smaller than 400 μm will be aspirated by the suction tube and removed by the mesh filter. When the mesh filter was checked after the perfusion process, minute aggregates and debris were deposited on the surface of the mesh filter.

[0093] After the 10-hour perfusion process at a flow rate of 6.7 mL / min was completed, a new suction tube with a diameter of φ10 was installed in the culture device. In addition, a circulation flow path for returning the culture solution to the culture vessel was connected to this suction tube. A recovery suction tube was provided in the middle of the circulation flow path as an aggregate recovery section. The recovery suction tube is provided with an inlet port and an outlet port on the side surface, and the diameter is φ20. Suction of the culture solution at a flow rate of 15.7 mL / min was started, and the perfusion process of returning the aspirated culture solution to the culture vessel was continued for 12 hours.

[0094] As shown in Figure 8, when the diameter of the suction tube is φ10 and the flow rate is 15.7 mL / min, the flow velocity of the culture solution becomes the antagonistic suction flow velocity of aggregates with a particle size of 700 μm. Therefore, it is expected that aggregates smaller than 700 μm will be aspirated by the suction tube and flow into the recovery section. In addition, the suction tube with a diameter of φ20 has a cross-sectional area 4 times that of the suction tube with a diameter of φ10. Therefore, the flow velocity of the culture solution decreases by 1 / 4. In this case, the flow velocity of the culture solution is lower than the antagonistic suction flow velocity of the aggregates flowing into the recovery suction tube. Therefore, it is expected that aggregates with a particle size of 400 μm or more and less than 700 μm will settle at the bottom of the recovery suction tube.

[0095] After the 12-hour perfusion treatment, a part of the aggregates was collected from the bottom of the recovery suction tube into a 24-well plate and imaged with an optical microscope (BZ-X500, Keyence Corporation) to obtain an optical microscope image. Fig. 14 is an optical microscope image of the aggregates deposited at the bottom of the recovery suction tube. As shown in Fig. 14, it was confirmed that a large number of aggregates with the assumed particle size were deposited at the bottom of the recovery suction tube. From the above, it was confirmed that according to the culture apparatus and the culture method according to the embodiment, aggregates with the target particle size can be recovered from a population of aggregates with various particle sizes. Thus, it can be understood that the yield of aggregates having a desired particle size can be increased.

Industrial Applicability

[0096] The present invention can be used in a culture apparatus and a culture method.

Explanation of Signs

[0097] 1 Culture apparatus, 2 Culture vessel, 4 Suction tube, 8 Suction part, 10 Management part, 14 Control part, S Aggregate, W Culture solution.

Claims

1. A culture vessel containing cell aggregates and a culture solution, A suction tube for sucking the culture solution in the culture vessel, A suction unit that generates a suction force in the suction tube, A control unit that controls the suction unit according to the antagonistic suction flow rate of the aggregate, which is a rate that antagonizes the sedimentation of the aggregate and the floating of the aggregate accompanying the suction of the culture solution, The control unit controls the suction unit to suck the culture solution at a flow rate equal to or lower than the antagonistic suction flow rate of at least some of the aggregates, The antagonistic suction flow rate is represented by the following formula (1): 【Number 1】 [In formula (1), W* is the dimensionless sedimentation velocity, ws is the sedimentation velocity of the aggregate [m / s], s is the specific gravity given by ρs / ρw, ρs is the density of the aggregate [kg / m3], ρw is the density of the culture solution [kg / m3], g is the gravitational acceleration [m / s2], and d is the particle diameter of the aggregate [m]], The following formula (2): 【Number 2】 [In formula (2), S* is the dimensionless particle parameter, ν is the kinematic viscosity of the culture solution given by μ / ρw [m2 / s], μ is the viscosity of the culture solution [Pa·s], ρw is the density of the culture solution [kg / m3], and d, s, and g are the same as in formula (1)], The following formula (3): 【Number 3】 [In formula (3), W* is the same as in formula (1), S* is the same as in formula (2), and A and B are constants determined from a straight line obtained by plotting the values for each particle diameter of the aggregate in a coordinate system with 1 / W* on the first axis and 1 / S* on the second axis], and, The following formula (4): 【Number 4】 [In formula (4), ws, s, g, d are the same as in formula (1), ν is the same as in formula (2), and A and B are the same as in formula (3)], taking a value according to the sedimentation velocity prediction curve of the aggregate obtained from the above, A culture device.

2. The control unit controls the suction unit to suck the culture solution at a first flow rate that exceeds the antagonistic suction flow rate for the aggregate of a predetermined first particle diameter and is equal to or lower than the antagonistic suction flow rate for the aggregate of a second particle diameter larger than the first particle diameter, The culture device according to claim 1.

3. After sucking the culture solution at the first flow rate, the control unit controls the suction unit to suck the culture solution at a second flow rate that exceeds the antagonistic suction flow rate for the aggregate of the second particle diameter, The culture device according to claim 2.

4. Comprising a management unit connected to the suction tube and performing at least one of analysis and adjustment of the components of the sucked culture solution, The culture device according to claim 1 or 2.

5. Having a plurality of combinations of the culture vessel and the suction tube, The plurality of combinations are connected to each other, The diameters of the respective suction tubes are different from each other, By driving the suction unit, the culture solution is sucked by each suction tube from the culture vessel on the upstream side to the culture vessel on the downstream side and moves in order, The culture device according to claim 1 or 2.

6. Comprising a recovery unit for the aggregate including the suction tube, The suction tube has an inlet port and an outlet port disposed above the inlet port, and the inlet port is connected to the culture vessel, By driving the suction unit, the culture solution is sucked by the suction tube, flows into the suction tube from the inlet port, rises in the suction tube, and is discharged from the outlet port, and the suction tube recovers the aggregate having a particle size corresponding to the tube diameter, The culture device according to claim 1 or 2.

7. The recovery unit includes a plurality of the suction tubes having different diameters, One outlet port and the other inlet port in two adjacent suction tubes are connected, and the plurality of suction tubes are connected to each other. The inlet port of the most upstream suction tube is connected to the culture vessel, By driving the suction unit, the culture solution is sucked by each suction tube from the suction tube on the upstream side to the suction tube on the downstream side and moves in order, and each suction tube recovers the aggregate having a particle size corresponding to its own tube diameter, The culture device according to claim 6.

8. Including sucking the culture solution at a flow rate corresponding to the antagonistic suction flow rate of the aggregate, which is a rate at which the sedimentation of the aggregate and the floating of the aggregate accompanying the suction of the culture solution are antagonistic, from a culture vessel containing the aggregate of cells and the culture solution, In the suction of the culture solution, including sucking the culture solution at a flow rate equal to or lower than the antagonistic suction flow rate of at least a part of the aggregate, The antagonistic suction flow rate is given by the following formula (1) 【Number 5】 [In formula (1), W* is the dimensionless sedimentation velocity, ws is the sedimentation velocity of the aggregate [m / s], s is the specific gravity given by ρs / ρw, ρs is the density of the aggregate [kg / m3], ρw is the density of the culture solution [kg / m3], g is the acceleration due to gravity [m / s2], and d is the particle diameter of the aggregate [m]]. The following formula (2) 【Number 6】 [In formula (2), S* is a dimensionless particle parameter, ν is the kinematic viscosity [m2 / s] of the culture solution given by μ / ρw, μ is the viscosity [Pa·s] of the culture solution, ρw is the density [kg / m3] of the culture solution, and d, s, and g are the same as in formula (1).], The following formula (3) 【Number 7】 [In formula (3), W* is the same as in formula (1), S* is the same as in formula (2), and A and B are constants determined from a straight line obtained by plotting the values for each particle size of the aggregate on a coordinate system with 1 / W* set as the first axis and 1 / S* set as the second axis.], and, The following formula (4) 【Number 8】 [In formula (4), ws, s, g, d are the same as in formula (1), ν is the same as in formula (2), and A and B are the same as in formula (3).] Taking a value corresponding to the sedimentation velocity prediction curve of the aggregate obtained from A culturing method.

9. A culture vessel containing cell aggregates and a culture solution, A suction tube for sucking the culture solution in the culture vessel, A suction unit for generating a suction force in the suction tube, A control unit for controlling the suction unit according to the antagonistic suction flow rate of the aggregate, which is a rate that antagonizes the sedimentation of the aggregate and the floating of the aggregate accompanying the suction of the culture solution, The control unit controls the suction unit to suck the culture solution at a flow rate equal to or lower than the antagonistic suction flow rate of at least some of the aggregates, The antagonistic suction flow rate takes a value according to a linear approximation formula obtained by plotting the measurement results of the sedimentation rate and the antagonistic suction flow rate of the aggregates of various particle sizes on a coordinate system with the sedimentation rate of the aggregate set as the first axis and the antagonistic suction flow rate set as the second axis and performing linear approximation. A culture apparatus.

10. Including sucking the culture solution from a culture vessel containing cell aggregates and a culture solution at a flow rate corresponding to the antagonistic suction flow rate of the aggregate, which is a rate that antagonizes the sedimentation of the aggregate and the floating of the aggregate accompanying the suction of the culture solution, In the suction of the culture solution, including sucking the culture solution at a flow rate equal to or lower than the antagonistic suction flow rate of at least some of the aggregates, The antagonistic suction flow rate takes a value according to a linear approximation formula obtained by plotting the measurement results of the sedimentation rate and the antagonistic suction flow rate of the aggregates of various particle sizes on a coordinate system with the sedimentation rate of the aggregate set as the first axis and the antagonistic suction flow rate set as the second axis and performing linear approximation. A culturing method.

Images